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

Abstract

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

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 587,164, filed October 2, 2023, and U.S. Provisional Application No. 63 / 623,697, filed January 22, 2024, the contents of which are incorporated herein by reference in their entirety.

[0002] References to sequence lists submitted electronically This application contains a computer-readable sequence list submitted with it in XML file format, the entire contents of which are incorporated herein by reference. The sequence list XML file submitted with this application is named "12656-206-228_SEQLISTING.xml", was created on September 23, 2024, and has a size of 93,406 bytes.

[0003] 1. Introduction This disclosure provides methods and formulations for treating or preventing hearing loss associated with mucopolysaccharidosis II (MPS II) using recombinant human iduronate-2-sulfatase (IDS). 2. Background Technology Hunter syndrome / MPS II is a rare X-linked recessive genetic disorder affecting 0.5 to 1.3 out of every 100,000 male live births. This progressive and devastating disease is caused by a mutation in the IDS gene, resulting in a deficiency of the lysosomal storage enzyme iduronate-2-sulfatase (I2S), an enzyme required for the lysosomal catabolism of heparan sulfate and dermatan sulfate. Due to the absence or near-absence of I2S, this protein is unable to perform its normal exolytic lysosomal hydrolase function, leading to the accumulation of ubiquitous polysaccharides (GAGs) in the tissues and organs of MPS II patients, resulting in characteristic storage lesions and a variety of disease sequelae. Morbidity and mortality are high in this patient population; the median age of death has been reported to be 11.7 years for those with the severe phenotype (characterized by neurocognitive deterioration) and 21.7 years for those with the 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). It has been reported that most (two-thirds) of patients 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(1):E37-E46). Although the disease primarily affects boys, it has been reported that females are also affected due to non-random X inactivation and / or mutations in both alleles of the gene. (Martin et al., 2008, Recognition and diagnosis of mucopolysaccharidosis II (Hunter Syndrome). Pediatrics 121:e377).

[0005] Patients with MPS II appear normal at birth, but the signs and symptoms of the disease typically present in a severe form between 18 months and 4 years of age, and in a diminishing form between 4 and 8 years of age. Common signs and symptoms in all affected patients include short stature, coarse facial features, macrocephaly, macroglossia, hearing loss, hepatosplenomegaly, multiple dysplasia of the bone, joint contractures, spinal stenosis, and carpal tunnel syndrome. Most patients frequently experience upper respiratory tract and ear infections, and progressive airway obstruction is common, leading to sleep apnea and often death. Cardiac disease is the leading cause of death in this group, characterized by valvular dysfunction, resulting in left and right ventricular hypertrophy and heart failure. Death is usually attributed to obstructive airway disease or heart failure.

[0006] In severe forms of the disease, early developmental milestones may be reached, but developmental delays become apparent by 18–24 months. Some patients fail hearing screening tests by age one, and other milestones are delayed, including the ability to sit without support, walk, and speak. Developmental progress begins to plateau between 3 and 5 years of age, and is reported to begin to regress around 6.5 years of age. Of the approximately 50% of children with MPS II who are toilet trained, most (if not all) will lose this ability as the disease progresses. (Wraith et al., 2007) Same as above Martin et al., 2008, Same as above ).

[0007] Patients with significant neurological involvement exhibit severe behavioral disorders, including hyperactivity, inertia, and aggression that begin at age two and persist until age eight or nine, even as neurodegeneration diminishes these behaviors. (Muenzer et al., 2009, Mucopolysaccharidosis I: Management and Treatment Guidelines, Pediatric 123(1): 19-29).

[0008] It has been reported that more than half of patients with severe infections over the age of 10 experience seizures, and most patients with CNS involvement die with severe mental disorders and require ongoing care. (Wraith et al., 2007) Same as above Martin et al., 2008, Same as above Although patients with MPS II exhibited normal intellectual function, MRI imaging revealed significant brain abnormalities in all patients, including white matter lesions, ventricular enlargement, and brain atrophy. (Muenzer et al., 2009) Same as above ).

[0009] Enzyme replacement therapy (ERT) using recombinant idoxurase® (Elaprase®, Shire Human Genetic Therapies) produced from HT1080 (fibrosarcoma) cells is the only approved product for the treatment of Hunter syndrome and is administered via weekly infusion. (ELAPRASE (idoxurase) Injection [Product Information]. 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 idoxurase) has been shown to improve the systemic performance of MPS II, weekly ERT infusions over a lifetime increase the burden on patients and their caregivers, thus impacting the patient's quality of life.

[0010] Currently administered ERT cannot cross the blood-brain barrier and therefore cannot address severe cases. Right now This addresses an unmet need in patients with CNS / neurocognitive and behavioral impairment (MPS II). In a recent clinical trial designed to address this issue, an intrathecal drug delivery device implanted in the spine (using an inlet inserted via an incision in the lower rib area, with a catheter inserted at the L4 / L5 level) was used to administer edentulase formulated for intrathecal administration monthly to pediatric patients. Patients also received intravenous edentulase weekly. See Muenzer et al., 2016, Genetics in Med 18: 73-81, especially page 74; abstract available at https: / / www.ncbi.nlm.nih.gov / pubmed / 25834948?dopt=Abstract). Of the 12 patients treated, 6 (50%) underwent partial repair, total surgical repair, or removal due to device malfunction. Notably, 12 of the 14 SAEs (serious adverse events) were device-related (device insertion complications, device dislocation / connection problems, device breakage / malfunction / failure, implantation site infection, procedural pain, and wound dehiscence). (Muenzer et al., 2016, page 75, column 2 and...) Figure 1 The high activity levels of this pediatric population exacerbate device breakage and catheter displacement from the spinal canal (Discussion on page 78, Muenzer et al., 2016). 3. Summary of the Invention This disclosure provides methods and formulations for the prevention or treatment of MPS II-related hearing loss in subjects in need using recombinant human iduronate-2-sulfatase (IDS). In some embodiments, the recombinant human IDS is delivered to the subject via a recombinant vector.

[0012] In one aspect, this disclosure provides a method for preventing mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, 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 no hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

[0013] In another aspect, this disclosure provides a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for the prevention of mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has no hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

[0014] In another aspect, this disclosure provides the use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) in the prevention of mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has no hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

[0015] In some implementations, the therapeutically effective amount of the rAAV prevents 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 application of the rAAV.

[0016] In some embodiments, hearing loss is indicated by an increase in the hearing threshold measured by an auditory brain response (ABR) test in the at least one ear, as before the application of the rAAV, compared to the hearing threshold measured by the ABR test in the at least one ear, as after the application of the rAAV. In some embodiments, the hearing threshold is an estimate of a behavioral threshold (eHL) derived from an ABR threshold (nHL). In some embodiments, the methods or uses disclosed herein further include performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

[0017] In some embodiments, the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by a tympanogram test. In some embodiments, the methods or uses disclosed herein also include performing the tympanogram test on the at least one ear before and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0018] In another aspect, this disclosure provides a method for stabilizing or preventing further hearing loss associated with mucopolysaccharidosis II (MPS II) in a subject of need, 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 experienced hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear.

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

[0020] In another aspect, this disclosure provides the 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)-related hearing loss in a subject in need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has experienced hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear.

[0021] In some implementations, 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 application of the rAAV, the therapeutically effective amount of the rAAV stabilizes the hearing loss or prevents further hearing loss in the at least one ear.

[0022] In some embodiments, stabilizing the hearing loss or preventing further hearing loss is indicated by no change or minimal change in the hearing threshold measured by the auditory brain response (ABR) test in at least one ear before the application of the rAAV, as well as by a hearing threshold measured by the ABR test in at least one ear after the application of the rAAV. In some embodiments, the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL). In some embodiments, stabilizing the hearing loss or preventing further hearing loss is indicated by a change of approximately 10 dB or less in the eHL of at least one ear after the application of the rAAV, compared to before the application of the rAAV.

[0023] In some embodiments, the methods or uses disclosed herein also include performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

[0024] In some embodiments, the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by a tympanogram test. In some embodiments, the methods or uses disclosed herein also include performing the tympanogram test on the at least one ear before and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0025] In another aspect, this disclosure provides a method for improving mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, 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 had hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

[0026] In another aspect, this disclosure provides a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for improving mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

[0027] In another aspect, this disclosure provides the 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)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has had hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

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

[0029] In some embodiments, the improvement in hearing is indicated by a reduction in the hearing threshold measured by an auditory brain response (ABR) test in the at least one ear, as before the application of the rAAV, or as after the application of the rAAV. In some embodiments, the therapeutically effective amount of the rAAV reduces the hearing threshold in the at least one ear by 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 before the application of the rAAV. In some embodiments, the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL). In some embodiments, the improvement in hearing is indicated by a reduction in the eHL of the at least one ear by more than about 10 dB after the application of the rAAV, compared to before the application of the rAAV. In some embodiments, the methods or uses disclosed herein also include performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

[0030] In some embodiments, the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by a tympanogram test. In some embodiments, the methods or uses disclosed herein also include performing the tympanogram test on the at least one ear before and after the administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0031] In some embodiments, the at least one ear includes either the left or right ear. In some embodiments, the at least one ear includes both the left and right ears.

[0032] In some embodiments, the methods or uses disclosed herein further include administering additional treatment to the subject. In some embodiments, the additional treatment is an enzyme replacement therapy comprising recombinant idoxurase. In some embodiments, the additional treatment is a second administration of the rAAV. In some embodiments, the second administration of the rAAV is at a higher dose than the first administration.

[0033] In some embodiments, the rAAV is administered into the subject's cerebrospinal fluid (CSF). In some embodiments, the rAAV is administered via intracisional (IC) administration. In some embodiments, the rAAV is administered via intraventricular (ICV) administration. In some embodiments, the rAAV is administered at a volume not exceeding 10% of the subject's total CSF volume.

[0034] In some implementations, the rAAV is administered intravenously to the subject.

[0035] In some implementations, the rAAV is administered intrathecally to the subject.

[0036] In some embodiments, the rAAV is administered to the subject in solution form, the solution comprising: (a) Sodium chloride at a concentration of approximately 8.77 g / L, (b) Magnesium chloride at a concentration of approximately 0.244 g / L, (c) Potassium chloride at a concentration of approximately 0.224 g / L, (d) Calcium chloride with a concentration of approximately 0.206 g / L. (e) Dextran at a concentration of approximately 0.793 g / L, (f) Poloxamer 188 at a concentration of approximately 0.010 g / L. (g) Sodium dihydrogen phosphate monohydrate with a concentration of approximately 0.0278 g / L, and (h) Anhydrous disodium hydrogen phosphate at a concentration of approximately 0.114 g / L.

[0037] In some implementations, the rAAV is approximately 1.3 × 10⁻⁶. 10 GC / g brain mass, approximately 6.5 × 10⁻⁶ 10 GC / g brain mass or approximately 2 × 10⁻⁶ 11 The dosage of GC / g brain mass, wherein the brain mass is determined by MRI.

[0038] In some implementations, the rAAV is applied to the at least one ear.

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

[0040] In some embodiments, the subject is receiving enzyme replacement therapy (ERT) at the time of administration of the rAAV. In some embodiments, the subject does not respond to the ERT. In some embodiments, the ERT comprises recombinant idoxurase.

[0041] In some implementations, the ERT is interrupted approximately 6 months, approximately 24 weeks, approximately 9 months, approximately 12 months, approximately 52 weeks, approximately 15 months, approximately 18 months, approximately 21 months, or approximately 24 months after the administration of the rAAV.

[0042] In some embodiments, the rAAV is recombinant adeno-associated virus serotype 9. In some 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 β-actin promoter (CB7). In some embodiments, the human IDS expression cassette comprises (i) an IDS transgene flanked by an inverted terminal repeat (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal. In some embodiments, the ITR is an AAV2 ITR. In some embodiments, the human IDS expression cassette comprises a nucleic acid containing the nucleotide sequence of SEQ ID NO: 45.

[0043] In some embodiments, the methods or uses disclosed herein further include administering immunosuppressive therapy to the subject before or simultaneously with the rAAV, and optionally continuing the immunosuppressive therapy thereafter. In some embodiments, the immunosuppressive therapy comprises administering at least one corticosteroid, sirolimus, and / or tacrolimus. In some embodiments, the at least one corticosteroid is methylprednisolone and / or prednisone.

[0044] In some embodiments, the methods or uses disclosed herein further include administering at least one antibiotic to the subject before or simultaneously with the rAAV. In some embodiments, the at least one antibiotic is trimethoprim, sulfamethoxazole, pentanemidine, dapsone, and / or atovaquinone.

[0045] In some embodiments, the methods or uses disclosed herein also include administering at least one antifungal therapy to the subject before or simultaneously with rAAV.

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

[0047] In some 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 β-actin promoter (CB7). In some embodiments, the human IDS expression cassette comprises (i) an IDS transgene flanked by an inverted terminal repeat (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal.

[0048] In some embodiments, the ITR is an AAV2 ITR. In some embodiments, the human IDS expression cassette contains a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45.

[0049] 3.1 Explanatory Implementation Plan 1. A method for preventing mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, 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 no hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

[0050] 2. The method as described in embodiment 1, wherein, 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 application of the rAAV, the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

[0051] 3. The method as described in embodiment 1 or 2, wherein hearing loss is indicated by an increase in hearing threshold measured by the auditory brain response (ABR) test in the at least one ear as before the application of the rAAV, or as after the application of the rAAV.

[0052] 4. The method as described in embodiment 3, wherein the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL).

[0053] 5. The method as described in embodiment 3 or 4, wherein the method further comprises performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

[0054] 6. The method of any one of embodiments 1-5, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanogram testing.

[0055] 7. The method of embodiment 6, wherein the method further comprises performing the tympanogram test on the at least one ear before and after the application of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0056] 8. A method for stabilizing or preventing further hearing loss associated with mucopolysaccharidosis II (MPS II) in a subject in need, 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 experienced hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear.

[0057] 9. The method of embodiment 8, wherein, 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 application of the rAAV, the therapeutically effective amount of the rAAV stabilizes the hearing loss or prevents further hearing loss in the at least one ear.

[0058] 10. The method of embodiment 9, wherein stabilizing the hearing loss or preventing further hearing loss is indicated by no change or minimal change in the hearing threshold measured by the auditory brain response (ABR) test in the at least one ear before the application of the rAAV, as well as by the hearing threshold measured by the ABR test in the at least one ear after the application of the rAAV.

[0059] 11. The method of embodiment 10, wherein the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL).

[0060] 12. The method of embodiment 11, wherein stabilizing the hearing loss or preventing further hearing loss is indicated by a change of about 10 dB or less in the eHL of the at least one ear after the application of the rAAV compared to before the application of the rAAV.

[0061] 13. The method of any one of embodiments 10-12, wherein the method further comprises performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

[0062] 14. The method of any one of embodiments 8-13, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanogram testing.

[0063] 15. The method of embodiment 14, wherein the method further comprises performing the tympanogram test on the at least one ear before and after the application of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0064] 16. A method for improving mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, 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 hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

[0065] 17. The method of embodiment 16, wherein, approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 24 weeks, approximately 6 months, approximately 48 weeks, approximately 52 weeks, or approximately 1 year after administration of the rAAV, the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

[0066] 18. The method of embodiment 16 or 17, wherein the improvement in hearing is indicated by a reduction in the hearing threshold measured by the auditory brain response (ABR) test in the at least one ear as before the application of the rAAV, as after the application of the rAAV.

[0067] 19. The method of embodiment 18, wherein the therapeutically effective amount of the rAAV reduces the hearing threshold of the at least one ear by 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 of the at least one ear before the application of the rAAV.

[0068] 20. The method as described in embodiment 18 or 19, wherein the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL).

[0069] 21. The method of embodiment 20, wherein the improvement in hearing is indicated by a reduction in eHL of at least one ear by more than about 10 dB after application of the rAAV compared to before application of the rAAV.

[0070] 22. The method of any one of embodiments 18-21, wherein the method further comprises performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

[0071] 23. The method of any one of embodiments 8-22, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanogram testing.

[0072] 24. The method of embodiment 23, wherein the method further comprises performing the tympanogram test on the at least one ear before and after the application of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0073] 25. The method of any one of embodiments 1-24, wherein the at least one ear includes the left ear or the right ear.

[0074] 26. The method of any one of embodiments 1-25, wherein the at least one ear comprises a left ear and a right ear.

[0075] 27. The method of any one of embodiments 1-26, wherein the method further comprises administering additional treatment to the subject.

[0076] 28. The method of embodiment 27, wherein the additional treatment is an enzyme replacement therapy comprising recombinant idoxime.

[0077] 29. The method of embodiment 27, wherein the additional treatment is a second application of the rAAV.

[0078] 30. The method of embodiment 29, wherein the second administration of the rAAV is at a higher dose compared to the first administration of the rAAV.

[0079] 31. The method of any one of embodiments 1-30, wherein the rAAV is administered intravenously to the subject or the subject's cerebrospinal fluid (CSF).

[0080] 32. The method of any one of embodiments 1-31, wherein the rAAV is administered via intracranial (IC) administration.

[0081] 33. The method of any one of embodiments 1-31, wherein the rAAV is administered via intraventricular (ICV) administration.

[0082] 34. The method of any one of embodiments 1-33, wherein the rAAV is administered at a volume not exceeding 10% of the total cerebrospinal fluid volume of the subject.

[0083] 35. The method of any one of embodiments 1-34, wherein the rAAV is administered intrathecally to the subject.

[0084] 36. The method of any one of embodiments 1-35, wherein the rAAV is applied to the subject in solution form, the solution comprising: (a) Sodium chloride at a concentration of approximately 8.77 g / L, (b) Magnesium chloride at a concentration of approximately 0.244 g / L, (c) Potassium chloride at a concentration of approximately 0.224 g / L, (d) Calcium chloride with a concentration of approximately 0.206 g / L. (e) Dextran at a concentration of approximately 0.793 g / L, (f) Poloxamer 188 at a concentration of approximately 0.010 g / L. (g) Sodium dihydrogen phosphate monohydrate with a concentration of approximately 0.0278 g / L, and (h) Anhydrous disodium hydrogen phosphate at a concentration of approximately 0.114 g / L.

[0085] 37. The method as described in any one of embodiments 1-36, wherein the rAAV is at approximately 1.3 × 10⁻⁶. 10 GC / g brain mass, approximately 6.5 × 10⁻⁶ 10 GC / g brain mass or approximately 2 × 10⁻⁶ 11 The dosage of GC / g brain mass, wherein the brain mass is determined by MRI.

[0086] 38. The method of any one of embodiments 1-30, wherein the rAAV is applied to the at least one ear.

[0087] 39. The method of any one of embodiments 1-38, wherein the subject is 5 years or older and less than 18 years old.

[0088] 40. The method of any one of embodiments 1-38, wherein the subject is 4 months or older and less than 5 years old.

[0089] 41. The method of any one of embodiments 1-40, wherein the subject is receiving enzyme replacement therapy (ERT) at the time of administration of the rAAV.

[0090] 42. The method of embodiment 41, wherein the subject does not respond to the ERT.

[0091] 43. The method as described in embodiment 41 or 42, wherein the ERT comprises recombinant idoxime.

[0092] 44. The method as described in any one of embodiments 41-43, wherein the ERT is interrupted approximately 6 months, approximately 24 weeks, approximately 9 months, approximately 12 months, approximately 52 weeks, approximately 15 months, approximately 18 months, approximately 21 months, or approximately 24 months after the administration of the rAAV.

[0093] 45. The method of any one of embodiments 1-44, wherein the rAAV is recombinant adeno-associated virus serotype 9.

[0094] 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 β-actin promoter (CB7).

[0095] 47. The method of embodiment 46, wherein the human IDS expression cassette comprises (i) an IDS transgene with a side-mounted inverted terminal repeat (ITR) sequence, (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal.

[0096] 48. The method as described in embodiment 47, wherein the ITR is an AAV2 ITR.

[0097] 49. The method of any one of embodiments 46-48, wherein the human IDS expression cassette comprises a nucleic acid containing the nucleotide sequence of SEQ ID NO: 45.

[0098] 50. The method of any one of embodiments 1-49, further comprising administering immunosuppressive therapy to the subject before or simultaneously with the rAAV, and optionally continuing the immunosuppressive therapy thereafter.

[0099] 51. The method of embodiment 50, wherein the immunosuppressive therapy comprises administration of at least one corticosteroid, sirolimus, and / or tacrolimus.

[0100] 52. The method of embodiment 51, wherein the at least one corticosteroid is methylprednisolone and / or prednisone.

[0101] 53. The method of any one of embodiments 1-52, further comprising administering at least one antibiotic to the subject before or simultaneously with the rAAV.

[0102] 54. The method of embodiment 53, wherein the at least one antibiotic is trimethoprim, sulfamethoxazole, pentanemid, dapsone, and / or atovaquinone.

[0103] 55. The method of any one of embodiments 1-54, further comprising administering at least one antifungal therapy to the subject before or simultaneously with rAAV.

[0104] 56. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for the prevention of mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has no hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

[0105] 57. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for stabilizing mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has experienced hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear.

[0106] 58. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for improving mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

[0107] 59. Use of a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) in the prevention of mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has no hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

[0108] 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)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

[0109] 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)-related hearing loss in a subject in need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has experienced hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear.

[0110] 62. A formulation suitable for application to said ear, comprising a therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS).

[0111] 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 β-actin promoter (CB7).

[0112] 64. The formulation of embodiment 63, wherein the human IDS expression cassette comprises (i) an IDS transgene with a side-mounted inverted terminal repeat (ITR) sequence, (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal.

[0113] 65. The formulation as described in embodiment 64, wherein the ITR is an AAV2 ITR.

[0114] 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. Description of the attached drawings Figure 1 The amino acid sequence of human IDS (SEQ ID NO: 1). C 84 Post-translational formicoglycine modification ( Figure 1 (shown in bold) are required for enzyme activity. Eight N-linked glycosylation sites (N...) 31 N 115 N 144 N 246 N 280 N 325 N 513 and N 537 () is in bold and boxed. A tyrosine-O-sulfation site (Y) is in bold, and the fully sulfated site sequence (PSSEKY) is also in bold. 165 ENTKTCRGPD (SEQ ID NO: 47) is boxed. The N-terminus of the mature 42 kDa and mature 14 kDa peptides are indicated by horizontal arrows. In the brain, the N-terminus of the mature 42 kDa form begins at position 34 or 36: T 34 DALNVLLI (SEQ ID NO: 54); and A 36 LNVLLIIV (SEQ ID NO: 55), such as Figure 1 As shown. See Sleat, 2005, Proteomics 5: 1520-1532, Table S2). Two of the eight N-linked glycosylation sites, namely N... 280 and N 116 In IDS obtained from the human brain, it is mannose-6-phosphorylated. (Sleat et al., 2006, Mol & Cell Proeomics 5.4:686-701, reported in Table V).

[0116] Figure 2Multiple sequence alignment of hIDS with known orthologs. The species names and protein IDs are as follows: SP|P22304|IDS_Human [Homo sapiens]; TR|K6ZGI9_PANTR [Chimpanzee (Pan troglodytes)]; TR|K7BKV4_PANTR [Chimpanzee (Pan troglodytes)]; TR|H9FTX2_MACMU [Rhesus macaque (Macaca mulatta)]; TRF7EJG2_CALJA [White-tufted-ear marmoset (Callithrix jacchus)]; TR|U3DTL8_CALJA [White-tufted-ear marmoset (Callithrix jacchus)]; TR|G7NRX7_MACMU [Rhesus macaque (Macaca mulatta)]; TR|G7Q1V9_MACFA [Crab-eating macaque (Macaca fascicularis)]; TR|H2PX10_PONAB [Sumatran orangutan (Pongoabelii)]; TR|A0A0D9R4D1_CHLSB [Green monkey (Chlorocebus sabaeus)]; TR|G1RST8|G1RST8_NOMLE [Northern white-cheeked gibbon (Nomascus leucogenys)]; UPI0000D9F625 [Rhesus macaque (Macaca fascicularis)] mulatta (Rhesus macaque)]; UPI000274358B (Pan paniscus) (Pygmy chimpanzee; Bonobo)]; UPI00027F6FC5 (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 NO: 27-44).

[0117] Figure 3 MPS II mutations in hIDS and their corresponding disease phenotypes (mild, moderate, or severe). (From Uniprot).

[0118] Figure 4 Millat et al., 1997, Exp. Cell. Res. 230: 362-367. Figure 7 The report states that IDS processing is being carried out by individuals.

[0119] Figure 5 A schematic diagram of builder 1.

[0120] Figure 6 Clustal multiple sequence alignments of AAV capsids 1–9 (SEQ ID NO: 16-26). Amino acid substitutions can be performed on AAV9 and AAV8 capsids by “recruiting” amino acid residues from corresponding positions in other aligned AAV capsids (shown in bold in the bottom row, SEQ ID NO: 48-53). The sequence region specified by “HVR” = hypervariable region.

[0121] Figure 7 Heparin sulfate (HS) is digested using heparinase.

[0122] Figure 8The graph shows the heparin sulfate biomarkers in cerebrospinal fluid (CSF) (ng / mL). The graph shows the sustained reduction of HS in CSF after administration of construct 1. The median change from baseline (N=6) at week 8 was -30.3% (p=0.03, as measured by Wilcoxon signed-rank test). The median change from baseline (N=6) at the last available time point was -35.8% (p=0.03, as measured by Wilcoxon signed-rank test). The graph shows the measurable CSF I2S enzyme concentrations in group 2, ranging from 1170 to 1940 pg / mL, after administration of construct 1.

[0123] Figure 9 The figure shows the cerebrospinal fluid (CSF) D2S6 biomarker (ng / mL). The median change from baseline at week 8 (N=6) was -44.2% (p=0.03, as measured by Wilcoxon signed-rank test). The median change from baseline at the last available time point (N=6) was -39.2% (p=0.03, as measured by Wilcoxon signed-rank test).

[0124] Figures 10A to 10C The chart shows a comparison of neurodevelopmental function at age equivalent (months) versus age (months). Figure 10A The chart shows that, during a follow-up period of more than 6 months, 4 out of 5 patients exhibited sustained cognitive development. Patients 1, 3, and 5 demonstrated sustained cognitive development within the normal range. Patients 2 and 4 exhibited significant cognitive delays at baseline. Patient 2 showed sustained cognitive development. Patient 4 acquired expressive and receptive language skills. Figure 10B and Figure 10C The cognitive development quotient and age equivalent quotient of patients in the construct 1 gene therapy clinical trial are shown respectively. Figure 10B and Figure 10C The charts show that 3 out of 4 patients had cognitive abilities above the normal range (-2SD) at study entry and maintained these abilities during a follow-up period of >6 months. One patient had significant neurocognitive developmental delay at baseline at study entry but showed relative stability after construct 1 administration at a later age (59 months) and continued to acquire expressive and receptive language skills.

[0125] Figures 11A to 11D The chart illustrates the neurodevelopmental functions related to language and motor skills. Figure 11A Demonstrates expressive communication; Figure 11B Demonstrates receptive communication; Figure 11C Demonstrates gross motor skills; and Figure 11DThey demonstrated fine motor skills. The chart shows that patients continued to acquire language and / or motor skills during a follow-up period of more than 6 months.

[0126] Figure 12 The graph illustrates the systemic efficacy obtained 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 administration of construct 1 (normal range (14,706 pg / mL to < 100,000 pg / mL)).

[0127] Figure 13 The chart illustrates the systemic efficacy of ERT by measuring total urinary GAG levels (g / mol CK) in patients. The chart shows a sustained decrease in urinary GAG levels in all patients receiving ERT.

[0128] Figure 14 The graph illustrates the systemic efficacy by measuring total urinary GAG levels (g / mol CK) in patients who did not receive ERT and those who did not receive ERT interruption. The graph shows that urinary GAG levels in patients who did not receive ERT decreased rapidly after administration of construct 1 (with no rebound in urinary GAG after ERT discontinuation).

[0129] Figure 15 Ultrasound of the liver or spleen in patients who did not receive ERT. 24 weeks after construct 1 administration, the liver and spleen sizes of patients who did not receive ERT decreased.

[0130] Figure 16 Proof-of-concept research activities.

[0131] Figure 17 Dosage principle of the first human clinical trial.

[0132] Figure 18 Total heparan sulfate in CSF samples from healthy subjects, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0133] Figures 19A to 19C The chart shows the DOS0 disaccharide (DOS0) levels in healthy subjects, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects. Figure 19A ), D0A6 disaccharide ( Figure 19B ) and D0A0 ( Figure 19C ) level.

[0134] Figure 20The chart shows the levels of D2S6 disaccharide in healthy subjects, MPS I (neuropathic and non-neuropathic) subjects, and MPSII (neuropathic and non-neuropathic) subjects.

[0135] Figure 21 The chart shows the percentage composition of heparan sulfate disaccharide in healthy subjects, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0136] Figure 22 The chart shows the summary of the MPS II Phase 1 / 2 clinical study.

[0137] Figure 23 The chart shows the extensive CNS and systemic biodistribution following IC administration of construct 1 in non-humans. The terms "LD" represent low dose, "IS" represent immunosuppression, and "HD" represent high dose.

[0138] Figures 24A to 24B The chart shows the cerebrospinal fluid (CSF) biomarker heparan sulfate (HS) in subjects during the phase 1 / 2 study. CSF HS measurements showed a dose-dependent reduction in groups 1–3 at weeks 8 and 24 (median shown as...). Figure 24A In the middle, individual participant data is shown in Figure 24B middle).

[0139] Figures 25A to 25B The chart shows D2S6 concentrations in patients during the phase 1 / 2 study. CSF D2S6 measurements showed dose-dependent reductions in groups 1–3 at weeks 8 and 24, with group 3 participants showing near-normal levels (median shown as...). Figure 25A In the middle, individual participant data is shown in Figure 25B middle).

[0140] Figures 26A to 26C The chart shows the cognition of Group 1 patients in the Phase 1 / 2 study ( Figure 26A ), expressive language ( Figure 26B ) and fine motor skills Figure 26C Neurodevelopmental function.

[0141] Figures 27A to 2 7C. Chart, which shows the cognition of patients in group 2 of the phase 1 / 2 study ( Figure 27A ), expressive language ( Figure 27B ) and fine motor (Fig. 27C) neurodevelopmental functions.

[0142] Figures 28A to 28B The chart shows the maladaptive behavior index of patients in the phase 1 / 2 study. Figure 28A) and toilet skills ( Figure 28B ).

[0143] Figures 29A to 29B The chart shows the plasma I2S protein levels in patients from the phase 1 / 2 study. Figure 29A ) and urine GAG ​​levels ( Figure 29B ).

[0144] Figure 30 The chart shows the summary of the MPS I Phase 1 / 2 clinical study.

[0145] Figures 31A to 31B The chart shows the participants in the MPS I Phase 1 / 2 study ( Figure 31A ) and individual participants ( Figure 31B The concentrations of cerebrospinal fluid (CSF) biomarkers and heparin sulfate were shown in the graph. The graph illustrates the concentrations up to the last available time point (e.g., week 24 of the phase 1 / 2 trial). Figure 31A ) and the 59th week of a single participant ( Figure 31B The study showed that most participants and individual participants in the phase 1 / 2 trial had measurable CSF heparin activity.

[0146] Figures 32A to 32F The chart shows the MPS I Phase 1 / 2 study and the single-participant study ( Figures 32D to 32F As cognition () Figure 32A ), expressive language ( Figure 32B ) and fine motor skills Figure 32C BSID-III, a neurodevelopmental function.

[0147] Figure 33 The chart illustrates neurodevelopmental function of BSID cognition in the MPS I single-participant study.

[0148] Figure 34 The table shows the neurodevelopmental function (WASI-II and VABS-III) of 13-year-old participants in the MPS I Phase 1 / 2 study.

[0149] Figures 35A to 35B The chart shows the participants in MPS I Phase 1 / 2 ( Figure 35A ) and single participant studies ( Figure 35B The ISO6 level in ).

[0150] Figures 36A to 36B The chart shows the participants in MPS I Phase 1 / 2 ( Figure 36A ) and single participant studies ( Figure 36B The total urinary GAG level in ).

[0151] Figure 37A and Figure 37B A composite audiogram showing the left ear ( Figure 37A ) and right ear ( Figure 37B The average air conduction ABR threshold at 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz, in dBeHL.

[0152] Figure 38 The chart shows the change in ABR air conduction threshold for each participant from baseline to the last available time point, with results indicating that hearing thresholds improved or stabilized in most ears.

[0153] Figure 39 This is a research flowchart of the currently disclosed Implementation Example 10, which shows the timeline of events. AAV9 = Adeno-associated virus vector 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 = Intracisional; ICV = Intraventricular; INR = International Normalized Ratio; IQ = Intelligence quotient; IT = Intrathecal; IV = Intravenous; MRI = Magnetic resonance imaging; PI = Principal investigator; PO = Oral; PT = Prothrombin time; PTT = Activated 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 cells = T lymphocytes. Visit 14 was intentionally omitted from the visit numbering scheme. The charts are labeled as follows: ● a The facility can receive subjects between day -2 and the morning of day 1. Therefore, according to institutional standards, some pre-drug tests can be performed on day -2 or day -1.

[0154] ● b For ET visits, lumbar puncture (and CSF assessment) is performed only if medication is discontinued before week 56, and cognitive function assessment is performed only if medication is discontinued before week 48.

[0155] ● c Researchers should consult with medical monitors to determine which assessments need to be performed during unscheduled visits.

[0156] ● dFrequent monitoring of vital signs and neurological assessments were performed, including every 45 ± 10 minutes for the first 3 hours after anesthesia in the post-anesthesia care room, then every hour ± 15 minutes for the next 4 hours, and then every 4 hours ± 30 minutes for the remainder of the 24 hours following administration.

[0157] ● e If the PI deems it appropriate, the subject may be discharged home approximately 1-2 days after administration. The subject / caregiver will receive instructions regarding neurological monitoring.

[0158] ● f The 64-week visit was performed only on subjects who had interrupted IV ERT.

[0159] ● g The assessments at weeks 20 and 28 (in italics in the table) will be limited to evaluating adverse events and concomitant therapies via telephone contact.

[0160] ● h Pre-drug neurological assessment can be performed at any time from day -2 to day 1.

[0161] ● i If there are clinical indications.

[0162] ● j It is executed according to the standards of each institution and can be executed before day 1.

[0163] ● k The results should be evaluated or reviewed by a neurologist.

[0164] ● kk Before using gadolinium for screening MRI, an estimated glomerular filtration rate (eGFR) based on creatinine must be measured. If the laboratory determines that creatinine is below the limit of validation or detection, a minimum cutoff value will be used to estimate eGFR. If eGFR < 30 mL / min / 1.73 m 2 If so, researchers must consult a medical monitor before performing an MRI.

[0165] ● l An evaluation of contraindications for IC or ICV injections should be performed according to the appropriate administration manual and completed before day -2. Approval for IC or ICV injections must be documented before initiating IS on day -2.

[0166] ● m MRI using gadolinium is performed only for screening assessment. Every effort should be made to perform an MRI after confirming the neurocognitive score. If a sedated MRI is performed prior to neurocognitive testing, there must be at least a 24-hour interval between the MRI and the time when the neurocognitive test can be performed.

[0167] ● n Genetic confirmation of MPS type 2. Pre-study genetic testing is acceptable if adequate documentation is available. Subjects who have not previously undergone genetic testing may be tested during the screening period.

[0168] ● nn Changes in auditory ability are measured using auditory brainstem response (ABR) and behavioral audiometry (BAUD) (if applicable). Tympanometry is performed upon completion of BAUD and / or ABR assessments. Hearing ability is assessed at the designated visit in this event timeline; additional assessments are permitted at the week 24 visit if the investigator deems them clinically necessary.

[0169] ● Plasma biomarkers: Collect GAG and I2S samples. Every effort should be made to perform I2S assessment at the trough associated with IV ERT, defined as at least 96 hours after ERT infusion until the start of subsequent infusions.

[0170] ● p Abdominal ultrasound was performed to assess the size of the liver and spleen. For subjects who discontinued ERT after week 24, plasma and urine biomarkers were collected. Abdominal ultrasound was performed at the same facility at each time point.

[0171] ● q AAV9 and I2S antibodies.

[0172] ● r Urinary biomarkers: Samples should be collected for GAG. Every effort should be made to collect urinary GAG at the trough associated with IV ERT, defined as at least 96 hours after ERT infusion until the start of subsequent infusions.

[0173] ● s IC or ICV injections are performed according to the appropriate administration manual.

[0174] ● t Tacrolimus: Adjust the dose to maintain a whole blood trough concentration of 2-4 ng / mL. Nesirolimus: Adjust the dose to maintain a whole blood trough concentration of 1-3 ng / mL. Subjects should continue with the new maintenance dose for at least 7-14 days, followed by further dose adjustments via concentration monitoring.

[0175] ● Methylprednisolone should be administered prior to lumbar puncture and IC or ICV injection of the investigational product. Preoperative use of acetaminophen and antihistamines is optional and at the investigator's discretion.

[0176] ● uScreening (days -35 to -2) lumbar punctures were used solely to assess CSF pressure and biomarkers. Iodized myelography was performed on day 1 prior to administration for comparison (see administration manual).

[0177] ● v CSF biomarkers: GAG and I2S samples were collected, and samples for future use were tested only from the remaining portion of the study samples (if available).

[0178] ● w CSF safety laboratory tests: CSF stress, red blood cell count, white blood cell differential count, total protein, and glucose.

[0179] ● x Cognitive abilities are assessed using BSID-III or KABC-II; adaptive functions are assessed using VABS-II. If a sedated MRI is performed prior to neurocognitive testing, there must be at least a 24-hour interval between the MRI and the time at which neurocognitive testing can be performed.

[0180] ● z For VZV: Antibody titer at baseline. For EBV and CMV: Viral genomic PCR and serial tests at baseline, as described in Table X. If positive at any time point, more frequent testing may be performed according to field care standards.

[0181] ● y If a participant’s relative has the same MPS II mutation, a separate informed consent form is provided for reviewing the appropriate kinship documentation.

[0182] ● aa If possible, the entire study should be conducted by the same caregiver.

[0183] ● bb If possible, the entire study should be conducted by the same clinician.

[0184] Figure 40 This is a table summarizing the hearing test data from Example 16. Hearing test data for 11 participants at the screening (baseline) and the last time point after treatment with Construct 1. 1 The average of the estimated behavioral thresholds (in dBeHL) obtained from the ABR. Threshold differences >10 dB were used to determine the change from baseline. * indicates participants with the last time point in long-term studies. 2 This indicates tube insertion or tympanic membrane perforation. 3 The selection was based on the first week of the year.

[0185] Figure 41Composite audiograms were constructed, showing the mean air conduction ABR threshold for each ear at 500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz, in dBeHL. Improvement in hearing thresholds was observed at the final time point after construct 1 treatment compared to baseline.

[0186] Figure 42 It is a graph showing the average change in ABR air conduction threshold for each participant from baseline to the last available time point.

[0187] Figure 43 This is a table summarizing the hearing test data from Example 17. Hearing test data for 11 participants at the last time point after screening (baseline) and construct 1. 1 The average value (in dBeHL) of the estimated behavioral thresholds obtained from the ABR measurement frequencies. Threshold differences of >10 dB are used to determine the variation compared to the baseline. 2 This indicates tube insertion or tympanic membrane perforation. 3 Conductive element with a frequency of 500 Hz.

[0188] Figure 44 Composite audiograms were constructed, showing the mean air conduction ABR threshold for each ear at 500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz, in dBeHL. Improvement in hearing thresholds was observed at the final time point after construct 1 treatment compared to baseline.

[0189] Figure 45 It is a graph showing the average change in ABR air conduction threshold for each participant from baseline to the last available time point. 5. Detailed Implementation This invention relates to the delivery of recombinant human iduronate-2-sulfatase (rhIDS) produced by human neurons or glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS) of human subjects diagnosed with mucopolysaccharidosis II (MPS II), including but not limited to patients diagnosed with Hunter syndrome. For the compositions and methods that can be used according to the invention described herein, see also International Patent Application No. PCT / US2017 / 027770, filed April 14, 2017 (published October 19, 2017 as WO / 2017 / 181113), which is incorporated herein by reference in its entirety.

[0191] In a preferred embodiment, the treatment is performed via gene therapy, for example... ，By administering a viral vector or other DNA expression construct encoding human IDS (hIDS) or an hIDS derivative to the CSF of patients diagnosed with MPSII (human subjects), a permanent reservoir of transduced neurons and / or glial cells is created, continuously supplying the transgene to the CNS. rhIDS secreted from the neuronal / glial cell reservoir into the CSF is endocytosed by cells in the CNS, resulting in “cross-correction” for enzymatic defects in the recipient cells. Furthermore, it was unexpectedly found that the transduced neuronal and glial cell reservoirs in the CNS can deliver the recombinant enzyme to both the CNS and the whole body, potentially reducing or eliminating the need for systemic therapy (e.g., weekly intravenous injections of the enzyme). This article also provides treatment of MPS I by administering to subjects a viral vector encoding human IDUA or other DNA expression constructs (e.g., a non-replicating recombinant AAV with a serotype 9 capsid containing an hIDUA expression cassette; construct 2; see PCT / US2021 / 014129; PCT / US2018 / 015910; and PCT / US2019 / 042205, each of which is incorporated herein by reference in its entirety).

[0192] In alternative embodiments, hIDS can be produced by human neurons or glial cells in cell cultures (e.g., bioreactors) and administered as enzyme replacement therapy (“ERT”), for example, by injecting the enzyme into the CSF, directly into the CNS, and / or systemically. However, gene therapy methods offer several advantages over ERT because systemic delivery of the enzyme does not result in treatment of the CNS, as the enzyme cannot cross the blood-brain barrier; and unlike the gene therapy methods of this invention, direct delivery of the enzyme to the CSF and / or CNS would require repeated injections, which is not only cumbersome but also carries the risk of infection.

[0193] The transgenic encoded hIDS may include, but is not limited to, human IDS (hIDS), which have the amino acid sequence of SEQ ID NO.1 (e.g., Figure 1 (as shown); and derivatives of hIDS that have amino acid substitutions, deletions, or additions, for example ， Including but not limited to those selected from Figure 2 The amino acid substitutions of corresponding non-conserved residues in orthologs of the IDS shown are provided that such mutations do not include the substitution of the cysteine ​​residue at position 84 (C84), which is required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or mutations already identified in severe, severe-moderate, moderate, or weakened MPS II phenotypes, such as... Figure 3Mutations shown or reported as follows: Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761 (reporting “attenuated” mutants R48P, A85T, W337R and truncated mutant Q531X; and “severe” mutants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406: 214-218 (reporting “attenuated” mutants P480L and P480Q; and “severe” mutant P86L); and Bonucelli et al., 2001, BBA 1537:233-238, each of which is incorporated herein by reference in its entirety.

[0194] For example, the amino acid substitutions at specific positions in hIDS can be selected from... Figure 2 The corresponding non-conserved amino acid residues present at the positions described in the IDS orthologs compared with the Chinese model. Prerequisites This type of replacement does not include Figure 3 Any harmful mutations shown or reported as follows: Sukegawa-Hayasaka et al., 2006, Same as above Millat et al., 1998, Same as above ; or Bonucelli et al., 2001, Same as above Each of the cited references is incorporated herein by reference in its entirety. The resulting transgenic products can be tested using routine assays in vitro, in cell cultures, or in test animals to ensure that the mutations do not impair IDS function. Selected preferred amino acid substitutions, deletions, or additions should be those that maintain or increase the enzymatic activity, stability, or half-life of the IDS, as tested using routine assays in vitro, in MPS II cell cultures, or in animal models. For example, the enzymatic activity of the transgenic products can be assessed using routine enzyme assays, such as with 4-methylumbelliferone α-L-idopyranuronic acid 2-sulfate or 4-methylumbelliferone sulfate as substrates (…). SeeFor example, exemplary IDS enzyme assays available in Lee et al., 2015, Clin. Biochem. 48(18):1350-1353, and Dean et al., 2006, Clin. Chem. 52(4):643-649 (each of which is incorporated herein by reference in its entirety). The ability of transgenic products to correct MPS II phenotypes can be assessed in cell cultures; for example, by transducing MPS II cells in cultures with a viral vector or other DNA expression construct encoding hIDS or derivatives; by adding transgenic products or derivatives to MPS II cells in cultures; or by co-culturing MPS II cells with human neuronal / glial host cells engineered to express and secrete rhIDS or derivatives, and determining the correction of defects in MPS II cultured cells, for example, by detecting IDS enzyme activity and / or a reduction in GAG storage in MPS II cells in cultures. See also, For example, Stroncek et al., 1999, Transfusion 39(4):343-350, which is incorporated into this paper in its entirety by reference.

[0195] MPS II animal models that can be used to evaluate the therapies described herein have been described. For example, by replacing the neomycin resistance gene... IDS Exons 4 and 5 of the gene were engineered to create a knockout mouse model of MPS II (IDS-knockout). (Garcia et al., 2007, J Inherit Metab Dis 30: 924-34). These IDS-knockout mice exhibited many characteristics of MPS II, including skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAG ​​levels, and brain storage lesions (Muenzer et al., 2001, Acta Paediatr Supplement 91:98-99) and were used to evaluate the efficacy of enzyme replacement therapy in MPS II in supporting ERT clinical trials. Therefore, this mouse model is a relevant model for investigating the efficacy of gene therapy delivery of rIDS generated by neurons or glial cells as a treatment for MPS II. See also, For example, Polito and Cosma, 2009, Am. J. Hum.Genet. 85(2):296-301, which is incorporated herein by reference in its entirety.

[0196] Preferably, the hIDS transgene produced by human neurons / glial cells should be controlled by expression control elements that function in neurons and / or glial cells (e.g., the CB7 promoter (chicken β-actin promoter and CMV enhancer)) and may include other expression control elements that enhance the expression of the vector-driven transgene (e.g., chicken β-actin introns and rabbit β-globin poly A signaling). The cDNA construct of the hIDS transgene should include the coding sequence of a signal peptide that ensures proper co-translation and post-translational processing (glycosylation and protein sulfation) by transduced CNS cells. Such signal peptides used by CNS cells may include, but are not limited to: - Oligodendrocyte-myelin glycoprotein (hOMG) signal peptide, which has the amino acid sequence MEYQILKMSLCLFILLFLTPGILC (SEQ ID NO:2). - The cell repressor (hCREG2) signal peptide of E1A-stimulated gene 2, which has the amino acid sequence MSVRRGRRPARPGTRLSWLLCCSALLSPAAG (SEQ ID NO:3). - A signal peptide containing a 2B group V and a transmembrane domain (hVSTM2B), which has the amino acid sequence MEQRNRLGALGYLPPLLLHALLLFVADA (SEQ ID NO:4). - Protocadherin α-1 (hPCADHA1) signal peptide, which has the amino acid sequence MVFSRRGGLGARDLLLWLLLLAAWEVGSG (SEQ ID NO:5). - FAM19A1 (TAFA1) signal peptide, which has the amino acid sequence MAMVSAMSWVLYLWISACA (SEQ ID NO:6). - Interleukin-2 signal peptide, which has the amino acid sequence MYRMQLLSCIALILALVTNS (SEQ ID NO:14). The signal peptide may also be referred to as the leader sequence or leader peptide in this article.

[0197] Recombinant vectors used for delivering transgenes should be tropistic to cells in the CNS (including, but not limited to, neurons and / or glial cells). Such vectors may include non-replicating recombinant adeno-associated virus vectors (“rAAV”), particularly those carrying AAV9 or AAVrh10 capsids. AAV variant capsids may be used, including, but not limited to, those described by Wilson in U.S. Patent No. 7,906,111, which is incorporated herein by reference in its entirety, with particular preference for AAV / hu.31 and AAV / hu.32; and AAV variant capsids described by Chatterjee in U.S. Patent Nos. 8,628,966, 8,927,514, and Smith et al., 2014, Mol Ther 22: 1625-1634, each of which is incorporated herein by reference in its entirety. However, other viral vectors may be used, including, but not limited to, lentiviral vectors, vaccinia virus vectors, or nonviral expression vectors known as “naked DNA” constructs.

[0198] Suitable pharmaceutical compositions for CSF administration comprise a suspension of the rhIDS carrier in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In some embodiments, the pharmaceutical composition is suitable for intrathecal administration. In some embodiments, the pharmaceutical composition is suitable for intracisional administration (injection into the cisterna magna). In some embodiments, the pharmaceutical composition is suitable for subarachnoid injection via C1-2 puncture. In some embodiments, the pharmaceutical composition is suitable for intraventricular administration. In some embodiments, the pharmaceutical composition is suitable for administration via lumbar puncture. In some embodiments, the pharmaceutical composition comprising the rAAV of this disclosure comprises sodium chloride at a concentration of about 8.77 g / L, magnesium chloride hexahydrate at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, anhydrous dextran at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.010 g / L, sodium dihydrogen phosphate monohydrate at a concentration of about 0.0278 g / L, and anhydrous disodium hydrogen phosphate at a concentration of about 0.114 g / L.

[0199] The therapeutically effective dose of the recombinant vector should be administered to the CSF via intrathecal administration (i.e., injection into the subarachnoid space, allowing the recombinant vector to distribute through the CSF and transduce cells in the CNS). In some embodiments, the recombinant vector is administered in solution containing approximately 8.77 g / L sodium chloride, approximately 0.244 g / L magnesium chloride hexahydrate, approximately 0.224 g / L potassium chloride, approximately 0.206 g / L calcium chloride dihydrate, approximately 0.793 g / L anhydrous dextran, approximately 0.010 g / L poloxamer 188, approximately 0.0278 g / L sodium dihydrogen phosphate monohydrate, and approximately 0.114 g / L anhydrous disodium hydrogen phosphate. This can be achieved in various ways, for example, by intracranial (cistern or ventricle) injection or injection into the lumbar cistern. For example, intracisional (IC) injection (injection into the cisterna magna) can be performed via CT-guided suboccipital puncture; or, where feasible for the patient, injection into the subarachnoid space can be performed via C1-2 puncture; or lumbar puncture (a diagnostic procedure typically performed to collect CSF samples) can be used to access the CSF. Alternatively, the recombinant vector can be directly instilled into the ventricles using intraventricular (ICV) administration (a more invasive technique for introducing anti-infective or anticancer drugs that do not cross the blood-brain barrier). Alternatively, the recombinant vector can be delivered to the CNS via intranasal administration.

[0200] Due to the relatively rapid brain growth that occurs in early childhood, the total dose of AAV9.hIDS administered via IC depends on the presumed brain mass at different age levels. See For example, see Table 2 below. For brain quality of study subjects by age, see, for example, AS Dekaban, Ann Neurol, October 1978; 4(4): 345-56.

[0201] Table 2. Total dosage administered by age * GC was determined using Poly-A specific PCR assay. CSF concentration can be monitored by directly measuring the rhIDS concentration in the CSF fluid obtained from occipital or lumbar puncture, or estimated by extrapolating the rhIDS concentration detected in the patient's serum.

[0202] With the help of background technology, human IDS is translated into a language containing Figure 1 Eight potential N-glycosylation sites (N) depicted in the paper 31 N 115 N 144 N 246 N 280 N 325N 513 and N 537 The polypeptide comprises 550 amino acids and includes a 25-amino acid signal sequence that is cleaved during processing. Following modification of the oligosaccharide chain of the initial 76 kDa intracellular precursor in the Golgi apparatus, the precursor is converted into a phosphorylated 90 kDa precursor. This precursor is then processed into its predominant 55 kDa form via various intracellular intermediates through glycosylation modification and proteolytic cleavage. In summary, proteolytic processing involves N after removal of the 25-amino acid signal sequence. 31 Downstream N-terminal proteolytic cleavage removes eight amino acid residues (residues 26-33) of the propeptide, as well as the N... 513 Upstream C-terminal proteolytic cleavage releases an 18 kDa polypeptide and generates a 62 kDa intermediate, which is converted to a 55 kDa mature form. Further proteolytic cleavage produces a 45 kDa mature form located in the lysosomal compartment. See Figure 4 Figures and tables are 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 herein by reference in its entirety.

[0203] C required for enzyme activity 84 Formicoylglycine modification ( Figure 1 (Highlighted in bold) This may occur as an early post-translational or co-translational event, most likely in the endoplasmic reticulum. See Millat 1997a, citing Schmidt et al., 1995, Cell 82: 271-278). Post-translational processing continues in the Golgi apparatus, involving the addition of complex sialic acid-containing glycans and the acquisition of mannose-6-phosphate residues, which label the enzyme for delivery to the lysosomal compartment. See A concise review by Clarke, 2008, Expert Opin Pharmacother 9: 311-317 (which is incorporated herein by reference in its entirety). While individual glycosylation sites are not important for IDS stability, position N... 280Glycosylation at the cellular level is crucial for internalization and lysosomal targeting via the mannose-6-phosphate (M6P) receptor (Chung et al., 2014, Glycoconj J 31:309-315, p. 310, column 1). Under normal physiological conditions, IDS are produced at very low levels, and the amount of enzymes secreted by the cell (if present) is also very small (Clarke, 2008). Same as above ).

[0204] This invention is partly based on the following principles: (i) Neurons and glial cells in the CNS are secretory cells possessing post-translational mechanisms for secreting proteins, including robust processes such as glycosylation, mannose-6-phosphorylation, and tyrosine-O-sulfation. Regarding post-translational modifications performed by human CNS cells, See also, For example, Sleat et al., 2005, Proteomics5: 1520-1532 and Sleat 1996, J Biol Chem 271: 19191-98, describe the human brain mannose-6-phosphoglycoprotein genome and point out that the brain contains far more proteins with individual isotypes 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, report the production of tyrosine-sulfated glycoproteins secreted by neuronal cells, each of which is incorporated herein by reference in its entirety.

[0205] (ii) The human brain produces multiple natural / naturally occurring IDS isotypes. Specifically, N-terminal sequencing of human brain mannose-6-phosphorylated glycoproteins revealed a change in the N-terminal sequence of the mature 42 kDa chain of hIDS in the brain, beginning at position 34 or 36: T 34 DALNVLLI (SEQ ID NO: 54); and A 36 LNVLLIIV (SEQ ID NO: 55). (Sleat, 2005, Proteomics 5: 1520-1532, Table S2). Two of the eight N-linked glycosylation sites were found, namely N... 280 and N 116 In IDS obtained from the human brain, it is mannose-6-phosphorylated. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported in Table V).

[0206] (iii) During hIDS processing, nerve cells and glial cells secrete two peptides, 76 kDa and 90 kDa, but only the 90 kDa peptide is phosphorylated with mannose-6-phosphorylated, which is necessary for cross-correction of the secreted form of the enzyme. See Millat, 1997 Figure 1 The results of transduced lymphoblasts were shown, as well as Froissart 1995. Figure 4 Similar results were shown for transduced fibroblasts, where only the 90 kDa form was phosphorylated in the culture medium. Interestingly, it has been demonstrated that recombinant IDS produced by neurons and glial cells may be more readily internalized by receptor CNS cells than recombinant IDS produced by other cells such as the kidney. Daniele 2002 demonstrated that M6P receptor-mediated internalization of recombinant IDS from conditioned mediated cultures of transduced neurons and glial cells by a receptor population of non-transduced neurons and glial cells, which properly processed the precursors into a mature, active form of 45 kDa. The uptake of recombinant IDS produced by neuronal and glial cell lines (74% internalization) far exceeded that of enzymes produced by kidney cell lines (5.6% internalization). In each case, uptake was inhibited by M6P, suggesting that the uptake of recombinant IDS is M6P receptor-mediated. (See the accompanying description in Tables 2 and 4 of Daniele 2002 and the results summarized in Table 3 below on pages 205–206).

[0207] Table 3. Summary of the results reported by Daniele in 2002 (iv) The gene therapy approach described herein should result in the continuous secretion of a 90 kDa hIDS glycoprotein precursor with enzymatic activity, as measured by polyacrylamide gel electrophoresis (depending on the assay used). First, C, required to induce IDS activity, is expressed in the cerebral cortex of the human brain. 84 The formicylglycine-modified enzyme, namely FGly-producing enzyme (FGE, also known as SUMF1) (gene expression data for SUMF1 can be found on GeneCards, accessible at http: / / www.genecards.org). Secondly, it should be absorbed by transduced neurons and glial cells. In situ The resulting secreted glycosylated / phosphorylated rIDs are correctly processed by untransduced neurons and glial cells in the CNS. Unbound by any theory, it seems that if applied to the CNS, gene therapy... In situ The resulting secretory rhIDS precursor may be more readily internalized by recipient cells in the CNS than conventional recombinases used for ERT. For example, Elaprase.® (Prepared in the fibrosarcoma cell line HT1080) is a purified protein reported to have a molecular weight of approximately 76 kDa, rather than the 90 kDa species that appears to be more phosphorylated and secreted by neurons and glial cells. Although it is reported that eight N-linked glycosylation sites completely occupy Elaprase. ® It contains two dismannose-6-phosphate-terminated polysaccharides and a highly sialylated polysaccharide complex, but C 84 Post-translational modification to FGly (which is the absolute requirement for enzyme activity) is only about 50%. (Clarke, 2008, Expert Opin Pharmacother 9:311-317; Elaprase) ® (All prescribing information and EMA application). Another recombinant product, Hunterase. ® Prepared in CHO cells. Although reported to be better than Elaprase. ® It exhibits higher FGly activity, but there is no difference in mannose 6-phosphorylation and uptake. (Chung, 2014, Glycoconj J 31:309-315).

[0208] (v) The efficacy of extracellular IDS in vivo depends on its uptake via mannose-6-phosphate (M6P) (cellular and lysozyme in vivo) and its active site, formylglyine (FGly), which is produced by an enzyme that generates formylglyine from C6P via post-translational modification. 84 Derived from transgenic IDS. As shown in Table 3 above, brain cells (neurons and glial cells) exhibited higher enzyme activity when incubated with IDS precursor medium secreted by transduced neurons and glial cells, rather than with IDS precursor medium secreted by genetically engineered kidney cells. The resulting five-fold increase in activity may be attributed to efficient IDS uptake (see Daniele 2002, Tables 2 and 4). Commercially available forms of IDS produced by CHO cells or HT-1080 cells have approximately 50% to 70% FGly content, which determines enzyme activity. However, neurons and glial cells can improve this activity due to improved IDS uptake.

[0209] (vi) Cellular and subcellular transport / uptake of lysosomal proteins (including IDS) occurs via M6P. IDS derived from brain cells may contain higher levels of M6P, as reported in Daniele 2002 and Sleat, Proteomics, 2005 (indicating that the human brain contains more Man6-P glycoprotein (in both quantitative and qualitative terms) than other tissues). The M6P content of IDS precursors can be measured, as in Daniele 2002. In the presence of inhibitory M6P (e.g., 5 mM), the uptake of IDS precursors produced by non-neuronal or non-glial cells (such as genetically engineered kidney cells in Daniele 2002) is predicted to decrease to near-control cell levels, as shown in Daniele 2002. Simultaneously, in the presence of inhibitory M6P, the uptake of IDS precursors produced by brain cells (such as neurons and glial cells) is predicted to remain at high levels, as shown in Daniele 2002, where uptake is four times higher than in control cells and comparable to the IDS activity (or uptake) levels of IDS precursors produced by genetically engineered kidney cells in the absence of inhibitory M6P. This assay allows for methods to predict the M6P content in IDS precursors produced by brain cells, particularly comparing the M6P content in IDS precursors produced by different cell types. The gene therapy approach described herein should result in the continuous secretion of hIDS precursors, in which, in the presence of inhibitory M6P, the hIDS precursors can be uptaken at high levels into neurons and glial cells.

[0210] (vii) The M6P content and uptake of IDS precursors can also be demonstrated using 90 kDa and 76 kDa gel bands (e.g., SDS-PAGE gel bands). The 90 kDa band is reported to be highly glycosylated / phosphorylated and contains M6P, while the 76 kDa band is not. Very wide gel bands ranging from 76 kDa to 95 kDa with an average MW of 80-85 kDa can be produced (similar to IDS precursor gel bands generated from genetically engineered kidney cells (Daniele 2002)). Figure 1 This is compared to an IDS precursor gel band produced by brain cells. In Daniele 2002, a gel band could not be obtained due to unsuccessful immunoprecipitation of the IDS precursor. The gene therapy method described herein should result in the continuous secretion of hIDS precursors, which are different from the IDS precursor gel bands produced by genetically engineered kidney cells.

[0211] (viii) The M6P content of commercial IDS precursors ranges from 2 to 2.5 mol / mol, with the majority existing as diphosphorylated glycans. Although, on average, each IDS precursor is phosphorylated, the normal distribution of glycans will result in some IDS precursors having 2, 1, and 0 diphosphorylated M6P glycans, exhibiting multiple phosphorylation sites. In the case of multiple phosphorylation, the uptake should be significantly higher.

[0212] (ix) Glycosylation of hIDS by human CNS cells results in the addition of glycans, which can improve the stability and half-life of the transgenic product and reduce its unwanted aggregation. Notably, the glycans added to the hIDS of this invention comprise 2,6-sialic acid, incorporating Neu5Ac (“NANA”) but not its hydroxylated derivative NeuGc (N-hydroxyacetylneuraminic acid, ... Right now "NGNA" or "Neu5Gc"). These glycans are used to prepare recombinant IDS products (such as Hunterase) in CHO cells. ® The presence of sialidyltransferase (SLT) in CHO cells is because CHO cells do not possess the 2,6-sialylate transferase required for this post-translational modification; and CHO cells do not produce bivariate GlcNAc, but they do add Neu5Gc (NGNA) instead of Neu5Ac (NANA) as a human atypical (and potentially immunogenic) sialic acid. See also, For example, Dumont et al., 2016, Critical Rev in Biotech 36(6):1110-1122 (Early online, page 5 of 1-13); and Hague et al., 1998 Electrophor 19:2612-2630 (“[t]the CHO cell line is considered 'phenotypically restricted,' in terms of glycosylation, due to the lack of an α2,6-sialyl-transferase”). Furthermore, CHO cells can produce immunogenic glycans, namely α-Gal antigens, which react with anti-α-Gal antibodies present in most individuals and can trigger allergic reactions at high concentrations. See also, For example, Bosques, 2010, Nat Biotech 28: 1153-1156. The human glycosylation mode of rhIDS in this invention should reduce the immunogenicity of transgenic products and improve efficacy.

[0213] (x) The immunogenicity of transgenic products can be induced by a variety of factors, including the patient's immune status, the structure and properties of the infused protein drug, the route of administration, and the duration of treatment. Process-related impurities (such as host cell proteins (HCPs), host cell DNA, and chemical residues) and product-related impurities (such as protein degradation products and structural features such as glycosylation, oxidation, and aggregation (subvisible particles)) can also enhance immunogenicity by acting as adjuvants to enhance the immune response. The amounts of process-related and product-related impurities can be affected by manufacturing processes such as cell culture, purification, formulation, storage, and handling, which can affect commercially manufactured IDS products. In gene therapy, proteins are produced in vivo, making process-related impurities absent, and protein products are unlikely to contain product-related impurities / protein-related degradation products resulting from recombinant technologies such as protein aggregation and protein oxidation. Aggregation is associated, for example, with protein production and storage at high protein concentrations, surfaces interacting with manufacturing equipment and containers, and purification processes using certain buffer systems. However, these conditions promoting aggregation are not present when transgenes are expressed in vivo. Oxidation (such as the oxidation of methionine, tryptophan, and histidine) is also associated with protein production and storage, for example, by stressful cell culture conditions, metal-air contact, and impurities in buffers and excipients. Proteins expressed in vivo can also be oxidized under stress conditions, but humans, like many organisms, are equipped with an antioxidant defense system that not only mitigates oxidative stress but also repairs and / or reverses oxidation. Therefore, proteins produced in vivo are unlikely to be in oxidized form. Both aggregation and oxidation can affect potency, pharmacokinetics (clearance), and may increase immunogenicity issues. The gene therapy approach described herein should result in the continuous secretion of hIDS precursors with reduced immunogenicity compared to commercially manufactured products.

[0214] (xi) In addition to N-linked glycosylation sites, hIDS also contain tyrosine (“Y”) sulfated sites (PSSEKY). 165 ENTKTCRGPD (SEQ ID NO: 47)). (Analysis of amino acids surrounding tyrosine residues that have undergone protein tyrosine sulfate treatment.) See also,For example, Yang et al., 2015, Molecules 20:2138-2164, especially page 2154, which is incorporated in its entirety by reference. The “rule” can be summarized as follows: Y residues with E or D are located in positions +5 to -5 of Y, and where position -1 of Y is a neutral or acidic charged amino acid rather than a basic amino acid (e.g., sulfation of R, K, or H). While not intended to be bound by any theory, sulfation at this site in hIDS can improve enzyme stability and substrate binding affinity. Tyrosine sulfation of hIDS (a robust post-translational process in human CNS cells) should lead to improved processing and activity of transgenic products. The significance of tyrosine sulfation of lysosomal proteins has not been elucidated; however, in other proteins, it has been shown to increase protein-protein interaction affinity (antibodies and receptors) and promote proteolytic processing (peptide hormones). (See Moore, 2003, J Biol. Chem. 278: 24243-46; and Bundegaard et al., 1995, The EMBO J 14: 3073-79). Tyrosyl protein sulfotransferase (TPST1), which induces tyrosine sulfation (which can occur as a final step in IDS processing), is expressed at significantly higher levels (based on mRNA) in the brain. (Gene expression data for TPST1 are available in, for example, the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home). This post-translational modification is at best underrepresented in CHO cell products. Unlike human CNS cells, CHO cells are not secretory cells and have limited capacity for post-translational tyrosine sulfation. See also, For example, Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537, especially the discussion on page 1537).

[0215] For the reasons stated above, rhIDS produced by human neurons and / or glial cells should result in a "biomodified" molecule for the treatment of MPS II, achieved via gene therapy, for example by administering a viral vector or other DNA expression construct encoding rhIDS to patients (human subjects) diagnosed with MPS II disease (including but not limited to Hunter's disease) to create a permanent reservoir in the CNS continuously supplied with a fully human glycosylated, mannose-6-phosphorylated, sulfated transgenic product secreted by transduced CNS cells. The hIDS transgenic product secreted from the reservoir into the CSF will be endocytosed by cells in the CNS, resulting in "cross-correction" of enzymatic defects in MPS II recipient cells.

[0216] Each rhIDS molecule produced in gene therapy or protein therapy methods need not be fully glycosylated, phosphorylated, and sulfated. Instead, the resulting glycoprotein population should have sufficient glycosylation (including 2,6-sialylation and mannose-6-phosphorylation) and sulfation to demonstrate efficacy. The goal of the gene therapy treatment of this invention is to slow or halt disease progression. Efficacy can be monitored by measuring: cognitive function (e.g., prevention or reduction of neurocognitive decline); reduction of disease biomarkers (such as GAG) in CSF and / or serum; and / or increase of IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events can also be monitored.

[0217] As an alternative or additional treatment to gene therapy, rhIDS glycoprotein can be produced in human neuronal or glial cell lines using recombinant DNA technology, and the glycoprotein can be administered systemically to patients diagnosed with MPS II and / or administered to CSF ​​for ERT. Human cell lines that can be used for the production of such recombinant glycoproteins include, but are not limited to, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM (for a review of human cell lines that can be used for recombinant production of rHuGlyIDS glycoprotein). See also, For example ， Dumont et al., 2016, Critical Rev in Biotech 36(6):1110-1122 “Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives” (incorporated in whole by reference). To ensure complete glycosylation, particularly sialylation and tyrosine sulfation, cell lines for production can be enhanced by engineering host cells to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferase) and / or TPST-1 and TPST-2 enzymes that induce tyrosine-O-sulfation.

[0218] While delivery of rhIDS should minimize immune responses, the most apparent potential source of toxicity associated with CNS-directed gene therapy is the development of immunity against the expressed rhIDS protein in human subjects who have IDS genetic defects and may therefore be intolerant to the protein and / or the vector used to deliver the transgene.

[0219] Therefore, in a preferred embodiment, co-treatment with immunosuppressive therapy is recommended, especially when treating patients with severe disease whose IDS levels are close to zero. Immunosuppressive therapy may be employed using regimens involving a combination of tacrolimus or rapamycin (sirolimus) and mycophenolic acid, or other immunosuppressive regimens used in tissue transplantation procedures. This immunosuppressive therapy may be administered during the gene therapy process, and in some embodiments, pre-treatment with immunosuppressive therapy may be preferred. Immunosuppressive therapy may be continued after gene therapy treatment based on the treating physician's judgment, and subsequently, for example, after 180 days, may be withdrawn during the induction of immune tolerance.

[0220] The methods of this invention encompass combinations of rhIDS delivery to the CSF and delivery of other available treatments. Additional treatments may be administered before, during, or after gene therapy. Available treatments for MPS II that can be combined with the gene therapy of this invention include, but are not limited to, the use of elaprase administered systemically or to the CSF. ® Enzyme replacement therapy; and / or HSCT therapy.

[0221] In some embodiments, this document describes a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) precursor produced by human neurons or human glial cells to the cerebrospinal fluid (CSF) of said human subject.

[0222] In some embodiments, this document describes a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPSII), comprising delivering a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor to the cerebrospinal fluid (CSF) of the human subject, said precursor being 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, in C10. 84 It contains formylglycine residues ( Figure 1 It is α2,6-sialylated, does not contain detectable NeuGc, and is mannose-6-phosphorylated.

[0223] In some embodiments, this document describes a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPSII), comprising delivering a therapeutically effective amount of a recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor to the cerebrospinal fluid (CSF) of the human subject, said precursor being 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, in C10. 84 It contains formylglycine residues ( Figure 1 It is α2,6-sialylated, does not contain detectable NeuGc and / or α-Gal antigens, and is mannose-6-phosphorylated.

[0224] In some embodiments, a human IDS precursor is delivered to the CSF from a genetically engineered cellular reservoir in the central nervous system designed to secrete the IDS precursor into the CSF. In some embodiments, the reservoir is formed in the brain of a subject. In some embodiments, the human subject has an IDS activity deficiency. In some embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO. 1.

[0225] In some embodiments, this document describes a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPSII), comprising administering a recombinant nucleotide expression vector encoding human iduronate-2-sulfatase (IDS) to the cerebrospinal fluid (CSF) of the human subject, wherein, when used for transduction of primary human neuronal cells in a culture, the expression vector directs the expression of a secretory human IDS glycoprotein precursor, said precursor being 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, at C1000 kDa. 84 It contains formylglycine residues ( Figure 1 ), is α2,6-sialylated and mannose-6-phosphorylated.

[0226] In some embodiments, this document describes a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPSII), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS to the cerebrospinal fluid of the human subject's brain, such that a reservoir is formed in the central nervous system of the subject secreting a precursor of a recombinant human IDS glycoprotein, said precursor being α2,6-sialylated and mannose-6-phosphorylated.

[0227] In some embodiments, the secretion of the α2,6-sialylated recombinant human IDS glycoprotein precursor is confirmed by transducing human neuronal cell lines in cell culture using the recombinant nucleotide expression vector. In some embodiments, the secretion of the mannose-6-phosphorylated recombinant human IDS glycoprotein precursor is confirmed by transducing human neuronal cell lines in cell culture using the recombinant nucleotide expression vector. In some embodiments, secretion is confirmed in the presence and absence of mannose-6-phosphate.

[0228] In some embodiments, this document describes a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPSII), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS to the cerebrospinal fluid of the human subject's brain, such that a reservoir is formed that secretes a glycosylated IDS precursor containing α2,6-sialylated glycan; wherein, when used to transduce human neuronal cells in a culture, the recombinant vector results in the secretion of the glycosylated IDS precursor containing α2,6-sialylated glycan in the cell culture.

[0229] In some embodiments, this document describes a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPSII), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS to the cerebrospinal fluid of the human subject's brain, thereby forming a reservoir that secretes a glycosylated IDS precursor containing mannose-6-phosphate; wherein, when used to transduce human neuronal cells in a culture, the recombinant vector results in the secretion of the glycosylated IDS precursor, which is mannose-6-phosphate, in the cell culture.

[0230] In some embodiments, this document describes a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPSII), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS to the cerebrospinal fluid of the human subject's brain, such that a reservoir is formed that secretes a glycosylated IDS precursor containing formylglycine; wherein, when used to transduce human neuronal cells in a culture, the recombinant vector results in the secretion of the glycosylated IDS precursor containing formylglycine in the cell culture.

[0231] In some embodiments, the human IDS comprises the amino acid sequence of SEQ ID NO. 1. In some embodiments, the IDS transgene encodes a leader peptide. In some embodiments, the expression vector is a replication-deficient AAV vector. In some embodiments, the expression vector is delivered to the subject's CSF via intrathecal (e.g., intracisional, C1-2 puncture (if feasible for the patient), or lumbar puncture), intraventricular, or intranasal administration. In some embodiments, the human subject has IDS activity deficiency. In some embodiments, the expression vector is delivered in solution form to the subject's CSF via intrathecal administration. The solution comprises approximately 8.77 g / L sodium chloride, approximately 0.244 g / L magnesium chloride hexahydrate, approximately 0.224 g / L potassium chloride, approximately 0.206 g / L calcium chloride dihydrate, approximately 0.793 g / L anhydrous dextrose, approximately 0.010 g / L poloxamer 188, approximately 0.0278 g / L sodium dihydrogen phosphate monohydrate, and approximately 0.114 g / L anhydrous disodium hydrogen phosphate.

[0232] In a preferred embodiment, the glycosylated IDS does not contain detectable NeuGc and / or α-Gal. As used herein, the phrase "detectable NeuGc and / or α-Gal" means the NeuGc and / or α-Gal moiety that can be detected by standard assays known in the art. For example, NeuGc can be detected by HPLC according to the following: Hara et al. The reference cited is incorporated herein by reference in 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. Alternatively, NeuGc can be detected by mass spectrometry. α-Gal can be detected using ELISA. See For example, Galili et al., 1998, "A sensitive assay for measuring alpha-Galepitope expression on cells by a monoclonal anti-Gal antibody." Transplantation. 65(8):1129-32, or mass spectrometry, See For example, Ayoub et al. , 2013, “Correctprimary structure assessment and extensive glyco-profiling of cetuximab by acombination of intact, middle-up, middle-down and bottom-up ESI and MALDImass spectrometry techniques.” Landes Bioscience. 5(5): 699–710. See also Platts-Mills et al. References cited in “Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal” Immunol Allergy Clin North Am. 35(2): 247–260, 2015.

[0233] In one aspect, this article provides a method for treating a human subject diagnosed with MPS II, comprising delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neurons or human glial cells to the CSF of the human subject, wherein the glycosylated recombinant human IDS precursor is delivered by administering a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose dependent on the brain mass of the human subject, and wherein the brain mass is determined by brain MRI of the human subject's brain.

[0234] In another aspect, this article provides a method for treating a human subject diagnosed with MPS II, comprising determining the brain mass of the human subject based on a brain MRI of the human subject, and subsequently delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal cells or human glial cells to the human subject's CSF, wherein the glycosylated recombinant human IDS precursor is delivered by administering a recombinant nucleotide expression vector encoding human IDS, and wherein the recombinant nucleotide expression vector is administered at a dose dependent on the brain mass of the human subject.

[0235] In another aspect, this article provides a method for treating a human subject diagnosed with MPS II, comprising (a) determining the brain mass of the human subject based on a brain MRI of the human subject, (b) calculating a dose based on the brain mass of the human subject, and (c) subsequently administering the dose of a recombinant nucleotide expression vector encoding human IDS to the subject's CSF.

[0236] In another aspect, this article provides a method for treating a human subject diagnosed with MPS II, comprising, in the following order: (a) delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neurons or human glial cells to the CSF of the human subject; (b) measuring the level of heparan sulfate in the CSF of the human subject; and (c) comparing the level of heparan sulfate in the CSF of the human subject with the level of heparan sulfate in a reference population; wherein the glycosylated recombinant human IDS precursor is delivered by administering a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose dependent on the brain mass of the human subject, and wherein the brain mass is determined by magnetic resonance imaging (MRI) of the brain of the human subject. In some implementations, the reference group consists of: (a) at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500 or 1000 healthy human individuals who do not have MPS II, preferably of similar age, weight and / or the same sex as the human subjects.

[0237] In another aspect, this document provides a method for treating a human subject diagnosed with MPS II, comprising, in the following order: (a) first measuring the level of heparan sulfate in the human subject's CSF; (b) delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neurons or human glial cells into the human subject's CSF; and (c) measuring the level of heparan sulfate a second time after a period of time; wherein the glycosylated recombinant human IDS precursor is delivered by administering a recombinant nucleotide expression vector encoding human IDS, wherein the recombinant nucleotide expression vector is administered at a dose dependent on the brain mass of the human subject, and wherein the brain mass is determined by magnetic resonance imaging (MRI) of the human subject's brain. In some embodiments, the time period is approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 11 months, or 1 year.

[0238] In a preferred embodiment, the glycosylated recombinant human IDS precursor is endocytosed by cells in the CNS. In a preferred embodiment, the glycosylated recombinant human IDS precursor is delivered to the lysosomes of cells in the CNS of a human subject.

[0239] In some embodiments of the treatment methods described herein, by using cm 3 The brain volume of the human subject is multiplied by a coefficient of 1.046 g / cm³. 3 The brain volume of human subjects was converted into the brain mass of human subjects, where the brain volume of human subjects was determined by brain MRI of the subjects' brains.

[0240] In some embodiments of the treatment methods described herein, the recombinant nucleotide expression vector is at approximately 1.3 × 10⁻⁶. 10 GC / g is the brain mass determined by MRI, or approximately 6.5 × 10⁻⁶. 10 The dosage of GC / g is determined by MRI of brain mass. In some embodiments of the method for treatment described herein, the recombinant nucleotide expression vector is administered at approximately 1.3 × 10⁻⁶ g / g. 10 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by Poly-A specific PCR assay). In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector is used at approximately 1.9 × 10⁻⁶ g / g brain mass. 10 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by transgene-specific PCR assay). In some embodiments of the method for treatment described herein, the recombinant nucleotide expression vector is used at approximately 6.5 × 10⁻⁶. 10 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by Poly-A specific PCR assay). In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector is used at approximately 9.6 × 10⁻⁶ g / g brain mass. 10 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by transgene-specific PCR assay). In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector is used at approximately 2.0 × 10⁻⁶ g / g brain mass. 11 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by Poly-A specific PCR assay). In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector is used at approximately 2.9 × 10⁻⁶ g / g brain mass. 11 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by transgene-specific PCR assay).

[0241] In various embodiments of the treatment methods described herein, the human subjects are 5 years of age or older and less than 18 years of age. In specific embodiments, the human subjects are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age. In specific embodiments, the human subjects are approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age. In specific embodiments, the human subjects 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 of age. In specific implementation schemes, the age of human subjects is approximately 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. In some implementation schemes, the recombinant nucleotide expression vector is at approximately 6.5 × 10⁻⁶. 10 The dosage of GC / g is determined by MRI to measure brain mass. In some embodiments, the recombinant nucleotide expression vector is administered at the dosage specified in Table 7.

[0242] In various embodiments of the treatment methods described herein, the human subjects are 4 months or older and less than 5 years old. In specific embodiments, the human subjects are 4, 5, 6, 7, 8, 9, 10, or 11 months old. In specific embodiments, the human subjects are about 4, 5, 6, 7, 8, 9, 10, or 11 months old. In specific embodiments, the human subjects are 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months old. In specific embodiments, the human subjects are 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 subjects are 1, 2, 3, 4, or 5 years old. In specific embodiments, the human subjects are about 1, 2, 3, 4, or 5 years old. In specific embodiments, the human subjects are 1-2, 2-3, 3-4, 4-5, or 5-6 years old. In specific implementation schemes, the age of human subjects is approximately 1–2, 2–3, 3–4, 4–5, or 5–6 years. In some implementation schemes, the recombinant nucleotide expression vector is used at approximately 1.3 × 10⁻⁶ ppm. 10 GC / g is administered based on the brain mass determined by MRI. In some embodiments, the recombinant nucleotide expression vector is administered at approximately 6.5 × 10⁻⁶. 10 GC / g is administered at a dose corresponding to brain mass determined by MRI. In some embodiments, the recombinant nucleotide expression vector is administered at approximately 2.0 × 10⁻⁶. 11The dosage of GC / g is determined by MRI to measure brain mass. In some embodiments, the recombinant nucleotide expression vector is administered at approximately 2.9 × 10⁻⁶ g / g. 11 GC / g is administered at a dose determined by MRI of brain mass. In some embodiments, the recombinant nucleotide expression vector is administered at approximately 1.9 × 10⁻⁶ g / g. 10 GC / g is administered based on the brain mass determined by MRI. In some embodiments, the recombinant nucleotide expression vector is administered at approximately 9.6 × 10⁻⁶ g / g. 10 GC / g is administered at a dose determined by MRI of the brain mass. In some embodiments, the recombinant nucleotide expression vector is administered at approximately 2.0 × 10⁻⁶. 11 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by Poly-A specific PCR assay). In some embodiments, the recombinant nucleotide expression vector is administered at approximately 2.9 × 10⁻⁶ g / g brain mass. 11 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by transgene-specific PCR assay). In some embodiments, the recombinant nucleotide expression vector is administered at approximately 1.3 × 10⁻⁶ g / g brain mass. 10 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by Poly-A specific PCR assay). In some embodiments, the recombinant nucleotide expression vector is administered at approximately 1.9 × 10⁻⁶ g / g brain mass. 10 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by transgene-specific PCR assay). In some embodiments, the recombinant nucleotide expression vector is administered at approximately 6.5 × 10⁻⁶ g / g brain mass. 10 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by Poly-A specific PCR assay). In some embodiments, the recombinant nucleotide expression vector is administered at approximately 9.6 × 10⁻⁶ g / g brain mass. 10 Dosage administration of GC / g brain mass (e.g., brain mass determined by MRI and genome counting determined by transgene-specific PCR assay). In some embodiments, the recombinant nucleotide expression vector is administered at a dose selected from dose 1 or dose 2 according to Table 5. In some embodiments, the recombinant nucleotide expression vector is administered at a dose according to Table 6.

[0243] In some embodiments of the treatment methods described herein, the recombinant nucleotide expression vector is administered via intracisional (IC) administration. In other embodiments of the treatment methods described herein, the recombinant nucleotide expression vector is administered via intraventricular (ICV) administration.

[0244] In some embodiments of the treatment method described herein, the recombinant nucleotide expression vector is administered at a volume not exceeding 10% of the total cerebrospinal fluid volume of the human subject.

[0245] In some embodiments of the treatment methods described herein, glycosylated recombinant human IDS precursors are secreted at detectable levels.

[0246] In some embodiments of the treatment methods described herein, human neurons or human glial cells carry at least one mutation in an endogenous gene encoding a human IDS precursor.

[0247] In some embodiments of the treatment methods described herein, human neurons or human glial cells are transduced using a recombinant adeno-associated virus vector (rAAV).

[0248] In a preferred embodiment, the recombinant nucleotide expression vector is an AAV9 or AAVrh10 vector.

[0249] In some embodiments of the treatment methods described herein, the glycosylated recombinant human IDS precursor is expressed under the control of the CB7 promoter.

[0250] In some embodiments of the treatment methods described herein, the glycosylated recombinant human IDS precursor is expressed by cDNA encoding the human IDS precursor.

[0251] In some embodiments of the treatment methods described herein, the glycosylated recombinant human IDS precursor is approximately 90 kDa, as measured by polyacrylamide gel electrophoresis.

[0252] In some embodiments of the treatment methods described herein, the glycosylated recombinant human IDS precursor contains formylglycine.

[0253] In some embodiments of the treatment methods described herein, the glycosylated recombinant human IDS precursor is (a) α2,6-sialylated; (b) free of detectable NeuGc; (c) free of detectable α-Gal antigen; (d) tyrosine-sulfated; and / or (e) mannose-6-phosphorylated.

[0254] In some embodiments of the treatment methods described herein, the glycosylated recombinant human IDS precursor comprises the amino acid sequence of SEQ ID NO. 1.

[0255] In some embodiments provided herein, the method further includes administering immunosuppressive therapy to a human subject prior to or concurrent with human IDS precursor treatment, and optionally continuing immunosuppressive therapy thereafter.

[0256] In some implementations, immunosuppressive therapy includes the administration of one or more corticosteroids, sirolimus, and / or tacrolimus. In specific implementations, one or more corticosteroids are methylprednisolone and / or prednisone.

[0257] In specific implementation schemes, immunosuppressive therapy includes doses of approximately 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, etc. Prednisone is administered at doses of 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 specific embodiments, immunosuppressive therapy includes administration of prednisone at doses ranging from about 0.10 mg / kg to about 0.20 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of prednisone at doses ranging from about 0.20 mg / kg to about 0.30 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of prednisone at doses ranging from about 0.30 mg / kg to about 0.40 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of prednisone at a dose ranging from about 0.40 mg / kg to about 0.50 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of prednisone at a dose ranging from about 0.50 mg / kg to about 1 mg / kg. In specific embodiments, the dose is administered daily. In specific embodiments, immunosuppressive therapy includes administration of prednisone at a dose of 0.5 mg / kg daily. In another specific embodiment, immunosuppressive therapy includes administration of prednisone at a dose of 0.5 mg / kg daily, gradually tapered and discontinued.

[0258] In specific implementation schemes, immunosuppressive therapy includes administration of methylprednisolone at doses of approximately 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 specific embodiments, immunosuppressive therapy includes administration of methylprednisolone at a dose ranging from about 0.50 mg / kg to about 1.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of methylprednisolone at a dose ranging from about 1.0 mg / kg to about 2.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of methylprednisolone at a dose ranging from about 2.0 mg / kg to about 3.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of methylprednisolone at a dose ranging from about 3.0 mg / kg to about 5.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of methylprednisolone at a dose ranging from about 5.0 mg / kg to about 10.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of methylprednisolone at a dose ranging from about 10.0 mg / kg to about 15.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of methylprednisolone at a dose ranging from about 15.0 mg / kg to about 20.0 mg / kg. In a particular embodiment, methylprednisolone is administered once. In certain embodiments, methylprednisolone is administered intravenously. In certain embodiments, up to 500 mg of methylprednisolone is administered. In certain embodiments, methylprednisolone is administered over at least 30 minutes. In certain embodiments, immunosuppressive therapy comprises administering methylprednisolone intravenously at a dose of 10 mg / kg over at least 30 minutes, up to a maximum of 500 mg.

[0259] In specific implementation methods, immunosuppressive therapy includes administration of sirolimus at a dose that maintains a target blood level of 1-3 ng / mL. In specific implementation methods, immunosuppressive therapy includes administration of approximately 0.25 mg / mL. 2 / day, 0.3 mg / m 2 / day, 0.4 mg / m 2 / day, 0.5 mg / m 2 / day, 0.6 mg / m 2 / day, 0.7 mg / m 2 / day, 0.8 mg / m 2 / day, 0.9 mg / m 2 / day, 1 mg / m 2 / day, 1.25 mg / m 2 / day, 1.5 mg / m 2 / day, 1.75 mg / m 2 / day, 2 mg / m 2 / day, 2.25 mg / m 2 / day, 2.5 mg / m 2 / day, 2.75 mg / m 2 / day, 3 mg / m 2 / day, 3.25 mg / m 2 / day, 3.5 mg / m 2 / day, 3.75 mg / m 2 / day, 4 mg / m 2 / day, 4.25 mg / m 2 / day, 4.5 mg / m 2 / day, 4.75 mg / m 2 / day, or 5 mg / m 2 Sirolimus is administered at a dose of approximately 0.25 mg / m² / day. In a specific implementation plan, immunosuppressive therapy includes administration of approximately 0.25 mg / m² / day. 2 / day to approximately 0.5mg / m 2 Sirolimus is administered at a dose within the range of / day. In a specific implementation plan, immunosuppressive therapy includes administration of approximately 0.50 mg / m². 2 / day to approximately 1.0mg / m² 2 Sirolimus is administered at a dose within the range of 1.0 mg / m². In a specific implementation plan, immunosuppressive therapy includes administration of approximately 1.0 mg / m². 2 / day to approximately 1.5mg / m 2 Sirolimus is administered at a dose within the range of 1.5 mg / m². In a specific implementation plan, immunosuppressive therapy includes administration of approximately 1.5 mg / m². 2 / day to approximately 2mg / m 2 Sirolimus is administered at a dose within the range of / day. In specific implementation methods, immunosuppressive therapy includes administration of approximately 2 mg / m². 2 / day to approximately 5mg / m 2Sirolimus is administered at a dose within the range of / day. In certain embodiments, the dose is divided into BIDs for administration. In certain embodiments, immunosuppressive therapy comprises administering 1 mg / m² every approximately every 4 hours. 2 Sirolimus is administered at a dose of approximately 0.5 mg / m² / day. In certain implementation regimens, immunosuppressive therapy includes administration of sirolimus at approximately 0.5 mg / m² / day. 2 Sirolimus is administered in BID doses per day.

[0260] In specific embodiments, immunosuppressive therapy includes administration of tacrolimus at a dose that maintains a target blood level of 2-4 ng / mL. In specific embodiments, immunosuppressive therapy includes administration of tacrolimus at doses 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 specific embodiments, immunosuppressive therapy includes administration of tacrolimus at doses ranging from 0.01 mg / kg to 0.02 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of tacrolimus at doses ranging from 0.02 mg / kg to 0.03 mg / kg. In specific embodiments, immunosuppressive therapy includes administration of tacrolimus at doses ranging from 0.03 mg / kg to 0.05 mg / kg. In certain embodiments, immunosuppressive therapy includes administration of tacrolimus at a dose ranging from 0.05 mg / kg to 0.07 mg / kg. In certain embodiments, immunosuppressive therapy includes administration of tacrolimus at a dose ranging from 0.07 mg / kg to 0.10 mg / kg. In certain embodiments, the dose is administered twice daily. In certain embodiments, immunosuppressive therapy includes administration of tacrolimus twice daily at a dose of approximately 0.05 mg / kg.

[0261] In some embodiments, the method further includes administering one or more antibiotics to a human subject before or concurrently with immunosuppressive therapy. In a specific embodiment, the one or more antibiotics are trimethoprim, sulfamethoxazole, pentanediol, dapsone, and / or atovaquinone. In another specific embodiment, the one or more antibiotics are trimethoprim and / or sulfamethoxazole. In yet another specific embodiment, the one or more antibiotics are pentanediol, dapsone, and / or atovaquinone. In a specific embodiment, 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 a specific embodiment, one or more antibiotics are administered at a dose in the range of about 1 mg / kg to 2 mg / kg. In a specific embodiment, one or more antibiotics are administered at a dose in the range of about 2 mg / kg to 3 mg / kg. In a specific embodiment, one or more antibiotics are administered at a dose in the range of about 3 mg / kg to 5 mg / kg. In specific implementations, one or more antibiotics are administered at a dose ranging from about 5 mg / kg to 7 mg / kg. In specific implementations, one or more antibiotics are administered at a dose ranging from about 7 mg / kg to 10 mg / kg. In specific implementations, one or more antibiotics are administered at a dose approximately three times per week. In some implementations, one or more antibiotics are administered to prevent Pneumocystis carinii pneumonia.

[0262] In some embodiments, the method further includes administering one or more antifungal therapies to a human subject before or concurrently with immunosuppressive therapy. In some embodiments, if the absolute neutrophil count is < 500 mmHg... 3 If so, one or more antifungal therapies will be initiated.

[0263] In some embodiments, the method further includes measuring one or more of the following biomarkers after administration of the recombinant nucleotide expression vector: (a) the level of glycosaminoglycans (GAG) in CSF; (b) the level of iduronate-2-sulfatase (I2S) in CSF; (c) the level of GAG in plasma; (d) the level of I2S in plasma; (e) the level of leukocyte I2S enzyme activity; and (f) the level of GAG in urine. In a specific embodiment, the GAG ​​in CSF comprises heparin sulfate in CSF. In another specific embodiment, the GAG ​​in CSF is heparin sulfate in CSF. In another specific embodiment, the GAG ​​in plasma comprises heparin sulfate in plasma. In another specific embodiment, the GAG ​​in plasma is heparin sulfate in plasma. In another specific embodiment, the GAG ​​in urine comprises heparin sulfate in urine. In another specific embodiment, the GAG ​​in urine is heparin sulfate in urine. In a specific embodiment, the measurement step includes measuring the level of heparin sulfate in CSF. In another specific embodiment, the measurement step includes measuring the level of leukocyte I2S enzyme activity.

[0264] In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector is a liquid composition. In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector is a frozen composition. In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector is a lyophilized composition or a reconstituted lyophilized composition. In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector provided herein can be formulated into various dosage forms for IC or ICV administration. In some embodiments of the methods for treatment described herein, the recombinant nucleotide expression vector provided herein can be provided in single-dose or multi-dose formulations. As used herein, a single-dose form refers to a physically independent unit suitable for administration to human and animal subjects and individually packaged in a manner known in the art. Each unit dose contains a predetermined amount of the recombinant nucleotide expression vector and / or other components sufficient to produce the desired therapeutic effect and contains the desired drug carrier or excipient. Examples of unit dosage forms include ampoules, vials, pre-filled syringes, or cartridges.

[0265] In some embodiments of the methods for treatment described herein, the unit dosage form may be administered in fractions or multiple times. In some embodiments of the methods for treatment described herein, multiple dosage forms are multiple identical unit dosage forms packaged in a single container and administered as separate unit dosage forms. Examples of multiple dosage forms include vials, pre-filled syringes, or cartridges. In some embodiments, the pre-filled syringe contains 8.5 × 10⁸ units. 12GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 9.8 × 10⁻⁶ nucleotides. 12 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.1 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.3 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.5 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.7 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 4.2 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 4.9 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 5.5 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 6.3 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 7.3 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 8.5 × 10⁸ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 9.0 × 10⁻⁶ nucleotides. 13 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.0 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.1 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.2 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.3 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.4 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.5 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.6 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.7 × 10⁻⁶ nucleotides. 14GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.8 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.9 × 10⁻⁶ nucleotides. 14 The recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.0 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.1 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.2 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.3 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.4 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.5 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.6 × 10⁻⁶ nucleotides. 14 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.3 × 10⁻⁶ nucleotides. 10 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 1.9 × 10⁻⁶ nucleotides. 10 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 6.5 × 10⁻⁶ nucleotides. 10 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 9.6 × 10⁻⁶ nucleotides. 10 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.0 × 10⁻⁶ nucleotides. 11 GC recombinant nucleotide expression vector. In some embodiments, the pre-filled syringe contains 2.9 × 10⁻⁶ nucleotides. 11 GC recombinant nucleotide expression vector.

[0266] As used herein, the term "about" means within ±10% of a given value or range. In some embodiments, the term "about" means within ±1% of a given value or range, where the value is a dose dependent on the brain mass of a human subject, and where the brain mass is determined by brain MRI of the human subject's brain. In some embodiments, the term "about" means within ±2% of a given value or range, where the value is a dose determined by brain MRI of a subject's brain, and where the brain mass is determined by brain MRI of the human subject's brain. In some embodiments, the term "about" means within ±5% of a given value or range, where the value is a dose dependent on the brain mass of a human subject, and where the brain mass is determined by brain MRI of the human subject's brain. In some embodiments, the term "about" means within ±7% of a given value or range, where the value is a dose dependent on the brain mass of a human subject, and where the brain mass is determined by brain MRI of the human subject's brain. In some implementations, the term "about" means within plus or minus 10% of a given value or range, where the value is a dose dependent on the brain mass of a human subject, and where the brain mass is determined by brain MRI of the human subject's brain. However, it should be understood that in this specification, the term "about" also provides support for a description of the precise value to which the term is associated. For example, "about 10" also precisely provides support for the number "10".

[0267] 5.1 Processing, N-glycosylation and tyrosine sulfation 5.1.1. Processing Human IDS comprises a 25-amino acid signal sequence that is cleaved during processing. Following modification of the oligosaccharide chain of the initial 76 kDa intracellular IDS precursor in the Golgi apparatus, the precursor is converted into a phosphorylated 90 kDa IDS precursor. This precursor is then processed into its predominant 55 kDa form via various intracellular intermediates through glycosylation modification and proteolytic cleavage. In summary, proteolytic processing involves N... 31 Downstream N-terminal proteolytic cleavage removes eight amino acid residues (residues 26-33) of the propeptide, as well as the N... 513 Upstream C-terminal proteolytic cleavage releases an 18 kDa polypeptide and generates a 62 kDa intermediate, which is converted to a 55 kDa mature form. Further proteolytic cleavage produces a 45 kDa mature form located in the lysosomal compartment. See Figure 4Figures and tables are reproduced from Millat et al., 1997, Exp CellRes 230: 362-367 (“Millat 1997”); Millat et al., 1997, Biochem J. 326: 243-247 (“Millat 1997a”); and Froissart et al., 1995, Biochem J. 309:425-430, each of which is incorporated herein by reference in its entirety.

[0268] C required for enzyme activity 84 Formicoylglycine modification ( Figure 1 (Highlighted in bold) This may occur as an early post-translational or co-translational event, most likely in the endoplasmic reticulum. See Millat 1997a, citing Schmidt et al., 1995, Cell 82: 271-278). Post-translational processing continues in the Golgi apparatus, involving the addition of complex sialic acid-containing glycans and the acquisition of mannose-6-phosphate residues, which label the enzyme for delivery to the lysosomal compartment. See A concise review by Clarke, 2008, Expert Opin Pharmacother 9: 311-317, which is incorporated herein by reference in its entirety.

[0269] In the specific implementation plan, when exist When expressed in vivo or in vitro in neurons or glial cells, the HuGlyIDS used according to the methods described herein can be an enzyme in 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. IDS produced by neurons and glial cells may contain higher levels of M6P, as reported in Daniele 2002 and Sleat, Proteomics, 2005 (indicating that the human brain contains more M6P glycoprotein (both quantitatively and qualitatively) than other tissues). The M6P content of IDS precursors can be measured, as performed in Daniele 2002.

[0270] Therefore, in some embodiments, when expressed in vivo or in vitro in neurons or glial cells, HuGlyIDS used according to the methods described herein is mannose-6-phosphorylated at a higher level than IDS expressed in non-neuronal or glial cells. Specifically, when expressed in vivo or in vitro in neurons or glial cells, HuGlyIDS used according to the methods described herein is mannose-6-phosphorylated at a higher level than IDS expressed in HT1080 or CHO cells. In some embodiments, the mannose-6-phosphorylation level of the expressed IDS is measured by the uptake of the IDS by human neuronal cells in the presence of M6P (e.g., 5 mM M6P). In some embodiments, when... exist When expressed in vivo or in vitro in neurons or glial cells, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% of the HuGlyIDS molecules used according to the methods described herein are mannose-6-phosphorylated.

[0271] 5.1.2. N-glycosylation Neurons and glial cells in the CNS are secretory cells possessing post-translational mechanisms for secreting proteins, including glycosylation and tyrosine-O-sulfation. hIDS has the ability to... Figure 1 Eight asparagine (“N”) glycosylation sites (N) identified in the study 31 ST;N 115 FS; N 144 HT;N 246 IT; N 280 IS; N 325 ST;N 513 FS; N 537 DS). Two of the eight N-linked glycosylation sites, namely N 280 and N 116 In IDS obtained from the human brain, it is mannose-6-phosphorylated. (Sleat et al., 2006, Mol & Cell Proeomics 5.4: 686-701, reported in Table V).

[0272] Although a single glycosylation site is not important for IDS stability, position N 280Glycosylation at the cellular level is crucial for internalization and lysosomal targeting via the mannose-6-phosphate (M6P) receptor (Chung et al., 2014, Glycoconj J 31:309-315, p. 310, column 1). Under normal physiological conditions, IDS are produced at very low levels, and the amount of enzymes secreted by the cell (if present) is also very small (Clarke, 2008). Same as above ).

[0273] Each molecule produced in gene therapy or protein therapy does not need to be fully glycosylated and sulfated. Instead, the resulting glycoprotein population should have sufficient glycosylation and sulfatedity to demonstrate efficacy.

[0274] In specific embodiments, when expressed in vivo or in vitro in neurons or glial cells, HuGlyIDS used according to the methods described herein can be glycosylated at 100% of its N-glycosylation sites. However, those skilled in the art will understand that not every N-glycosylation site of HuGlyIDS needs to be N-glycosylated to obtain the benefits of glycosylation. Rather, the benefits of glycosylation can be achieved only when a certain percentage of N-glycosylation sites are glycosylated and / or only when a certain percentage of the expressed IDS molecules are glycosylated. Therefore, in some embodiments, when exist When expressed in vivo or in vitro in neurons or glial cells, HuGlyIDS used according to the methods described herein is glycosylated at 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% of its available N-glycosylation sites. In some embodiments, when exist When expressed in vivo or in vitro in neurons or glial cells, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, or 90%–100% of the HuGlyIDS molecule used according to the methods described herein are glycosylated at at least one of its available N-glycosylation sites.

[0275] In a specific implementation, when HuGlyIDS used according to the method described herein is expressed in vivo or in vitro in neurons or glial cells, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in the HuGlyIDS are glycosylated at Asn residues (or other relevant residues) present in the N-glycosylation sites. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resulting HuGlyIDS are glycosylated.

[0276] In another specific embodiment, when HuGlyIDS used according to the methods described herein is expressed in vivo or in vitro in neurons or glial cells, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in the HuGlyIDS molecule are glycosylated with the same attached glycans linked to the Asn residues (or other related residues) present in the N-glycosylation sites. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resulting HuGlyIDS have the same attached glycans.

[0277] Importantly, when the IDS protein used according to the methods described herein is expressed in neurons or glial cells, it avoids expression in prokaryotic host cells (e.g., Escherichia coli). E. coli The need for in vitro production in eukaryotic host cells (e.g., CHO cells) is not present. Instead, due to the methods described herein (e.g., using neurons or glial cells to express IDS), it is advantageous to use therapeutically relevant and beneficial glycans to modify the N-glycosylation sites of IDS proteins, and, particularly, the therapeutic target sites. This advantage is not available when CHO cells or E. coli are used for protein production because, for example, CHO cells (1) do not express 2,6-sialyltransferase and therefore cannot add 2,6-sialic acid during N-glycosylation, and (2) Neu5Gc can be added instead of Neu5Ac as sialic acid; and because E. coliNaturally, it does not contain the components required for N-glycosylation. Furthermore, this advantage may not be realized when human cells, which are not neurons or glial cells, are used for protein production. Therefore, in one embodiment, the IDS protein expressed in neurons or glial cells to produce the HuGlyIDS used in the treatment methods described herein is glycosylated in a manner that allows for N-glycosylation of the protein in human neurons or glial cells, but not in a manner that allows for glycosylation of the protein in CHO cells. In another embodiment, the IDS protein expressed in neurons or glial cells to produce the HuGlyIDS used in the treatment methods described herein is glycosylated in a manner that allows for N-glycosylation of the protein in neurons or glial cells, wherein such glycosylation is naturally impossible using prokaryotic host cells (e.g., using *E. coli*). In one embodiment, the IDS protein expressed in human neurons or glial cells to produce the HuGlyIDS used in the treatment methods described herein is glycosylated in a manner that allows for N-glycosylation of the protein in human neurons or glial cells, but not in a manner that allows for glycosylation of the protein in human cells, which are not neurons or glial cells.

[0278] Determination of protein glycosylation patterns is known in the art. For example, hydrazine hydrolysis can be used to analyze glycans. First, by incubation with hydrazine, the glycan is released from its associated protein (using the Ludger Liberate Hydrazine Hydrolysis Glycan Release Kit, Oxfordshire, UK). The nucleophilic hydrazine attacks the glycosidic bond between the glycan and the carrier protein, allowing the release of the attached glycan. N-acetyl groups are lost during this treatment and must be reconstituted by re-N-acetylation. The free glycan can be purified on a carbon column and subsequently labeled at the reducing end with the fluorophore 2-aminobenzamide. The labeled glycan can be separated on a GlycoSep-N column (GLSciences) according to the HPLC protocol described by Royle et al., Anal Biochem 2002, 304(1):70-90. The resulting fluorescence chromatograms indicate the length of the glycan and the number of repeating units. Structural information can be collected by collecting the individual peaks and then performing MS / MS analysis. Thus, the monosaccharide composition and the sequence of repeating units can be confirmed, and the homogeneity of the glycan composition can also be identified. Specific peaks at low molecular weights can be analyzed by MALDI-MS / MS, and the results are used to confirm the glycan sequence. Each peak corresponds to a polymer composed of a certain number of repeating units and their fragments. Therefore, the chromatogram allows for the measurement of polymer length distribution. Elution time is an indicator of polymer length, while fluorescence intensity is correlated with the molar abundance of the corresponding polymer.

[0279] Because the homogeneity of glycan patterns is related to glycan length and the number of glycans present at glycosylation sites, the homogeneity of glycan patterns associated with proteins can be assessed using methods known in the art, such as methods for measuring glycan length and hydrodynamic radius. Size exclusion HPLC allows for the measurement of hydrodynamic radius. A greater number of glycosylation sites in a protein results in a larger variation in hydrodynamic radius compared to carriers with fewer glycosylation sites. However, when analyzing individual glycan chains, they may be more homogeneous due to their longer controlled lengths. Glycan length can be measured by hydrazine hydrolysis, SDS-PAGE, and capillary gel electrophoresis. Furthermore, homogeneity can also refer to a change in the pattern of some glycosylation sites, resulting in a wider / narrower range. These factors can be measured by glycopeptide LC-MS / MS.

[0280] N-glycosylation imparts numerous benefits to the HuGlyIDs used in the methods described herein. These benefits cannot be obtained through... big Enterobacteriaceae The benefits are not achieved through protein production in humans, as *E. coli* does not naturally possess the components required for N-glycosylation. Furthermore, some benefits cannot be obtained through protein production in, for example, CHO cells, because CHO cells lack the components required for the addition of certain glycans (e.g., 2,6-sialic acid), and because CHO cells can add glycans atypical for humans (e.g., Neu5Gc) and α-Gal antigens, which are immunogenic in most individuals and can trigger allergic reactions at high concentrations. Additionally, some benefits cannot be achieved through protein production in human cells that are not neurons or glial cells. Therefore, expression of IDS in human neurons or glial cells leads to the production of HuGlyIDS containing beneficial glycans, which would otherwise not associate with proteins if produced in CHO cells, *E. coli*, or human cells that are not neurons or glial cells.

[0281] 5.1.3. Tyrosine sulfation In addition to N-linked glycosylation sites, hIDS also contain tyrosine ("Y") sulfation sites (PSSEKY). 165 ENTKTCRGPD (SEQ ID NO: 47)). (Analysis of amino acids surrounding tyrosine residues that have undergone protein tyrosine sulfate treatment.) See also, For example, Yang et al., 2015, Molecules 20:2138-2164, especially page 2154, which is incorporated in its entirety by reference. The “rule” can be summarized as follows: Y residues with E or D are in positions +5 to -5 of Y, and where position -1 of Y is a neutral or acidic charged amino acid rather than a basic amino acid, such as an eliminated sulfated R, K, or H).

[0282] Importantly, tyrosine-sulfated proteins cannot... E. coli Because *E. coli* does not naturally possess the enzyme required for tyrosine sulfation, *CHO* cells lack tyrosine sulfation; they are not secretory cells and have limited post-translational tyrosine sulfation capacity. See, for example, Mikkelsen & Ezban, 1991, *Biochemistry* 30:1533-1537. Advantageously, the methods provided herein require the expression of an IDS, such as HuGlyIDS, in neurons or glial cells that are secretory and do possess tyrosine sulfation capacity. Assays for detecting tyrosine sulfation are known in the art. See, for example, Yang et al., 2015, *Molecules* 20:2138-2164.

[0283] Tyrosine sulfated hIDS (a robust post-translational process in human CNS cells) should lead to improved processing and activity of transgenic products. The significance of tyrosine sulfated lysosomal proteins is not yet fully elucidated; however, in other proteins, it has been shown to increase protein-protein interaction affinity (antibodies and receptors) and promote proteolytic processing (peptide hormones). (See Moore, 2003, J Biol. Chem. 278:24243-46; and Bundegaard et al., 1995, TheEMBO J 14: 3073-79). Tyrosyl protein sulfotransferase (TPST1), which causes tyrosine sulfated tyrosine (which can occur as a final step in IDS processing), is expressed at significantly higher levels (mRNA-based) in the brain. (Gene expression data for TPST1 are available, for example, in the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home).

[0284] 5.2 Components and Formulations The methods provided herein are used with viral vectors or other DNA expression constructs encoding iduronate-2-sulfatase (IDS) (e.g., human IDS (hIDS)). The methods provided herein are used with viral vectors or other DNA expression constructs encoding glycosylated (HuGly)α-L-iduronase (IDUA) (e.g., human IDUA (hIDUA)). The viral vectors and other DNA expression constructs provided herein include any suitable method for delivering transgenes to cerebrospinal fluid (CSF). Transgene delivery methods include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetically modified mRNA, unmodified mRNA, small molecules, non-bioactive molecules (e.g., gold particles), polymeric molecules (e.g., dendritic polymers), naked DNA, plasmids, bacteriophages, transposons, granules, or episomes. In some embodiments, the vector is a targeting vector, such as a vector targeting neuronal cells.

[0285] In some aspects, this disclosure provides 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-1α promoter, UB6 promoter, chicken β-actin promoter, CAG promoter, RPE65 promoter, and opsin promoter.

[0286] In some embodiments, this document provides recombinant vectors comprising one or more nucleic acids (e.g., polynucleotides). Nucleic acids may include DNA, RNA, or a combination of DNA and RNA. In some embodiments, the DNA comprises one or more sequences selected from the group consisting of: a promoter sequence, a sequence of the gene of interest (transgenic, such as an IDS), an untranslated region, and a termination sequence. In some embodiments, the viral vectors provided herein comprise a promoter operatively linked to the gene of interest.

[0287] In some embodiments, the nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein may be codon-optimized, for example via any codon optimization technique known to those skilled in the art. See For example, a review by Quax et al., 2015, Mol Cell 59:149-161.

[0288] In another aspect, this disclosure provides a formulation comprising a recombinant nucleotide expression vector encoding human IDS, wherein the formulation is suitable for administration to cerebrospinal fluid of a human brain, such that a reservoir is formed in the central nervous system of a human who secretes a recombinant human IDS glycoprotein precursor, said precursor being 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, containing formylglycine, being α2,6-sialylated, not containing detectable NeuGc, not containing α-Gal antigen, and / or mannose-6-phosphorylated. For example, the formulation may contain a buffer (such as a buffer with a specific pH or a buffer containing specific components) that makes it suitable for administration to the human brain via cerebrospinal fluid, such that a reservoir is formed in the central nervous system of a person secreting a recombinant human IDS glycoprotein precursor, said precursor being 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, containing formylglycine, being α2,6-sialylated, not containing detectable NeuGc, not containing α-Gal antigen, and / or mannose-6-phosphorylated. In a specific embodiment, the buffer comprises a physiologically compatible aqueous buffer, a surfactant, and optional excipients.

[0289] In another aspect, this disclosure provides a kit comprising a recombinant nucleotide expression vector encoding human IDS and a pharmaceutically acceptable carrier, wherein the recombinant nucleotide expression vector is suitable for administration to cerebrospinal fluid (CSF) of the human brain, such that a reservoir is formed in the central nervous system of a person secreting a recombinant human IDS glycoprotein precursor, said precursor being 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, containing formylglycine, being α2,6-sialylated, not containing detectable NeuGc, not containing detectable α-Gal antigen, and / or mannose-6-phosphorylated. In another aspect, this disclosure provides a kit comprising a formulation containing a recombinant nucleotide expression vector encoding a human IDS, wherein the formulation is suitable for administration to the CSF of the human brain, such that a reservoir is formed in the central nervous system of a person secreting a recombinant human IDS glycoprotein precursor, said precursor being 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, containing formylglycine, being α2,6-sialylated, not containing detectable NeuGc, not containing detectable α-Gal antigen, and / or mannose-6-phosphorylated. The kits described herein comprise a recombinant nucleotide expression vector or a formulation in one or more containers. Optionally combined with such one or more containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, reflecting approval for human administration by the manufacturing, using, or selling agency.

[0290] The formulations and kits covered herein can be used according to the methods for treating human patients provided in this disclosure.

[0291] 5.2.1. mRNA In some embodiments, the vector provided herein is a modified mRNA encoding a gene of interest (e.g., a transgene, such as an IDS). The synthesis of modified and unmodified mRNAs for delivering transgenes to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is incorporated herein by reference in its entirety. In some embodiments, this document provides a modified mRNA encoding an IDS (e.g., hIDS).

[0292] 5.2.2. Viral Vector Viral vectors include adenoviruses, adeno-associated viruses (AAVs, such as AAV9 and AAVrh10), lentiviruses, helper-dependent adenoviruses, herpes simplex viruses, poxviruses, hemagglutinin virus of Japan (HVJ), alphaviruses, vaccinia viruses, and retroviral vectors. Retroviral vectors include vectors based on murine leukemia virus (MLV) and human immunodeficiency virus (HIV). Alphavirus vectors include semliki forestvirus (SFV) and sindbis virus (SIN). In some embodiments, the viral vectors provided herein are recombinant viral vectors. In some embodiments, the viral vectors provided herein are modified to be replication-deficient in humans. In some embodiments, the viral vectors are hybrid vectors, such as AAV vectors contained in a "helpless" adenovirus vector. In some embodiments, the viral vectors provided herein comprise a viral capsid from a first virus and a viral envelope protein from a second virus. In a specific implementation, the second virus is vesicular stomatitis virus (VSV). In a more specific implementation, the envelope protein is the VSV-G protein.

[0293] In some embodiments, the viral vector provided herein is an HIV-based viral vector. In some embodiments, the HIV-based vector provided herein contains at least two polynucleotides, wherein the gag and pol genes are derived from the HIV genome and the env gene is derived from another virus.

[0294] In some embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In some embodiments, the herpes simplex virus-based vectors provided herein are modified so that they do not contain one or more immediate early (IE) genes, thereby making them non-cytotoxic.

[0295] In some embodiments, the viral vectors provided herein are MLV-based viral vectors. In some embodiments, the MLV-based vectors provided herein contain up to 8 kb of heterologous DNA in place of the viral genes.

[0296] In some embodiments, the viral vector provided herein is a lentivirus-based viral vector. In some embodiments, the lentivirus vector provided herein is derived from human lentivirus. In some embodiments, the lentivirus vector provided herein is derived from non-human lentivirus. In some embodiments, the lentivirus vector provided herein is packaged into a lentivirus capsid. In some embodiments, the lentivirus vector provided herein comprises one or more of the following elements: long terminal repeat sequence, primer binding site, polypurine tract, att site, and capsidation site.

[0297] In some embodiments, the viral vector provided herein is an alphavirus-based viral vector. In some embodiments, the alphavirus vector provided herein is a recombinant replication-defective alphavirus. In some embodiments, the alphavirus replicon in the alphavirus vector provided herein targets a specific cell type by displaying a functional heterologous ligand on its virion surface.

[0298] In some embodiments, the viral vectors provided herein are AAV-based viral vectors. In a preferred embodiment, the viral vectors provided herein are AAV9 or AAVrh10-based viral vectors. In some embodiments, the AAV9 or AAVrh10-based viral vectors provided herein retain tropism towards CNS cells. Multiple AAV serotypes have been identified. In some embodiments, the AAV-based vectors provided herein comprise components from one or more AAV serotypes. In some embodiments, the AAV-based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV10, or AAV11. In a preferred embodiment, the AAV-based vectors provided herein comprise components from one or more of AAV8, AAV9, AAVrh10, AAV10, or AAV11 serotypes. AAV9-based viral vectors are used in the methods described herein. Nucleic acid sequences of AAV-based viral vectors and methods for preparing recombinant AAV and AAV capsids are taught in, for example, U.S. Patent Nos. 7,282,199 B2, 7,790,449 B2, 8,318,480 B2, 8,962,332 B2, and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety. In one aspect, this document provides AAV-based (e.g., AAV9 or AAVrh10) viral vectors encoding transgenes (e.g., IDS). In a particular embodiment, this document provides AAV9-based viral vectors encoding IDS. In a more particular embodiment, this document provides AAV9-based viral vectors encoding hIDS.

[0299] In certain embodiments, an AAV9 vector comprising an artificial genome is provided, the artificial genome comprising: (i) an expression cassette containing a transgene controlled by a regulatory element and side-linked to an ITR; and (ii) a viral capsid having the amino acid sequence of the AAV9 capsid protein or having at least 95%, 96, 97%, 98%, 99%, or 99.9% identity with the amino acid sequence of the AAV9 capsid protein (SEQ ID NO: 26), while retaining the biological function of the AAV9 capsid. In some embodiments, the encoded AAV9 capsid has the sequence of SEQ ID NO: 26, having substitutions of 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 acids, and retaining the biological function of the AAV9 capsid. Figure 6Comparative alignments of amino acid sequences of capsid proteins from different AAV serotypes are provided, where potential amino acids that can be substituted at certain positions in the aligned sequences are based on comparisons in rows labeled SUBS. Therefore, in a specific embodiment, the AAV9 vector comprises an AAV9 capsid variant having... Figure 6 The 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 line, wherein the amino acid substitutions are not present at the said positions in the natural AAV9 sequence.

[0300] In some embodiments, the AAV used in the methods described herein is Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated herein by reference in its entirety. In some embodiments, the AAV used in the methods described herein comprises one of the following amino acid inserts: LGETTRP (SEQ ID NO: 56) or LALGETTRP (SEQ ID NO: 57), as described in U.S. Patent Nos. 9,193,956; 9,458,517; and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, the AAV used in the methods described herein is AAV.7m8, such as that described in U.S. Patent Nos. 9,193,956; 9,458,517; and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, the AAV used in the methods described herein is any AAV disclosed in U.S. Patent No. 9,585,971, such as AAV-PHP.B. In some embodiments, the AAV 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: U.S. Patent Nos. 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; ... 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282; U.S. Patent Application Publication Nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0301] In some embodiments, single-stranded AAV (ssAAV) can be used, as described in the previous patent. In some embodiments, self-complementary vectors, such as scAAV ( SeeFor example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al., 2001, Gene Therapy, Vol. 8, No. 16, pp. 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.

[0302] In some embodiments, the viral vector used in the methods described herein is an adenovirus-based viral vector. Recombinant adenovirus vectors can be used for transfer in IDS. The recombinant adenovirus can be a first-generation vector with an E1 deletion, with or without an E3 deletion, and an expression cassette inserted into either deleted region. The recombinant adenovirus can be a second-generation vector containing all or part of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus inverted terminal repeat sequence and the packaging signal (φ). With or without a filler sequence that maintains the artificial genome approximately 36 kb in size (wild-type), the transgene is inserted between the packaging signal and the 3' ITR. Exemplary protocols for producing adenovirus vectors can be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for genetherapy,” Gene Therapy 12:S18-S27, which is incorporated herein by reference in its entirety.

[0303] In some embodiments, the viral vector used in the methods described herein is a lentivirus-based viral vector. Recombinant lentivirus vectors can be used for transfer in IDS. Four types of plasmids are used to prepare the constructs: a plasmid containing the Gag / pol sequence, a plasmid containing the Rev sequence, a plasmid containing the envelope protein (i.e., VSV-G), and a Cis plasmid containing packaging elements and the IDS gene.

[0304] For the production of lentiviral vectors, four plasmids are co-transfected into cells (i.e., HEK293-based cells), with polyethyleneimine or calcium phosphate being particularly suitable transfection agents. Lentiviral virus is then harvested from the supernatant (lentiviruses require budding from cells to become active, therefore cell harvesting is not required / should not be performed). The supernatant is filtered (0.45 μm), and then magnesium chloride and a totipotent nuclease (benzonase) are added. Further downstream processes can vary considerably, with the use of TFF and column chromatography being the most GMP-compliant. Other processes use ultracentrifugation and / or do not use column chromatography. Exemplary protocols for producing lentiviral vectors can 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 herein by reference in their entirety.

[0305] In specific embodiments, the vector used in the methods described herein is a vector encoding an IDS (e.g., hIDS) such that, upon transduction of cells in the CNS or related cells (e.g., in vivo or in vitro neuronal cells), the transduced cells express a glycosylated variant of the IDS. In specific embodiments, the vector used in the methods described herein is a vector encoding an IDS (e.g., hIDS) such that, upon transduction of cells in the CNS or related cells (e.g., in vivo or in vitro neuronal cells), the cells express a sulfated variant of the IDS.

[0306] 5.2.3. Promoters and Modifiers of Gene Expression In some embodiments, the vectors provided herein contain components that regulate gene delivery or gene expression (e.g., "expression control elements"). In some embodiments, the vectors provided herein contain components that regulate gene expression. In some embodiments, the vectors provided herein contain components that influence binding to or targeting cells. In some embodiments, the vectors provided herein contain components that influence the localization of polynucleotides (e.g., transgenes) within cells after uptake. In some embodiments, the vectors provided herein contain components, for example, detectable or selectable markers for detecting or selecting cells that absorb polynucleotides.

[0307] In some embodiments, the viral vector provided herein contains one or more promoters. In some embodiments, the promoter is a constitutive promoter. In alternative embodiments, the promoter is an inducible promoter. As with most housekeeping genes, native IDS genes primarily use GC-rich promoters. In a preferred embodiment, a strong constitutive promoter that provides sustained hIDS expression is used. Such promoters include the synthetic “CAG” promoter containing: “C”, an early enhancer element of cytomegalovirus (CMV); “A”, a promoter of the chicken β-actin gene along with its first exon and intron; and “G”, a splice acceptor of the rabbit β-globin gene (see Miyazaki et al., 1989, Gene 79: 269-277; and Niwa et al., Gene 108: 193-199).

[0308] In some implementations, the promoter is the CB7 promoter ( See Dinculescu et al., 2005, HumGene Ther 16: 649-663 (which is incorporated herein by reference in its entirety). In some embodiments, the CB7 promoter includes other expression control elements that enhance vector-driven transgene expression. In some embodiments, other expression control elements include chicken β-actin introns and / or rabbit β-globin polA signaling. In some embodiments, the promoter contains a TATA cassette. In some embodiments, the promoter contains one or more elements. In some embodiments, one or more promoter elements may be anti-reverse or movable relative to each other. In some embodiments, the elements of the promoter are positioned to function synergistically. In some embodiments, the elements of the promoter are positioned to function independently. In some embodiments, the viral vectors provided herein contain one or more promoters selected from the group consisting of: human CMV early gene promoter, SV40 early promoter, Rous sarcoma virus (RS) long terminal repeat sequence, and rat insulin promoter. In some 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 some embodiments, the vectors provided herein comprise one or more tissue-specific promoters (e.g., neuron cell-specific promoters).

[0309] In some embodiments, the viral vector provided herein includes one or more regulatory elements in addition to a promoter. In some embodiments, the viral vector provided herein includes an enhancer. In some embodiments, the viral vector provided herein includes a repressor. In some embodiments, the viral vector provided herein includes an intron or a chimeric intron. In some embodiments, the viral vector provided herein includes a polyadenylated sequence.

[0310] 5.2.4. Signal peptide In some embodiments, the vectors provided herein contain components that regulate protein delivery. In some embodiments, the viral vectors provided herein contain one or more signal peptides. In some embodiments, the signal peptide allows the transgenic product (e.g., IDS) to achieve proper packaging (e.g., glycosylation) in the cell. In some embodiments, the signal peptide allows the transgenic product (e.g., IDS) to achieve proper localization in the cell. In some embodiments, the signal peptide allows the transgenic product (e.g., IDS) to be secreted from the cell. Examples of signal peptides used in conjunction with the vectors and transgenes provided herein are shown in Table 4. The signal peptide may also be referred to herein as a leader sequence or leader peptide.

[0311] Table 4. Signal peptides used with the vectors provided herein. 5.2.5. Non-translated area In some embodiments, the viral vector provided herein comprises one or more untranslated regions (UTRs), such as 3' and / or 5' UTRs. In some embodiments, the UTRs are optimized for desired protein expression levels. In some embodiments, the UTRs are optimized for the half-life of the transgenic mRNA. In some embodiments, the UTRs are optimized for the stability of the transgenic mRNA. In some embodiments, the UTRs are optimized for the secondary structure of the transgenic mRNA.

[0312] 5.2.6. Inverted terminal repeat sequences In some embodiments, the viral vector provided herein contains one or more inverted terminal repeat (ITR) sequences. The ITR sequences can be used to package recombinant gene expression cassettes into the virion of the viral vector. In some embodiments, the ITRs are derived from AAV, such as AAV9 (…). See also,For example, Yan et al., 2005, J. Virol., 79(1):364-379; U.S. Patent Nos. 7,282,199 B2, 7,790,449 B2, 8,318,480 B2, 8,962,332 B2 and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety.

[0313] 5.2.7. Genetically Modified Organisms In some embodiments, the vector provided herein encodes an IDS transgene. In specific embodiments, the IDS is controlled by an appropriate expression control element for expression in neuronal cells: in some embodiments, the IDS (e.g., hIDS) transgene comprises the amino acid sequence of SEQ ID NO: 1. In some 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 shown in SEQ ID NO: 1.

[0314] Transgenic HuGlyIDS may include, but is not limited to, human IDS (hIDS), which have the amino acid sequence of SEQ ID NO.1 (e.g., Figure 1 (as shown); and derivatives of hIDS that have amino acid substitutions, deletions, or additions, for example ， Including but not limited to those selected from Figure 2 The amino acid substitutions of corresponding non-conserved residues in orthologs of the IDS shown are provided that such mutations do not include the substitution of the cysteine ​​residue at position 84 (C84), which is required for enzyme activity (Millat et al., 1997, Biochem J 326: 243-247); or mutations already identified in severe, severe-moderate, moderate, or weakened MPS II phenotypes, such as... Figure 3Mutations shown or reported as follows: Sukegawa-Hayasaka et al., 2006, J Inhert Metab Dis 29: 755-761 (reporting “attenuated” mutants R48P, A85T, W337R and truncated mutant Q531X; and “severe” mutants P86L, S333L, S349I, R468Q, R468L); Millat et al., 1998, BBA 1406: 214-218 (reporting “attenuated” mutants P480L and P480Q; and “severe” mutant P86L); and Bonucelli et al., 2001, BBA 1537:233-238, each of which is incorporated herein by reference in its entirety.

[0315] For example, the amino acid substitutions at specific positions in hIDS can be selected from... Figure 2 The corresponding non-conserved amino acid residues present at the positions described in the IDS orthologs compared with the Chinese model. Prerequisites This type of replacement does not include Figure 3 Any harmful mutations shown or reported as follows: Sukegawa-Hayasaka et al., 2006, Same as above Millat et al., 1998, Same as above ; or Bonucelli et al., 2001, Same as above Each of the cited references is incorporated herein by reference in its entirety. The resulting transgenic products can be tested using routine assays in vitro, in cell cultures, or in test animals to ensure that the mutations do not impair IDS function. Selected preferred amino acid substitutions, deletions, or additions should be those that maintain or increase the enzymatic activity, stability, or half-life of the IDS, as tested using routine assays in vitro, in MPS II cell cultures, or in animal models. For example, the enzymatic activity of the transgenic products can be assessed using routine enzyme assays, such as with 4-methylumbelliferone α-L-idopyranuronic acid 2-sulfate or 4-methylumbelliferone sulfate as substrates (…). SeeFor example, exemplary IDS enzyme assays available in Lee et al., 2015, Clin. Biochem. 48(18):1350-1353, and Dean et al., 2006, Clin. Chem. 52(4):643-649 (each of which is incorporated herein by reference in its entirety). The ability of transgenic products to correct MPS II phenotypes can be assessed in cell cultures; for example, by transducing MPS II cells in cultures with a viral vector or other DNA expression construct encoding hIDS or derivatives; by adding transgenic products or derivatives to MPS II cells in cultures; or by co-culturing MPS II cells with human neuronal / glial host cells engineered to express and secrete rhIDS or derivatives, and determining the correction of defects in MPS II cultured cells, for example, by detecting IDS enzyme activity and / or a reduction in GAG storage in MPS II cells in cultures. See also, For example, Stroncek et al., 1999, Transfusion 39(4):343-350, which is incorporated into this paper in its entirety by reference.

[0316] In some embodiments, a PCR assay is used to determine the dosage of the recombinant AAV of this disclosure. 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 dosage determined in one assay differs from the dosage determined in another assay (e.g., higher or lower). For example, the dosage determined using a transgene-specific PCR assay is higher than the dosage determined using a Poly-A specific PCR assay (e.g., about 50% higher). In some embodiments, the dosage determined by a transgene-specific PCR assay is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than about 75% higher than the dosage determined by a Poly-A specific PCR assay. In some embodiments, 2.0 × 10 11 The dose of GC / g brain mass (where the genome copy number was determined using Poly-A specific PCR assay) is equivalent to 2.9 × 10⁻⁶. 11 The dose is measured in GC / g brain mass (where the genome copy number is determined using transgene-specific PCR assay). In some embodiments, the total dose administered to the subject takes into account the subject's estimated brain mass, which can be determined using magnetic resonance imaging (MRI) screening.

[0317] 5.2.8. Constructs In some embodiments, the viral vector provided herein comprises the following elements in the following order: a) a first ITR sequence, b) a first adapter sequence, c) a promoter sequence, d) a second adapter sequence, e) an intron sequence, f) a third adapter sequence, g) a sequence encoding a transgene (e.g., IDS), h) a fourth adapter sequence, i) a poly A sequence, j) a fifth adapter sequence, and k) a second ITR sequence.

[0318] In some embodiments, the viral vector provided herein comprises the following elements in the following order: a) a promoter sequence, and b) a sequence encoding a transgene (e.g., IDS). In some embodiments, the viral vector provided herein comprises the following elements in the following order: a) a promoter sequence, and b) a sequence encoding a transgene (e.g., IDS), wherein said transgene comprises a signal peptide.

[0319] In some embodiments, the viral vector provided herein comprises the following elements in the following order: a) a first ITR sequence, b) a first adapter sequence, c) a promoter sequence, d) a second adapter sequence, e) an intron sequence, f) a third adapter sequence, g) a first UTR sequence, h) a sequence encoding a transgene (e.g., IDS), i) a second UTR sequence, j) a fourth adapter sequence, k) a poly A sequence, l) a fifth adapter sequence, and m) a second ITR sequence.

[0320] In some embodiments, the viral vector provided herein comprises the following elements in the following order: a) a first ITR sequence, b) a first adapter sequence, c) a promoter sequence, d) a second adapter sequence, e) an intron sequence, f) a third adapter sequence, g) a first UTR sequence, h) a sequence encoding a transgene (e.g., IDS), i) a second UTR sequence, j) a fourth adapter sequence, k) a poly A sequence, l) a fifth adapter sequence, and m) a second ITR sequence, wherein the transgene comprises a signal peptide, and wherein the transgene encodes hIDS.

[0321] In specific implementation schemes, the viral vectors described herein include, for example: Figure 5 The components and their order are shown.

[0322] 5.2.9. Carrier Manufacturing and Testing The viral vectors provided in this article can be manufactured using host cells. Specifically, they can be manufactured using mammalian host cells such as A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. The viral vectors provided in this article can also be manufactured using host cells derived from humans, monkeys, mice, rats, rabbits, or hamsters.

[0323] Host cells are stably transformed using sequences encoding transgenes and related elements (i.e., the vector genome) and building blocks for viral production in host cells, such as replication and capsid genes (e.g., the rep and cap genes of AAV). For methods to produce recombinant AAV vectors with an AAV8 capsid, See Part IV of the detailed description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. The genomic copy titer of the vector can be determined, for example, by TAQMAN® analysis. The virion can be recovered, for example, by CsCl2 sedimentation.

[0324] In vitro assays (e.g., cell culture assays) can be used to measure transgene expression in the vectors described herein, thereby indicating, for example, the efficacy of the vector. For example, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM cell lines, or other cell lines derived from neurons or glial cells or neuronal or glial cell progenitor cells, can be used to assess transgene expression. Once expressed, the expressed product (i.e., HuGlyIDS) can be characterized, including identifying the glycosylation and tyrosine sulfation patterns associated with HuGlyIDS.

[0325] 5.2.10. Composition A composition is described comprising a vector encoding the transgene described herein and a suitable carrier. Those skilled in the art will readily select a suitable carrier (e.g., for application to CSF, and for example, for application to neuronal cells).

[0326] 5.3 Gene Therapy Methods for administering a therapeutically effective amount of a transgenic construct to a subject with MPS II to prevent, improve, or stabilize MPS II-related hearing loss are described. More specifically, methods for administering (particularly for administration to CSF) a therapeutically effective amount of the transgenic construct to a patient with MPS II are described. In certain embodiments, such methods for administering a therapeutically effective amount of the transgenic construct to CSF ​​can be used to treat patients with Hunter syndrome to prevent, improve, or stabilize MPS II-related hearing loss.

[0327] 5.3.1. Target Patient Group In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to a patient diagnosed with MPS II. In some embodiments, the patient has MPS II-related hearing loss. In specific embodiments, the patient has been diagnosed with mild MPS II. In specific embodiments, the patient has been diagnosed with severe MPS II. In specific embodiments, the patient has been diagnosed with Hunter syndrome. In specific embodiments, the patient has been diagnosed with neuropathic MPS II. In some embodiments, the patient has been diagnosed with hepatosplenomegaly, has symptoms associated with hepatosplenomegaly, is suspected of having hepatosplenomegaly, and / or has a predisposition to hepatosplenomegaly. Examples of symptoms associated with hepatosplenomegaly include, but are not limited to, brown urine, clay-colored stools, abdominal enlargement or swelling, fever, itching, jaundice or yellowing of the eyes and skin, nausea, pain (e.g., the upper right part of the stomach), fatigue, and / or vomiting. In some embodiments, the patient diagnosed with MPS II has hepatosplenomegaly. In some embodiments, the patient has hepatosplenomegaly associated with MPS II. In some embodiments, the patient is receiving or has received ERT treatment.

[0328] In some implementations, a therapeutically effective dose of the recombinant vector is administered to a patient diagnosed with MPS II who has been identified as responding to IDS (e.g., hIDS) treatment.

[0329] In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to pediatric patients. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients under three years of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 2 to 4 years. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 4 months or older but under 5 years of age. In a specific embodiment, a therapeutically effective dose of the recombinant carrier is administered to patients with severe MPS II aged 4 months or older but under 5 years of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 5 years or older but under 18 years of age. In a specific embodiment, a therapeutically effective dose of the recombinant carrier is administered to patients with neuropathic MPS II aged 5 years or older but under 18 years of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 18 months or older but under 8 years of age. In a specific embodiment, a therapeutically effective dose of the recombinant carrier is administered to pediatric male patients aged 18 months or older but under 8 years of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 3 to 8 years of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 8 to 16 years. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 5 to 18 years. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 10 years or younger. In a specific embodiment, a therapeutically effective dose of the recombinant carrier is administered to patients with severe MPS II who are 10 years or younger. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 18 years or younger. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients older than 5 years. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients older than 10 years.

[0330] In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 4, 5, 6, 7, 8, 9, 10, or 11 months. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients approximately 4, 5, 6, 7, 8, 9, 10, or 11 months of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients approximately 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 1, 2, 3, 4, or 5 years of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients approximately 1, 2, 3, 4, or 5 years of age. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 1-2, 2-3, 3-4, 4-5, or 5-6 years. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged approximately 1-2, 2-3, 3-4, 4-5, or 5-6 years. In some embodiments, a therapeutically effective dose of the recombinant carrier is administered to patients aged 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. In some implementations, a therapeutically effective dose of the recombinant carrier is administered to patients aged approximately 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.

[0331] In some embodiments, a therapeutically effective dose of the recombinant vector is administered to adolescent patients. In some embodiments, a therapeutically effective dose of the recombinant vector is administered to adult patients. In some embodiments, a therapeutically effective dose of the recombinant vector is administered to male patients. In other embodiments, a therapeutically effective dose of the recombinant vector is administered to female patients.

[0332] In some implementations, a therapeutically effective dose of the recombinant vector is administered to a patient diagnosed with MPS II who has been identified as responding to IDS treatment (e.g., hIDS injected into CSF ​​prior to gene therapy).

[0333] 5.3.2. Dosage and administration method In some implementations, the therapeutically effective dose of the recombinant carrier is administered via intrathecal administration. Right nowThe recombinant vector is administered to the CSF (cerebral sac) via injection into the subarachnoid space, allowing it to distribute through the CSF and transduce cells in the CNS. This can be achieved in various ways, such as intracranial (cistern or ventricle) injection or injection into the lumbar cistern. In some embodiments, intrathecal administration is performed via intracisional (IC) injection (e.g., injection into the cisterna magna). In a specific embodiment, intracisional injection is performed via CT-guided suboccipital puncture. In a specific embodiment, intrathecal injection is performed via lumbar puncture. In a specific embodiment, if feasible for the patient, injection into the subarachnoid space is performed via C1-2 puncture. Alternatively, the recombinant vector can be infused directly into the ventricle using intraventricular (ICV) administration (a more invasive technique for introducing anti-infective or anticancer drugs that do not penetrate the blood-brain barrier) (e.g., image-assisted ICV injection). In a specific embodiment, the recombinant vector is administered via a single image-assisted ICV injection. In another specific embodiment, the recombinant vector is administered via a single image-assisted ICV injection, and the administration catheter is immediately removed. In some embodiments, a therapeutically effective dose of the recombinant vector is administered to the CNS via intranasal administration. In some embodiments, a therapeutically effective dose of the recombinant vector is administered to the CNS via intraparenchymal injection. In some embodiments, the intraparenchymal injection targets the striatum. In some embodiments, the intraparenchymal injection targets the white matter. In some embodiments, a therapeutically effective dose of the recombinant vector is administered to the CSF by any means known in the art, such as by any means disclosed in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496, which is hereby incorporated in its entirety by reference.

[0334] In a preferred embodiment, for intrathecal administration (including IC and ICV administration), a therapeutically effective dose of the recombinant carrier is administered to the CSF at an injection volume not exceeding 10% of the total CSF volume, which is approximately 50 mL for infants and approximately 150 mL for adults. A carrier suitable for intrathecal injection, such as Elliott B solution or a modified Elliott B solution, should be used as the medium for the recombinant carrier. Elliott B solution (generic name: sodium chloride, sodium bicarbonate, anhydrous dextran, magnesium sulfate, potassium chloride, calcium chloride, and sodium phosphate) is a sterile, non-pyrogenic, isotonic solution free of antibacterial preservatives and is used as a diluent for intrathecal administration of chemotherapeutic agents. The modified Elliott B solution comprises 8.77 g / L sodium chloride, 0.244 g / L magnesium chloride, 0.0278 g / L sodium dihydrogen phosphate monohydrate, 0.114 g / L anhydrous disodium hydrogen phosphate, 0.224 g / L potassium chloride, 0.206 g / L calcium chloride, 0.793 g / L dextrose, and 0.010 g / L poloxamer 188, pH 7.26. In some embodiments, the AAV of this disclosure or a composition containing AAV is provided in the modified Elliott B solution for intrathecal administration.

[0335] In one embodiment, a non-replicating recombinant AAV9 vector expressing human iduronate-2-sulfatase (IDS) is used for treatment. In some embodiments, the IDS expression cassette is side-joined with an inverted terminal repeat (ITR) sequence and expression is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken β-actin promoter (CB7). In some embodiments, the transgene includes a chicken β-actin intron and a rabbit β-globin polyadenylation (polyA) signal.

[0336] In some embodiments, the recombinant nucleotide expression vector is administered in a dose dependent on the brain mass of a human subject. In a preferred embodiment, brain mass is determined by magnetic resonance imaging (MRI) of the human subject's brain. In some embodiments, brain mass is determined by measuring the brain mass in cm⁻¹. 3 The brain volume of a human subject is converted to brain mass by multiplying the brain volume of the human subject by a factor of 1.046 g / cm³, wherein the brain volume of the human subject is obtained from a brain MRI of the human subject. In some embodiments, the dose is the number of genome copies per unit brain mass. In some embodiments, the number of genome copies in the dose (e.g., the dose of the recombinant nucleotide expression vector) is determined by Poly-A specific PCR assay. In some embodiments, the number of genome copies in the dose (e.g., the dose of the recombinant nucleotide expression vector) is determined by transgene-specific PCR assay. In some embodiments, the weight of the brain mass is determined by MRI.

[0337] In some embodiments, rAAV9.hIDS is administered as a single fixed-dose IC (via suboccipital injection), the dose ranging from approximately 1.4 × 10⁻⁶ in a volume of about 5 to 20 ml. 13 GC (1.1 × 10 10 (GC / g brain mass) up to 7.0 × 10 13 GC (5.6 × 10 10 (GC / g brain mass). If the patient has neutralizing antibodies against AAV, a high range of doses can be used. In some embodiments, a single dose of rAAV encoding hIDS is administered to the subject's central nervous system (e.g., cerebrospinal fluid), and surprisingly, therapeutic effects are observed outside the CNS. For example, changes in organ size (e.g., spleen or liver) are observed outside the CNS after administration of the rAAV of this disclosure to the subject's CNS. In some embodiments, changes in biomarker levels (e.g., D2S6, HS, total GAG, and / or anti-IDS antibodies) are detected outside the CNS after administration of the rAAV of this disclosure to cerebrospinal fluid (e.g., changes in biomarker levels detected in the liver, spleen, urine, plasma, or blood). In some embodiments, no additional therapy of MPS II is administered to the subject outside the CNS.

[0338] In some embodiments, the recombinant vector described herein can be administered intrathecally in a single fixed dose, the dose ranging from about 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 6.5 × 10⁻⁶ 10 GC / g brain weight (e.g., for human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 1.3 × 10⁻⁶ g. 10 Intrathecal administration of GC / g brain mass (e.g., when human patients are 4 months or older and less than 5 years old). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 1.9 × 10⁻⁶ g / g brain mass. 10 Intrathecal administration of GC / g brain mass (e.g., when human patients are 4 months or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein can be administered in a single fixed dose of approximately 6.5 × 10⁻⁶. 10 Intrathecal administration of GC / g brain mass (e.g., when human patients are 4 months or older and less than 5 years old). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 9.6 × 10⁻⁶ g / g brain mass. 10 Intrathecal administration of GC / g brain mass (e.g., when human patients are 4 months or older and less than 5 years old). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 2.0 × 10⁻⁶ g / g brain mass.11 Intrathecal administration of GC / g brain mass (e.g., when human patients are 4 months or older and less than 5 years old). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 2.9 × 10⁻⁶ g / g brain mass. 11 GC / g brain weight intrathecal administration (e.g., when human patients are 4 months or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein can be administered intrathecally in a single fixed dose as listed in Table 5 below and according to dose 1 or dose 2 of Table 5 below (e.g., when human patients are 4 months or older and less than 5 years old).

[0339] In some embodiments, the recombinant vector described herein can be administered intrathecally in a single fixed dose, the dose ranging from about 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 2.0 × 10⁻⁶ 11 GC / g brain weight (e.g., for human patients aged 4 months or older and less than 5 years). In some embodiments, the recombinant vector described herein can be administered intrathecally in a single fixed dose, the dose ranging from about 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 2.9 × 10⁻⁶ 11 GC / g brain weight (e.g., when the human patient is 4 months or older and less than 5 years old). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 2.0 × 10⁻⁶ g. 11 Intrathecal administration of GC / g brain mass (e.g., when human patients are 4 months or older and less than 5 years old). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 2.9 × 10⁻⁶ g / g brain mass. 11 GC / g brain weight intrathecal administration (e.g., when human patients are 4 months or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein can be administered intrathecally in a single fixed dose as listed in Table 6 below and according to the dose 3 in Table 6 below (e.g., when human patients are 4 months or older and less than 5 years old).

[0340] In some embodiments, the recombinant vector described herein can be administered via a single fixed dose via intracardiac infusion, the dose ranging from approximately 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 6.5 × 10⁻⁶ 10 GC / g brain weight (e.g., for human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 1.3 × 10⁻⁶ g. 10GC / g brain mass is administered via intracellular injection (IC) (e.g., in human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 1.9 × 10⁻⁶ g / g brain mass. 10 GC / g brain mass is administered via intracellular injection (IC) (e.g., in human patients aged 4 months or older and less than 5 years). In another specific embodiment, the recombinant vector described herein can be administered in a single fixed dose of approximately 6.5 × 10⁻⁶. 10 GC / g brain mass is administered via intracellular injection (IC) (e.g., in human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 9.6 × 10⁻⁶ g / g brain mass. 10 GC / g brain mass is administered via IC (e.g., when human patients are 4 months or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein can be administered via IC in a single fixed dose as listed in Table 5 below and according to dose 1 or dose 2 in Table 5 below (e.g., when human patients are 4 months or older and less than 5 years old).

[0341] In some embodiments, the recombinant vector described herein can be administered via a single fixed dose via intracardiac infusion, the dose ranging from approximately 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 2.0 × 10⁻⁶ 11 GC / g brain weight (e.g., for human patients aged 4 months or older and less than 5 years). In some embodiments, the recombinant vector described herein can be administered via IC in a single fixed dose, the dose ranging from about 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 2.9 × 10⁻⁶ 11 GC / g brain weight (e.g., when the human patient is 4 months or older and less than 5 years old). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 2.0 × 10⁻⁶ g. 11 GC / g brain mass is administered via intracellular injection (IC) (e.g., in human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 2.9 × 10⁻⁶ g. 11 GC / g brain mass is administered via IC (e.g., when human patients are 4 months or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein can be administered via IC in a single fixed dose as listed in Table 6 below and according to dose 3 in Table 6 below (e.g., when human patients are 4 months or older and less than 5 years old).

[0342] In some embodiments, the recombinant vector described herein can be administered via ICV in a single fixed dose, the dose ranging from approximately 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 6.5 × 10⁻⁶ 10 GC / g brain weight (e.g., for human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 1.3 × 10⁻⁶ g. 10 GC / g brain mass is administered via ICV (e.g., in human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 1.9 × 10⁻⁶ g. 10 GC / g brain mass is administered via ICV (e.g., in human patients aged 4 months or older and less than 5 years). In another specific embodiment, the recombinant vector described herein can be administered in a single fixed dose of approximately 6.5 × 10⁻⁶. 10 GC / g brain mass is administered via ICV (e.g., in human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 9.6 × 10⁻⁶ g / g brain mass. 10 GC / g brain mass is administered via ICV (e.g., when human patients are 4 months or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein can be administered via ICV in a single fixed dose as listed in Table 5 below and according to dose 1 or dose 2 in Table 5 below (e.g., when human patients are 4 months or older and less than 5 years old).

[0343] In some embodiments, the recombinant vector described herein can be administered via ICV in a single fixed dose, the dose ranging from approximately 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 2.0 × 10⁻⁶ 11 GC / g brain weight (e.g., for human patients aged 4 months or older and less than 5 years). In some embodiments, the recombinant vector described herein can be administered via ICV in a single fixed dose, the dose ranging from about 1.3 × 10⁻⁶. 10 GC / g brain mass approximately 2.9 × 10⁻⁶ 11 GC / g brain weight (e.g., when the human patient is 4 months or older and less than 5 years old). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 2.0 × 10⁻⁶ g. 11 GC / g brain mass is administered via ICV (e.g., in human patients aged 4 months or older and less than 5 years). In a specific implementation, the recombinant vector described herein can be administered in a single fixed dose of approximately 2.9 × 10⁻⁶ g.11 GC / g brain mass is administered via ICV (e.g., when human patients are 4 months or older and less than 5 years old). In another specific embodiment, the recombinant vector described herein can be administered via ICV in a single fixed dose as listed in Table 6 below and according to dose 3 in Table 6 below (e.g., when human patients are 4 months or older and less than 5 years old).

[0344] Table 5. Total dosage determined by brain mass (Dose 1 or Dose 2) *GC is determined based on Poly-A specific PCR assay. Table 6. Total dosage determined by brain mass (dose 3) In a specific implementation plan, the recombinant vector described herein can be used in a single fixed dose of approximately 6.5 × 10⁻⁶. 10 GC / g brain weight intrathecal administration (e.g., when human patients are 5 years or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein can be administered intrathecally in a single fixed dose as listed in Table 7 below (e.g., when human patients are 5 years or older and less than 18 years old).

[0345] In a specific implementation plan, the recombinant vector described herein can be used in a single fixed dose of approximately 6.5 × 10⁻⁶. 10 GC / g brain mass is administered via intracardiac (IC) administration (e.g., when human patients are 5 years or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein can be administered via IC administration in a single fixed dose as listed in Table 7 below and in accordance with Table 7 below (e.g., when human patients are 5 years or older and less than 18 years old).

[0346] In a specific implementation plan, the recombinant vector described herein can be used in a single fixed dose of approximately 6.5 × 10⁻⁶. 10 GC / g brain mass is administered via ICV (e.g., when the human patient is 5 years or older and less than 18 years old). In another specific embodiment, the recombinant vector described herein can be administered via ICV in a single fixed dose as listed in Table 7 below and according to Table 7 below (e.g., when the human patient is 5 years or older and less than 18 years old).

[0347] Table 7. Total dosage determined by brain mass 5.3.3. Prevention and treatment of MPS II-related hearing loss This disclosure also provides methods and formulations for the prevention or treatment of MPS II-related hearing loss in subjects in need using recombinant human iduronate-2-sulfatase (IDS). In some embodiments, the recombinant human IDS is delivered to the subject via a currently disclosed recombinant vector (see Section 5.2). In some embodiments, the subject is 4 months or older and less than 5 years old. In some embodiments, the subject is 5 years or older and less than 18 years old.

[0348] This disclosure is partly based on the surprising finding that therapeutically effective amounts of the currently disclosed recombinant vector can stabilize and improve hearing in subjects with MPS II-related hearing loss. Furthermore, in subjects without hearing loss (normal hearing), therapeutically effective amounts of the currently disclosed recombinant vector can prevent hearing loss for at least 3 or 4 years after administration.

[0349] In one aspect, this disclosure provides a method for preventing MPS II-related hearing loss in a subject of need, wherein the method comprises administering to the subject a therapeutically effective amount of the recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) disclosed herein (see Section 5.2), wherein the subject has not experienced hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear. In some embodiments, the at least one ear includes the left or right ear. In some embodiments, the at least one ear includes both the left and right ears.

[0350] In some embodiments, the therapeutically effective amount of the rAAV prevents hearing loss in 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 administration of the rAAV. In some embodiments, the therapeutically effective amount of rAAV prevents hearing loss in at least one ear for at least about 3 years. In some embodiments, the therapeutically effective amount of rAAV prevents hearing loss in at least one ear for at least about 4 years. In some embodiments, the therapeutically effective amount of rAAV prevents hearing loss in at least one ear for at least about 5 years.

[0351] In some embodiments, prevention of hearing loss is indicated by no change or minimal change in the hearing threshold measured by an auditory brain response (ABR) test in at least one ear before the application of the rAAV, compared to the hearing threshold measured by the ABR test in at least one ear after the application of the rAAV. In some embodiments, the hearing threshold is an estimate of the behavioral threshold (eHL) derived from an ABR threshold (nHL) determined by the ABR test. In some embodiments, prevention of hearing loss is indicated by a change in eHL of 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 in at least one ear after the application of the rAAV, compared to the change before the application of the rAAV. In some embodiments, prevention of hearing loss is indicated by a change in eHL of about 25 dB or less in at least one ear after the application of the rAAV, compared to the change before the application of the rAAV. In some implementations, prevention of hearing loss is indicated by a change in eHL of about 10 dB or less in at least one ear after rAAV application compared to before rAAV application.

[0352] In some implementations, the hearing loss is conductive, sensorineural, or a combination of both, as determined by tympanogram testing.

[0353] In another aspect, this disclosure provides a method for treating mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, 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 experienced hearing loss in at least one ear (e.g., unilateral or bilateral) prior to administration of the rAAV. In some embodiments, the therapeutically effective amount of rAAV stabilizes the hearing loss or improves hearing in at least one ear. In some embodiments, the at least one ear includes either the left or right ear. In some embodiments, the at least one ear includes both the left and right ears.

[0354] In another aspect, this disclosure provides a method for stabilizing or preventing further hearing loss associated with mucopolysaccharidosis II (MPS II) in a subject of need, 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 prior hearing loss in at least one ear (e.g., unilateral or bilateral) before administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear (e.g., worsening or exacerbation of hearing impairment, further hearing decline). In some embodiments, the at least one ear includes the left or right ear. In some embodiments, the at least one ear includes both the left and right ears. As used herein, stabilizing MPS II-related hearing loss refers to preventing the worsening or exacerbation of hearing impairment.

[0355] In some embodiments, the therapeutically effective amount of the rAAV stabilizes the hearing loss or prevents further hearing loss in the at least one ear for at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 24 weeks, 48 ​​weeks, 1 year, 52 weeks, 1.5 years, 2 years, 3 years, 4 years, or 5 years after administration of the rAAV. In some embodiments, the therapeutically effective amount of rAAV prevents further hearing loss in the at least one ear for at least about 16 weeks after administration of the rAAV. In some embodiments, the therapeutically effective amount of rAAV prevents further hearing loss in the at least one ear for at least about 24 weeks after administration of the rAAV. In some embodiments, the therapeutically effective amount of rAAV prevents further hearing loss in the at least one ear for at least about 48 weeks after administration of the rAAV.

[0356] In some embodiments, stabilizing the hearing loss or preventing further hearing loss is indicated by an increase in the hearing threshold measured by an ABR test in at least one ear, as before the application of the rAAV, or as after the application of the rAAV. In some embodiments, the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL). In some embodiments, stabilizing the hearing loss or preventing further hearing loss is indicated by a change in eHL of 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 in at least one ear after the application of the rAAV, compared to before the application of the rAAV. In some embodiments, stabilizing the hearing loss or preventing further hearing loss is indicated by a change in eHL of about 10 dB or less in at least one ear after the application of the rAAV, compared to before the application of the rAAV.

[0357] In some implementations, the hearing loss is conductive, sensorineural, or a combination of both, as determined by tympanogram testing.

[0358] In another aspect, this disclosure provides a method for improving mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, 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 experienced 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 hearing in the at least one ear. In some embodiments, the at least one ear includes the left or right ear. In some embodiments, the at least one ear includes both the left and right ears.

[0359] In some embodiments, the therapeutically effective amount of the rAAV improves hearing in at least one ear 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 administration of the rAAV. In some embodiments, the therapeutically effective amount of the rAAV improves hearing in at least one ear less than 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 administration of the rAAV. In some embodiments, the therapeutically effective amount of the rAAV improves hearing in at least one ear less than about 16 weeks after administration of the rAAV. In some embodiments, the therapeutically effective amount of the rAAV improves hearing in at least one ear less than about 24 weeks after administration of the rAAV. In some implementations, at least approximately 48 weeks after rAAV administration, a therapeutically effective dose of rAAV improves hearing in at least one ear.

[0360] In some embodiments, the improvement in hearing is indicated by a reduction in the hearing threshold measured by an ABR test in the at least one ear, as before the application of the rAAV, compared to the hearing threshold measured by the ABR test in the at least one ear, as before the application of the rAAV. In some embodiments, the therapeutically effective amount of the rAAV reduces the hearing threshold of the at least one ear, as measured by the ABR test, by 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 measured by the ABR test in the at least one ear before the application of the rAAV. In some embodiments, the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL). In some embodiments, the improvement in hearing is indicated by a reduction in the eHL of at least one ear by 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 after the application of the rAAV, compared to before the application of the rAAV. In some embodiments, the improvement in hearing is indicated by a reduction in the eHL of at least one ear by more than about 10 dB after the application of the rAAV, compared to before the application of the rAAV.

[0361] In some implementations, the hearing loss is conductive, sensorineural, or a combination of both, as determined by tympanogram testing.

[0362] In some embodiments, the methods disclosed herein further include performing an ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV. The ABR test is a physiological procedure used to estimate the behavioral hearing threshold of an individual for whom behavioral audiometry cannot be reliably performed. The ABR measures the electrical activity response to a variety of sound stimuli.

[0363] Those skilled in the art will know how to perform ABR testing on subjects. In some embodiments, ABR testing is performed by delivering tonal pulse stimulation via an insertable earpiece to measure the ABR threshold for each ear in nHL at the following frequencies: 500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz. Once the nHL threshold is obtained, an appropriate correction factor is applied by frequency to obtain an estimate of the behavioral threshold (eHL). The mean of the measured thresholds is then used to classify the average degree of hearing loss in each ear as mild, moderate, severe, or profound. ABR testing is also performed using high-intensity (80–90 dBnHL) click stimulation at a rate of 17–30 clicks / second. Absolute waveform delay values ​​and comments on waveform morphology quality are then recorded. These measurements are performed to assess the integrity of the auditory brainstem pathway.

[0364] In some embodiments, the methods disclosed herein also include performing tympanogram testing on at least one ear before and after rAAV administration to determine whether the hearing loss is conductive, sensorineural, or a combination of both. Tympanogram testing is performed to assess middle ear function.

[0365] Those skilled in the art will know how to perform tympanic pressure testing on a subject. In some embodiments, a tympanogram is performed on both ears, combined with an ABR test, to help differentiate between conductive and sensorineural hearing loss. The following parameters are recorded: equivalent ear canal volume (cc), static admittance (mmho), tympanic cavity width (daPa), and peak tympanic cavity pressure (daPa). The tympanogram results are then categorized using the following legend: A = normal, B1 = flat tympanogram with normal equivalent volume (suggesting middle ear dysfunction), B2 = T-tube or tympanic membrane perforation, C = negative middle ear pressure.

[0366] In some embodiments, this disclosure provides a method for preventing MPS II-related hearing loss in a subject of need, wherein the method comprises (a) administering to the subject a therapeutically effective amount of the recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) disclosed herein (see Section 5.2), and (b) performing an ABR test on the at least one ear before and after administration of the rAAV to determine the hearing threshold of the at least one ear before and after administration of the rAAV; wherein the subject did not have hearing loss in at least one ear before administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear. In some embodiments, the method further comprises (c) performing a tympanograph test on the at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixture of conductive and sensorineural.

[0367] In some embodiments, this disclosure provides a method for treating mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the method comprises (a) administering to the subject a therapeutically effective amount of the recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) disclosed herein (see Section 5.2), and (b) performing an ABR test on the at least one ear before and after administration of the rAAV to determine the hearing threshold of the at least one ear before and after administration of the rAAV; wherein the subject had hearing loss in at least one ear (e.g., unilateral or bilateral) before administration of the rAAV. In some embodiments, the therapeutically effective amount of rAAV stabilizes the hearing loss or improves hearing in at least one ear. In some embodiments, the method further comprises (c) performing a tympanograph test on the at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixture of conductive and sensorineural.

[0368] In some embodiments, this disclosure provides a method for stabilizing MPS II-related hearing loss in a subject of need, wherein the method comprises (a) administering to the subject a therapeutically effective amount of the recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) disclosed herein (see Section 5.2), and (b) performing an ABR test on the at least one ear before and after administration of the rAAV to determine the hearing threshold of the at least one ear before and after administration of the rAAV; wherein the subject had hearing loss in at least one ear before administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear. In some embodiments, the method further comprises (c) performing a tympanograph test on the at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixture of conductive and sensorineural.

[0369] In some embodiments, this disclosure provides a method for improving mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the method comprises (a) administering to the subject a therapeutically effective amount of the recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) disclosed herein (see Section 5.2), and (b) performing an ABR test on the at least one ear before and after administration of the rAAV to determine the hearing threshold of the at least one ear before and after administration of the rAAV; wherein the subject had hearing loss in at least one ear (e.g., unilateral or bilateral) before administration of the rAAV, and the therapeutically effective amount of the rAAV improves the hearing in the at least one ear. In some embodiments, the method further comprises (c) performing a tympanograph test on the at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a mixture of conductive and sensorineural.

[0370] In another aspect, this disclosure provides a formulation suitable for administration to said ear (e.g., inner ear) comprising a therapeutically effective amount of a currently disclosed recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) (see Section 5.2). In some embodiments, said formulation is suitable for intratympanic delivery, intracochlear delivery, or intralabyrinthine delivery. See, for example(Liu et al., Front Neurosci. 022; 16: 867453, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the formulation is suitable for targeted delivery of rAAV to the cochlea. In some embodiments, the formulation is suitable for intracochlear or intralabyrinthine delivery. In some embodiments, the formulation comprises an adjuvant (e.g., histamine, benzyl alcohol, saponins, and N-methyl-2-pyrrolidone (NMP)) to increase drug permeability through the round window membrane (RWM). See, for example (See Saber et al., Round Window Membrane and Delivery of Biologically Active Agents into the Cochlea. Solna: Karolinska Institutet.; Creber et al., Eur. J. Pharm. Sci. 126 69–81.; Zhang et al., Adv. Funct. Mater. 31:2008701, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the formulation comprises nanocarriers, such as liposomes, micelles, polymer nanoparticles, and dendritic polymers, to deliver rAAV. In some embodiments, the formulation comprises hydrogels, nanocarriers, penetration enhancers, or microfluidic systems to improve drug penetration and sustained release.

[0371] 5.4 Combination Therapy The methods of this invention encompass combinations of administering HuGlyIDS to a patient in conjunction with other available treatments. Additional treatments may be administered before, during, or after gene therapy. Available treatments for MPSII that can be combined with the gene therapy of this invention include, but are not limited to, enzyme replacement therapy (ERT) using idoxime enzymes administered systemically or to the CSF; and / or HSCT therapy. In another embodiment, ERT may be administered using rHuGlyIDS glycoprotein produced in human neuronal and glial cell lines via recombinant DNA technology. Human neuronal and glial cell lines suitable for the production of such recombinant glycoproteins include, but are not limited to, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM, to name just a few. To ensure complete glycosylation, particularly sialylation and tyrosine sulfation, the cell lines used for production can be enhanced by engineering host cells to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferase) and / or TPST-1 and TPST-2 enzymes that induce tyrosine-O-sulfation.

[0372] 5.5 Biomarker / Sampling / Monitoring Efficacy Efficacy can be monitored by measuring the following data: cognitive function (e.g., prevention or reduction of neurocognitive decline); reduction of disease biomarkers (such as GAGs, including heparan sulfate and dermatan sulfate) in CSF and / or serum; and / or increase of IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events can also be monitored.

[0373] In one aspect, this document provides a method for monitoring efficacy by determining whether a subject undergoing or receiving ERT treatment can discontinue ERT treatment after administration of a gene therapy of this disclosure (e.g., rAAV encoding hIDS). For example, this document provides a method for treating and / or identifying a subject diagnosed with MPS II (e.g., a subject who may respond to discontinuation of ERT treatment), comprising: (a) administering to the subject a therapeutically effective amount of a gene therapy of this disclosure (e.g., rAAV encoding hIDS), wherein the subject has received or is receiving ERT treatment; (b) identifying the subject who may respond to discontinuation of 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 who may respond to discontinuation of ERT treatment if the level of the biomarker in the biological sample differs from a reference value (e.g., a reference level for at least one biomarker) (e.g., higher or lower); and (c) discontinuing the subject's ERT treatment. In some implementations, the biomarkers are D2S6, HS, total GAG, and / or anti-IDS antibodies. In some implementations, ERT is recombinant iduxitase. In some implementations, subjects diagnosed with MPS II have hepatosplenomegaly.

[0374] In another aspect, this document provides a method for selectively treating a subject with MPS II, comprising administering a therapeutically effective amount of rAAV encoding hIDS to the subject, wherein the subject has received or is receiving ERT treatment, and wherein the subject has been identified as potentially responsive to discontinuation of ERT treatment 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 identified as potentially responsive to discontinuation of ERT treatment when the level of at least one biomarker differs from a reference value (e.g., higher or lower). In some embodiments, the biomarkers are D2S6, HS, total GAG, and / or anti-IDS antibodies. In some embodiments, the ERT is recombinant idoxurase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[0375] In another aspect, this document provides a method for identifying or diagnosing a subject with neuropathic or non-neuropathic MPS II or MPS I. In some embodiments, the method includes determining the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, D2S6) in a biological sample from the subject. In some embodiments, the method includes determining the level of undegraded glycosaminoglycans (GAGs) in a biological sample from the subject. In some embodiments, the subject is identified or diagnosed with neuropathic MPS II or MPS I if the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) is elevated compared to a reference level. In some embodiments, the subject is identified or diagnosed with neuropathic MPS II or MPS I if the level of the GAG ​​heparin sulfate (HS) is elevated compared to a reference level (e.g., in the brain). In some embodiments, the total amount of heparin sulfate (t-HS) is the sum of the four disaccharides (D2S6, D0AO, D0SO, D0A6) in the cerebrospinal fluid (CSF) after enzymatic digestion (e.g., determined by bioanalytical mass spectrometry). In some embodiments, elevated D2S6 levels in the subject's (e.g., presymptomatic subject) CSF indicate neuropathic MPS II or MPS I. In some embodiments, D2S6 levels indicate the enzymatic activity of iduronic acid-2-sulfatase and can be used for therapeutic monitoring. In some embodiments, one or more disaccharides include one or more of D0AO, D0SO, D0A6, D2S6, or combinations thereof. In another aspect, this document provides a method for identifying or diagnosing a subject with neuropathic or non-neuropathic MPSII or MPSI, wherein the subject is identified or diagnosed with neuropathic MPSII if the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from the subject is elevated compared to a reference level. In some embodiments, the subject is presymptomatic or does not have visible or detectable MPSII or MPSI symptoms. In some embodiments, the subject has or has been diagnosed with MPSI or MPSII. 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-neuropathic subjects. In some embodiments, the reference level is the level of D2S6 in a biological sample (e.g., a CSF sample) from one or more healthy subjects and / or from one or more non-neuropathic subjects. In some embodiments, the reference level is a predetermined level.In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, D2S6) is about or at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, or more than 40% of the total heparan sulfate disaccharide (HS) in a biological sample from a subject (e.g., a subject with MPS I or MPS II). In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 20% of the total heparan sulfate disaccharide (HS) in a biological sample from a subject (e.g., a subject with MPS I or MPS II). In some implementations, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from a subject (e.g., a subject with MPS I or MPS II) is about or at least about 5%, 10%, 115%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 90% higher than the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample from a reference (e.g., a healthy subject). In some implementations, the levels of total heparin sulfate (e.g., D0A0, D0S0, D0A6, D2S6) are approximately or at least approximately 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 The levels are 900 ng / mL, 925 ng / mL, 950 ng / mL, 975 ng / mL, 1000 ng / mL, or greater than 1000 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 500 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 600 ng / mL.In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6 and / or D2S6) is about or at least about 700 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6 and / or D2S6) is about or at least about 800 ng / mL. In some embodiments, the levels of total heparin sulfate (e.g., DOA0, DOS0, DOA6 and / or D2S6) are 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, about 500 ng / mL to about 900 ng / mL, about 300 ng / mL to about 900 ng / mL, about 400 ng / mL to about 900 ng / mL, about 550 ng / mL to about 900 ng / mL, about 600 ng / mL to about 900 ng / mL, or about 700 ng / mL to about 900 ng / mL. In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6 and / or D2S6) is about or at least about 50 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 200 ng / mL, etc. ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 270 ng / mL, 280 ng / mL, 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL or greater than 400 ng / mL.In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, or greater than 200 ng / mL. In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 80 ng / mL (e.g., for D0S0 or D0A6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 100 ng / mL (e.g., for D0S0, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 90 ng / mL (e.g., for D0S0, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 110 ng / mL (e.g., for D0S0, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 120 ng / mL (e.g., for D0S0, D0A6, or D2S6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 160 ng / mL (e.g., for D2S6 or D0A6). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 250 ng / mL (e.g., for D2S6 or D0A0). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 300 ng / mL (e.g., for D2S6 or D0A0). In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6 and / or D2S6) is about or at least about 350 ng / mL (e.g., for D2S6 or D0A0).In some embodiments, the level of at least one heparin sulfate (e.g., D0A0, D0S0, 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, D0S0, 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, D0S0, 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, D0S0, D0A6, and / or D2S6) is about or at least about 200 ng / mL (e.g., for D2S6). In some implementations, compared to a reference level (e.g., the level of one or more heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) or the level of D2S6 in biological samples from one or more healthy subjects and / or one or more non-neurotic subjects, or a predetermined value), the level of one or more heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) or the level of D2S6 in the biological samples from the subjects is increased 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, etc.) in the biological samples from the subjects. 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, 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 greater than 500 ng / mL.In some implementations, compared to a reference level (e.g., the total level of heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and D2S6) in biological samples from one or more healthy subjects and / or one or more non-neurotic subjects, or a predetermined value), the total level of heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in the subjects' biological samples is increased 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, etc.) 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, 350ng / 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, 580ng / 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, 850ng / mL, 900 ng / mL, 950 ng / mL, 1000 ng / mL or greater than 1000 ng / mL.

[0376] In some embodiments, a subject's response to the treatment of this disclosure (e.g., rAAV9 encoding hIDUA for treating MPS I; construct 2) is determined based on IOS6 levels in biological samples from the subject. In some embodiments, an increase in IOS6 levels in biological samples from a subject (e.g., a subject with MPS I) compared to a reference value indicates a response to the treatment of this disclosure (or treatment of MPS I with rAAV9 encoding hIDUA). In some embodiments, the reference value is the IOS6 level in biological samples from healthy subjects or a population of healthy subjects. In some embodiments, the reference value is the IOS6 level in biological samples from subjects with MPS I or a population of subjects with MPS I. In some embodiments, the reference value is the IOS6 level in biological samples from subjects who do not have MPS I or have not been diagnosed with MPS I or a population of subjects who have not been diagnosed with MPS I. In some embodiments, the reference value is the IOS6 level in biological samples from the same subject but taken from different time points (e.g., obtained at an earlier time point). In some implementations, the reference value is a predetermined value.

[0377] 5.5.1. Disease biomarkers In some embodiments, the therapeutic efficacy of the recombinant nucleotide expression vector is monitored by measuring the levels of disease biomarkers in patients. In some embodiments, the levels of disease biomarkers are measured in the patient's CSF. In some embodiments, the levels of disease biomarkers are measured in the patient's serum. In some embodiments, the levels of disease biomarkers are measured in the patient's plasma. In some embodiments, the levels of disease biomarkers are measured in the patient's urine. In some embodiments, the disease biomarker is GAG. In some embodiments, the disease biomarker is IOS6. In a preferred embodiment, the disease biomarker is heparan sulfate. In some embodiments, the disease biomarker is D2S6. The I2S enzyme cleaves sulfate from HS in lysosomes, and the loss of I2S leads to the accumulation of long chains of fully sulfated D2S6. In some embodiments, quantitative measurement of D2S6 reflects the level of I2S enzyme activity, and elevated levels of HS and D2S6 are closely associated with the neuropathic phenotype of MPS II. In some embodiments, the level of D2S6 is negatively correlated with neurocognitive development. In some embodiments, the disease biomarker is an anti-AAV antibody (e.g., an anti-AAV9 antibody). In some embodiments, the disease biomarker is dermatin sulfate. In some embodiments, the disease biomarker is IDS enzyme activity. In some embodiments, the disease biomarker is inflammation. In some embodiments, the disease biomarker is a safety event.

[0378] In some embodiments, the therapeutic efficacy of the recombinant nucleotide expression vector is monitored by measuring one or more of the following biomarkers in samples from patients: (a) GAG levels in CSF; (b) I2S levels in CSF; (c) GAG levels in plasma; (d) I2S levels in plasma; (e) leukocyte I2S enzyme activity levels; (f) GAG levels in urine; (g) heparan sulfate levels in CSF; and (h) dermatan sulfate levels in CSF. In some embodiments, the therapeutic efficacy of the recombinant nucleotide expression vector is monitored by measuring I2S and / or GAG in CSF, urine, and / or plasma. In some embodiments, the therapeutic efficacy of the recombinant nucleotide expression vector is monitored by measuring heparan sulfate in CSF, urine, and / or plasma. In some embodiments, the therapeutic efficacy of the recombinant nucleotide expression vector is monitored by measuring non-reduced heparan sulfate. In some embodiments, heparan sulfate measured in CSF is the primary endpoint for determining therapeutic efficacy. In some embodiments, the therapeutic efficacy of 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 (e.g., CSF, urine, and / or plasma) for detecting D2S6. In some embodiments, the therapeutic efficacy of the recombinant nucleotide expression vector is monitored by measuring total urinary GAG, urinary HS, and / or plasma I2S enzyme activity. In some embodiments, urinary GAG indicates systemic effects and / or is independent of ERT treatment. In some embodiments, the therapeutic efficacy of this disclosure is determined based on the I2S protein concentration level in a sample from the subject (e.g., an increase in I2S protein concentration level indicates efficacy). In some embodiments, heparan sulfate (HS) and D2S6 (glycosaminoglycan (GAG)) in cerebrospinal fluid (CSF) are measured at baseline and / or after administration of the recombinant vector of this disclosure.In some embodiments, the determination or monitoring of the efficacy of MPS II treatment in a subject is made by detecting the level of at least one biomarker (e.g., D2S6) in a biological sample from the subject (e.g., and comparing said level to a reference), said biological sample being 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, 31 The MPS of a subject is determined after 0, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 days, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, or 104 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the MPS of a subject is determined by detecting the level of at least one biomarker (e.g., ISO6) in a biological sample from the subject (e.g., and comparing said level to a reference). The determination or monitoring of the efficacy of treatment I, wherein the biological sample is obtained after administration of the rAAV of this disclosure to a subject at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days, or 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years later. In some embodiments, after administration of the recombinant vector of this disclosure, the levels of HS and / or D2S6 in the subject are reduced compared to reference values ​​(e.g., compared to the subject's HS and / or D2S6 levels before administration of the recombinant vector of this disclosure, or compared to baseline or predetermined values). In some embodiments, after administration of the recombinant vector of this disclosure, the level of HS is reduced by about or at least about 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or greater than 100%. In some embodiments, after administration of the recombinant vector of this disclosure, the level of D2S6 is reduced by 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 greater than 100%. In some embodiments, after administration of the recombinant vector of this disclosure, the level of IOS6 in the subject is reduced compared to a reference value (e.g., compared to the subject's IOS6 level before administration of the recombinant vector of this disclosure, or compared to baseline or a predetermined value). In some embodiments, after administration of the recombinant vector of this disclosure, the ISO6 level is reduced by 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 greater than 100%.In some embodiments, biomarkers (e.g., HS, D2S6, IOS6, total GAG, and / or I2S enzyme) are detected before administration of the recombinant vector of this disclosure, on the same day as administration of the recombinant vector of this disclosure, on the day after administration of the recombinant vector of this disclosure, and / or approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or 1 day after administration of the recombinant vector of this disclosure. Measurements were taken at 3 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. In some embodiments, biomarkers (e.g., HS, D2S6, IOS6, total GAG, and / or I2S enzyme) are measured before administration of the recombinant vector of this disclosure, on the same day as administration of the recombinant vector of this disclosure, and at 8, 16, 24, 32, 40, 48, 56, 72, 104 weeks, 1 year, 2 years, or more than about 2 years after administration of the recombinant vector of this disclosure. In some embodiments, HS and / or D2S6 in CSF are measured in ng / ml. In some embodiments, following administration of the recombinant vector of this disclosure, biomarkers (e.g., HS, IOS6, and / or D2S6) are reduced 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, etc., compared to reference values ​​(e.g., compared to baseline or pre-treatment levels with the recombinant vector of this disclosure). 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 greater than 300 ng / ml. In some embodiments, total GAG (e.g., GAG in urine) is measured in g / mol, CK.In some embodiments, after administration of the recombinant vector of this disclosure, the biomarker (e.g., total GAG) is reduced by about or at least about 5 g / mol, 10 g / mol, 15 g / mol, 20 g / mol, 25 g / mol, 30 g / mol, 35 mg / ml, 40 mg / ml, 45 g / mol, 50 g / mol, 55 g / mol, 60 g / mol, 65 g / mol, 70 g / mol, 75 g / mol, 80 g / mol, 85 g / mol, 90 g / mol, 95 g / mol, 100 g / mol, 110 g / mol, 120 g / mol, 130 g / mol, 140 g / mol, 150 g / mol, 160 g / mol, 170 g / mol, 200 g / mol, or greater than 200 g / mol compared to a reference value (e.g., the amount compared to baseline or the amount before treatment with the recombinant vector of this disclosure). In some embodiments, after administration of the recombinant vector of this disclosure, compared with reference values ​​(e.g., before ERT interruption) (e.g., compared with baseline levels or levels before treatment with the recombinant vector of this disclosure), biomarkers (e.g., HS, D2S6, IOS6, and / or total GAG) are reduced and / or hepatosplenomegaly is reduced by about or at least about 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or greater than 100%. In some embodiments, the determination of an increase or decrease in biomarkers in a subject is made before the subject's ERT is interrupted.In some embodiments, after administration of the recombinant vector of this disclosure, compared with reference values ​​(e.g., compared with the biomarker levels of the subject before administration of the recombinant vector of this disclosure, or compared with the biomarker levels of a previously obtained biological sample from the subject, or compared with baseline, or compared with the biomarker levels of a healthy subject), biomarkers (e.g., HS, IOS6, D2S6, and / or total GAG) are reduced and / or hepatosplenomegaly is reduced by approximately, approximately after, at least approximately or at most approximately 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, etc.) 54 weeks, 56 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, over 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. For example, in some embodiments, after administration of the recombinant vector of this disclosure (e.g., compared with baseline biomarker levels or hepatosplenomegaly or biomarker levels or hepatosplenomegaly prior to treatment with the recombinant vector of this disclosure, or compared with biomarker levels or hepatosplenomegaly obtained from the subject at an earlier time point), biomarkers (e.g., HS, D2S6, IOS6 and / or total GAG) are reduced and / or hepatosplenomegaly is reduced by about or at least about 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, for about, at least about or at most about 2 weeks, 4 weeks, 6 weeks, 8 weeks, 16 weeks, 24 weeks, 48 ​​weeks, 56 weeks, 104 weeks, 2 years or more).In some embodiments, after administration of the recombinant vector of this disclosure, a decrease in biomarkers (e.g., HS, D2S6, IOS6, and / or total GAG), a decrease in hepatosplenomegaly, and / or an increase in biomarkers (e.g., I2S) persists 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, and 95 weeks. 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.

[0379] In some embodiments, after administration of the recombinant vector of this disclosure, the level of a biomarker (e.g., I2S enzyme) increases 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 greater than 100%. In some embodiments, after administration of the recombinant vector of this disclosure, compared with reference values ​​(e.g., compared with the subject's biomarker levels before administration of the recombinant vector of this disclosure), the biomarker (e.g., I2S enzyme) increases by approximately, approximately after, at least approximately or at most approximately 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, etc. 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. In some embodiments, after administration of the recombinant vector of this disclosure (e.g., compared to a baseline amount or an amount prior to treatment with the recombinant vector of this disclosure), a biomarker (e.g., I2S enzyme) increases by about or at least about 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50%, lasting for about, at least about or at most 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.In some embodiments, after administration of the recombinant vector of this disclosure (e.g., compared to a baseline amount or an amount prior to treatment with the recombinant vector of this disclosure), the biomarker (e.g., I2S enzyme) increases 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 greater than 50000 pg / ml.

[0380] In some embodiments, bioanalytical LC / MS is used to measure heparan sulfate (HS) glycosaminoglycans (CAG) in CSF. In some embodiments, bioanalytical LC / MS is used to measure the non-reduced end of heparan sulfate and total heparan sulfate in CNS. In some embodiments, bioanalytical LC / MS is used to measure the non-reduced end of heparan sulfate and total heparan sulfate CAG in plasma. In some embodiments, bioanalytical LC / MS is used to measure HS CAG in urine. In some embodiments, a colorimetric assay is used to measure the total CAG concentration in urine. In some embodiments, further details regarding the assays that can be used are provided in Part 6 of this disclosure.

[0381] In some embodiments, if, after administration of the recombinant vector of this disclosure to a subject, an increase in biomarkers (e.g., D2S6, IOS6, HS, GAG (e.g., total GAG in urine)) and / or an increase in hepatosplenomegaly and / or a decrease in biomarkers (e.g., I2S), the subject is administered another dose (e.g., the same dose, a lower dose, or a higher dose compared to the previous dose) of the recombinant vector of this disclosure. In some embodiments, if, after administration of the recombinant vector of this disclosure to a subject, an increase in biomarkers (e.g., D2S6, HS, GAG (e.g., total GAG in urine)) and / or an increase in hepatosplenomegaly and / or a decrease in biomarkers (e.g., I2S), the subject is administered another therapy or treatment (e.g., a therapy or treatment for treating MPSII, a therapy or treatment for treating symptoms suffered by the subject, a therapy or treatment for hepatosplenomegaly, and / or a therapy or treatment for treating or preventing symptoms of MPSII). In some embodiments, if, after administration of the recombinant vector of this disclosure to a subject, an increase in biomarkers (e.g., IOS6) and / or a decrease in hepatosplenomegaly and / or a decrease in biomarkers (e.g., I2S), another therapy or treatment is administered to the subject (e.g., a therapy or treatment for treating MPS I, a therapy or treatment for treating symptoms suffered by the subject, a therapy or treatment for hepatosplenomegaly, and / or a therapy or treatment for treating or preventing symptoms of MPS I). For example, if, compared to a reference value, a biomarker (e.g., D2S6, IOS6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly increases and / or a biomarker (e.g., I2S) decreases by about or 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 greater than 100%, the subject is given another dose (e.g., the same dose, a lower dose, or a higher dose compared to the previous dose) of the recombinant vector of this disclosure and / or given another therapy or treatment (e.g., ERT treatment). In some embodiments, the reference value is a control. In some embodiments, the reference value is the level of biomarkers and / or hepatosplenomegaly in a previous biological sample or from images (e.g., ultrasound) obtained from the subject. In some embodiments, the reference values ​​are the biomarker levels and / or hepatosplenomegaly of the subject at baseline (e.g., before administration of the recombinant vector of this disclosure to the subject). In some embodiments, the reference values ​​are the biomarker levels and / or hepatosplenomegaly of healthy subjects or subjects who do not have or have not been diagnosed with MPS II.In some embodiments, the reference values ​​are the levels of at least one biomarker and / or hepatosplenomegaly in biological samples obtained from subjects diagnosed with MPS II who have never received ERT or are not currently receiving ERT. In some embodiments, the reference values ​​are the levels of biomarkers and / or hepatosplenomegaly in subjects or subject groups with MPS II. In some implementations, if, after application of the recombinant vector of this disclosure, within approximately, at least approximately, or at most approximately 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, greater than 104 weeks, or 1 month, Biomarkers (e.g., D2S6, IOS6, HS, GAG) were observed over periods of 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. An increase in total GAG in urine (e.g., an increase in hepatosplenomegaly) and / or a decrease in biomarkers (e.g., I2S) may result in the administration of another dose (e.g., the same dose, a lower dose, or a higher dose compared to the previous dose) of the recombinant vector of this disclosure to the subject and / or administration of another therapy or treatment to the subject.

[0382] In some embodiments, the subject is receiving or has received enzyme replacement therapy (ERT) prior to and / or continuously before administration of the recombinant vector of this disclosure. In some embodiments, the subject's ERT treatment is interrupted prior to administration of the recombinant vector of this disclosure. In some embodiments, the subject's ERT treatment is interrupted approximately 52 weeks after administration of the recombinant vector of this disclosure. In some embodiments, the subject's ERT treatment is interrupted approximately 56 weeks after administration of the recombinant vector of this disclosure. In some implementations, ERT treatment in subjects is interrupted approximately 52 weeks after administration of the recombinant vector of this disclosure, or approximately 52 weeks before or after (e.g., approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 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 administration of the recombinant vector of this disclosure). In some embodiments, determining biomarker levels and / or hepatosplenomegaly is used to determine whether a patient can discontinue enzyme replacement therapy (ERT) after administration of the recombinant vector of this disclosure to a subject. In some embodiments, an increase in a biomarker (e.g., I2S) after administration of the recombinant vector of this disclosure to a subject (e.g., compared to baseline) indicates that the subject can discontinue ERT. In some embodiments, an increase in a biomarker (e.g., I2S) over a period of time after administration of the recombinant vector of this disclosure to a subject indicates that the subject can discontinue ERT. In some embodiments, if the subject is negative for anti-AAV antibodies (e.g., a biological sample from the subject does not show detectable levels of anti-AAV9 antibodies), ERT is discontinued before administration of the recombinant vector of this disclosure to the subject. In some embodiments, ERT is discontinued approximately or at least 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 administration of the recombinant vector of this disclosure to the subject. In some embodiments, a reduction in biomarkers (e.g., D2S6, HS, GAG (e.g., total GAG in urine)) and / or a reduction in hepatosplenomegaly (e.g., compared to baseline) after administration of the recombinant vector of this disclosure to the subject indicates that the subject can discontinue ERT treatment.In some embodiments, a decrease in biomarkers (e.g., D2S6, IOS6, HS, GAG (e.g., total GAG in urine)) and / or a decrease in hepatosplenomegaly over a period of time after administration of the recombinant vector of this disclosure to the subject indicates that the subject may discontinue ERT treatment. In some implementations, after application of the recombinant vector of this disclosure, approximately, at least approximately, or at most approximately 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, greater than 104 weeks, 1 month, or 2 months... Biomarkers (e.g., D2S6, IOS6, HS, GAG) over a period of 1 month, 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. A decrease in the level of a biomarker (e.g., total GAG in urine) and / or a decrease in hepatosplenomegaly and / or an increase in a biomarker (e.g., I2S) indicates that the subject may discontinue or stop ERT treatment. In some implementations, ERT treatment is discontinued or interrupted if, compared to reference levels, the levels of biomarkers (e.g., D2S6, IOS6, HS, GAG (e.g., total GAG in urine)) decrease and / or the hepatosplenomegaly decreases and / or the biomarker (e.g., I2S) increases by about or 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 greater than 100%.In some embodiments, ERT treatment is discontinued or interrupted if, compared to a reference level, biomarker levels (e.g., D2S6, IOS6, HS, GAG (e.g., total GAG in urine)) decrease and / or hepatosplenomegaly decreases and / or biomarkers (e.g., I2S) increase by about or 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 greater than 100 times (e.g., after administration of the recombinant vector of this disclosure). In some embodiments, ERT is interrupted indefinitely. In some implementations, the ERT is interrupted or stopped for approximately, at least approximately, or at most approximately 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, 1 00 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 approximately or at least approximately 52 weeks after administration of the recombinant vector of this disclosure.

[0383] In some embodiments, biomarkers (e.g., I2S, IOS6, D2S6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly are monitored after ERT treatment is discontinued. For example, in some embodiments, biomarkers are monitored approximately, at least approximately, 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, or 22 weeks after ERT treatment is stopped or discontinued and / or after administration of the recombinant vector of this disclosure to the subject. Weeks 24, 26, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100 Measure biomarkers (e.g., I2S, IOS6, D2S6, HS, GAG) at 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. (e.g., total GAG in urine) and / or hepatosplenomegaly (e.g., size of the liver or spleen).

[0384] In some implementations, if biomarkers (e.g., D2S6, IOS6, HS, GAG (e.g., total GAG in urine)) increase and / or hepatosplenomegaly increases and / or biomarkers (e.g., I2S) decrease after ERT treatment is stopped or interrupted, ERT treatment is re-administered to the subject. For example, if, compared to reference values, biomarkers (e.g., D2S6, IOS6, HS, GAG (e.g., total GAG in urine)) increase and / or hepatosplenomegaly increases and / or biomarkers (e.g., I2S) decrease by about or 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 greater than 100% (e.g., after administering the recombinant vector of this disclosure to the subject), then ERT treatment is re-administered to the subject (e.g., for a period of time until biomarkers (e.g., D2S6, IOS6, HS, GAG (e.g., total GAG in urine)) decrease or are controlled and / or hepatosplenomegaly decreases or is controlled and / or biomarkers (e.g., I2S) increase or are controlled). In some embodiments, the reference value is a control. In some embodiments, the reference value is the level of biomarkers and / or hepatosplenomegaly in a previous biological sample or from images (e.g., ultrasound) obtained from the subject. In some embodiments, the reference value is the level of biomarkers and / or hepatosplenomegaly in the subject at baseline (e.g., before administration of the recombinant vector of this disclosure to the subject). In some embodiments, the reference value is the level of biomarkers and / or hepatosplenomegaly in healthy subjects or subjects who do not have or have not been diagnosed with MPS II. In some embodiments, the reference value is the level of biomarkers and / or hepatosplenomegaly in subjects or subject groups who have MPS II. In some embodiments, the reference value is the level of biomarkers and / or hepatosplenomegaly in healthy subjects or subjects who do not have or have not been diagnosed with MPS I. In some embodiments, the reference value is the level of biomarkers and / or hepatosplenomegaly in subjects or subject groups who have MPS I. In some embodiments, the reference value is the level of biomarkers or hepatosplenomegaly in subjects or subject groups who are receiving or have received ERT treatment.

[0385] In some embodiments, ERT treatment is interrupted after administration of the recombinant vector of this disclosure. For example, ERT treatment is stopped after administration of the recombinant vector of this disclosure to a subject who has received ERT treatment and has an anti-IDS antibody level (e.g., an anti-IDS antibody level as a result of ERT treatment). In some embodiments, the subject immediately stops ERT treatment when or after administration of the recombinant vector of this disclosure to a subject with an anti-IDS antibody level. In some embodiments, the anti-IDS antibody level in a biological sample (e.g., a serum sample) from the subject is determined. In some embodiments, the anti-IDS antibody level in a biological sample obtained from the subject is determined before administration of the recombinant vector of this disclosure to the subject. In some embodiments, the anti-IDS antibody level is monitored before and / or after administration of the recombinant vector of this disclosure to the subject. In some embodiments, ERT treatment is stopped or interrupted if the anti-IDS antibody level is higher than a reference level. In some embodiments, the reference level is a predetermined level. In some embodiments, the reference level is the anti-IDS antibody level in a previous biological sample obtained from the subject. In some embodiments, the reference level is the anti-IDS antibody level from the subject at baseline (e.g., prior to administration of the recombinant vector of this disclosure to the subject). In some embodiments, the reference level is the anti-IDS antibody level in healthy subjects or subjects who do not have or have not been diagnosed with MPS II. In some embodiments, the reference level is the anti-IDS antibody level in subjects or subject groups who have MPS II. In some embodiments, the reference level is the anti-IDS antibody level in subjects or subject groups who are receiving or have received ERT treatment. In some embodiments, ERT treatment is interrupted if the anti-IDS antibody level is higher or increases by 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 greater than 1000% compared to the reference level. In some implementations, ERT treatment is stopped or interrupted if the level of anti-IDS antibody is higher or increases by about or 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 greater than 1000 times compared to the reference level.In some implementations, the recombinant vector of this disclosure is administered to the subject after ERT treatment, after ERT treatment is stopped or interrupted, before the subject is administered the recombinant vector of this disclosure, and / or after the subject is administered the recombinant vector of this disclosure, approximately, at least approximately, 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, or 70 weeks. Anti-IDS antibody levels were measured at 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.

[0386] In some embodiments, therapeutic efficacy or monitoring of therapeutic efficacy (e.g., with rAAV9 encoding hIDUA) is based on IOS6 levels. In some embodiments, IOS6 levels are determined from a biological sample from a subject. In some embodiments, the biological sample is plasma. In some embodiments, a decrease in IOS6 levels after treatment with the recombinant vector of this disclosure (e.g., rAAV9 encoding hIDUA) compared to a reference value indicates therapeutic efficacy. In some embodiments, the reference value is the IOS6 level in a biological sample from a subject obtained prior to administration of the recombinant AAV of this disclosure. In some embodiments, the reference value is a predetermined value. In some embodiments, the reference value is the IOS6 level in a biological sample from another subject with MPS I or from a group of subjects with MPS I. In some embodiments, IOS6 levels decrease after administration of the recombinant AAV of this disclosure compared to before rAAV administration. In some embodiments, the subject is pretreated with ERT prior to rAAV administration and / or receives ERT after rAAV administration. In some implementations, the reduction in ISO6 levels is defined as a reduction of approximately or at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to a reference value. In some implementations, the efficacy of MPS I treatment is defined as an improvement in at least one subtest of the Belle Baby Development Scales, Version 3 (BSID-III) compared to a reference value. In some implementations, at least one subtest is an age-equivalent score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. In some implementations, the efficacy of MPS I treatment is defined as an improvement in WASI and / or VABS scores. In some embodiments, rAAV9 encoding hIDUA is administered to a human subject in solution form, the 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 dihydrogen phosphate monohydrate at a concentration of about 0.0278 g / L, and (h) anhydrous disodium hydrogen phosphate at a concentration of about 0.114 g / L.

[0387] 5.5.2. Hepatomegaly and splenomegaly In some implementations, a subject diagnosed with MPS II has hepatosplenomegaly or is experiencing symptoms associated with hepatosplenomegaly. In some implementations, hepatosplenomegaly is diagnosed through physical examination. For example, a physician or healthcare professional may observe the abdominal region for signs of organ enlargement. In some implementations, a physician or healthcare professional feels or palpates the subject's abdominal region to examine the sensitivity or enlargement of the liver and / or spleen. In some implementations, diagnostic tests are performed to detect hepatosplenomegaly. For example, blood tests (e.g., liver function tests, blood cell counts, and / or coagulation factor tests) may be used to detect hepatosplenomegaly. In some implementations, imaging scans (such as computed tomography (CT) scans or ultrasound) are used to detect hepatosplenomegaly. In some implementations, hepatosplenomegaly is detected via biopsy.

[0388] In some embodiments, hepatosplenomegaly is measured before, during, and / or after treatment with the recombinant vector of this disclosure. In some embodiments, hepatosplenomegaly is measured at different time points before, during, and / or after treatment with the recombinant vector of this disclosure. In some embodiments, a reduction in hepatosplenomegaly indicates that the treatment is effective. In some embodiments, assessing or monitoring hepatosplenomegaly is used to determine the amount of dose, whether to increase the dose, whether to decrease the dose, and / or to determine the frequency of dose administration.

[0389] In some embodiments, hepatosplenomegaly is determined before and / or after the subject begins treatment with the therapies or treatments of this disclosure. In some embodiments, hepatosplenomegaly is determined between dose administrations (e.g., between the administration of the first dose of the recombinant vector of this disclosure to the subject (i.e., before treatment with the recombinant vector of this disclosure begins) and after the first dose of the recombinant vector of this disclosure, 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 after dose administration or after the start of treatment (e.g., after administration of the recombinant vector of this disclosure to the subject and / or after ERT treatment). 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, 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 Hepatomegaly and splenomegaly are determined within 10 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.

[0390] In some embodiments, after administration of the recombinant vector of this disclosure to a subject, hepatosplenomegaly (e.g., the size of the liver or spleen) is reduced 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 greater than 200%. In some embodiments, following administration of the recombinant vector of this disclosure to a subject, hepatosplenomegaly is reduced by about 1.5 times, 2 times, 2.5 times, 3 times, 5 times, 7 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 97 times, 100 times, 200 times, or greater than 200 times. In some embodiments, the reduction in hepatosplenomegaly corresponds to a reduction in the size of the liver and / or spleen. In some embodiments, the reduction in hepatosplenomegaly corresponds to a reduction in the mass or diameter of the liver and / or spleen. In some embodiments, the reduction in hepatosplenomegaly corresponds to a reduction in the size of the liver and / or spleen. In some embodiments, the reduction in hepatosplenomegaly occurs after administration of the recombinant vector of this disclosure to a subject and / or between dose administrations. In some embodiments, hepatosplenomegaly is reduced after administration of the recombinant vector of this disclosure to the subject and / or after dose 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, after administration of the recombinant vector of this disclosure to the subject, hepatosplenomegaly is reduced for about, about, at least about or at most about 1 day, 3 days, 5 days, 7 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, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 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.In some implementations, after administration of the recombinant vector of this disclosure to a subject, the reduction in hepatosplenomegaly is maintained for at least about 1 day, 3 days, 5 days, 7 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, 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, 1 The duration of treatment is 8 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 greater than 10 years. In some implementations, the reduction in hepatosplenomegaly is greater in subjects undergoing the treatment of this disclosure compared to reductions in hepatosplenomegaly in subjects undergoing another treatment for treating hepatosplenomegaly or MPS II. In some embodiments, the reduction in hepatosplenomegaly in subjects receiving the recombinant vector of this disclosure is greater than the reduction in hepatosplenomegaly in comparable subjects undergoing another treatment for treating hepatosplenomegaly or MPS II. In some embodiments, the reduction in hepatosplenomegaly is greater than 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 greater than 200% (e.g., compared to the reduction in hepatosplenomegaly in subjects undergoing another treatment for treating hepatosplenomegaly or MPS II).

[0391] 5.5.3. Neurocognitive and Neurodegenerative Functional Tests In some embodiments, the therapeutic efficacy of the recombinant vector is monitored by measuring the level of a patient's cognitive function. Cognitive function can be measured by any method known to those skilled in the art. In some embodiments, cognitive function is measured via a validated instrument for measuring intelligence quotient (IQ). In specific embodiments, IQ is measured using the Wechsler Abbreviated Scale of Intelligence, Second Edition (WASI-II). In some embodiments, neurocognition is determined by measuring IQ, such as using the Bayley's Infantile Development Scale. In some embodiments, developmental quotient (DQ) is measured using the Bayley's Infantile Development Scale (BSID-III). In some embodiments, the Bayley's Infantile Development Scale is used to measure the motor, cognitive, linguistic, socio-emotional, and / or adaptive behavioral development of infants, toddlers, or patients of this disclosure. In some embodiments, the efficacy of MPS II treatment according to the methods of this disclosure (e.g., by administering rAAV encoding hIDS to a subject) is determined by analyzing at least one subtest of BSID-III. In some embodiments, the efficacy of MPS I treatment according to the methods of this disclosure (e.g., by administering rAAV encoding hIDUA to the subject) is determined by analyzing at least one subtest of BSID-III. In some embodiments, the efficacy of MPS II or MPS I treatment according to the methods of this disclosure (e.g., by administering rAAV encoding hIDS or rAAV encoding hIDUA to the subject) is determined by analyzing age-equivalent scores, 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 the improvement in at least one subtest of the Bailey Infant Development Scale, Version 3 (BSID-III) compared to a reference value. In some embodiments, at least one subtest is an age-equivalent score, cognitive developmental quotient (DQ), expressive language DQ, receptive language DQ, gross motor DQ, and / or fine motor DQ. In some implementations, 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 greater than 100. In some implementations, the age equivalent score increases by about or at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, or greater than 24 months compared to the reference value.In some embodiments, the reference value is a score of at least one subtest of BSID-III obtained prior to administration of the rAAV of this disclosure. In some embodiments, the reference value is the mean score of at least one subtest of BSID-III obtained from subjects with MPS II of the same age as the subjects receiving the rAAV of this disclosure. In some embodiments, the reference value is the mean score of at least one subtest of BSID-III obtained from subjects with MPS I of the same age as the subjects receiving the rAAV of this disclosure. In some embodiments, the level of the biomarker of this disclosure indicates the efficacy of MPS II treatment. For example, in some embodiments, the level 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 the level of D2S6 in a biological sample is associated with improvement in at least one subtest of BSID-III. In some embodiments, a decrease in the level of IOS6 in a biological sample is associated with improvement in at least one subtest of BSID-III. In some embodiments, a decrease in D2S6 levels in biological samples obtained from subjects after administration of rAAV encoding hIDS (e.g., compared to a reference value or D2S6 levels before...

Claims

1. A method for preventing mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, 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 no hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

2. The method of claim 1, wherein, 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 application of the rAAV, the therapeutically effective amount of the rAAV prevents hearing loss in the at least one ear.

3. The method of claim 1 or 2, wherein hearing loss is indicated by an increase in hearing threshold measured by auditory brain response (ABR) test in at least one ear as before the application of the rAAV, or as after the application of the rAAV.

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

5. The method of claim 3 or 4, wherein the method further comprises performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

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

7. The method of claim 6, wherein the method further comprises performing the tympanogram test on the at least one ear before and after the application of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

8. A method for stabilizing or preventing further hearing loss associated with mucopolysaccharidosis II (MPS II) in a subject in need, 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 experienced hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear.

9. The method of claim 8, wherein, 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, the therapeutically effective amount of the rAAV stabilizes the hearing loss or prevents further hearing loss in the at least one ear.

10. The method of claim 9, wherein stabilizing the hearing loss or preventing further hearing loss is indicated by no change or minimal change in the hearing threshold measured by the auditory brain response (ABR) test in the at least one ear before the application of the rAAV, compared to the hearing threshold measured by the ABR test in the at least one ear after the application of the rAAV.

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

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

13. The method of any one of claims 10-12, wherein the method further comprises performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

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

15. The method of claim 14, wherein the method further comprises performing the tympanogram test on the at least one ear before and after the application of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

16. A method for improving mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, 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 hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

17. The method of claim 16, wherein, after 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 following the administration of the rAAV, the therapeutically effective amount of the rAAV improves hearing in the at least one ear.

18. The method of claim 16 or 17, wherein the improvement in hearing is indicated by a reduction in the hearing threshold measured by the auditory brain response (ABR) test in the at least one ear, as before the application of the rAAV, compared to the hearing threshold measured by the ABR test in the at least one ear.

19. The method of claim 18, wherein the therapeutically effective amount of the rAAV reduces the hearing threshold of the at least one ear by 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 of the at least one ear before the application of the rAAV.

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

21. The method of claim 20, wherein the improvement in hearing is indicated by a reduction in eHL of more than about 10 dB in at least one ear after application of the rAAV compared to before application.

22. The method of any one of claims 18-21, wherein the method further comprises performing the ABR test on the at least one ear before and after the application of the rAAV to determine the hearing threshold of the at least one ear before and after the application of the rAAV.

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

24. The method of claim 23, wherein the method further comprises performing the tympanogram test on the at least one ear before and after the application of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination 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 a left ear and a right ear.

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

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

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

30. The method of claim 29, wherein the second administration of the rAAV is at a higher dose 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 subject's cerebrospinal fluid (CSF).

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

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

34. The method of any one of claims 1-33, wherein the rAAV is administered at a volume not exceeding 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 applied to the subject in solution form, the solution comprising: (a) Sodium chloride at a concentration of approximately 8.77 g / L, (b) Magnesium chloride at a concentration of approximately 0.244 g / L, (c) Potassium chloride at a concentration of approximately 0.224 g / L, (d) Calcium chloride with a concentration of approximately 0.206 g / L. (e) Dextran at a concentration of approximately 0.793 g / L, (f) Poloxamer 188 at a concentration of approximately 0.010 g / L. (g) Sodium dihydrogen phosphate monohydrate with a concentration of approximately 0.0278 g / L, and (h) Anhydrous disodium hydrogen phosphate at a concentration of approximately 0.114 g / L.

37. The method of any one of claims 1-36, wherein the rAAV is at about 1.3 × 10 10 GC / g brain mass, approximately 6.5 × 10⁻⁶ 10 GC / g brain mass or approximately 2 × 10⁻⁶ 11 The dosage of GC / 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 applied to the at least one ear.

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

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

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

42. The method of claim 41, wherein the subject does not respond to the ERT.

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

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

45. The method of any one of claims 1-44, wherein the rAAV is 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 β-actin promoter (CB7).

47. The method of claim 46, wherein the human IDS expression cassette comprises (i) an IDS transgene with a side-mounted inverted terminal repeat (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal.

48. The method of claim 47, wherein the ITR is an AAV2 ITR.

49. The method of any one of claims 46-48, wherein the human IDS expression cassette comprises a nucleic acid containing the nucleotide sequence of SEQ IDNO:

45.

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

51. The method of claim 50, wherein the immunosuppressive 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 antibiotic to the subject before or simultaneously with the rAAV.

54. The method of claim 53, wherein the at least one antibiotic is trimethoprim, sulfamethoxazole, pentanediol, dapsone, and / or atovaquinone.

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

56. A therapeutically effective amount of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for the prevention of mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has no hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents 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 stabilizing mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has experienced hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss 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 improving mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV improves 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 the prevention of mucopolysaccharidosis II (MPS II)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has no hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV prevents 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)-related hearing loss in a subject of need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has hearing loss in at least one ear prior to administration of the rAAV, and the therapeutically effective amount of the rAAV improves 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)-related hearing loss in a subject in need, wherein the therapeutically effective amount of the rAAV can be administered to the subject, wherein the subject has experienced hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective amount of the rAAV prevents further hearing loss in the at least one ear.

62. A formulation suitable for application to said 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 β-actin promoter (CB7).

64. The formulation of claim 63, wherein the human IDS expression cassette comprises (i) an IDS transgene with a side-mounted inverted terminal repeat (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal.

65. The formulation of claim 64, wherein the ITR is an AAV2 ITR.

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 IDNO: 45.

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