Method and preparation for the treatment of hearing loss associated with mucopolysaccharidosis type II using recombinant human iduronate-2-sulfatase

Abstract

This disclosure provides methods and formulations for treating or preventing hearing loss associated with mucopolysaccharidosis type II (MPS II) using recombinant human iduronide-2-sulfatase (IDS).

Description

[Technical Field]

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

[0002] Reference to electronically submitted sequence listings This application includes a computer-readable sequence listing filed with this application in XML format, the contents of which are incorporated herein by reference. The XML sequence listing filed with this application is named "12656-206-228_SEQLISTING.xml", created on 23 September 2024, and is 93,406 bytes in size.

[0003] 1. Introduction This disclosure provides methods and formulations for treating or preventing hearing loss associated with mucopolysaccharidosis type II (MPS II) using recombinant human iduronide-2-sulfatase (IDS). [Background technology]

[0004] Hunter syndrome / MPS II is a rare X-linked recessive genetic disorder affecting 0.5 to 1.3 males per 100,000 births. This progressive and serious disease is caused by a gene mutation in the IDS gene that leads to a deficiency in iduronate-2-sulfatase (I2S), an enzyme necessary for the lysosomal catabolism of heparan sulfate and dermatan sulfate, as well as a lysosomal storage enzyme. When I2S is absent or almost absent, this protein cannot perform its normal lysosomal exohydrolase function, and these common polysaccharides called GAGs (glycosaminoglycans) accumulate in the tissues and organs of MPS II patients, resulting in characteristic storage lesions and a variety of disease sequelae. The morbidity and mortality rates in this patient population are high, with an average life expectancy of 11.7 years for patients with a severe phenotype (characterized by impaired neurocognition) and 21.7 years for patients with a mild or weak phenotype (Young et al., 1982, A clinical and genetic study of Hunter's syndrome.2 Differences between the mild and severe forms. J. Medical Genetics 19:408-411). The majority of patients (two-thirds) are reported to have a severe form of the disease (Wraith JE, et al., 2007, Enzyme replacement therapy in patients who have mucopolysaccharidosis I and are younger than 5 years: Results of a multinational study of recombinant human alpha-L-Iduronidase (Laronidase). Pediatrics 120(1):E37-E46).This disease primarily affects boys, but in affected women, it has been reported to be due to non-random X inactivation and / or mutations in both alleles of the gene (Martin et al., 2008, Recognition and diagnosis of mucopolysaccharidosis II (Hunter Syndrome). Pediatrics 121:e377).

[0005] Patients with MPS II appear normal at birth, but signs and symptoms of the disease generally appear between 18 months and 4 years of age in severe cases, and between 4 and 8 years of age in mild cases. Common signs and symptoms in all affected patients include short stature, rough facial surface, macrocephaly, macroglossia, hearing loss, hepatomegaly and splenomegaly, polyostosis, articular contractures, spinal stenosis, and carpal tunnel syndrome. Upper respiratory tract and ear infections are common in most patients, and progressive airway obstruction is common, often leading to sleep apnea and death. Cardiac disease is the leading cause of death in this population, characterized by bilateral ventricular hypertrophy and valvular dysfunction leading to heart failure. Common causes of death are obstructive airway disease or heart failure.

[0006] In severe forms of the disease, important stages of early development may be reached, but developmental delays are easily observed between 18 and 24 months of age. Some patients fail the hearing screening test at 1 year of age, and delays are observed in other developmental stages, such as the ability to sit independently, walk, and speak. Developmental progress is reported to begin stagnating between 3 and 5 years of age, with regression starting around 6.5 years of age. Of the approximately 50% of children with MPS II who were toilet trained, the vast majority, if not all, become unable to use the toilet as the disease progresses (Wraith et al., 2007 (above), Martin et al., 2008 (above)).

[0007] Patients with significant neurological disorders exhibit severe behavioral problems such as hyperactivity, stubbornness, and aggression, which begin at age 2 and continue until age 8-9 when neurodegeneration weakens these behaviors (Muenzer, et al., 2009, Mucopolysaccharidosis I: Management and Treatment Guidelines, Pediatric 123(1):19-29).

[0008] Seizures are reported in more than half of severely ill patients who reach the age of 10, and by the time of death, most patients with CNS disease have severe mental disorders and require constant care (Wraith et al., 2007 (above), Martin et al., 2008 (above)). Patients with less severe disease have normal intellectual function, but MRI images reveal overall brain abnormalities such as white matter lesions, ventricular hypertrophy, and brain atrophy in all patients with MPS II (Muenzer, et al., 2009 (above)).

[0009] Enzyme replacement therapy (ERT) using recombinant idursulfase produced by HT1080 (fibrosarcoma) cells (Elaprase®, Shire Human Genetic Therapies) is the only product approved for the treatment of Hunter syndrome, and it is administered by weekly infusion (ELAPRASE (idursulfase) injection [package insert], Lexington, MA: Shire Human Genetic Therapies, Inc; 2013, available from http: / / pi.shirecontent.com / PI / PDFs / Elaprase_USA_ENG.pdf). While weekly intravenous (IV) ERT (recombinant idursulfase) treatment has shown improvement in systemic symptoms of MPS II, it is a burden for patients and their caregivers to live with such weekly ERT infusions for life, and this has negatively impacted the patient's quality of life.

[0010] Current ERTs do not cross the blood-brain barrier and therefore cannot treat patients with severe forms of the disease, namely MPS II, which affects the CNS / neurocognition and behavior. In a recent clinical trial designed to address this problem, pediatric patients received subarachnoid-formulated idursulfase (Elaprase) once a month using a spinal-implanted subarachnoid drug delivery device (incision of the lower rib cage to implant an access port and insertion of a catheter at the L4 / L5 level). Patients also received idursulfase intravenously once a week. See Muenzer et al., 2016, Genetics in Med 18:73-81, esp. p. 74. The abstract is available at https: / / www.ncbi.nlm.nih.gov / pubmed / 25834948?dopt=Abstract. Device malfunctions occurred, resulting in partial device modification, complete surgical modification, or exclusion in 6 out of 12 treated patients (50%). Notably, 12 out of 14 SAEs (Severe Adverse Events) were device-related (complications due to device insertion, device migration / connection problems, device damage / malfunction / failure, implant site infection, procedural pain, and wound dehiscence). (Muenzer et al., 2016, p.75 (second paragraph and Figure 1)). Device damage and catheter migration from the spinal canal were exacerbated by the high activity levels in this pediatric population (Muenzer et al., 2016 at p.78 Discussion). [Overview of the Initiative]

[0011] This disclosure provides methods and formulations for preventing or treating MPS II-associated hearing loss in subjects requiring prevention or treatment of MPS II-associated hearing loss, using recombinant human iduronate-2-sulfatase (IDS). In certain embodiments, recombinant human IDS is delivered to the subject by a recombinant vector.

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

[0013] In another aspect, the Disclosure provides a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in subjects requiring prevention of mucopolysaccharidosis type II (MPS II) associated hearing loss, wherein the therapeutically effective dose of rAAV is administerable to a subject who has not developed hearing loss in at least one ear prior to administration of rAAV, and the therapeutically effective dose of rAAV prevents the development of hearing loss in at least one ear.

[0014] In another aspect, the disclosure provides the use of a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) to prevent hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring prevention of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject, the subject has not developed hearing loss in at least one ear prior to administration of rAAV, and the therapeutically effective dose of rAAV prevents the development of hearing loss in at least one ear.

[0015] In certain embodiments, a therapeutically effective dose of rAAV prevents the development of hearing loss in 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 administration of rAAV.

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

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

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

[0019] In another aspect, the disclosure provides a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in subjects requiring stabilization of mucopolysaccharidosis type II (MPS II) associated hearing loss, wherein the therapeutically effective dose of rAAV is administerable to subjects who have developed hearing loss in at least one ear prior to administration of rAAV, and the therapeutically effective dose of rAAV prevents further progression of hearing loss in at least one ear.

[0020] In another aspect, the disclosure provides the use of a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) to stabilize hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring stabilization of MPS II-associated hearing loss, wherein the therapeutically effective dose of rAAV is administerable to subjects who have developed hearing loss in at least one ear prior to rAAV administration, and the therapeutically effective dose of rAAV prevents further progression of hearing loss in at least one ear.

[0021] In certain embodiments, a therapeutically effective dose of rAAV stabilizes hearing loss or prevents further progression of hearing loss in at least one ear after rAAV administration 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.

[0022] In certain embodiments, stabilization of hearing loss or prevention of further hearing loss progression is indicated by the hearing threshold measured by auditory brainstem response (ABR) testing in at least one ear after rAAV administration showing no change or only a slight change compared to the hearing threshold measured by ABR testing in at least one ear before rAAV administration. In certain embodiments, the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL). In certain embodiments, stabilization of hearing loss or prevention of further hearing loss progression is indicated by the change in eHL in at least one ear after rAAV administration being approximately 10 dB or less compared to before rAAV administration.

[0023] In certain embodiments, the methods or uses disclosed herein further include performing ABR testing in at least one ear before and after administration of rAAV to determine the hearing threshold of at least one ear before and after administration of rAAV.

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

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

[0026] In another aspect, the Disclosure provides a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) for use in improving hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring improvement of hearing loss associated with MPS II, wherein the therapeutically effective dose of rAAV is administerable to a subject who has developed hearing loss in at least one ear prior to administration of rAAV, and the therapeutically effective dose of rAAV improves hearing in at least one ear.

[0027] In another aspect, the disclosure provides the use of a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) to improve hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring improvement of hearing loss associated with MPS II, wherein the therapeutically effective dose of rAAV is administerable to the subject, the subject has developed hearing loss in at least one ear prior to administration of rAAV, and the therapeutically effective dose of rAAV improves hearing in at least one ear.

[0028] In certain embodiments, a therapeutically effective dose of rAAV improves hearing in at least one ear 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 after administration of rAAV.

[0029] In certain embodiments, improved hearing is indicated by a decrease in the auditory brainstem response (ABR) threshold measured in at least one ear after rAAV administration compared to the auditory threshold measured in the ABR test in at least one ear before rAAV administration. In certain embodiments, a therapeutically effective dose of rAAV reduces the auditory threshold in 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% compared to the auditory threshold in at least one ear before rAAV administration. In certain embodiments, the auditory threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL). In certain embodiments, improved hearing is indicated by a decrease of at least about 10 dB in eHL in at least one ear after rAAV administration compared to before rAAV administration. In certain embodiments, the methods or uses disclosed herein further include performing ABR testing in at least one ear before and after administration of rAAV to determine the hearing threshold of at least one ear before and after administration of rAAV.

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

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

[0032] In certain embodiments, the methods or uses disclosed herein further include providing additional treatment to the subject. In certain embodiments, the additional treatment is enzyme replacement therapy comprising recombinant idursulfase. In certain embodiments, the additional treatment is a second dose of rAAV. In certain embodiments, the second dose of rAAV is a higher dose than the first dose of rAAV.

[0033] In certain embodiments, rAAV is administered into the cerebrospinal fluid (CSF) of the subject. In certain embodiments, rAAV is administered via intracisional (IC) administration. In certain embodiments, rAAV is administered via intraventricular (ICV) administration. In certain embodiments, rAAV is administered in an amount not exceeding 10% of the total volume of the cerebrospinal fluid of the subject.

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

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

[0036] In certain embodiments, rAAV is administered to the subject as a solution, and this solution is (a) Sodium chloride with a concentration of approximately 8.77 g / L (b) Magnesium chloride hexahydrate 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) Dextrose at a concentration of approximately 0.793 g / L (f) Poloxamer 188 with a concentration of approximately 0.010 g / L, (g) Sodium phosphate monobasic monohydrate at a concentration of approximately 0.0278 g / L, and (h) Contains dibasic anhydrous sodium phosphate at a concentration of approximately 0.114 g / L.

[0037] In certain embodiments, rAAV is approximately 1.3 × 10⁻⁶ 10 Dosage per GC / brain weight in grams: approximately 6.5 × 10 10GC / brain weight in grams, or approximately 2 × 10⁻⁶ 11 The drug is administered at a dose of GC / brain weight in grams, and brain weight is measured by MRI.

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

[0039] In certain embodiments, the target population is between 5 years of age and under 18 years of age. In certain embodiments, the target population is between 4 months of age and under 5 years of age.

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

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

[0042] In certain embodiments, rAAV is recombinant adeno-associated virus serotype 9. In certain embodiments, rAAV comprises a human IDS expression cassette, where human IDS expression is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken β-actin promoter (CB7). In certain embodiments, the human IDS expression cassette comprises (i) an IDS transgene flanked by an inverted terminal repeat sequence (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (PolyA) signal. In certain embodiments, the ITR is an AAV2 ITR. In certain embodiments, the human IDS expression cassette comprises a nucleic acid containing the nucleotide sequence of Sequence ID No. 45.

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

[0044] In certain embodiments, the methods or uses disclosed herein further include administering to the subject at least one antibiotic before or concurrently with the administration of rAAV. In certain embodiments, the at least one antibiotic is trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atobacuone.

[0045] In certain embodiments, the methods or uses disclosed herein further include administering at least one antifungal therapy to the subject before or concurrently with the administration of rAAV.

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

[0047] In certain embodiments, rAAV comprises a human IDS expression cassette, in which human IDS expression is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken β-actin promoter (CB7). In certain embodiments, the human IDS expression cassette comprises (i) an IDS transgene flanked by an inverted terminal repeat sequence (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (PolyA) signal.

[0048] In certain embodiments, the ITR is an AAV2 ITR. In certain embodiments, the human IDS expression cassette contains a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 45. 3.1 Typical Embodiments 1. A method for preventing hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring prevention of such hearing loss, comprising administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), The method wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents the development of hearing loss in at least one ear. 2. The method according to Embodiment 1, wherein the therapeutically effective dose of rAAV prevents the onset of hearing loss in 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 administration of the rAAV. 3. The method according to Embodiment 1 or 2, wherein the onset of hearing loss is indicated by an increase in the hearing threshold measured by auditory brainstem response (ABR) testing in at least one ear after rAAV administration compared to the hearing threshold measured by ABR testing in at least one ear before rAAV administration. 4. The method according to Embodiment 3, wherein the hearing threshold is an estimated value of the behavioral threshold (eHL) obtained from the ABR threshold (nHL). 5. The method according to Embodiment 3 or 4, further comprising performing an ABR test in at least one ear before and after administration of the rAAV to determine the hearing threshold of at least one ear before and after administration of the rAAV. 6. The method according to any one of Embodiments 1 to 5, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic hearing test. 7. The method according to Embodiment 6, further comprising performing the tympanic audiometry test in at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural. 8. A method for stabilizing hearing loss associated with mucopolysaccharidosis type II (MPS II) or preventing further progression of hearing loss associated with MPS II in a subject requiring stabilization of MPS II-related hearing loss or prevention of further progression of MPS II-related hearing loss, the method comprising administering to the subject a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS). The method wherein the subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents further progression of hearing loss in the at least one ear. 9. The method according to Embodiment 8, wherein the therapeutically effective dose of rAAV stabilizes the hearing loss or prevents further progression of the hearing loss in 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 administration of the rAAV. 10. The method according to Embodiment 9, wherein stabilization of the hearing loss or prevention of further hearing loss is indicated by the hearing threshold measured by auditory brainstem response (ABR) testing in at least one ear after rAAV administration showing no change or only a slight change compared to the hearing threshold measured by ABR testing in at least one ear before rAAV administration. 11. The method according to Embodiment 10, wherein the hearing threshold is an estimated value of the behavioral threshold (eHL) obtained from the ABR threshold (nHL). 12. The method according to Embodiment 11, wherein stabilization of the hearing loss or prevention of further hearing loss is indicated by a change in eHL in at least one ear being approximately 10 dB or less after rAAV administration compared to before rAAV administration. 13. The method according to any one of Embodiments 10 to 12, further comprising performing an ABR test in at least one ear before and after administration of the rAAV to determine the hearing threshold of at least one ear before and after administration of the rAAV. 14. The method according to any one of Embodiments 8 to 13, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic audiometry. 15. The method according to Embodiment 14, further comprising performing the tympanic audiometry test in at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural. 16. A method for improving hearing loss associated with mucopolysaccharidosis type II (MPS II) in a subject requiring improvement of such hearing loss, comprising administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), The method wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV improves the hearing loss in at least one ear. 17. The method according to Embodiment 16, wherein the therapeutically effective dose of rAAV improves hearing in at least one ear about 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 after administration of the rAAV. 18. The method according to Embodiment 16 or 17, wherein the improvement of hearing is indicated by a decrease in the hearing threshold measured by auditory brainstem response (ABR) testing in at least one ear after rAAV administration compared to the hearing threshold measured by ABR testing in at least one ear before rAAV administration. 19. The method according to Embodiment 18, wherein the therapeutically effective dose of rAAV reduces the hearing threshold of 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 at least one ear before administration of rAAV. 20. The method according to Embodiment 18 or 19, wherein the hearing threshold is an estimated value of the behavioral threshold (eHL) obtained from the ABR threshold (nHL). 21. The method according to Embodiment 20, wherein the improvement of hearing is indicated by a decrease of approximately 10 dB or more in eHL in at least one ear after rAAV administration compared to before rAAV administration. 22. The method according to any one of embodiments 18 to 21, further comprising performing an ABR test in at least one ear before and after administration of the rAAV to determine the hearing threshold of at least one ear before and after administration of the rAAV. 23. The method according to any one of Embodiments 8 to 22, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic audiometry. 24. The method according to Embodiment 23, further comprising performing the tympanic audiometry test in at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural. 25. The method according to any one of embodiments 1 to 24, wherein the at least one ear includes either the left ear or the right ear. 26. The method according to any one of embodiments 1 to 25, wherein the at least one ear includes a left ear and a right ear. 27. The method according to any one of embodiments 1 to 26, further comprising performing an additional treatment on the subject. 28. The method according to Embodiment 27, wherein the additional treatment is enzyme replacement therapy containing recombinant idursulfase. 29. The method according to Embodiment 27, wherein the additional treatment is a second dose of the rAAV. 30. The method according to Embodiment 29, wherein the second dose of rAAV is administered in a larger amount than the first dose of rAAV. 31. The method according to any one of Embodiments 1 to 30, wherein the rAAV is administered intravenously to the subject or into the cerebrospinal fluid (CSF) of the subject. 32. The method according to any one of Embodiments 1 to 31, wherein the rAAV is administered via intracision cell (IC) administration. 33. The method according to any one of Embodiments 1 to 31, wherein the rAAV is administered via intraventricular (ICV) administration. 34. The method according to any one of Embodiments 1 to 33, wherein the rAAV is administered in an amount not exceeding 10% of the total volume of cerebrospinal fluid of the subject. 35. The method according to any one of Embodiments 1 to 34, wherein the rAAV is administered intrathecally to the subject. 36. The rAAV is administered to the subject as a solution, and the solution is (a) Sodium chloride with a concentration of approximately 8.77 g / L (b) Magnesium chloride hexahydrate 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) Dextrose at a concentration of approximately 0.793 g / L (f) Poloxamer 188 with a concentration of approximately 0.010 g / L, (g) Sodium phosphate monobasic monohydrate at a concentration of approximately 0.0278 g / L, and (h) The method according to any one of Embodiments 1 to 35, comprising a dibasic anhydride of sodium phosphate at a concentration of approximately 0.114 g / L. 37. The rAAV is approximately 1.3 × 10⁻⁶ 10 Dosage per GC / brain weight in grams: approximately 6.5 × 10 10 GC / brain weight in grams, or approximately 2 × 10⁻⁶ 11 The method according to any one of embodiments 1 to 36, wherein the GC is administered in g / brain weight, and the brain weight is measured by MRI. 38. The method according to any one of Embodiments 1 to 30, wherein the rAAV is administered to at least one of the ears. 39. The method according to any one of Embodiments 1 to 38, wherein the subject is 5 years of age or older and under 18 years of age. 40. The method according to any one of Embodiments 1 to 38, wherein the subject is 4 months of age or older and under 5 years of age. 41. The method according to any one of Embodiments 1 to 40, wherein the subject is receiving enzyme replacement therapy (ERT) at the time of administration of the rAAV. 42. The method according to Embodiment 41, wherein the subject does not respond to the ERT. 43. The method according to embodiment 41 or 42, wherein the ERT comprises a recombinant idursulfase. 44. The method according to any one of embodiments 41 to 43, wherein the ERT is discontinued 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 administration of the rAAV. 45. The method according to any one of Embodiments 1 to 44, wherein the rAAV is recombinant adeno-associated virus serotype 9. 46. ​​The method according to Embodiment 45, wherein the rAAV comprises a human IDS expression cassette, and 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 according to Embodiment 46, wherein the human IDS expression cassette comprises (i) an IDS transgene adjacent to an inverted terminal repeat sequence (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal. 48. The method according to embodiment 47, wherein the ITR is an AAV2 ITR. 49. The method according to any one of embodiments 46 to 48, wherein the human IDS expression cassette comprises a nucleic acid containing the nucleotide sequence of SEQ ID NO: 45. 50. The method according to any one of Embodiments 1 to 49, further comprising administering immunosuppressive therapy to the subject before or simultaneously with the administration of rAAV, and optionally continuing the immunosuppressive therapy thereafter. 51. The method according to Embodiment 50, wherein the immunosuppressive therapy comprises administering at least one corticosteroid, sirolimus, and / or tacrolimus. 52. The method according to Embodiment 51, wherein the at least one corticosteroid is methylprednisolone and / or prednisone. 53. The method according to any one of Embodiments 1 to 52, further comprising administering at least one antibiotic to the subject before or concurrently with the administration of rAAV. 54. The method according to Embodiment 53, wherein the at least one antibiotic is trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atobacuone. 55. The method according to any one of Embodiments 1 to 54, further comprising administering at least one antifungal therapy to the subject before or concurrently with the administration of the rAAV. 56. A therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), for use in subjects requiring prevention of hearing loss associated with mucopolysaccharidosis type II (MPS II), wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents the development of the hearing loss in at least one ear. 57. A therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), used to stabilize hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring stabilization of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents further progression of hearing loss in at least one ear. 58. A therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), used to improve hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring improvement of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV improves hearing in at least one ear. 59. Use of a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) to prevent hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring prevention of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents the development of the hearing loss in at least one ear, as described above. 60. Use of a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) to improve hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring improvement of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV improves the hearing in at least one ear, according to the use described above. 61. Use of a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) to stabilize hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring stabilization of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents further progression of hearing loss in the at least one ear, as described above. 62. A formulation suitable for administration to the ear, comprising a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS). 63. The formulation according to Embodiment 62, wherein the rAAV comprises a human IDS expression cassette, and 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 according to Embodiment 63, wherein the human IDS expression cassette comprises (i) an IDS transgene adjacent to an inverted terminal repeat sequence (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (PolyA) signal. 65. The formulation according to Embodiment 64, wherein the ITR is AAV2 ITR. 66. The formulation according to any one of embodiments 63 to 65, wherein the human IDS expression cassette comprises a nucleic acid containing the nucleotide sequence of SEQ ID NO: 45. [Brief explanation of the drawing]

[0049] [Figure 1] This is the amino acid sequence of human IDS (SEQ ID NO: 1). The post-translational formylglycine modification at C84 (shown in bold in Figure 1) is necessary for enzyme activity. The eight N-linked glycosylation sites (N31, N115, N144, N246, N280, N325, N513, and N537) are shown in bold and enclosed in boxes. One tyrosine-O-sulfation site (Y) is shown in bold, and the entire sulfated site sequence (PSSEKY165ENTKTCRGPD (SEQ ID NO: 47)) is enclosed in a box. The mature 42kDa and 14kDa polypeptides at the N-terminus are indicated by horizontal arrows. In the brain, the mature 42kDa form of the N-terminus begins at position 34 or 36, as shown in Figure 1, as follows: T34DALNVLLI (SEQ ID NO: 54) and A36LNVLLIIV (SEQ ID NO: 55) (see Sleat, 2005, Proteomics 5:1520-1532, Table S2). Two of the eight N-linked glycosylation sites, namely N280 and N116, undergo mannose-6-phosphorylation in IDS obtained from human brain (reported in Sleat et al., 2006, Mol & Cell Proeomics 5.4:686-701, Table V).

[0050] [Figure 2]This is a multiple sequence alignment of hIDS with known orthologs. The species names and protein IDs are as follows: SP|P22304|IDS_HUMAN[Human];TR|K6ZGI9_PANTR[Pan troglodytes (Chimpanzee)];TR|K7BKV4_PANTR[Pan troglodytes (Chimpanzee)];TR|H9FTX2_MACMU[Macaca mulatta (Rhesus macaque)], TRF7EJG2_CALJA[Callithrix jacchus (Snow-eared marmoset)], TR|U3DTL8_CALJA[Callithrix jacchus (Snow-eared marmoset)], TR|G7NRX7_MACMU[Macaca mulatta (Rhesus macaque)], TR|G7Q1V9_MACFA[Macaca fascicularis (Cynomolgus macaque)], TR|H2PX10_PONAB[Pongo [abelii (Sumatran orangutan)], TR|A0A0D9R4D1_CHLSB [Chlorocebus sabaeus (green monkey)], TR|G1RST8|G1RST8_NOMLE [Nomascus leucogenys (Northern white-handed gibbon)], UPI0000D9F625 [Macaca mulatta (rhesus monkey)], UPI000274358B [Pan paniscus (pygmy chimpanzee, bonobo)], UPI00027F6FC5 [Papio Anubis (olive baboon)], UPI00027FAE03 [Saimiri boliviensis (Bolivian squirrel monkey)], UPI0003ABBF28 [Macaca fascicularis (crab-eating macaque, cinomoro monkey)], UPI000533297F [Rhinopithecus] roxellana (Golden snub-nosed monkey, Pygathrix roxellana), UPI0005F40BD2 [Colobus angolensis palliates (Peters' Angola Colobus)] (Sequence IDs 27-44).

[0051] [Figure 3]hIDS and the corresponding mild, moderate, or severe disease phenotypes are MPS II mutations (derived from Uniprot).

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

[0053] [Figure 5] This is a schematic diagram of Construct 1.

[0054] [Figure 6] This is a culal multiplex sequence alignment of AAV capsids 1-9 (SEQ ID NOs: 16-26). By "mobilizing" amino acid residues from corresponding positions in other aligned AAV capsids, amino acid substitutions can be performed on AAV9 and AAV8 capsids (SEQ ID NOs: 48-53, shown in bold in the bottom row). Sequence regions labeled "HVR" are hypervariable regions.

[0055] [Figure 7] Digestion of heparan sulfate (HS) using heparinase.

[0056] [Figure 8] Graph showing cerebrospinal fluid (CSF) heparan sulfate biomarker (ng / mL). The graph shows a consistent decrease in heparan sulfate in CSF after administration of Construct 1. The median change from baseline at week 8 (N=6) was -30.3% (p-value = 0.03, measured by Wilcoxon's signed-rank test). The median change from baseline at the last available time point (N=6) was -35.8% (p-value = 0.03, measured by Wilcoxon's signed-rank test). This graph shows that the measurable CSF I2S enzyme concentration in Cohort 2 after administration of Construct 1 was in the range of 1170–1940 pg / mL.

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

[0058] [Figure 10] Graphs showing neurodevelopmental function compared to age equivalence (months) and age (months). Graph 10A shows that in follow-up studies exceeding 6 months, 4 out of 5 patients showed continuous cognitive development. Patients 1, 3, and 5 showed continuous cognitive development within the normal range. Patients 2 and 4 showed significant cognitive delay at baseline. Patient 2 continued to show cognitive development. Patient 4 acquired expressive and receptive language skills. Figures 10B and 10C show the cognitive development index and age equivalence of patients in the Construct-1 gene therapy clinical trial, respectively. Graphs 10B and 10C show that 3 out of 4 patients who participated in the trial with cognitive skills exceeding -2SD (normal range) remained above -2SD at follow-up exceeding 6 months. One patient participated in the trial with significant neurocognitive developmental delay at baseline, but after receiving Construct-1 at a higher age (59 months), showed relative stability and continued to acquire expressive and receptive language skills.

[0059] [Figure 11-1] A graph illustrating neurodevelopmental function from the perspectives of language ability and motor ability. A represents expressive communication. B represents receptive communication. [Figure 11-2] This graph illustrates neurodevelopmental function from the perspectives of language and motor skills. C represents gross motor skills, and D represents fine motor skills. The graph shows the continuous acquisition of language and / or motor skills in patients with a follow-up period of more than 6 months.

[0060] [Figure 12]This graph shows systemic efficacy obtained by measuring plasma I2S protein concentration (pg / mL). This graph shows a general increase in plasma I2S enzyme levels in 5 out of 6 patients after administration of Construct 1 (within the normal range (14,706 pg / mL to less than 100,000 pg / mL)).

[0061] [Figure 13] This graph shows the systemic effectiveness obtained by measuring the total urinary GAG levels (g / mol CK) in patients treated with ERT. The graph shows a sustained decrease in urinary GAG levels in all patients treated with ERT.

[0062] [Figure 14] This graph shows systemic efficacy obtained by measuring total urinary GAG levels (g / mol CK) in patients who were not treated with ERT and those who discontinued ERT. The graph shows a rapid decrease in urinary GAG in patients who were not treated with ERT after administration of Construct 1 (no urinary GAG rebound after ERT discontinuation).

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

[0064] [Figure 16] Proof of Concept (PCO) testing activity.

[0065] [Figure 17] Dosage rationale for the initial human clinical trial.

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

[0067] [Figure 19]Figure 19A is a graph showing the levels of D0S0 disaccharide in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects. Figure 19B is a graph showing the levels of D0A6 disaccharide in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects. Figure 19C is a graph showing the levels of D0A0 in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0068] [Figure 20] Graph showing D2S6 disaccharide levels in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0069] [Figure 21] Graph showing the compositional percentage of heparan sulfate disaccharide in healthy individuals, MPS I (neuropathic and non-neuropathic) subjects, and MPS II (neuropathic and non-neuropathic) subjects.

[0070] [Figure 22] Schematic diagram of the MPS II Phase 1 / 2 clinical trial.

[0071] [Figure 23] A graph showing the broad CNS and systemic biodistribution of Construct 1 after IC administration in nonhuman subjects. The terms "LD" represent low dose, "IS" represent immunosuppression, and "HD" represent high dose.

[0072] [Figure 24] Graph showing cerebrospinal fluid (CSF) biomarker heparan sulfate (HS) in subjects of the Phase 1 / 2 trial. CSF HS measurements showed a dose-dependent decrease in cohorts 1-3 at weeks 8 and 24 (median values ​​are shown in A, and individual participant data are shown in B).

[0073] [Figure 25]Graph showing D2S6 concentrations in patients during the Phase 1 / 2 trial. CSF D2S6 measurements showed a dose-dependent decrease in cohorts 1-3 at weeks 8 and 24. Participants in cohort 3 approached normal levels (median values ​​are shown in A, and individual participant data are shown in B).

[0074] [Figure 26] Graphs showing the neurodevelopmental functions of Cognitive (A), Expressive Language (B), and Fine Motor (C) skills in Cohort 1 patients in a Phase 1 / 2 trial.

[0075] [Figure 27] Graphs showing the neurodevelopmental functions of cognition (A), expressive language (B), and fine motor skills (C) in Cohort 2 patients in a Phase 1 / 2 trial.

[0076] [Figure 28] Graph showing patients' maladaptive behavior index (A) and elimination skills (B) in a Phase 1 / 2 trial.

[0077] [Figure 29] Graph showing patients' plasma I2S protein levels (A) and urinary GAG levels (B) in a Phase 1 / 2 trial.

[0078] [Figure 30] Schematic diagram of the MPS I Phase 1 / 2 clinical trial.

[0079] [Figure 31] Graphs showing cerebrospinal fluid (CSF) biomarkers and heparin sulfate concentrations in participants (A) and single participants (B) in the MPS I Phase 1 / 2 trial. The graphs show the decrease in CSF heparin sulfate for all participants at the last available time point (e.g., week 24 for the Phase 1 / 2 trial (A) and week 59 for single participants (B)). Measurable CSF IDUA enzyme activity was observed in the majority of participants in the Phase 1 / 2 trial and in single participants in the trial.

[0080] [Figure 32] This graph shows the neurodevelopmental function BSID-III as cognition (A), expressive language (B), and fine motor skills (C) in the MPS I Phase 1 / 2 trial, and as cognition (D), expressive language (E), and fine motor skills (F) in the single-participant trial.

[0081] [Figure 33] This graph shows the neurodevelopmental function BSID-III cognition in the MPS I single-participant trial.

[0082] [Figure 34] This table shows the neurodevelopmental function (WASI-II and VABS-III) of 13-year-old participants in the MPS I Phase 1 / 2 trial.

[0083] [Figure 35] Graph showing I0S6 levels in MPS I Phase 1 / 2 participants (A) and single-participant trials (B).

[0084] [Figure 36] Graph showing urinary total GAG levels in MPS I Phase 1 / 2 participants (A) and single-participant trials (B).

[0085] [Figure 37] A composite audiogram showing the mean air conduction ABR thresholds in dBeHL units for 500Hz, 1000Hz, 2000Hz, and 4000Hz in the left ear (A) and right ear (B).

[0086] [Figure 38] According to graphs showing the change in each participant's ABR air conduction threshold from baseline to the last available point, hearing thresholds improved or stabilized in most ears.

[0087] [Figure 39]This is a test flowchart of Example 10 disclosed herein, illustrating the process flow. AAV9 = Recombinant adeno-associated virus serotype 9, ABR = Auditory brainstem response, CSF = Cerebrospinal fluid, CT = Computed tomography, DNA = Deoxyribonucleic acid, ECG = Electrocardiogram, ELISPOT = Enzyme-linked immunosuppressant, EOS = End of study, ERT = Enzyme replacement therapy, ET = Early termination, GAG = Glycosaminoglycan, Hep = Hepatitis, HIV = Human immunodeficiency virus, IC = Intraventricular, ICV = Intravenous, INR = International normalized ratio, IQ = Intelligence quotient, IT = Intrathecal, IV = Intravenous, MRI = Magnetic resonance imaging, PI = Principal Investigator, PO = Oral, PT = Prothrombin time, PTT = Partial thromboplastin time, QD = Once daily, qPCR = Quantitative polymerase chain reaction, Construct 1 = Recombinant adeno-associated virus serotype 9 capsid containing human iduronate-2-sulfatase expression cassette, sAg = Surface antigen, T cell = T lymphocyte. In assigning visit numbers, the 14th visit is intentionally omitted. The notes for the figures and tables are as follows: ·a Each facility can accept subjects from two days prior to the morning of the first day. Therefore, some pre-administration tests can be performed two days or one day prior, according to the facility's standards. ·b At ET visits, lumbar puncture (and CSF assessment) is performed only if the subject drops out before week 56, and cognitive function assessment is performed only if the subject drops out before week 48. ·c The principal investigator should consult with the medical monitor to determine which assessments should be performed at unscheduled visits. ·d Vital signs and neurological assessments are monitored frequently, with assessments performed every 45 ± 10 minutes for the first 3 hours after starting in the post-anesthetic recovery room, every 1 hour ± 15 minutes for the following 4 hours, and every 4 hours ± 30 minutes for the remaining 24 hours immediately following administration. ·If ePI is deemed appropriate, subjects can be discharged approximately 1-2 days after administration. Subjects / caregivers should be informed about neurological function monitoring. • The 64-week follow-up visit will only be performed for patients who have discontinued IV ERT. • The evaluations at weeks 20 and 28, indicated in italics in Table g, will be limited to evaluation of adverse events and concomitant therapies via telephone contact.• h Neurological evaluation before administration can be performed at any time between 2 days prior to day 1. • i When clinically required. • j Performed according to the standards of each institution and may be performed before day 1. • k Results must be evaluated or reviewed by a neurologist. • kk Before performing MRI with gadolinium, the estimated glomerular filtration rate (eGFR) based on creatinine levels must be measured. If the creatinine level is determined to be below the assay validation or detection limit in the laboratory, that lower limit will be used to estimate the eGFR. If the eGFR is less than 30 mL / min / 1.73m2, the principal investigator must consult with the medical monitor before performing the MRI. • l Tests to evaluate contraindications for IC or ICV injection must be performed according to the appropriate implementation manual and completed at least 2 days prior to administration. - Before starting IS on day 2, written approval for the administration of IC or ICV injection must be obtained. • m MRI with gadolinium should be performed only as a screening test. After confirming the neurocognitive function score, MRI should be performed whenever possible. If MRI under sedation is performed before neurocognitive function testing, there must be at least a 24-hour interval between the time of the MRI and the time when the neurocognitive function testing can be performed. · Genetic confirmation of nMPS2 type. Genetic testing performed before the study is permitted if there is proper documentation. Subjects who have not previously undergone genetic testing can have it done at the time of screening. · Changes in auditory function are measured by auditory brainstem response (ABR) and, if possible, behavioral audiometry (BAUD). Tympanotomy is performed when BAUD and / or ABR testing is performed. Auditory function is assessed at the visits specified in this protocol. Additional assessments are permitted at the 24-week visit if the principal investigator deems it clinically necessary. · Plasma biomarkers: Samples are taken for GAG and I2S. IV ERT-related I2S assessments should be performed within the period defined as at least 96 hours after ERT administration and before the start of the next administration. · p Abdominal ultrasound is performed to assess the size of the liver and spleen.For subjects who discontinue ERT after week 24, plasma and urinary biomarkers will be collected. Abdominal ultrasound examinations will be performed at the same facility at all times. · Antibodies against qAAV9 and I2S. ·r Urinary biomarkers: Samples will be collected for GAG. IV ERT-related urinary GAG collection should be performed within the defined period from at least 96 hours after ERT administration until the start of the next administration. ·s IC or ICV injection will be performed according to the applicable administration manual. ·t Tacrolimus: Dose adjustments will be made to maintain a minimum whole blood concentration within 2-4 ng / mL. Sirolimus: Dose adjustments will be made to maintain a minimum whole blood concentration within 1-3 ng / mL. Subjects should continue the new maintenance dose for at least 7-14 days before further dose adjustments while monitoring concentrations. · Methylprednisolone should be administered before lumbar puncture and IC or ICV injection of the investigational drug. Pre-medication with acetaminophen and antihistamines is optional and left to the discretion of the principal investigator. ·u Lumbar puncture during screening (35 to 2 days prior to administration) is for the sole purpose of evaluating CSF and biomarkers. Following the pre-administration lumbar puncture on day 1, iodomielography contrast agent is administered via IOT (see administration manual). ·vCSF biomarkers: Samples are collected for GAG and I2S, and sample testing for future use is performed using only residual test samples, if available. ·wCSF safety laboratory tests: CSF pressure, red blood cell count, white blood cell count (including fractions), total protein, and blood glucose. ·x Cognition is assessed using BSID-III or KABC-II, and adaptive function is assessed using VABS-II. If MRI under sedation is performed before neurocognitive function testing, there must be at least a 24-hour interval between the time of MRI and the time when neurocognitive function testing can be performed. For zVZV, the baseline Ab antibody titer is used. For EBV and CMV, the baseline viral genome PCR test and a series of tests as described in Table X are used. If a positive result is obtained at any point in time, the frequency of testing may be increased in accordance with the facility's standard treatment policy.• If the subject has relatives with the same MPS II mutation, a separate consent form will be provided to verify the appropriate relevant documents. • If possible, the same caregiver should complete the form throughout the trial period. • If possible, the same clinician should complete the form throughout the trial period.

[0088] [Figure 40] This table summarizes the audiometry data from Example 16. It shows audiometry data from 11 participants at screening (baseline) and at the final time point after Construct 1 treatment. 1. Average value of estimated behavioral threshold in dBeHL units obtained from ABR for each measured frequency. A threshold difference of more than 10 dB was used to determine the change from baseline. *Indicates participants for whom data is available at the final time point in long-term studies. 2. Indicates tube placement or tympanic membrane perforation. 3. Screening was at week 1.

[0089] [Figure 41] This is a composite audiogram showing the mean air conduction ABR thresholds in dBeHL units at 500Hz, 1000Hz, 2000Hz, and 4000Hz for each ear. At the final time point after treatment with Construct 1, improvement in hearing thresholds was observed compared to baseline.

[0090] [Figure 42] This graph shows the mean change in each participant's ABR air conduction threshold from baseline to the last available time point.

[0091] [Figure 43] This table summarizes the audiometry data from Example 17. It includes audiometry data from 11 participants at the screening (baseline) and the final point after Construct 1. 1. Average value of the estimated behavioral threshold in dBeHL units obtained from ABR for each measured frequency. A threshold difference exceeding 10 dB was used to determine the change from baseline. 2. Indicates tube placement or tympanic membrane perforation. 3. Conduction element at 500 Hz.

[0092] [Figure 44] This is a composite audiogram showing the mean air conduction ABR thresholds in dBeHL units at 500Hz, 1000Hz, 2000Hz, and 4000Hz for each ear. At the final time point after treatment with Construct 1, improvement in hearing thresholds was observed compared to baseline.

[0093] [Figure 45] This graph shows the mean change in each participant's ABR air conduction threshold from baseline to the last available time point. [Modes for carrying out the invention]

[0094] The present invention includes, but is not limited to, the delivery of recombinant human iduronide-2-sulfatase (rhIDS) produced by human nerve cells or human glial cells to the cerebrospinal fluid (CSF) of the central nervous system (CNS) of human subjects diagnosed with mucopolysaccharidosis type II (MPS II), including patients diagnosed with Hunter syndrome. For compositions and methods that can be used in accordance with the invention described herein, please also refer to International Patent Application PCT / US2017 / 027770, filed on 14 April 2017 (published as WO / 2017 / 181113 on 19 October 2017), which is incorporated herein by reference in its entirety.

[0095] In a preferred embodiment, treatment is carried out via gene therapy, for example, by administering a viral vector or other DNA expression construct encoding human IDS (hIDS) or a derivative of hIDS to the CSF of a patient diagnosed with MPS II (human subject) to create a permanent depot of transduced neurons and / or glial cells, thereby continuously supplying the transgene product to the CNS. The rhIDS secreted from the neuronal / glial cell depot into the CSF is taken up by cells in the CNS to "cross-correct" the enzyme deficiency in the recipient cells. Furthermore, unexpectedly, it has been found that the depot of transduced neurons and glial cells in the CNS allows for the delivery of recombinant enzymes both to the CNS and systemically, potentially mitigating or eliminating the need for systemic treatment, such as weekly intravenous injections of the enzyme. Furthermore, this specification also provides for the treatment of MPS I by administering a viral vector encoding human IDUA or other DNA expression construct to a subject (see, for example, non-replicating recombinant AAV with serotype 9 capsid containing hIDUA expression cassette; construct 2; PCT / US2021 / 014129; PCT / US2018 / 015910; and PCT / US2019 / 042205 (each of these is incorporated herein by reference in its entirety)).

[0096] In alternative embodiments, hIDS can be produced in cell cultures of human nerve cells or human glial cells (e.g., a bioreactor) and administered as enzyme replacement therapy ("ERT"), for example, by injection, directly to the CSF, the CNS, and / or systemically. However, enzymes cannot cross the blood-brain barrier, systemic delivery of enzymes does not result in treatment of the CNS, and unlike the gene therapy methods of the present invention, direct delivery of enzymes to the CNS requires repeated injections which not only imposes a heavy burden but also carries a risk of infection, thus the gene therapy methods offer several advantages over ERT.

[0097] The hIDS encoded by the transgene may include, but are not limited to, human IDS (hIDS) having the amino acid sequence of Sequence ID No. 1 (shown in Figure 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, such as, but are not limited to, amino acid substitutions selected from non-conserved residues corresponding to orthologs of IDS shown in Figure 2, provided that such mutations do not include substitutions of the cysteine ​​residue at position 84 (C84) required for enzyme activity (Millat et al., 1997, Biochem J326:243-247), or, for example, as shown in Figure 3, or Sukegawa-Hayasaka et al., 2006, J Inhert Metabolism. Provided that the variants do not include those identified in severe, severe-moderate, moderate, or weak MPS II phenotypes, such as those reported in Dis29:755-761 (which reports the “weak” variants R48P, A85T, W337R, and the truncated variant Q531X, and the “severe” variants P86L, S333L, S349I, R468Q, and R468L), Millat et al., 1998, BBA1406:214-218 (which reports the “weak” variants P480L and P480Q; and the “severe” variant P86L), and Bonucelli et al., 2001, BBA1537:233-238 (each of these is incorporated herein by reference in whole).

[0098] For example, amino acid substitutions at specific sites in hIDS can be selected from the corresponding non-conservative amino acid residues found at that site in the IDS ortholog shown in Figure 2, provided that they do not contain any harmful mutations such as those shown in Figure 3, or those reported in Sukegawa-Hayasaka et al., 2006 (above); Millat et al., 1998 (above); and Bonucelli et al., 2001 (above) (each of which is incorporated herein by reference in whole). The resulting transgene product can be tested in vitro in cell culture or in test animals using conventional assays to ensure that the mutations do not impair IDS function. The preferred amino acid substitutions, deletions, or additions selected should maintain or increase the enzymatic activity, stability, or half-life of IDS when tested in vitro with conventional assays for MPS II in cell culture or animal models. For example, the enzymatic activity of the transgene product can be evaluated using conventional enzyme assays with substrates such as 4-methylumbelliferyl α-L-idopyranocisuronic acid 2-sulfate or 4-methylumbelliferyl sulfate (see, for example, Lee et al., 2015, Clin. Biochem. 48(18):1350-1353, Dean et al., 2006, Clin. Chem. 52(4):643-649 (each of these is incorporated herein by reference in its entirety)). The ability of the transgene product to modify the MPS II phenotype can be evaluated in cell culture.For example, by transducing cultured MPS II cells with a viral vector or other DNA expression construct encoding hIDS or derivatives, adding transgene products or derivatives to cultured MPS II cells, or co-culturing MPS II cells with human neural host cells / human glial host cells genetically engineered to express and secrete rhIDS or derivatives to determine the correction of defects in cultured MPS II cells, for example by detecting a decrease in IDS enzyme activity and / or GAG storage in cultured MPS II cells. (See, e.g., Stroncek et al., 1999, Transfusion 39(4):343-350 (which is incorporated herein by reference in its entirety)).

[0099] This specification describes animal models for MPS II that can be used to evaluate the therapeutic agents described herein. For example, a knockout mouse model of MPS II (IDS knockout) was genetically engineered by replacing exons 4 and 5 of the IDS gene with a neomycin resistance gene (Garcia et al., 2007, J Inherit Metab Dis30:924-34). This IDS knockout mouse exhibited many of the features of MPS II, including skeletal abnormalities, hepatosplenomegaly, elevated urinary and tissue GAGs, and cerebral storage lesions (Muenzer et al., 2001, Acta Paediatr Suppl91:98-99), and was used to evaluate the efficacy of enzyme replacement therapy in MPS II to support clinical trials of ERT. Therefore, this mouse model is a suitable model for testing the efficacy of gene therapy that delivers rIDS produced by neurons or glial cells as a treatment for MPS II (see, for example, Polito and Cosma, 2009, Am.J.Hum.Genet. 85(2):296-301 (which is incorporated herein by reference in its entirety)).

[0100] Preferably, the hIDS transgene produced by human neurons / human glial cells should be regulated by expression regulatory elements that function in neurons and / or glial cells, such as the CB7 promoter (chicken β-actin promoter and CMV enhancer), and may include other expression regulatory elements that enhance vector-driven transgene expression (e.g., chicken β-actin introns and rabbit β-globin polyA signaling). The cDNA construct for the hIDS transgene should include a coding sequence for a signal peptide to ensure proper in-translational and post-translational processing (glycosylation and protein sulfated nucleotides) by transduced CNS cells. Examples of such signal peptides for use in CNS cells include, but are not limited to, the following: - An oligodendrocyte-myelin glycoprotein (hOMG) signal peptide with the amino acid sequence MEYQILKMSLCLFILLFLTPGILC (SEQ ID NO: 2). - Cell repressor 2 (hCREG2) signal peptide of the E1A-stimulated gene having the amino acid sequence MSVRRGRRPARPGTRLSWLLCCSALLSPAAG (SEQ ID NO: 3) -MEQRNRLGALGYLPPLLLHALLLFVADA (SEQ ID NO: 4) is a V-set and transmembrane domain-containing 2B (hVSTM2B) signal peptide with an amino acid sequence. - Protocadherin alpha-1 (hPCADHA1) signal peptide having the amino acid sequence -MVFSRRGGLGARDLLLWLLLLAAWEVGSG (SEQ ID NO: 5) -MAMVSAMSWVLYLWISACA (SEQ ID NO: 6) is a signal peptide containing the amino acid sequence FAM19A1(TAFA1). - An interleukin-2 signal peptide having the amino acid sequence of MYRMQLLSCIALILALVTNS (SEQ ID NO: 14).

[0101] The signal peptide may also be referred to herein as the leader sequence or leader peptide.

[0102] Recombinant vectors used to deliver the transgene should have tropism to cells in the CNS, including, but are not limited to, nerve cells and / or glial cells. Such vectors may include non-replicating recombinant adeno-associated virus vectors ("rAAV"), with viral vectors having AAV9 or AAVrh10 capsids being particularly preferred. AAV variant capsids may be used, but are not limited to, AAV variant capsids having AAV / hu.31 and AAV / hu.32, particularly preferred, as described by Wilson in U.S. Patent No. 7,906,111 (which is incorporated herein by reference in its entirety), and AAV variant capsids described by Chatterjee in U.S. Patent No. 8,628,966, U.S. Patent No. 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 referred to as "naked DNA" constructs.

[0103] A pharmaceutical composition suitable for administration to CSF ​​comprises a suspension of an rhIDS vector in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant, and an optional excipient. In certain embodiments, the pharmaceutical composition is suitable for intra-arachnoid administration. In certain embodiments, the pharmaceutical composition is suitable for intracisterna magna administration (injection into the cisterna magna). In certain embodiments, the pharmaceutical composition is suitable for intra-arachnoid injection via C1-2 puncture. In certain embodiments, the pharmaceutical composition is suitable for intraventricular administration. In certain embodiments, the pharmaceutical composition is suitable for administration via lumbar puncture. In some embodiments, a pharmaceutical composition comprising rAAV of the present disclosure comprises sodium chloride at a concentration of about 8.77 g / L, magnesium chloride hexahydrate at a concentration of about 0.244 g / L, potassium chloride at a concentration of about 0.224 g / L, calcium chloride dihydrate at a concentration of about 0.206 g / L, dextrose anhydride at a concentration of about 0.793 g / L, poloxamer 188 at a concentration of about 0.010 g / L, monobasic phosphate monohydrate at a concentration of about 0.0278 g / L, and dibasic phosphate anhydride at a concentration of about 0.114 g / L.

[0104] The therapeutically effective dose of the recombinant vector should be administered to the CSF via intra-abracranial administration (i.e., by injection into the subarachnoid space, thereby allowing the recombinant vector to diffuse through the CSF and transduce cells of the CNS). In some embodiments, the recombinant vector is administered in a solution containing approximately 8.77 g / L of sodium chloride, approximately 0.244 g / L of magnesium chloride hexahydrate, approximately 0.224 g / L of potassium chloride, approximately 0.206 g / L of calcium chloride dihydrate, approximately 0.793 g / L of dextrose anhydride, approximately 0.010 g / L of poloxamer 188, approximately 0.0278 g / L of monobasic phosphate monohydrate, and approximately 0.114 g / L of dibasic phosphate anhydride. This can be achieved by several methods, such as intracranial (cisterna magnum or ventricle) injection, or lumbar cisternal injection. For example, intracisional (IC) injection (into the cisterna magna) can be performed by CT-guided suboccipital puncture, or, if feasible for the patient, intra-arachnoid injection can be performed via C1-2 puncture, or lumbar puncture (a diagnostic procedure commonly performed to collect CSF samples) can be used to access the CSF. Alternatively, recombinant vectors can be directly administered into the ventricles using intraventricular (ICV) administration (a more invasive technique used for introducing anti-infective or anticancer agents that do not cross the blood-brain barrier). Alternatively, recombinant vectors can be delivered to the CNS using intranasal administration.

[0105] Due to the relatively rapid brain growth that occurs early in childhood development, the total dose of AAV9.hIDS administered in IC varies depending on the estimated brain mass in different age groups. See, for example, Table 2 below. For brain mass in the age groups of the subjects in the study, see, for example, AS Dekaban, Ann Neurol, 1978 Oct;4(4):345-56. [Table 1]

[0106] The CSF concentration can be monitored by directly measuring the concentration of rhIDS in CSF fluid obtained from an occipital or lumbar puncture, or can be estimated by extrapolation from the concentration of rhIDS detected in the patient's serum.

[0107] In the background, human IDS is a 550-amino acid polypeptide containing 8 potential N-glycosylation sites as described in Figure 1 (N 31 , N 115 , N 144 , N 246 , N 280 , N 325 , N 513 , and N 537 ), and contains a 25-amino acid signal sequence that is cleaved during processing. The initial 76 kDa intracellular precursor is converted to a phosphorylated 90 kDa precursor after its oligosaccharide chains are modified in the Golgi apparatus. This precursor is processed through various intracellular intermediates to the predominant 55 kDa form via glycosylation modifications and proteolytic cleavage. In summary, after removal of the 25-amino acid signal sequence, proteolytic processing involves N-terminal proteolytic cleavage downstream of N31 to remove the 8-amino acid (residues 26 to 33) propeptide, and C-terminal proteolytic cleavage upstream of N513 to release the 18 kDa polypeptide and generate the 62 kDa intermediate, which is converted to the 55 kDa mature form. Further proteolytic cleavage generates the 45 kDa mature form located in the lysosomal compartment (see Figure 4 for the drawing excerpted from Millat et al., 1997, Exp Cell Res 230:362-367 ("Millat 1997"); see also Millat et al. 1997, Biochem J. 326:243-247 ("Millat 1997a"), and Froissart et al., 1995, Biochem J. 309:425-430. Each of these is incorporated herein by reference in its entirety).

[0108] C required for enzymatic activity 84The formylglycine modification (shown in bold in Figure 1) likely occurs after early translation or as a cotranslational event, almost certainly in the endoplasmic reticulum (see Millat 1997a, citing Schmidt et al., 1995, Cell 82:271-278). Posttranslational processing continues in the Golgi apparatus, incorporating the complex sialic acid-containing glycan and obtaining a mannose-6-phosphate residue that binds to an enzyme for delivery to the lysosomal compartment (see Clarke, 2008, Expert Opin Pharmacother 9:311-317, which is incorporated herein by reference in its entirety). While there is no single glycosylation site essential for IDS stability, glycosylation at position N280 is important for cellular internalization via the mannose-6-phosphate (M6P) receptor and for lysosomal targeting (Chung et al., 2014, Glycoconj J31:309-315 p.310, first paragraph). Under normal physiological conditions, IDS is produced at very low levels, and the enzyme is hardly secreted from cells, if present (Clarke, 2008 (above)).

[0109] This invention is based in part on the following principles. (i) Neuronal and glial cells of the CNS are secretory cells that have cellular mechanisms for post-translational processing of secretory proteins, including glycosylation, mannose-6-phosphorylation, and tyrosine-O-sulfation, which are robust processes in the CNS. For example, see Sleat et al., 2005, Proteomics 5:1520-1532, and Sleat 1996, J Biol Chem 271:19191-98, which describe the human brain mannose-6-phosphate glycoproteome and note that the brain contains more proteins and mannose-6-phosphorylated proteins with far more individual isoforms than are found in other tissues; and Kanan et al., 2009, Exp. Eye Res. 89:559-567, and Kanan & Al-Ubaidi, 2015, Exp. Eye Res. 133:126-131, which report the production of tyrosine sulfated glycoproteins secreted by nerve cells. Regarding post-translational modifications induced by human CNS cells, each of these is incorporated as a whole by reference. (ii) The human brain generates multiple isoforms of native IDS. In particular, N-terminal sequencing of human brain mannose-6-phosphorylated glycoprotein revealed that the N-terminal sequence of the mature 42kDa chain of hIDS changes in the brain from position 34 or 36 as follows: 34 DALNVLLI(sequence number 54); and A 36 LNVLLIIV (Sequence ID 55) (Sleat, 2005, Proteomics 5:1520-1532, Table S2). Two of the eight N-linked glycosylation sites, i.e., N 280 and N 116 It was found that mannose-6-phosphorylated iodine (IDS) obtained from human brain was found to be present (reported in Sleat et al., 2006, Mol & Cell Proeomics 5.4:686-701, Table V). (iii) During hIDS processing, neurons and glial cells secrete two polypeptides, 76 kDa and 90 kDa, but only the 90 kDa polypeptide undergoes mannose-6 phosphorylation, and this secreted form of enzyme is necessary for cross-collection (Millat, 1997, results in Figure 1 for transduced lymphoblastoid cells, and Froissart, 1995, similar results in Figure 4 for transduced fibroblasts - only the 90 kDa form is phosphorylated in culture medium). Interestingly, recombinant IDS produced by neurons and glial cells are demonstrated to be more readily endocytosizable by recipient CNS cells than recombinant IDS produced by other cells such as kidney cells. Daniele 2002 demonstrated M6P receptor-mediated endocytosis of recombinant IDS derived from conditioned media of transduced neuronal and glial cell cultures in recipient populations of untransduced neurons and glial cells appropriately treated with precursors for the 45kDa mature active form. The uptake of recombinant IDS produced by neuronal and glial cell lines (74% endocytosis) far exceeded the uptake of enzymes produced by kidney cell lines (5.6% endocytosis). In both cases, uptake was inhibited by M6P, indicating that the uptake of recombinant IDS is mediated by the M6P receptor (see Daniele 2002, Tables 2 and 4; an explanation related to the results on pp. 205-206 is summarized in Table 3 below). [Table 2] (iv) The gene therapy methods described herein will result in the continuous secretion of an enzymatically active hIDS glycoprotein precursor of approximately 90 kDa, as measured by polyacrylamide gel electrophoresis (depending on the assay used). First, the C required for IDS activity 84The enzyme involved in formylglycine modification, FGly-producing enzyme (FGE, also known as SUMF1), is expressed in the cerebral cortex of the human brain (SUMF1 gene expression data is accessible, for example, at GeneCards' http: / / www.genecards.org). Next, glycosylated / phosphorylated rIDS produced and secreted by in situ transduced neurons and glial cells will be taken up by untransduced neurons and glial cells in the CNS and processed appropriately. While not bound by any theory, rhIDS precursors produced and secreted in situ by gene therapy, when administered to the CNS, can be endocytotic more greedily by recipient cells in the CNS than conventional recombinant enzymes used in ERT. For example, Elaprase® (prepared using the fibrosarcoma cell line HT1080) is a purified protein with a reported molecular weight of approximately 76 kDa, unlike the highly phosphorylated 90 kDa type secreted by nerve cells and glial cells. Eight N-linked glycosylation sites are completely occupied by Elaprase®, and it has been reported to contain two bismannose-6-phosphate terminal glycans, as well as a highly sialylated glycan complex, although C, an absolute requirement for enzyme activity, is absent. 84 Post-translational modification from to FGly occurs in only about 50% of cases (Clarke, 2008, Expert Opin Pharmacother 9:311-317; Elaprase® Full Prescribing Information and EMA filing). Another recombinant product, Hunterase®, is produced in CHO cells. Although it has been reported to produce more FGly and have higher activity than Elaprase®, there were no differences in mannose-6-phosphorylation or uptake (Chung, 2014, Glycoconj J31:309-315). (v) The extracellular efficacy of IDS in vivo is due to mannose-6-phosphate (M6P) and its active site formylglycine (FGly), i.e., post-translational modification by formylglycine-producing enzymes. 84It depends on the uptake (internalization by cells and lysosomes) via FGly, which is converted from . As shown in Table 3 above, brain cells (neurons and glial cells) show higher enzyme activity when incubated with IDS precursor medium secreted by transduced neurons and glial cells than when incubated with IDS precursor medium secreted by genetically modified kidney cells. The resulting five-fold increase in activity is likely due in large part to the efficient uptake of IDS (see Daniele 2002, Tables 2 and 4). The FGly content of commercially available IDS produced by CHO cells or HT-1080 cells is approximately 50% to 70%, and this determines the enzyme activity. However, neurons and glial cells can increase this activity because they have improved IDS uptake. (vi) The cellularity and intracellular transport / uptake of lysosomal proteins such as IDS are mediated by M6P. As reported by Daniele 2002 and Sleat, Proteomics, 2005, brain cell-derived IDS may contain high levels of M6P (indicating that the human brain contains more Man6-P glycoprotein (in both quantitative and qualitative senses) than other tissues). Measuring the M6P content of IDS precursors is possible, as done 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 levels close to those of control cells, as shown in Daniele 2002. 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. This uptake is four times higher than that of control cells and is comparable to the level of IDS activity (or uptake) of IDS precursors produced by genetically engineered kidney cells without inhibitory M6P. This assay provides a method for predicting the M6P content of IDS precursors produced by brain cells, and in particular, allows for the comparison of M6P content in IDS precursors produced by different cell types. The gene therapy techniques described herein, as a result, continuously secrete hIDS precursors that can be taken up at high levels by neurons and glial cells in such assays, even in the presence of inhibitory M6P. (vii) The M6P content and uptake of IDS precursors can be demonstrated in 90 kDa and 76 kDa gel bands (e.g., SDS-PAGE gel bands). The 90 kDa gel band is reported to be highly glycosylated / phosphorylated and contain M6P, but the 76 kDa gel band is not. A very broad range of gel bands, ranging from 76 kDa to 95 kDa and having an average molecular weight of 80 to 85 kDa, can be compared to gel bands of IDS precursors generated from brain cells, similar to gel bands of IDS precursors generated from genetically engineered kidney cells (Daniele 2002, Figure 1). In Daniele 2002, immunoprecipitation of the IDS precursor failed, so gel bands could not be obtained. The gene therapy techniques described herein will result in the continuous secretion of hIDS precursors that differ from the IDS precursor gel bands generated from genetically engineered kidney cells. (viii) The M6P content of commercially available IDS precursors is 2-2.5 mol / mol, and the majority of it exists in the form of diphosphorylated glycans. On average, all IDS precursors are phosphorylated, but the normal distribution of glycans, assuming multiple phosphorylation sites, means that some IDS precursors have 2, 1, and 0 diphosphorylated M6P glycans. The uptake rate will increase significantly with multiple phosphorylations. (ix) Glycosylation of hIDS by human cells in the CNS adds a glycan that enhances stability and half-life and reduces undesirable aggregation of the transgene product. Importantly, the glycan added to the hIDS of the present invention contains 2,6-sialic acid and incorporates Neu5Ac ("NANA"), but does not incorporate its hydroxylated derivative, NeuGc (N-glycolylneuraminic acid, i.e., "NGNA" or "Neu5Gc"). Such a glycan is not present in recombinant IDS products such as Hunterase® produced in CHO cells. This is because CHO cells do not possess the 2,6-sialyltransferase required for this post-translational modification; and CHO cells do not produce bisected GlcNAc, instead adding Neu5Gc (NGNA) as a sialic acid that is uncommon in humans (and potentially immunogenic) instead of Neu5Ac (NANA). See, for example, Dumont et al., 2016, Critical Rev in Biotech 36(6):1110-1122 (Early Online pp.1-13, p.5), and Hague et al., 1998 Electrophor 19:2612-2630 ("CHO cell lines are thought to be 'phenotypically limited' in relation to glycosylation due to the lack of α2,6-sialyltransferase"). Furthermore, CHO cells can also produce α-Gal antigen, an immunogenic glycan that can react with anti-α-Gal antibodies present in most individuals to induce anaphylaxis at high concentrations. See, for example, Bosques, 2010, Nat Biotech 28:1153-1156. The human glycosylation pattern of rhIDS according to the present invention will reduce the immunogenicity of the transgene product and improve its efficacy. (x) The immunogenicity of the transgene product can be induced by various factors, including the patient's immune state, the structure and characteristics of the injected protein drug, the route of administration, and the duration of treatment. Process-related impurities, such as host cell proteins (HCPs), host cell DNA, and chemical residues, as well as product-related impurities, such as protein degradation products and structural features, such as glycosylation, oxidation, and aggregation (invisible particles), can also enhance immunogenicity by acting as adjuvants that enhance the immune response. The amount of process-related and product-related impurities can be affected by the manufacturing process, i.e., cell culture, purification, formulation, storage, and handling, and these can affect commercially produced IDS products. In gene therapy, proteins are produced in vivo, and as a result, process-related impurities are absent, and the protein product is unlikely to contain product-related impurities / degradation products associated with proteins produced by recombinant technology, such as protein aggregates and protein oxides. Aggregation is associated with protein production and storage, for example, and can result from high protein concentrations, surface interactions with production equipment and containers, and purification processes using certain buffering systems. However, these conditions that promote aggregation are absent when transgenes are expressed in vivo. Oxidation, such as the oxidation of methionine, tryptophan, and histidine, is also associated with protein production and storage and can occur, for example, due to stressed cell culture conditions, contact with metals and air, and impurities in buffers and excipients. While proteins expressed in vivo can oxidize even under stressed conditions, humans, like many organisms, possess an antioxidant defense system that not only reduces oxidative stress but also repairs and / or reverses oxidation. Therefore, it is unlikely that proteins produced in vivo are in an oxidized form. Both aggregation and oxidation can affect potency, pharmacokinetics (clearance), and increase immunogenicity concerns. The gene therapy techniques described herein will result in the continuous secretion of hIDS precursors with reduced immunogenicity compared to commercially produced products. (xi) In addition to the N-linked glycosylation site, hIDS also have a tyrosine ("Y") sulfated site (PSSEKY 165 (ENTKTCRGPD (SEQ ID NO: 47)) is included (e.g., Yang et al., 2015, Molecules 20:2138-2164, in particular p.2154, regarding the analysis of amino acids surrounding tyrosine residues for protein tyrosine sulfate, this whole is incorporated by reference. The "rules" can be summarized as follows: Y residues having E or D within +5 to -5 positions of Y, and Y position -1 being neutral or an acid-charged amino acid; however, not basic amino acids, e.g., R, K, or H which discard sulfate). Although not bound by any theory, sulfation of this site in hIDS may improve enzyme stability and binding affinity to substrates. Tyrosine sulfate of hIDS, a robust post-translational process in human CNS cells, would improve the processing and activity of transgene products. The importance of tyrosine sulfate in lysosomal proteins remains unclear, but in other proteins, it has been shown to enhance the affinity of protein-protein interactions (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 J14:3073-79). Tyrosyl protein sulfotransferase (TPST1), which is involved in tyrosine sulfate (which can occur as the final step of IDS processing), is expressed at even higher levels in the brain (based on mRNA) (TPST1 gene expression data can be found, for example, in the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home). Such post-translational modifications are, at best, underestimated in CHO cell products. Unlike human CNS cells, CHO cells are not secretory cells and have limited post-translational tyrosine sulfate capacity (see, for example, Mikkelsen & Ezban, 1991, Biochemistry 30:1533-1537, particularly the discussion on p. 1537).

[0110] For the reasons stated above, the production of rhIDS by human neurons and / or glial cells would provide a “bio-better” molecule for treating MPS II, achieving gene therapy by creating a permanent depot in the CNS, which continuously supplies fully human glycosylated, mannose-6-phosphorylated, sulfated transgene products secreted by transduced CNS cells. This depot is created by administering a viral vector or other DNA expression construct encoding rhIDS to the CSF of patients (human subjects) diagnosed with MPS II disease (including, but not limited to, Hunter's). The hIDS transgene products secreted from the depot into the CSF are endocytized by cells in the CNS, “cross-correcting” the enzymatic deficiency in MPS II recipient cells.

[0111] It is not essential that all rhIDS molecules produced in gene therapy or protein therapy methods be completely glycosylated, phosphorylated, and sulfated. Rather, the population of glycoproteins produced should undergo sufficient glycosylation (including 2,6-sialylation and mannose-6-phosphorylation) and sulfated to exhibit efficacy. The objective of the gene therapy treatment of the present invention is to delay or halt disease progression. Efficacy can be monitored by measuring cognitive function (e.g., prevention or suppression of decline in neurocognitive function); a decrease in disease biomarkers (e.g., GAG) in CSF and / or serum; and / or an increase in IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events can also be monitored.

[0112] As an alternative or add-on therapy to gene therapy, rhIDS glycoproteins can be produced by recombinant DNA technology in human neuronal or glial cells, and the glycoproteins can be administered systemically and / or to CSF ​​in patients diagnosed with MPS II for ERT. Human cell lines that can be used for such recombinant glycoprotein production include, but are not limited to, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM (see, 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,” for a review of human cell lines that can be used for recombinant production of rHuGlyIDS glycoproteins, which is incorporated herein by reference in its entirety). To ensure complete glycosylation, particularly sialylation and tyrosine sulfate formation, the cell line used for production can be enhanced by genetically engineering the host cells to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) and / or TPST-1 and TPST-2 enzymes, which are responsible for tyrosine-O-sulfation.

[0113] While rhIDS delivery would likely minimize the immune response, the most obvious potential source of toxicity with respect to CNS-related gene therapy is the development of immunity to the expressed rhIDS protein in human subjects who are genetically deficient in IDS and therefore may not be tolerant of the vector used to deliver the protein and / or the transgene.

[0114] Therefore, in preferred embodiments, co-treatment of patients with immunosuppressive therapy is appropriate, particularly when treating patients with severe disease where the level of IDS is close to zero. Immunosuppressive therapies involving tacrolimus or rapamycin (sirolimus) in combination with mycophenolic acid, or other immunosuppressive regimens used in tissue transplantation procedures, can be utilized. Such immunosuppressive therapy may be administered in the course of gene therapy, and in certain embodiments, pre-treatment with immunosuppressive therapy may be preferred. Immunosuppressive therapy may be continued after gene therapy procedures at the discretion of the attending physician and may then be discontinued, for example, after 180 days, if immune tolerance has been induced.

[0115] The method of the present invention includes a combination of delivery of rhIDS to CSF, accompanied by delivery of other available therapies. Further therapies can be administered before, concurrently with, or after the gene therapy procedure. Available therapies for MPS II that can be used in combination with the gene therapy of the present invention include, but are not limited to, enzyme replacement therapy with systemic or to CSF ​​administration of Elaprase®, and / or HSCT therapy.

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

[0117] In certain embodiments, the method described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering a therapeutically effective amount of recombinant human iduronate-2-sulfatase (IDS) precursor to the cerebrospinal fluid (CSF) of the aforementioned human subject, wherein the precursor is approximately 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, and C 84 It contains a formylglycine residue (Figure 1), undergoes α2,6-sialylation, does not contain detectable NeuGc, and undergoes mannose-6-phosphorylation.

[0118] In certain embodiments, the method described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising delivering a therapeutically effective amount of recombinant human iduronate-2-sulfatase (IDS) glycoprotein precursor to the cerebrospinal fluid (CSF) of the aforementioned human subject, wherein the precursor is approximately 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, and C 84 It contains a formylglycine residue (Figure 1), is α2,6-sialylated, does not contain detectable NeuGc and / or α-Gal, and is mannose-6-phosphorylated.

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

[0120] In certain embodiments, the method described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering a recombinant nucleotide expression vector encoding human iduronate-2-sulfatase (IDS) to the cerebrospinal fluid (CSF) of the aforementioned human subject, wherein the expression vector, when used to transduce primary human neurons in culture, is approximately 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, has a formylglycine residue at C84 (Figure 1), is α2,6-sialylated, and is mannose-6-phosphorylated.

[0121] In certain embodiments, the method described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS into the cerebrospinal fluid of the brain of the aforementioned human subject, thereby forming a depot in the subject's central nervous system that secretes a recombinant human IDS glycoprotein precursor that is α2,6-sialylated and mannose-6-phosphorylated.

[0122] In certain embodiments, the secretion of the aforementioned recombinant human IDS glycoprotein precursor α2,6-sialylated is confirmed in cell culture by transducing the aforementioned recombinant nucleotide expression vector into a human nerve cell line. In certain embodiments, the secretion of the aforementioned recombinant human IDS glycoprotein precursor mannose-6-phosphate is confirmed in cell culture by transducing the aforementioned recombinant nucleotide expression vector into a human nerve cell line. In certain embodiments, secretion is confirmed in the presence and absence of mannose-6-phosphate.

[0123] In certain embodiments, the method described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS into the cerebrospinal fluid of the brain of the aforementioned human subject, thereby forming a depot that secretes a glycosylated IDS precursor containing α2,6-sialylated glycan, and when the recombinant vector is used in culture for transduction of human nerve cells, the aforementioned cell culture results in the secretion of the aforementioned glycosylated IDS precursor containing α2,6-sialylated glycan.

[0124] In certain embodiments, the method described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding human IDS into the cerebrospinal fluid of the brain of the aforementioned human subject, thereby forming a depot that secretes a glycosylated IDS precursor containing mannose-6-phosphate, the recombinant vector being used in cell culture for transduction of human neurons, resulting in the secretion of the aforementioned glycosylated IDS precursor containing mannose-6-phosphate in the aforementioned cell culture.

[0125] In certain embodiments, the method described herein is a method for treating a human subject diagnosed with mucopolysaccharidosis type II (MPS II), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding a human IDS into the cerebrospinal fluid of the brain of the aforementioned human subject, thereby forming a depot that secretes a glycosylated IDS precursor containing formylglycine, the recombinant vector, when used in cell culture for transduction of human nerve cells, results in the secretion of the aforementioned glycosylated IDS precursor containing formylglycine in the aforementioned cell culture.

[0126] In certain embodiments, the human IDS contains the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the IDS transgene encodes a leader peptide. In certain embodiments, the expression vector is a replication-deficient AAV vector. In certain embodiments, the expression vector is delivered to the target CSF by subarachnoid space (e.g., intracisional, C1-2 puncture if feasible for the patient, or lumbar puncture), intraventricular, or intranasal administration. In certain embodiments, the human subject is deficient in IDS activity. In some embodiments, the expression vector is delivered to the target CSF by intraarachnoid administration of a solution containing approximately 8.77 g / L of sodium chloride, approximately 0.244 g / L of magnesium chloride hexahydrate, approximately 0.224 g / L of potassium chloride, approximately 0.206 g / L of calcium chloride dihydrate, approximately 0.793 g / L of dextrose anhydride, approximately 0.010 g / L of poloxamer 188, approximately 0.0278 g / L of monobasic phosphate monohydrate, and approximately 0.114 g / L of dibasic phosphate anhydride.

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

[0128] In one aspect, the present specification provides a method of treating a human subject diagnosed with MPS II, comprising delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal cells or glial cells to the CSF of the human subject, wherein the glycosylated recombinant human IDS precursor is induced by administering a recombinant nucleotide expression vector encoding human IDS, the recombinant nucleotide expression vector is administered at a dose corresponding to the brain mass of the human subject, and the brain mass is determined by brain MRI of the human subject.

[0129] In another aspect, the present specification provides a method of treating a human subject diagnosed with MPS II, comprising determining the brain mass of the human subject by brain MRI of the human subject, and subsequently delivering a therapeutically effective amount of a glycosylated recombinant human IDS precursor produced by human neuronal cells 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, and the recombinant nucleotide expression vector is administered at a dose corresponding to the brain mass of the human subject.

[0130] In another aspect, the present specification provides a method of treating a human subject diagnosed with MPS II, comprising (a) determining the brain mass of the human subject from 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 the recombinant nucleotide expression vector encoding human IDS to the CSF of the subject.

[0131] In another embodiment, this specification provides a method for treating a human subject diagnosed with MPS II, comprising, in this 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 to the level of heparan sulfate in a reference population, wherein the glycosylated recombinant human IDS precursor is induced by administration of a recombinant nucleotide expression vector encoding human IDS, the recombinant nucleotide expression vector is administered in a dose corresponding to the brain mass of the human subject, and the brain mass of the human subject is determined by magnetic resonance imaging (MRI) of the brain. In certain embodiments, the reference population consists of (a) at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, or 1000 healthy individuals who are not MPS II, preferably healthy individuals of the same age, weight, and / or sex as the human subjects.

[0132] In another embodiment, this specification provides a method for treating a human subject diagnosed with MPS II, comprising, in this order: (a) performing a first measurement of heparan sulfate levels in the human subject's CSF; (b) delivering a therapeutically effective dose of a glycosylated recombinant human IDS precursor produced by human neurons or human glial cells to the human subject's CSF; and (c) performing a second measurement of heparan sulfate levels after a certain period of time, wherein the glycosylated recombinant human IDS precursor is induced by administration of a recombinant nucleotide expression vector encoding human IDS, the recombinant nucleotide expression vector is administered in a dose corresponding to the brain mass of the human subject, and the brain mass of the human subject is determined by magnetic resonance imaging (MRI) of the brain. In certain embodiments, the 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, 6 months, 7 months, 8 months, 11 months, or 1 year.

[0133] In a preferred embodiment, the glycosylated recombinant human IDS precursor is taken up by cells in the human target CNS. In a preferred embodiment, the glycosylated recombinant human IDS precursor is delivered to the lysosomes of cells in the human target CNS.

[0134] In certain embodiments of the therapeutic methods described herein, 1.046 g / cm³ is added to the brain volume of a human subject. 3 Multiply by the coefficient to obtain the human brain volume in cm². 3 The brain mass is converted to that of a human subject, and the brain volume of a human subject is obtained from a human brain MRI.

[0135] In certain embodiments of the therapeutic methods described herein, recombinant nucleotide expression vectors are administered at a concentration of approximately 1.3 × 10⁶ per gram of brain mass determined by MRI. 10 Approximately 6.5 × 10⁶ units of brain mass per gram, as determined by GC or MRI. 10 Administer in a GC dose. In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered at approximately 1.3 × 10⁶ doses. 10 The drug is administered in a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by polyA-specific PCR assay). In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered in a dose of approximately 1.9 × 10⁶ 10 The drug is administered at a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by a transgene-specific PCR assay). In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered at a dose of approximately 6.5 × 10⁶ 10 The drug is administered in a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by polyA-specific PCR assay). In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered in a dose of approximately 9.6 × 10⁶ 10 The drug is administered at a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by a transgene-specific PCR assay). In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered at a dose of approximately 2.0 × 10⁶ 11The drug is administered in a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by polyA-specific PCR assay). In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered in a dose of approximately 2.9 × 10⁶ 11 The drug is administered at a dose equal to GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by a transgene-specific PCR assay).

[0136] In various embodiments of the treatment methods described herein, the human subjects are 5 years of age or older and under 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 embodiments, the human subjects are approximately 5-6 years old, 6-7 years old, 7-8 years old, 8-9 years old, 9-10 years old, 10-11 years old, 11-12 years old, 12-13 years old, 13-14 years old, 14-15 years old, 15-16 years old, 16-17 years old, 17-18 years old, or 18-19 years old. In certain embodiments, the recombinant nucleotide expression vector is approximately 6.5 × 10⁶ per gram of brain mass determined by MRI. 10 Administer at the GC dose. In certain embodiments, the recombinant nucleotide expression vector is administered at the doses listed in Table 7.

[0137] In various embodiments of the treatment methods described herein, the human subjects are 4 months of age or older and under 5 years of age. In specific embodiments, the human subjects are 4, 5, 6, 7, 8, 9, 10, or 11 months of age. In specific embodiments, the human subjects are approximately 4, 5, 6, 7, 8, 9, 10, or 11 months of age. 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 of age. In specific embodiments, the human subjects are approximately 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months of age. In specific embodiments, the human subjects are 1 year, 2 years, 3 years, 4 years, or 5 years of age. In specific embodiments, the human subjects are approximately 1 year, 2 years, 3 years, 4 years, or 5 years of age. In specific embodiments, human subjects are 1-2 years old, 2-3 years old, 3-4 years old, 4-5 years old, or 5-6 years old. In specific embodiments, human subjects are approximately 1-2 years old, 2-3 years old, 3-4 years old, 4-5 years old, or 5-6 years old. In specific embodiments, the recombinant nucleotide expression vector is approximately 1.3 × 10⁶ per gram of brain mass determined by MRI. 10 Administer at the GC dose. In certain embodiments, the recombinant nucleotide expression vector is expressed at approximately 6.5 × 10⁶ per gram of brain mass determined by MRI. 10 Administer at the GC dose. In certain embodiments, the recombinant nucleotide expression vector is approximately 2.0 × 10⁶ per gram of brain mass determined by MRI. 11 Administer at the GC dose. In certain embodiments, the recombinant nucleotide expression vector is approximately 2.9 × 10⁶ per gram of brain mass determined by MRI. 11 Administer at the GC dose. In certain embodiments, the recombinant nucleotide expression vector is approximately 1.9 × 10⁶ per gram of brain mass determined by MRI. 10 Administer at the GC dose. In certain embodiments, the recombinant nucleotide expression vector is approximately 9.6 × 10⁶ per gram of brain mass determined by MRI. 10 Administer at a GC dose. In certain embodiments, the recombinant nucleotide expression vector is administered at approximately 2.0 × 10⁶ doses. 11The drug is administered at a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by polyA-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of approximately 2.9 × 10⁶ 11 The drug is administered at a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at approximately 1.3 × 10⁶ doses. 10 The patient is administered at a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by polyA-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of approximately 1.9 × 10⁶. 10 The drug is administered at a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of approximately 6.5 × 10⁶ 10 The patient is administered at a dose of GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by polyA-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered at a dose of approximately 9.6 × 10⁶. 10 The recombinant nucleotide expression vector is administered in doses equal to GC / g brain mass (for example, brain mass is determined by MRI, and genome number is determined by a transgene-specific PCR assay). In certain embodiments, the recombinant nucleotide expression vector is administered in doses selected from dose 1 or dose 2 according to Table 5. In certain embodiments, the recombinant nucleotide expression vector is administered in doses listed in Table 6.

[0138] In some embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is administered via intracisional (IC) administration. In other embodiments of the methods described herein, the recombinant nucleotide expression vector is administered via intraventricular (ICV) administration.

[0139] In certain embodiments of the methods described herein, the recombinant nucleotide expression vector is administered in a volume not exceeding 10% of the total volume of cerebrospinal fluid of the human subject.

[0140] In certain embodiments of the therapeutic methods described herein, glycosylated recombinant human IDS precursors are secreted at detectable levels.

[0141] In certain embodiments of the therapeutic methods described herein, human nerve cells or human glial cells have at least one mutation in an endogenous gene encoding a human IDS precursor.

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

[0143] In a preferred embodiment, the recombinant nucleotide expression vector is an AAV9 or AAVrh10 vector.

[0144] In certain embodiments of the therapeutic methods described herein, the glycosylated recombinant human IDS precursor is expressed under the control of the CB7 promoter.

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

[0146] In certain embodiments of the therapeutic methods described herein, the glycosylated recombinant human IDS precursor is approximately 90 kDa as measured by polyacrylamide gel electrophoresis.

[0147] In certain embodiments of the therapeutic methods described herein, the glycosylated recombinant human IDS precursor contains formylglycine.

[0148] In certain embodiments of the therapeutic methods described herein, the glycosylated recombinant human IDS precursor is (a) subjected to α2,6-sialylation, (b) free of detectable NeuGc, (c) free of detectable α-Gal antigen, (d) comprises tyrosine sulfation, and / or (e) subjected to mannose-6-phosphorylation.

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

[0150] In certain embodiments provided herein, the method further comprises administering immunosuppressive therapy to the human subject prior to or concurrently with the human IDS precursor treatment, and optionally continuing the immunosuppressive therapy thereafter.

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

[0152] In a specific embodiment, immunosuppressive therapy involves prednisone at 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, This includes administering doses of 0.32 mg / kg, 0.33 mg / kg, 0.34 mg / kg, 0.35 mg / kg, 0.36 mg / kg, 0.37 mg / kg, 0.38 mg / kg, 0.39 mg / kg, 0.40 mg / kg, 0.41 mg / kg, 0.42 mg / kg, 0.43 mg / kg, 0.44 mg / kg, 0.45 mg / kg, 0.46 mg / kg, 0.47 mg / kg, 0.48 mg / kg, 0.49 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1 mg / kg. In specific embodiments, immunosuppressive therapy includes administering prednisone in doses ranging from approximately 0.10 mg / kg to approximately 0.20 mg / kg. In specific embodiments, immunosuppressive therapy includes administering prednisone in doses ranging from about 0.20 mg / kg to about 0.30 mg / kg. In specific embodiments, immunosuppressive therapy includes administering prednisone in doses ranging from about 0.30 mg / kg to about 0.40 mg / kg. In specific embodiments, immunosuppressive therapy includes administering prednisone in doses ranging from about 0.40 mg / kg to about 0.50 mg / kg. In specific embodiments, immunosuppressive therapy includes administering prednisone in doses ranging from about 0.50 mg / kg to about 1 mg / kg. In certain embodiments, these doses are administered daily. In certain embodiments, immunosuppressive therapy includes administering prednisone in a dose of 0.5 mg / kg / day. In another specific embodiment, immunosuppressive therapy includes administering prednisone in a dose of 0.5 mg / kg / day, tapering and discontinuing.

[0153] In specific embodiments, immunosuppressive therapy includes administering methylprednisolone in 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 administering methylprednisolone in doses ranging from about 0.50 mg / kg to about 1.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administering methylprednisolone in doses ranging from about 1.0 mg / kg to about 2.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administering methylprednisolone in doses ranging from about 2.0 mg / kg to about 3.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administering methylprednisolone in doses ranging from about 3.0 mg / kg to about 5.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administering methylprednisolone in doses ranging from about 5.0 mg / kg to about 10.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administering methylprednisolone in doses ranging from about 10.0 mg / kg to about 15.0 mg / kg. In specific embodiments, immunosuppressive therapy includes administering methylprednisolone in doses ranging from approximately 15.0 mg / kg to approximately 20.0 mg / kg. In certain embodiments, methylprednisolone is administered as a single dose. In certain embodiments, methylprednisolone is administered intravenously. In certain embodiments, methylprednisolone is administered up to a maximum of 500 mg. In certain embodiments, methylprednisolone is administered over a period of at least 30 minutes.In certain embodiments, immunosuppressive therapy includes administering methylprednisolone at a dose of 10 mg / kg IV over a period of at least 30 minutes, up to a maximum of 500 mg.

[0154] In specific embodiments, immunosuppressive therapy includes administering sirolimus at a dose that maintains a target blood level of 1–3 ng / mL. In specific embodiments, immunosuppressive therapy includes administering sirolimus at a dose of approximately 0.25 mg / m². 2 / day, 0.3mg / m 2 / day, 0.4mg / m 2 / day, 0.5mg / m 2 / day, 0.6mg / m 2 / day, 0.7mg / m 2 / day, 0.8mg / m 2 0.9 mg / m² per day 2 / day, 1mg / m 2 / day, 1.25mg / m 2 / day, 1.5mg / m 2 / day, 1.75mg / m 2 / day, 2mg / m 2 / day, 2.25mg / m 2 / day, 2.5mg / m 2 / day, 2.75mg / m 2 / day, 3mg / m 2 / day, 3.25mg / m 2 / day, 3.5mg / m 2 / day, 3.75mg / m 2 / day, 4mg / m 2 / day, 4.25mg / m 2 / day, 4.5mg / m 2 / day, 4.75mg / m 2 / day, or 5mg / m² 2 This includes administering a dose of sirolimus at a dose of approximately 0.25 mg / m². In specific embodiments, immunosuppressive therapy involves administering sirolimus at a dose of approximately 0.25 mg / m². 2 / day~about 0.5mg / m 2 This includes administering doses in the range of / day. In specific embodiments, immunosuppressive therapy involves administering sirolimus at approximately 0.50 mg / m². 2 / day ~ approximately 1.0 mg / m² 2This includes administering doses in the range of / day. In specific embodiments, immunosuppressive therapy involves sirolimus at approximately 1.0 mg / m². 2 / day~about 1.5mg / m 2 This includes administering doses in the range of / day. In specific embodiments, immunosuppressive therapy involves administering sirolimus at approximately 1.5 mg / m². 2 / day ~ approx. 2mg / m 2 This includes administering doses in the range of / day. In specific embodiments, immunosuppressive therapy involves administering sirolimus at approximately 2 mg / m². 2 / day~about 5mg / m 2 This includes administering doses in the range of / day. In certain embodiments, the dose is divided for BID administration. In certain embodiments, immunosuppressive therapy involves sirolimus at approximately 1 mg / m². 2 The administration involves a daily dose, administered every four hours. In certain embodiments, the immunosuppressive therapy involves sirolimus divided into approximately 0.5 mg / m² doses for BID administration. 2 This includes administering the dose per day.

[0155] In specific embodiments, immunosuppressive therapy includes administering tacrolimus in doses that maintain a target blood level of 2–4 ng / mL. In particular embodiments, immunosuppressive therapy includes administering tacrolimus in doses of approximately 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 particular embodiments, immunosuppressive therapy includes administering tacrolimus in doses ranging from 0.01 mg / kg to 0.02 mg / kg. In particular embodiments, immunosuppressive therapy includes administering tacrolimus in doses ranging from 0.02 mg / kg to 0.03 mg / kg. In certain embodiments, immunosuppressive therapy includes administering tacrolimus in doses ranging from 0.03 mg / kg to 0.05 mg / kg. In certain embodiments, immunosuppressive therapy includes administering tacrolimus in doses ranging from 0.05 mg / kg to 0.07 mg / kg. In certain embodiments, immunosuppressive therapy includes administering tacrolimus in doses ranging from 0.07 mg / kg to 0.10 mg / kg. In certain embodiments, this dose is administered twice daily. In certain embodiments, immunosuppressive therapy includes administering tacrolimus at a dose of approximately 0.05 mg / kg twice daily.

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

[0157] In some embodiments, the method further comprises administering one or more antifungal therapies to a human subject before or concurrently with immunosuppressive therapy. In certain embodiments, one or more antifungal therapies are administered when the absolute neutrophil count reaches 500 mm. 3 Start if it is less than [a certain value].

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

[0159] In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is a liquid composition. In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is a frozen composition. In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vector is a lyophilized composition, or a reconstituted lyophilized composition. In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vectors provided herein may be formulated in various dosage forms for IC or ICV administration. In certain embodiments of the therapeutic methods described herein, the recombinant nucleotide expression vectors provided herein may be provided in a unit dose formulation or a multi-dose formulation. As used herein, a unit dose formulation refers to physically discrete units suitable for administration to human and animal subjects, which are individually packaged using packaging methods known in the art. Each unit dose formulation contains a predetermined amount of the aforementioned recombinant nucleotide expression vector, and / or other component(s), sufficient to achieve the desired therapeutic effect in association with the required pharmaceutical carrier or excipient. Examples of unit dose formulations include ampoules, vials, pre-filled syringes, or cartridges.

[0160] In certain embodiments of the therapeutic methods described herein, a unit dose may be administered in a dose that is a fraction or a multiple thereof. In certain embodiments of the therapeutic methods described herein, a multi-unit dose refers to a plurality of identical unit doses contained in a single container, which are administered separately as divided unit doses. Examples of multi-unit doses include vials, pre-filled syringes, or cartridges. In certain embodiments, the pre-filled syringe contains 8.5×10 12 GC of the recombinant nucleotide expression vector. In certain embodiments, the pre-filled syringe contains 9.8×10 12 GC of the recombinant nucleotide expression vector. In certain embodiments, the pre-filled syringe contains 1.1×10 13 GC of the recombinant nucleotide expression vector. In certain embodiments, the pre-filled syringe contains 1.3×10 13comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.5×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.7×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 4.2×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 4.9×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 5.5×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 6.3×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 7.3×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 8.5×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 9.0×10 13 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.0×10 14 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.1×10 14 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.2×10 14 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.3×10 14 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.4×10 14 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.5×10 14 comprises the recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.6×1014 It contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.7 × 10⁻⁶ 14 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.8 × 10⁶ 14 It contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.9 × 10⁶ 14 It contains a recombinant nucleotide expression vector. In certain embodiments, the pre-filled syringe contains 2.0 × 10⁶ 14 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 2.1 × 10⁻⁶ 14 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 2.2 × 10⁻⁶ 14 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 2.3 × 10⁻⁶ 14 It contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 2.4 × 10⁻⁶ 14 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 2.5 × 10⁻⁶ 14 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 2.6 × 10⁶ 14 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.3 × 10⁻⁶ 10 It contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 1.9 × 10⁶ 10 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 6.5 × 10⁻⁶ 10 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 9.6 × 10⁶ 10 Contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 2.0 × 10⁶ 11It contains a recombinant nucleotide expression vector for GC. In certain embodiments, the pre-filled syringe contains 2.9 × 10⁻⁶ 11 Contains a recombinant nucleotide expression vector for GC.

[0161] As used herein, the term "approximately" means within ±10% of a given value or range. In certain embodiments, the term "approximately" means within ±1% of a given value or range, and this value is a dose corresponding to the brain mass of a human subject, and the brain mass is determined by brain MRI of the human subject's brain. In certain embodiments, the term "approximately" means within ±2% of a given value or range, and this value is a dose determined by brain MRI of the subject's brain, and the brain mass is determined by brain MRI of the human subject's brain. In certain embodiments, the term "approximately" means within ±5% of a given value or range, and this value is a dose corresponding to the brain mass of a human subject, and the brain mass is determined by brain MRI of the human subject's brain. In certain embodiments, the term "approximately" means within ±7% of a given value or range, and this value is a dose corresponding to the brain mass of a human subject, and the brain mass is determined by brain MRI of the human subject's brain. In certain embodiments, the term "about" means within ±10% of a given value or range, and this number represents a dose corresponding to the brain mass of a human subject, which is determined by brain MRI of the human subject's brain. However, it should also be understood that in this specification, the term "about" can also indicate the exact numerical details associated with that term. For example, "about 10" precisely provides details about the number "10".

[0162] 5.1 Processing, N-glycosylation, and tyrosine sulfated 5.1.1. Processing Human IDS contains a 25-amino acid signal sequence that is cleaved during processing. The initial 76kDa intracellular IDS precursor is converted to a phosphorylated 90kDa IDS precursor after its oligosaccharide chain is modified within the Golgi apparatus. This precursor is processed through various intracellular intermediates via glycosylation modification and proteolytic cleavage to reach the major 55kDa form. In summary, after the removal of the 25-amino acid signal sequence, proteolytic processing is carried out. 31 Downstream, N-terminal proteolytic cleavage is performed to remove a propeptide consisting of 8 amino acids (residues 26-33), and N 513 Upstream, C-terminal proteolytic cleavage occurs, releasing an 18kDa polypeptide to produce a 62kDa intermediate, which is converted to a 55kDa mature form. Further proteolytic cleavage produces a 45kDa mature form located in the lysosomal compartment (see Figure 4 for a diagram extracted from Millat et al., 1997, Exp Cell Res230:362-367 ("Millat1997"); see Millat et al. 1997, Biochem J.326:243-247 ("Millat1997a"), and Froissart et al., 1995, Biochem J.309:425-430 (each of these is incorporated herein by reference in its entirety)).

[0163] C required for enzyme activity 84 The formylglycine modification (shown in bold in Figure 1) likely occurs after early translation or as a cotranslational event, almost certainly in the endoplasmic reticulum (see Millat 1997a, citing Schmidt et al., 1995, Cell 82:271-278). Posttranslational processing continues in the Golgi apparatus, incorporating the complex sialic acid-containing glycan and obtaining a mannose-6-phosphate residue that binds to an enzyme for delivery to the lysosomal compartment (see Clarke, 2008, Expert Opin Pharmacother 9:311-317, which is incorporated herein by reference in its entirety).

[0164] In specific embodiments, HuGlyIDS used according to the methods described herein may be in the mannose-6-phosphorylated form of the enzyme at 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) when expressed in vivo or in vitro in neurons or glial cells. IDS produced from neurons and glial cells may contain higher levels of M6P, as reported by Daniele 2002 and Sleat, Proteomics, 2005 (this indicates that the human brain contains more M6P glycoprotein (both quantitatively and qualitatively) than other tissues). Measuring the M6P content of IDS precursors is possible, as done in Daniele 2002.

[0165] Therefore, in certain embodiments, HuGlyIDS used according to the methods described herein undergoes higher levels of mannose-6-phosphorylation in vivo or in vitro than IDS expressed in non-neuronal or glial cells when expressed in neuronal or glial cells. In particular, HuGlyIDS used according to the methods described herein undergoes higher levels of mannose-6-phosphorylation in vivo or in vitro than IDS expressed in HT1080 or CHO cells when expressed in neuronal or glial cells. In certain embodiments, the mannose-6-phosphorylation level of the expressed IDS is measured by the uptake of the IDS by human neurons in the presence of M6P (e.g., 5 mM M6P). In certain embodiments, when expressed in vivo or in vitro in nerve cells 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 undergo mannose-6-phosphorylation.

[0166] 5.1.2. N-glycosylation Neurons and glial cells in the CNS are secretory cells that possess cellular mechanisms for post-translational processing of secretory proteins, such as glycosylation and tyrosine-O-sulfation. hIDS have eight asparagine ("N") glycosylation sites as shown in Figure 1 (N 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 It undergoes mannose-6-phosphorylation in IDS obtained from human brain (reported in Sleat et al., 2006, Mol & Cell Proeomics 5.4:686-701, Table V).

[0167] While there is no single glycosylation site essential for IDS stability, glycosylation at position N280 is important for cellular internalization via the mannose-6-phosphate (M6P) receptor and for lysosomal targeting (Chung et al., 2014, Glycoconj J31:309-315p.310, paragraph 1). Under normal physiological conditions, IDS is produced at very low levels, and the enzyme is hardly secreted from cells, if present (Clarke, 2008 (above)).

[0168] It is not essential that all molecules produced in gene therapy or protein therapy methods are completely glycosylated and sulfated. Rather, the resulting population of glycoproteins will likely exhibit sufficient glycosylation and sulfatedness to demonstrate efficacy.

[0169] In specific embodiments, HuGlyIDS used according to the methods described herein can be glycosylated at 100% of their N-glycosylation sites when expressed in vivo or in vitro in nerve cells or glial cells. However, those skilled in the art will recognize that it is not necessarily required that all N-glycosylation sites of HuGlyIDS be N-glycosylated in order to achieve the benefits of glycosylation. Rather, the benefits of glycosylation can be realized when only a certain percentage of the N-glycosylation sites are glycosylated, and / or when only a certain percentage of the expressed IDS molecules are glycosylated. Therefore, in certain embodiments, HuGlyIDS used according to the methods described herein, when expressed in vivo or in vitro in nerve cells or glial cells, glycosylates 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% of its available N-glycosylation sites. In certain embodiments, when expressed in vivo or in vitro in nerve cells or glial cells, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% of the HuGlyIDS molecules used according to the methods described herein are glycosylated at at least one of their available N-glycosylation sites.

[0170] In specific embodiments, when HuGlyIDS is expressed in vivo or in vitro in nerve cells 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 HuGlyIDS used according to the methods described herein are glycosylated by 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.

[0171] In another specific embodiment, when HuGlyIDS is expressed in vivo or in vitro in nerve cells 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 used according to the method described herein are glycosylated by the same bound glycan that binds to the Asn residue (or other relevant residue) present in the N-glycosylation site. 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 bound glycan.

[0172] Importantly, when the IDS protein used according to the methods described herein is expressed in nerve cells or glial cells, the need for in vitro production in prokaryotic host cells (e.g., E. coli) or eukaryotic host cells (e.g., CHO cells) is avoided. Instead, as a result of the therapeutic methods described herein (e.g., the use of nerve cells or glial cells to express IDS), the N-glycosylation site of the IDS protein is advantageously modified with glycans suitable and beneficial for human therapy, particularly at the therapeutic target site. Such advantages are not obtained 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) can add Neu5Gc instead of Neu5Ac as sialic acid, and E. coli does not naturally contain the components necessary for N-glycosylation. Furthermore, these advantages cannot be achieved by using human cells other than nerve cells or glial cells for protein production. Therefore, in one embodiment, the IDS protein expressed in nerve cells or glial cells to produce HuGlyIDS for use in the therapeutic methods described herein undergoes glycosylation in the manner of N-glycosylation in human nerve cells or human glial cells, but does not undergo glycosylation in the manner of glycosylation in CHO cells. In another embodiment, the IDS protein expressed in nerve cells or glial cells to produce HuGlyIDS for use in the therapeutic methods described herein undergoes glycosylation in the manner of N-glycosylation in nerve cells or glial cells, but such glycosylation cannot be achieved naturally when using prokaryotic host cells, such as E. coli.In one embodiment, the IDS protein, which is expressed in human nerve cells or human glial cells to produce HuGlyIDS for use in the therapeutic method described herein, undergoes glycosylation in a manner in which the protein is N-glycosylated in human nerve cells or human glial cells, but does not undergo glycosylation in a manner in which the protein is glycosylated in human cells other than nerve cells or glial cells.

[0173] Assays for determining the glycosylation patterns of proteins are known in the art. For example, hydrazine degradation can be used to analyze glycans. First, a polysaccharide is incubated with hydrazine to release it from the associated protein (the Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK can be used). The nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein, releasing the bound glycan. The N-acetyl group is lost during this process and must be reconstituted by re-N-acetylation. The free glycan can be purified on a carbon column and subsequently labeled at the reducing end using the fluorescent dye molecule 2-aminobenzamide. The labeled polysaccharide can be separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al, Anal Biochem 2002, 304(1):70-90. The resulting fluorescence chromatogram shows the length of the polysaccharide and the number of repeating units. Structural information can be collected by gathering individual peaks, followed by MS / MS analysis. This allows for the confirmation of the composition and sequence of monosaccharide repeating units, and further, the identification of the homogeneity of polysaccharide composition. Specific peaks of low molecular weight molecules can be analyzed by MALDI-MS / MS, and the results can be used to confirm the glycan sequence. Each peak corresponds to a polymer consisting of a specific number of repeating units and their fragments. Thus, chromatograms can be used to measure the distribution of polymer length. Elution time is an indicator of polymer length, while fluorescence intensity correlates with the molar abundance for each polymer.

[0174] The uniformity of the glycan pattern associated with a protein is related to the glycan length and the number of glycans present across the glycosylation sites. Therefore, this uniformity can be evaluated 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 leads to greater variability in hydrodynamic radius compared to carriers with fewer glycosylation sites. However, when analyzing a single glycan chain, they can be more uniform because their length is more precisely controlled. Glycan length can be measured by hydrazine lysis, SDS-PAGE, and capillary gel electrophoresis. In addition, uniformity may also mean that the utilization pattern of a particular glycosylation site varies over a broader / narrower range. These factors can be measured by glycopeptide LC-MS / MS.

[0175] N-glycosylation provides numerous benefits to HuGlyIDS used in the methods described herein. Such benefits are not obtained in protein production in E. coli, because E. coli does not naturally possess the components necessary for N-glycosylation. Furthermore, some benefits are not obtained in protein production in CHO cells, for example, because CHO cells lack the components necessary for the addition of certain glycans (e.g., 2,6-sialic acid) and can add glycans uncommon to humans, such as Neu5Gc, and the α-Gal antigen, which is immunogenic in most individuals and can induce anaphylaxis at high concentrations. Moreover, some benefits are not obtained in protein production in human cells other than neurons or glial cells. Therefore, while IDS expression in human neurons or human glial cells produces HuGlyIDS containing beneficial glycans, this is not related to proteins produced in CHO cells, or E. coli, or human cells other than neurons or glial cells.

[0176] 5.1.3. Tyrosine Sulfation In addition to the N-linked glycosylation site, hIDS also have a tyrosine ("Y") sulfated site (PSSEKY 165 This includes ENTKTCRGPD (SEQ ID NO: 47) (for example, Yang et al., 2015, Molecules 20:2138-2164, in particular p.2154, regarding the analysis of amino acids surrounding tyrosine residues subject to protein tyrosine sulfate, this whole is incorporated by reference. The "rules" can be summarized as follows: Y residues having E or D within the +5 to -5 positions of Y, and Y at position -1 being neutral or an acid-charged amino acid, but not a basic amino acid, e.g., R, K, or H which discard sulfate).

[0177] Importantly, tyrosine sulfated proteins cannot be produced in E. coli, which does not naturally possess the enzyme required for tyrosine sulfated proteins. Furthermore, CHO cells are defective in terms of tyrosine sulfated proteins. These cells are not secretory cells and have limited capacity for post-translational tyrosine sulfated proteins. See, for example, Mikkelsen & Ezban, 1991, Biochemistry 30:1533-1537. Advantageously, the method provided herein requires the expression of an IDS, e.g., HuGlyIDS, in secretory neuronal or glial cells that possess the capacity for tyrosine sulfated proteins. Assays for detecting tyrosine sulfated proteins are known in the art. See, for example, Yang et al., 2015, Molecules 20:2138-2164.

[0178] Tyrosine sulfate of hIDs, a robust post-translational process in human CNS cells, would likely improve the processing and activity of transgene products. While the importance of tyrosine sulfate in lysosomal proteins remains unclear, in other proteins, it has been shown to enhance the affinity of protein-protein interactions (antibody-receptor) and promote proteolytic processing (peptide hormones) (see Moore, 2003, J Biol. Chem. 278:24243-46; and Bundegaard et al., 1995, The EMBO J14:3073-79). Tyrosyl protein sulfotransferase (TPST1), involved in tyrosine sulfate (which may occur as the final step in IDS processing), is expressed at even higher levels in the brain (based on mRNA) (TPST1 gene expression data can be found, for example, in the EMBL-EBI Expression Atlas, accessible at http: / / www.ebi.ac.uk / gxa / home).

[0179] 5.2 Constructs and Formulations Viral vectors encoding iduronate-2-sulfatase (IDS), e.g., human IDS (hIDS), or other DNA expression constructs are provided for use in the methods provided herein. Viral vectors encoding glycosylated (HuGly)αL-idulonidase (IDUA), e.g., human IDUA (hIDUA), or other DNA expression constructs are provided for use in the methods provided herein. The viral vectors and other DNA expression constructs provided herein include any preferred method for delivering the transgene into cerebrospinal fluid (CSF). Means of transgene delivery include viral vectors, liposomes, complexes containing other lipids, other macromolecular complexes, synthetically modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g., gold particles), polymer molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a target vector, e.g., a vector targeting nerve cells.

[0180] In some embodiments, the present disclosure provides nucleic acids for use which encode hIDS operably linked to promoters selected from the group consisting of IDSs, such as cytomegalovirus (CMV) promoter, Roussarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter, and opsin promoter.

[0181] In certain embodiments, this specification provides recombinant vectors comprising one or more nucleic acids (e.g., polynucleotides). The nucleic acids may include DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more sequences selected from the group consisting of a promoter sequence, a gene sequence of interest (a transgene, e.g., an IDS), an untranslated region, and a stop sequence. In certain embodiments, the viral vectors provided herein include a promoter functionally linked to the gene of interest.

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

[0183] In another embodiment, the present disclosure provides a formulation comprising a recombinant nucleotide expression vector encoding a human IDS, the formulation being suitable for administration into the cerebrospinal fluid of a human brain, thereby forming a depot in the human central nervous system, the depot being approximately 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, and secreting a recombinant human IDS glycoprotein precursor containing formylglycine, α2,6-sialyzed, undetectable NeuGc-free, unalpha-Gal antigen-free, and / or mannose-6-phosphorylated. For example, the formulation may contain a buffer suitable for administration into the cerebrospinal fluid of a human brain (e.g., a buffer having a specific pH, or a buffer containing a specific component), thereby forming a depot in the human central nervous system that secretes a recombinant human IDS glycoprotein precursor that is approximately 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, contains formylglycine, is α2,6-sialyzed, does not contain detectable NeuGc, does not contain α-Gal antigen, and / or is mannose-6-phosphorylated. In specific embodiments, the buffer comprises a physiologically suitable aqueous buffer, a surfactant, and optional excipients.

[0184] In another embodiment, the Disclosure provides a kit comprising a recombinant nucleotide expression vector encoding human IDS and a pharmaceutically acceptable carrier, the recombinant nucleotide expression vector suitable for administration into the cerebrospinal fluid (CSF) of the human brain, thereby forming a depot in the human central nervous system, which, as measured by polyacrylamide gel electrophoresis, is approximately 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa), and secretes a recombinant human IDS glycoprotein precursor containing formylglycine, α2,6-sialyzed, undetectable NeuGc-free, α-Gal antigen-free, and / or mannose-6-phosphorylated. In another embodiment, the Disclosure provides a kit comprising a formulation comprising a recombinant nucleotide expression vector encoding a human IDS, the formulation being suitable for administration to the CSF of human brain, thereby a depot formed in the human central nervous system, the depot being approximately 90 kDa (e.g., 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, or 95 kDa) as measured by polyacrylamide gel electrophoresis, and secreting a recombinant human IDS glycoprotein precursor containing formylglycine, α2,6-sialyzed, undetectable NeuGc-free, unalpha-Gal antigen-free, and / or mannose-6-phosphorylated. The kits described herein comprise a recombinant nucleotide expression vector or formulation in one or more containers. Optionally, one or more such containers may be affixed with a warning in a form prescribed by a government agency that regulates the manufacture, use, or sale of a pharmaceutical or biological product, indicating that the agency has approved its manufacture, use, or sale for human administration.

[0185] The formulations and kits contained herein can be used in accordance with the methods for treating human patients provided herein.

[0186] 5.2.1. mRNA In certain embodiments, the vectors provided herein are modified mRNA encoding a gene of interest (e.g., a transgene, e.g., an IDS). The synthesis of modified and unmodified mRNA for transgene delivery to the CSF is taught, for example, in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496 (which is incorporated herein by reference in its entirety). In certain embodiments, modified mRNA encoding an IDS, e.g., hIDS, is described herein.

[0187] 5.2.2. Viral Vectors Viral vectors include adenoviruses, adeno-associated viruses (AAVs, e.g., AAV9, 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 mouse leukemia virus (MLV) and human immunodeficiency virus (HIV). Alphaviral vectors include Semryki forest virus (SFV) and Sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors provided herein are modified to be replication-deficient in humans. In certain embodiments, the viral vector is a hybrid vector, e.g., an AAV vector placed in a "helpless" adenovirus vector. In certain embodiments, viral vectors comprising a viral capsid from a first virus and a viral envelope protein from a second virus are provided herein. In a specific embodiment, the second virus is vesicular stomatitis virus (VSV). In a more specific embodiment, the envelope protein is the VSV-G protein.

[0188] In certain embodiments, the viral vector provided herein is an HIV-based viral vector. In certain embodiments, the HIV-based vector provided herein comprises 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.

[0189] In certain embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In certain embodiments, the herpes simplex virus-based vectors provided herein are modified to be non-cytotoxic by not containing one or more pre-initial (IE) genes.

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

[0191] In certain embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In certain embodiments, the lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are derived from non-human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are packaged in a lentiviral capsid. In certain embodiments, the lentiviral vectors provided herein include one or more of the following elements: a long terminal repeat sequence, a primer binding site, a polyprint lactate, an att site, and a capsidation site.

[0192] In certain embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In certain embodiments, the alphavirus vectors provided herein are recombinant, replication-deficient alphaviruses. In certain embodiments, the alphavirus replicons of the alphavirus vectors provided herein target specific cell types by presenting functional heterologous ligands on their virion surfaces.

[0193] In certain embodiments, the viral vectors provided herein are AAV-based viral vectors. In preferred embodiments, the viral vectors provided herein are AAV9 or AAVrh10-based viral vectors. In certain embodiments, the AAV9 or AAVrh10-based viral vectors provided herein retain tropism to CNS cells. Multiple AAV serotypes have been identified. In certain embodiments, the AAV-based vectors provided herein contain components derived from one or more AAV serotypes. In certain embodiments, the AAV-based vectors provided herein contain components derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV10, or AAV11 serotypes. In preferred embodiments, the AAV-based vectors provided herein contain components derived 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 AAVs and AAV capsids are taught, for example, in 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 embodiment, a viral vector based on an AAV (e.g., AAV9 or AAVrh10) encoding a transgene (e.g., IDS) is provided herein. In a specific embodiment, a viral vector based on AAV9 encoding an IDS is provided herein. In a more specific embodiment, a viral vector based on AAV9 encoding hIDS is provided herein.

[0194] In certain embodiments, an AAV9 vector is provided, comprising an artificial genome containing (i) an expression cassette containing a transgene that is under the control of a regulatory element and adjacent to an ITR, and (ii) a viral capsid having the amino acid sequence of the AAV9 capsid protein, or being at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of the AAV9 capsid protein (SEQ ID NO: 26), while simultaneously retaining the biological function of the AAV9 capsid. In certain embodiments, the encoded AAV9 capsid has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions in the sequence of SEQ ID NO: 26, while retaining the biological function of the AAV9 capsid. Figure 6 provides a comparative alignment of amino acid sequences of capsid proteins of different AAV serotypes that have potential amino acids that can be substituted at specific positions in the sequence, based on the comparison in the column labeled SUBS. Thus, in a specific embodiment, the AAV9 vector includes an AAV9 capsid variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions, which are identified in the SUBS column of Figure 6 and are not present at this position in the native AAV9 sequence.

[0195] In certain embodiments, the AAV used in the methods described herein is Anc80 or Anc80L65 as described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068 (which is incorporated herein in its entirety). In certain embodiments, the AAV used in the methods described herein includes one of the following amino acid insertions 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 in its entirety): LGETTRP (SEQ ID NO: 56) or LALGETTRP (SEQ ID NO: 57). In certain embodiments, the AAV used in the manner described herein is AAV.7m8 as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323 (each of which is incorporated herein by reference in whole). In certain embodiments, the AAV used in the manner described herein is any AAV disclosed in U.S. Patent No. 9,585,971, for example, AAV-PHP.B. In certain embodiments, the AAV used in the manner described herein is the AAV disclosed in any of the following patents and patent applications: U.S. Patent Nos. 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809; U.S. Patent Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, and 9458. Patent Nos. 517 and 9,587,282, U.S. Patent Application Publications 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335 (each of these is incorporated herein by reference in its entirety).

[0196] In certain embodiments, single-stranded AAVs (ssAAVs) can be used as described above. In certain embodiments, self-complementary vectors, such as scAAVs, can be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82, McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16 (pp. 1248-1254), and U.S. Patents 6,596,535, 7,125,717, and 7,456,683 (each of which is incorporated herein by reference in whole)).

[0197] In certain embodiments, the viral vector used in the methods described herein is an adenovirus-based viral vector. Recombinant adenovirus vectors can be used for introducing IDS. Recombinant adenoviruses may be first-generation vectors having an E1 deletion, or having or not having an E3 deletion, with an expression cassette inserted in either of the deleted regions. Recombinant adenoviruses may be second-generation vectors containing complete or partial deletions of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus reverse terminal repeat and the packaging signal (phi). The transgene may or may not contain a stuffer sequence inserted between the packaging signal and the 3'ITR, maintaining the size of the artificial genome close to the wild-type size of approximately 36 kb. An exemplary protocol for the production of adenovirus vectors can be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12: S18-S27 (which is incorporated herein by reference in its entirety).

[0198] In certain embodiments, the viral vector used in the methods described herein is a lentivirus-based viral vector. Recombinant lentiviral vectors can be used for the introduction of IDS. Four plasmids are used to construct: a plasmid containing a Gag / pol sequence, a plasmid containing a Rev sequence, a plasmid containing an envelope protein (i.e., VSV-G), and a packaging element, and a Cis plasmid containing the IDS gene.

[0199] For lentiviral vector production, four plasmids are co-transfected with cells (i.e., HEK293-based cells), thereby allowing the use of polyethyleneimine or calcium phosphate as transfection agents. Subsequently, the lentivirus is collected in the supernatant (cell recovery is unnecessary / should not be performed, as the lentivirus needs to be budding from the cells to maintain its activity). The supernatant is filtered (0.45 μm), followed by the addition of magnesium chloride and benzonase. Further downstream processes are highly variable, with the use of TFF and column chromatography being the most GMP-compliant process. Other processes use ultracentrifugation with or without column chromatography. Exemplary protocols for the production of lentiviral vectors can be found in Lesch et al., 2011, “Production and purification of lentiviral 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 these are incorporated herein by reference in their entirety).

[0200] In specific embodiments, the vector used in the methods described herein is a vector encoding an IDS (e.g., hIDS), and when transduced into CNS cells or suitable cells (e.g., nerve cells in vivo or in vitro), 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), and when transduced into CNS cells or related cells (e.g., nerve cells in vivo or in vitro), the cells express a sulfated variant of the IDS.

[0201] 5.2.3. Gene Expression Promoters and Modifiers In certain embodiments, the vectors provided herein include components that regulate gene delivery or gene expression (e.g., “expression regulatory elements”). In certain embodiments, the vectors provided herein include components that regulate gene expression. In certain embodiments, the vectors provided herein include components that affect cell binding or targeting to cells. In certain embodiments, the vectors provided herein include components that, after uptake, affect the localization of polynucleotides (e.g., transgenes) within cells. In certain embodiments, the vectors provided herein include components that can be used, for example, as detectable or selectable markers for detecting or selecting cells that have taken up polynucleotides.

[0202] In certain embodiments, the viral vectors provided herein include one or more promoters. In certain embodiments, the promoter is a constitutive promoter. In alternative embodiments, the promoter is an inductive promoter. Native IDS genes, like most housekeeping genes, primarily use GC-rich promoters. In preferred embodiments, a potent constitutive promoter that provides sustained expression of hIDS is used. Such promoters include the "CAG" synthetic promoter, which includes the "C" - cytomegalovirus (CMV) initial enhancer element, the "A" - promoter and first exon and intron of the chicken beta-actin gene, and the "G" - splice acceptor of the rabbit beta-globin gene (see Miyazaki et al., 1989, Gene 79:269-277, and Niwa et al., Gene 108:193-199).

[0203] In certain embodiments, the promoter is the CB7 promoter (see Dinculescu et al., 2005, Hum Gene Ther 16:649-663 (which is incorporated herein by reference in its entirety)). In some embodiments, the CB7 promoter includes other expression regulatory elements that enhance the expression of the vector-driven transgene. In certain embodiments, the other expression regulatory elements include chicken β-actin introns and / or rabbit β-globin polA signaling. In certain embodiments, the promoter includes a TATA box. In certain embodiments, the promoter includes one or more elements. In certain embodiments, one or more promoter elements may be oriented opposite to each other or repositioned. In certain embodiments, the elements of the promoter are arranged to function cooperatively. In certain embodiments, the elements of the promoter are arranged to function independently. In certain embodiments, the viral vectors provided herein include one or more promoters selected from the group consisting of the human CMV pre-initial gene promoter, the SV40 initial promoter, the Roussarcoma virus (RS) long-chain terminal repeat sequence, and the rat insulin promoter. In certain embodiments, the vectors provided herein include one or more long-terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2LTR. In certain embodiments, the vectors provided herein include one or more tissue-specific promoters (e.g., neuron-specific promoters).

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

[0205] 5.2.4. Signal Peptides In certain embodiments, the vectors provided herein include components that regulate protein delivery. In certain embodiments, the viral vectors provided herein include one or more signal peptides. In certain embodiments, the signal peptides enable proper intracellular packaging (e.g., glycosylation) of the transgene product (e.g., IDS). In certain embodiments, the signal peptides enable proper intracellular localization of the transgene product (e.g., IDS). In certain embodiments, the signal peptides enable the secretion of the transgene product (e.g., IDS) from the cell. Examples of signal peptides used in conjunction with vectors, and the transgenes provided herein, can be found in Table 4. Signal peptides may also be referred to herein as leader sequences or leader peptides. [Table 3]

[0206] 5.2.5. Untranslated Areas In certain embodiments, the viral vector provided herein includes one or more untranslated regions (UTRs) (e.g., 3' and / or 5' UTRs). In certain embodiments, the UTRs are optimized with respect to a desired level of protein expression. In certain embodiments, the UTRs are optimized with respect to the half-life of the transgene mRNA. In certain embodiments, the UTRs are optimized with respect to the stability of the transgene mRNA. In certain embodiments, the UTRs are optimized with respect to the secondary structure of the transgene mRNA.

[0207] 5.2.6. Reverse terminal repetition In certain embodiments, the viral vectors provided herein include one or more reverse-terminal repeat (ITR) sequences. The ITR sequences can be used to package recombinant gene expression cassettes into the virions of the viral vector. In certain embodiments, the ITRs are derived from AAV (e.g., AAV9) (see, for example, Yan et al., 2005, J. Virol., 79(1):364-379, U.S. Patent No. 7,282,199 B2, U.S. Patent No. 7,790,449 B2, U.S. Patent No. 8,318,480 B2, U.S. Patent No. 8,962,332 B2, and International Patent Application No. PCT / EP2014 / 076466 (each of which is incorporated herein by reference in whole)).

[0208] 5.2.7. Transgenes In certain embodiments, the vectors provided herein encode an IDS transgene. In specific embodiments, the IDS is controlled by a suitable expression regulatory element for expression in nerve cells. In certain embodiments, the IDS (e.g., hIDS) transgene comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the IDS (e.g., hIDS) transgene comprises an amino acid sequence that is identical to the sequence described in SEQ ID NO: 1 by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0209] The HuGlyIDS encoded by the transgene may include, but are not limited to, human IDS (hIDS) having the amino acid sequence of Sequence ID No. 1 (shown in Figure 1), and derivatives of hIDS having amino acid substitutions, deletions, or additions, such as, but are not limited to, amino acid substitutions selected from non-conserved residues corresponding to orthologs of IDS shown in Figure 2, provided that such mutations do not include substitutions of the cysteine ​​residue at position 84 (C84) required for enzyme activity (Millat et al., 1997, Biochem J326:243-247), or, for example, as shown in Figure 3, or Sukegawa-Hayasaka et al., 2006, J Inhert Metabolism. Provided that the variants do not include those identified in severe, severe-moderate, moderate, or weak MPS II phenotypes, such as those reported in Dis29:755-761 (which reports the “weak” variants R48P, A85T, W337R, and the truncated variant Q531X, and the “severe” variants P86L, S333L, S349I, R468Q, and R468L), Millat et al., 1998, BBA1406:214-218 (which reports the “weak” variants P480L and P480Q; and the “severe” variant P86L), and Bonucelli et al., 2001, BBA1537:233-238 (each of these is incorporated herein by reference in whole).

[0210] For example, amino acid substitutions at specific sites in hIDS can be selected from the corresponding non-conservative amino acid residues found at that site in the IDS ortholog shown in Figure 2, provided that they do not contain any harmful mutations such as those shown in Figure 3, or those reported in Sukegawa-Hayasaka et al., 2006 (above); Millat et al., 1998 (above); and Bonucelli et al., 2001 (above) (each of which is incorporated herein by reference in whole). The resulting transgene product can be tested in vitro in cell culture or in test animals using conventional assays to ensure that the mutations do not impair IDS function. The preferred amino acid substitutions, deletions, or additions selected should maintain or increase the enzymatic activity, stability, or half-life of IDS when tested in vitro with conventional assays for MPS II in cell culture or animal models. For example, the enzymatic activity of the transgene product can be evaluated using conventional enzyme assays with substrates such as 4-methylumbelliferyl α-L-idopyranocisuronic acid 2-sulfate or 4-methylumbelliferyl sulfate (see, for example, Lee et al., 2015, Clin. Biochem. 48(18):1350-1353, Dean et al., 2006, Clin. Chem. 52(4):643-649 (each of these is incorporated herein by reference in its entirety)). The ability of the transgene product to modify the MPS II phenotype can be evaluated in cell culture.For example, by transducing cultured MPS II cells with a viral vector or other DNA expression construct encoding hIDS or derivatives, adding transgene products or derivatives to cultured MPS II cells, or co-culturing MPS II cells with human neural host cells / human glial host cells genetically engineered to express and secrete rhIDS or derivatives to determine the correction of defects in cultured MPS II cells, for example by detecting a decrease in IDS enzyme activity and / or GAG storage in cultured MPS II cells. (See, e.g., Stroncek et al., 1999, Transfusion 39(4):343-350 (which is incorporated herein by reference in its entirety)).

[0211] In some embodiments, the dose of recombinant AAV of this disclosure is determined using a PCR assay. In some embodiments, the PCR assay is a poly(A) PCR assay. In some embodiments, the PCR assay is a transgene-specific PCR assay. In some embodiments, the dose determined by one assay is different from (e.g., higher or lower than) the dose determined by another assay. For example, the dose determined using a transgene-specific PCR assay is higher (e.g., about 50% higher) than the dose determined using a poly(A)-specific PCR assay. In some embodiments, the dose 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 dose determined by a poly(A)-specific PCR assay. In some embodiments, the genome copy number is 2.0 × 10⁶ when determined using a poly(A)-specific PCR assay. 11 The GC / g brain mass dose was determined by the number of genome copies using a transgene-specific PCR assay, resulting in a value of 2.9 × 10⁶. 11 This is equivalent to the dose of GC / g brain mass. In some embodiments, the total dose administered to the subject accounts for the subject's estimated brain mass, which can be determined using magnetic resonance imaging (MRI) screening.

[0212] 5.2.8. Construct In certain embodiments, the viral vector provided herein comprises the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a sequence encoding a transgene (e.g., an IDS), h) a fourth linker sequence, i) a poly(A) sequence, j) a fifth linker sequence, and k) a second ITR sequence.

[0213] In certain 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., an IDS). In certain 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., an IDS), where the transgene comprises a signal peptide.

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

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

[0216] In specific embodiments, the viral vector described herein includes the elements illustrated in Figure 5 in the order shown.

[0217] 5.2.9. Manufacturing and Testing of Vectors The viral vectors provided herein can be produced using host cells. The viral vectors provided herein can be produced using mammalian host cells, such as A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. The viral vectors provided herein can be produced using host cells derived from humans, monkeys, mice, rats, rabbits, or hamsters.

[0218] The host cell is stably transformed using the transgene and associated elements (i.e., the vector genome) and means for producing the virus in the host cell, such as sequences encoding replication and capsid genes (e.g., the rep and cap genes of AAV). For a method of producing a recombinant AAV vector having the AAV8 capsid, see Section IV of the detailed description in U.S. Patent No. 7,282,199 B2 (which is incorporated herein by reference in its entirety). The genomic copy titer of the above vector can be determined, for example, by TAQMAN® analysis. The virions can be recovered, for example, by CsCl2 precipitation.

[0219] In vitro assays, such as cell culture assays, can be used to measure transgene expression from the vectors described herein, and thus, for example, to demonstrate the efficacy of the vectors. For example, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM cell lines, or other cell lines derived from neuronal or glial cells, or neuronal or glial cell progenitor cells, can be used to evaluate transgene expression. Once expressed, the characteristics of the expression product (i.e., HuGlyIDS) can be determined, including the determination of glycosylation and tyrosine sulfate patterns associated with HuGlyIDS.

[0220] 5.2.10. Composition This specification describes a composition comprising a vector encoding the transgene described herein and a suitable carrier. Suitable carriers (e.g., CSF, and, for example, for administration to nerve cells) will be readily selected by those skilled in the art.

[0221] 5.3 Gene Therapy This paper describes a method for administering a therapeutically effective dose of an transgene construct to human subjects with MPS II in order to prevent, improve, or stabilize hearing loss associated with MPS II. More specifically, it describes a method for administering a therapeutically effective dose of an transgene construct to patients with MPS II, in particular a method for administration to the CSF. In certain embodiments, such a method for administering a therapeutically effective dose of an transgene construct to the CSF can be used to treat patients with Hunter syndrome in order to prevent, improve, or stabilize hearing loss associated with MPS II.

[0222] 5.3.1. Target patient population In certain embodiments, a therapeutically effective dose of recombinant vector is administered to a patient diagnosed with MPS II. In certain embodiments, the patient has hearing loss associated with MPS II. 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 is predisposed to hepatosplenomegaly. Examples of symptoms associated with hepatosplenomegaly, but not limited to, include dark urine, clay-colored stools, abdominal enlargement or edema, fever, itching, jaundice or yellowing of the eyes and skin, nausea, pain (e.g., upper right 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 being treated with or has been treated with ERT.

[0223] In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to patients diagnosed with MPS II, i.e., patients who have been shown to respond to treatment using an IDS, such as hIDS.

[0224] In certain embodiments, a therapeutically effective dose of recombinant vector is administered to pediatric patients. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients under 3 years of age. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 2 to 4 years. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 4 months or older but under 5 years of age. In specific embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 4 months or older but under 5 years of age who have severe MPS II. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 5 years or older but under 18 years of age. In specific embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 5 years or older but under 18 years of age who have neuropathic MPS II. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 18 months or older but under 8 years of age. In specific embodiments, a therapeutically effective dose of recombinant vector is administered to pediatric male patients aged 18 months or older but under 8 years of age. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 3 to 8 years of age. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 8 to 16 years. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 5 to 18 years. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 10 years or younger. In specific embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 10 years or younger with severe MPS II. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 18 years or younger. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients over 5 years of age. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients over 10 years of age.

[0225] In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 4, 5, 6, 7, 8, 9, 10, or 11 months. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged approximately 4, 5, 6, 7, 8, 9, 10, or 11 months. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged approximately 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, or 11-12 months. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged 1, 2, 3, 4, or 5 years. In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients aged approximately 1, 2, 3, 4, or 5 years. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to patients aged 1-2, 2-3, 3-4, 4-5, or 5-6 years. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to patients approximately 1-2, 2-3, 3-4, 4-5, or 5-6 years. In certain embodiments, a therapeutically effective dose of the recombinant vector 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 certain embodiments, a therapeutically effective dose of the recombinant vector is administered to patients 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 of age.

[0226] In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to adolescent patients. In certain 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.

[0227] In certain embodiments, a therapeutically effective dose of recombinant vector is administered to patients diagnosed with MPS II who have been confirmed to respond to treatment with an IDS, such as hIDS, infused into the CSF prior to gene therapy.

[0228] 5.3.2. Dosage and Administration Method In certain embodiments, a therapeutically effective dose of recombinant vector is administered to the CSF via intra-abracranial administration (i.e., injection into the subarachnoid space to allow the recombinant vector to diffuse through the CSF and transduce cells of the CNS). This can be achieved by several methods, e.g., intracranial (cisterna magnum or intraventricular) injection, or lumbar cisterna injection. In certain embodiments, intra-abracranial administration is performed by intracisterna magnum (IC) injection (e.g., into the cisterna magnum). In specific embodiments, intracisterna magnum injection is performed by CT-guided suboccipital puncture. In specific embodiments, intra-abracranial injection is performed by lumbar puncture. In specific embodiments, if feasible for the patient, intra-abracranial injection is performed by C1-2 puncture. Alternatively, the recombinant vector can be directly injected into the cerebral cavity using intraventricular (ICV) administration (a more invasive technique used to introduce anti-infective or anticancer drugs that do not cross the blood-brain barrier), e.g., image-assisted ICV injection. In specific embodiments, the recombinant vector is administered via a single image-assisted ICV infusion. In further specific embodiments, the recombinant vector is administered via a single image-assisted ICV infusion while immediately removing the administration catheter. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the CNS via intranasal administration. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the CNS by intraparenchymal infusion. In certain embodiments, the intraparenchymal infusion targets the striatum. In certain embodiments, the intraparenchymal infusion targets the white matter. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the CSF by any means known in the Art, e.g., any means disclosed in Hocquemiller et al., 2016, Human Gene Therapy 27(7):478-496 (which is incorporated in its entirety by reference).

[0229] In a preferred embodiment, for subarachnoid administration (such as IC and ICV administration), a therapeutically effective dose of recombinant vector is administered to the CSF in an infusion volume not exceeding 10% of the total CSF volume, with the total CSF volume being approximately 50 ml in infants and approximately 150 ml in adults. A carrier suitable for subarachnoid injection, such as Elliott's B solution or a modified Elliott's B solution, should be used as the vehicle for the recombinant vector. Elliott's B solution (generic name: sodium chloride, sodium bicarbonate, anhydrous dextrose, magnesium sulfate, potassium chloride, calcium chloride, and sodium phosphate) is a sterile, non-pyrogenic isotonic solution that does not contain bacteriostatic preservatives and is used as a diluent for subarachnoid administration of chemotherapeutic drugs. The modified Elliott's B solution contains 8.77 g / L sodium chloride, 0.244 g / L magnesium chloride, 0.0278 g / L monobasic phosphate monohydrate, 0.114 g / L dibasic anhydride phosphate, 0.224 g / L potassium chloride, 0.206 g / L calcium chloride, 0.793 g / L dextrose, 0.010 g / L poloxamer 188, and pH 7.26. In some embodiments, the AAV or AAV-containing composition of this disclosure is provided in the modified Elliott's B solution for subarachnoid administration.

[0230] In one embodiment, a non-replicating recombinant AAV9 vector expressing human iduronate-2-sulfatase (IDS) is used for therapeutic purposes. In a particular embodiment, the IDS expression cassette is flanked by a reverse terminal repeat (ITR), and expression is driven by a hybrid of a cytomegalovirus (CMV) enhancer and a chicken beta-actin promoter (CB7). In a particular embodiment, the transgene contains a chicken β-actin intron and a rabbit β-globin polyadenylation (PolyA) signal.

[0231] In certain embodiments, a recombinant nucleotide expression vector is administered in a dose corresponding to the brain mass of the human subject. In preferred embodiments, brain mass is determined by magnetic resonance imaging (MRI) of the subject's brain. In certain embodiments, the brain volume of the human subject is measured in cm². 3 The brain volume of a human subject is converted to brain mass of a human subject by multiplying it by a coefficient of 1.046 g / cm³, and the brain volume of a human subject is obtained from a human subject brain MRI. In some embodiments, the dose is the number of genome copies per unit weight of brain mass. In some embodiments, the number of genome copies at a given dose (e.g., dose of recombinant nucleotide expression vector) is determined by a polyA-specific PCR assay. In some embodiments, the number of genome copies at a given dose (e.g., dose of recombinant nucleotide expression vector) is determined by a transgene-specific PCR assay. In some embodiments, the weight of brain mass is determined by MRI.

[0232] In certain embodiments, rAAV9.hIDS contains 1.4 × 10¹³ ions in a volume of approximately 5–20 ml. 13 GC (1.1 × 10⁻¹⁰ per gram of brain mass) 10 GC) ~7.0×10 13 GC (5.6 × 10⁻¹⁰ per gram of brain mass) 10 A single fixed dose within the range of GC) is administered by IC (suboccipital injection). If the patient has neutralizing antibodies against AAV, a higher dose range may be used. In some embodiments, when a single dose of rAAV encoding hIDS is administered to the target central nervous system (e.g., cerebrospinal fluid), surprisingly, therapeutic effects are observed outside the CNS. For example, changes in organ size are observed outside the CNS (e.g., spleen or liver) after administration of the rAAV disclosed herein to the target CNS. In some embodiments, changes in biomarker levels (e.g., D2S6, HS, total GAG, and / or anti-IDS antibodies) are detected outside the CNS (e.g., changes in biomarker levels detected in the liver, spleen, urine, plasma, or blood) after administration of the rAAV disclosed in cerebrospinal fluid. In some embodiments, additional therapy for MPS II is not administered outside the target CNS.

[0233] In certain embodiments, the recombinant vector described herein provides approximately 1.3 × 10¹⁶ units per gram of brain mass (for example, when the human patient is between 4 months and 5 years old). 10 GC ~ Approximately 6.5 × 10⁻¹⁶ units of brain mass per gram 10 It can be administered intraarachnoidally as a single fixed dose within the GC range. In specific embodiments, the recombinant vector described herein is administered at approximately 1.3 × 10¹⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 10 GC can be administered intraarachnoidally as a single fixed dose. In specific embodiments, the recombinant vector described herein is administered at a dose of approximately 1.9 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 10 GC may be administered intraarachnoidally as a single fixed dose. In another specific embodiment, the recombinant vector described herein may be administered at approximately 6.5 × 10¹⁶ times per gram of brain mass (for example, when the human patient is between 4 months and 5 years of age). 10 GC can be administered intraarachnoidally as a single fixed dose. In specific embodiments, the recombinant vector described herein is administered at approximately 9.6 × 10⁶ times per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 10 GC can be administered intraarachnoidally as a single fixed dose. In specific embodiments, the recombinant vector described herein is administered at a dose of approximately 2.0 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 11 GC can be administered intraarachnoidally as a single fixed dose. In specific embodiments, the recombinant vector described herein is administered at a dose of approximately 2.9 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 11 GC may be administered intra-arachnoid as a single fixed dose. In another specific embodiment, the recombinant vector described herein may be administered intra-arachnoid as a single fixed dose of dose 1 or dose 2 as described in Table 5 below (for example, if the human patient is 4 months of age or older but less than 5 years of age).

[0234] In certain embodiments, the recombinant vector described herein provides approximately 1.3 × 10¹⁶ units per gram of brain mass (for example, when the human patient is between 4 months and 5 years old). 10 GC ~ Approximately 2.0 × 10⁻¹⁴ per gram of brain mass 11 It may be administered intraarachnoidally as a single fixed dose within the GC range. In certain embodiments, the recombinant vector described herein may be administered at approximately 1.3 × 10¹⁶ per g of brain mass (for example, when the human patient is between 4 months and 5 years of age). 10 GC ~ Approximately 2.9 × 10⁻¹⁶ units per gram of brain mass 11 It can be administered intraarachnoidally as a single fixed dose within the GC range. In specific embodiments, the recombinant vector described herein is administered at approximately 2.0 × 10¹⁴ per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 11 GC can be administered intraarachnoidally as a single fixed dose. In specific embodiments, the recombinant vector described herein is administered at a dose of approximately 2.9 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 11 GC may be administered intra-arachnoid as a single fixed dose. In another specific embodiment, the recombinant vector described herein may be administered intra-arachnoid as a single fixed dose of dose 3 as shown in Table 6 below (for example, when the human patient is 4 months of age or older but under 5 years of age).

[0235] In certain embodiments, the recombinant vector described herein provides approximately 1.3 × 10¹⁶ units of brain mass per gram (for example, when the human patient is between 4 months and 5 years old). 10 GC brain mass ~ approx. 6.5×10 10 It can be administered by IC administration as a single fixed dose within the range of GC / g brain mass. In specific embodiments, the recombinant vector described herein is administered at approximately 1.3 × 10⁶ per gram of brain mass (for example, when the human patient is between 4 months and 5 years old). 10 It can be administered as a single fixed dose of GC brain mass via IC administration. In specific embodiments, the recombinant vector described herein is administered at approximately 1.9 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older and under 5 years of age).10 It can be administered as a single fixed dose of GC brain mass by IC administration. In another specific embodiment, the recombinant vector described herein is administered at approximately 6.5 × 10¹⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 10 GC can be administered as a single fixed dose via IC administration. In specific embodiments, the recombinant vector described herein is administered at approximately 9.6 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older and under 5 years of age). 10 The recombinant vector may be administered by IC as a single fixed dose of GC brain mass. In another specific embodiment, the recombinant vector described herein may be administered by IC as a single fixed dose of dose 1 or dose 2 as shown in Table 5 below (for example, when the human patient is 4 months of age or older but under 5 years of age).

[0236] In certain embodiments, the recombinant vector described herein provides approximately 1.3 × 10¹⁶ units of brain mass per gram (for example, when the human patient is between 4 months and 5 years old). 10 GC brain mass ~ approx. 2.0×10 11 It can be administered by IC administration as a single fixed dose within the range of GC / g brain mass. In certain embodiments, the recombinant vector described herein is administered at approximately 1.3 × 10⁶ per gram of brain mass (for example, when the human patient is between 4 months and 5 years old). 10 GC brain mass ~ approx. 2.9×10 11 It can be administered by IC administration as a single fixed dose within the range of GC / g brain mass. In specific embodiments, the recombinant vector described herein is administered at approximately 2.0 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older and under 5 years of age). 11 It can be administered as a single fixed dose of GC brain mass via IC administration. In specific embodiments, the recombinant vector described herein is administered at a dose of approximately 2.9 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older and under 5 years of age). 11It may be administered by IC as a single fixed dose of GC brain mass. In another specific embodiment, the recombinant vector described herein may be administered by IC as a single fixed dose of dose 3 as shown in Table 6 below (for example, when the human patient is 4 months of age or older but under 5 years of age).

[0237] In certain embodiments, the recombinant vector described herein provides approximately 1.3 × 10¹⁶ units of brain mass per gram (for example, when the human patient is between 4 months and 5 years old). 10 GC brain mass ~ approx. 6.5×10 10 It can be administered by ICV as a single fixed dose within the range of GC / g brain mass. In specific embodiments, the recombinant vector described herein is administered at approximately 1.3 × 10⁶ per gram of brain mass (for example, when the human patient is between 4 months and 5 years old). 10 It can be administered by ICV as a single fixed dose of GC brain mass. In specific embodiments, the recombinant vector described herein is administered at approximately 1.9 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older and under 5 years of age). 10 It can be administered by ICV as a single fixed dose of GC brain mass. In another specific embodiment, the recombinant vector described herein is administered at approximately 6.5 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older but under 5 years of age). 10 GC can be administered as a single fixed dose via ICV. In specific embodiments, the recombinant vector described herein is administered at approximately 9.6 × 10⁶ times per gram of brain mass (for example, when the human patient is 4 months of age or older and under 5 years of age). 10 The recombinant vector may be administered via ICV as a single fixed dose of GC brain mass. In another specific embodiment, the recombinant vector described herein may be administered via ICV as a single fixed dose of dose 1 or dose 2 as shown in Table 5 below (for example, when the human patient is 4 months of age or older but under 5 years of age).

[0238] In certain embodiments, the recombinant vector described herein provides approximately 1.3 × 10¹⁶ units of brain mass per gram (for example, when the human patient is between 4 months and 5 years old). 10GC brain mass ~ approx. 2.0×10 11 It can be administered by ICV as a single fixed dose within the GC / g brain mass range. In certain embodiments, the recombinant vector described herein is administered at approximately 1.3 × 10⁶ per gram of brain mass (for example, when the human patient is between 4 months and 5 years old). 10 GC brain mass ~ approx. 2.9×10 11 It can be administered by ICV as a single fixed dose within the range of GC / g brain mass. In specific embodiments, the recombinant vector described herein is administered at approximately 2.0 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older and under 5 years of age). 11 It can be administered by ICV as a single fixed dose of GC brain mass. In specific embodiments, the recombinant vector described herein is administered at a dose of approximately 2.9 × 10⁶ per gram of brain mass (for example, when the human patient is 4 months of age or older and under 5 years of age). 11 The recombinant vector may be administered via ICV as a single fixed dose of GC brain mass. In another specific embodiment, the recombinant vector described herein may be administered via ICV as a single fixed dose of dose 3 as shown in Table 6 below (for example, when the human patient is 4 months of age or older but under 5 years of age). [Table 4] [Table 5] TIFF2026534516000007.tif154165

[0239] In specific embodiments, the recombinant vector described herein provides approximately 6.5 × 10¹⁶ units per gram of brain mass (for example, if the human patient is between 5 and 18 years of age). 10 GC may be administered intra-arachnoid as a single fixed dose. In another specific embodiment, the recombinant vector described herein may be administered intra-arachnoid as a single fixed dose as shown in Table 7 below (for example, when the human patient is 5 years of age or older and under 18 years of age), and in accordance with Table 7.

[0240] In specific embodiments, the recombinant vector described herein provides approximately 6.5 × 10¹⁶ units per gram of brain mass (for example, when the human patient is between 5 and 18 years of age). 10 GC may be administered as a single fixed dose via IC administration. In another specific embodiment, the recombinant vector described herein may be administered as a single fixed dose as shown in Table 7 below, and in accordance with Table 7, (for example, when the human patient is 5 years of age or older and under 18 years of age) via IC administration.

[0241] In specific embodiments, the recombinant vector described herein provides approximately 6.5 × 10¹⁶ units per gram of brain mass (for example, when the human patient is between 5 and 18 years of age). 10 GC can be administered by ICV as a single fixed dose. In another specific embodiment, the recombinant vector described herein may be administered by ICV as a single fixed dose as shown in Table 7 below, and in accordance with Table 7, (for example, when the human patient is 5 years of age or older and under 18 years of age). [Table 6]

[0242] 5.3.3. Prevention and treatment of hearing loss associated with MPS II This disclosure further provides methods and formulations for preventing or treating MPS II-associated hearing loss in subjects requiring prevention or treatment of MPS II-associated hearing loss, using recombinant human iduronate-2-sulfatase (IDS). In certain embodiments, recombinant human IDS is delivered to the subject by the recombinant vector of this disclosure (see Section 5.2). In certain embodiments, the subject is between 4 months and 5 years of age. In certain embodiments, the subject is between 5 years and 18 years of age.

[0243] This disclosure is in part based on the remarkable finding that therapeutically effective doses of the recombinant vector of this disclosure were able to stabilize and improve hearing in subjects suffering from MPS II-associated hearing loss. Furthermore, in subjects without hearing loss (with normal hearing), therapeutically effective doses of the recombinant vector of this disclosure prevented hearing loss for at least 3–4 years after administration.

[0244] In one embodiment, the Disclosure provides a method for preventing MPS II-related hearing loss in a subject requiring prevention of MPS II-related hearing loss, the method comprising administering to the subject a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS) as disclosed herein (see Section 5.2), wherein the subject has not developed hearing loss in at least one ear prior to the administration of rAAV, and the therapeutically effective dose of rAAV prevents the development of hearing loss in at least one ear. In certain embodiments, at least one ear includes the left ear or the right ear. In certain embodiments, at least one ear includes the left ear and the right ear.

[0245] In certain embodiments, a therapeutically effective dose of rAAV prevents the onset of hearing loss in at least one ear (e.g., one or both) 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 rAAV administration. In certain embodiments, a therapeutically effective dose of rAAV prevents the onset of hearing loss in at least one ear for at least about 3 years. In certain embodiments, a therapeutically effective dose of rAAV prevents the onset of hearing loss in at least one ear for at least about 4 years. In certain embodiments, a therapeutically effective dose of rAAV prevents the onset of hearing loss in at least one ear for at least about 5 years.

[0246] In certain embodiments, prevention of hearing loss is indicated by the hearing threshold measured by auditory brainstem response (ABR) testing in at least one ear after rAAV administration showing no change or only a slight change compared to the hearing threshold measured by ABR testing in at least one ear before rAAV administration. In certain embodiments, the hearing threshold is an estimate of the behavioral threshold (eHL) obtained from the ABR threshold (nHL) determined by ABR testing. In certain embodiments, prevention of hearing loss is indicated by the change in eHL in at least one ear after rAAV administration being approximately 25 dB or less, approximately 20 dB or less, approximately 15 dB or less, approximately 10 dB or less, approximately 5 dB or less, or approximately 1 dB or less compared to before rAAV administration. In certain embodiments, prevention of hearing loss is indicated by the change in eHL in at least one ear after rAAV administration being approximately 25 dB or less compared to before rAAV administration. In certain embodiments, prevention of hearing loss is indicated by a change in eHL in at least one ear after rAAV administration being approximately 10 dB or less compared to before rAAV administration.

[0247] In certain embodiments, the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic audiometry.

[0248] In another embodiment, the Disclosure provides a method for treating mucopolysaccharidosis type II (MPS II) associated hearing loss in a subject requiring treatment for MPS II associated hearing loss, the method comprising administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear (e.g., one or both) prior to the administration of rAAV. In certain embodiments, the therapeutically effective dose of rAAV stabilizes the hearing loss in at least one ear or improves hearing. In certain embodiments, at least one ear includes the left ear or the right ear. In certain embodiments, at least one ear includes the left ear and the right ear.

[0249] In another embodiment, the Disclosure provides a method for stabilizing hearing loss associated with mucopolysaccharidosis type II (MPS II) or preventing the progression of further hearing loss associated with MPS II in a subject requiring stabilization of MPS II-associated hearing loss or prevention of further progression of MPS II-associated hearing loss, the method comprising administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear (e.g., one or both) prior to the administration of rAAV, and the therapeutically effective dose of rAAV prevents further progression of hearing loss in at least one ear (e.g., worsening or progression of hearing impairment, further hearing loss). In certain embodiments, at least one ear includes the left ear or the right ear. In certain embodiments, at least one ear includes the left ear and the right ear. As used herein, "stabilizing hearing loss associated with MPS II" means preventing the worsening or progression of hearing loss.

[0250] In certain embodiments, a therapeutically effective dose of rAAV stabilizes hearing loss or prevents further progression of hearing loss in 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 rAAV administration. In certain embodiments, a therapeutically effective dose of rAAV prevents further progression of hearing loss in at least one ear for at least about 16 weeks after rAAV administration. In certain embodiments, a therapeutically effective dose of rAAV prevents further progression of hearing loss in at least one ear for at least about 24 weeks after rAAV administration. In certain embodiments, a therapeutically effective dose of rAAV prevents further progression of hearing loss in at least one ear for at least about 48 weeks after rAAV administration.

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

[0252] In certain embodiments, the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic audiometry.

[0253] In another embodiment, the Disclosure provides a method for improving hearing loss associated with mucopolysaccharidosis type II (MPS II) in a subject requiring improvement of hearing loss associated with MPS II, the method comprising administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS), wherein the subject has developed hearing loss in at least one ear (e.g., one or both) prior to administration of rAAV, and the therapeutically effective dose of rAAV improves hearing in at least one ear. In certain embodiments, at least one ear includes the left ear or the right ear. In certain embodiments, at least one ear includes the left ear and the right ear.

[0254] In certain embodiments, a therapeutically effective dose of rAAV improves hearing in at least one ear 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 after administration of rAAV. In certain embodiments, a therapeutically effective dose of rAAV improves hearing in at least one ear 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 after administration of rAAV. In certain embodiments, a therapeutically effective dose of rAAV improves hearing in at least one ear approximately 16 weeks after administration of rAAV. In certain embodiments, a therapeutically effective dose of rAAV improves hearing in at least one ear approximately 24 weeks after administration of rAAV. In certain embodiments, a therapeutically effective dose of rAAV improves hearing in at least one ear approximately 48 weeks after administration of rAAV.

[0255] In certain embodiments, improved hearing is indicated by a decrease in the hearing threshold measured by ABR testing in at least one ear after rAAV administration compared to the hearing threshold measured by ABR testing in at least one ear before rAAV administration. In certain embodiments, a therapeutically effective dose of rAAV reduces the hearing threshold measured by ABR testing in 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% compared to the hearing threshold in at least one ear before rAAV administration. In certain embodiments, the hearing threshold is an estimate of the behavioral threshold (eHL) derived from the ABR threshold (nHL). In certain embodiments, improved hearing is indicated by a decrease in eHL of approximately 5 dB or more, approximately 10 dB or more, approximately 15 dB or more, approximately 20 dB or more, approximately 25 dB or more, or approximately 30 dB or more in at least one ear after rAAV administration compared to before rAAV administration. In certain embodiments, improved hearing is indicated by a decrease in eHL of approximately 10 dB or more in at least one ear after rAAV administration compared to before rAAV administration.

[0256] In certain embodiments, the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic audiometry.

[0257] In certain embodiments, the methods disclosed herein further include performing ABR testing in at least one ear before and after administration of rAAV to determine the hearing threshold of at least one ear before and after administration of rAAV. ABR testing is a physiological method used to estimate the behavioral hearing threshold in individuals for whom behavioral audiometry cannot be reliably performed. ABR measures electrical activity in response to the presentation of various acoustic stimuli.

[0258] Those skilled in the art will know how to perform ABR testing on a subject. In certain embodiments, ABR testing is performed by presenting tone burst stimuli through an earplug-type earphone and measuring the ABR thresholds of nHL for each ear at the following frequencies: 500 Hz, 1000 Hz, 2000 Hz, and 4000 Hz. Once the nHL thresholds are obtained, appropriate correction factors are applied for each frequency to calculate an estimate of the behavioral threshold (eHL). The mean of the measured hearing thresholds is then used to classify the average degree of hearing loss for each ear as mild, moderate, severe, or very severe. ABR testing is also performed using high-intensity (80-90 dBnHL) click stimuli at a rate of 17-30 clicks / second. The absolute delay time of the waveform is then recorded, along with comments on the quality of the waveform shape. These tests are performed to assess the integrity of the auditory brainstem pathway.

[0259] In certain embodiments, the methods disclosed herein further include performing tympanotomy in at least one ear before and after administration of rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural. The tympanotomy is performed to assess the function of the middle ear.

[0260] Those skilled in the art will know how to perform tympanotomy on a subject. In certain embodiments, tympanotomy is performed bilaterally in conjunction with ABR testing to distinguish between conductive and sensorineural hearing loss. The following parameters are recorded: equivalent external auditory canal volume (cc), static admittance (mmho), tympanic membrane width (daPa), and tympanic membrane peak pressure (daPa). The results of the tympanotomy are then classified according to the following criteria: 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.

[0261] In certain embodiments, the Disclosure provides a method for preventing MPS II-related hearing loss in subjects requiring prevention of MPS II-related hearing loss, the method comprising (a) administering to a subject a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS) as disclosed herein (see Section 5.2), and (b) performing ABR testing in at least one ear before and after administration of rAAV to determine the hearing threshold of at least one ear before and after administration of rAAV, wherein the subject has not developed hearing loss in at least one ear prior to administration of rAAV, and the therapeutically effective dose of rAAV prevents the development of hearing loss in at least one ear. In certain embodiments, the method further comprises (c) performing tympanic audiometry in at least one ear before and after administration of rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0262] In certain embodiments, the Disclosure provides a method for treating MPS II-associated hearing loss in a subject requiring treatment for MPS II-associated hearing loss, the method comprising (a) administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS) as disclosed herein (see Section 5.2), and (b) performing ABR testing in at least one ear before and after administration of rAAV to determine the hearing threshold of at least one ear before and after administration of rAAV, wherein the subject has developed hearing loss in at least one ear (e.g., one or both) prior to administration of rAAV. In certain embodiments, the therapeutically effective dose of rAAV stabilizes the hearing loss in at least one ear or improves hearing. In certain embodiments, the method further includes (c) performing tympanic audiometry in at least one ear before and after administration of rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0263] In certain embodiments, the Disclosure provides a method for stabilizing MPS II-associated hearing loss in a subject requiring stabilization of MPS II-associated hearing loss, the method comprising (a) administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) as disclosed herein (see Section 5.2), and (b) performing ABR testing in at least one ear before and after administration of rAAV to determine the hearing threshold of at least one ear before and after administration of rAAV, wherein the subject has developed hearing loss in at least one ear prior to administration of rAAV, and the therapeutically effective dose of rAAV prevents further progression of hearing loss in at least one ear. In certain embodiments, the method further comprises (c) performing tympanic audiometry in at least one ear before and after administration of rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0264] In certain embodiments, the Disclosure provides a method for improving hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring improvement of hearing loss associated with MPS II, the method comprising (a) administering to a subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS) as disclosed herein (see Section 5.2), and (b) performing ABR testing in at least one ear before and after administration of rAAV to determine the hearing threshold of at least one ear before and after administration of rAAV, wherein the subject has developed hearing loss in at least one ear (e.g., one or both) prior to administration of rAAV, and a therapeutically effective dose of rAAV improves hearing in at least one ear. In certain embodiments, the method further includes (c) performing tympanic audiometry in at least one ear before and after administration of rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

[0265] In another embodiment, the Disclosure provides a formulation suitable for administration into the ear (e.g., the inner ear), comprising a therapeutically effective dose of a recombinant adeno-associated virus vector (rAAV) encoding human iduronate-2-sulfatase (hIDS) (see Section 5.2). In certain embodiments, the formulation is suitable for intratympanic, intracochlear, or intracochlear delivery (see, e.g., Liu et al., Front Neurosci. 2022;16:867453 (this content is incorporated in its entirety by reference)). In certain embodiments, the formulation is suitable for targeted delivery of rAAV to the cochlea. In certain embodiments, the formulation is suitable for intracochlear or intracochlear delivery. In certain embodiments, the formulation includes an adjuvant (e.g., histamine, benzyl alcohol, saponin, and N-methyl-2-pyrrolidone (NMP)) to enhance the permeability of the drug through a round window membrane (RWM) (see, for example, 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 each of these are incorporated in whole by reference)). In certain embodiments, the formulation includes nanocarriers such as liposomes, micelles, polymer nanoparticles, and dendrimers to deliver rAAV. In certain embodiments, the formulation includes a hydrogel, nanocarrier, penetration enhancer, or microfluidic system to enhance the penetration and sustained release of the drug.

[0266] 5.4 Combination Therapy The method of the present invention encompasses the administration of HuGlyIDS to a patient in combination with the implementation of other available therapies. Further therapies may be administered before, concurrently with, or after the gene therapy procedure. Available therapies for MPS II that can be used in combination with the gene therapy of the present invention include, but are not limited to, enzyme replacement therapy (ERT) using idursulfase administered systemically or to the CSF, and / or HSCT therapy. In another embodiment, ERT may be administered using rHuGlyIDS glycoprotein produced by human neuronal / human glial cell lines by recombinant DNA technology. Human neuronal / human glial cell lines that can be used for such recombinant glycoprotein production include, but are not limited to, HT-22, SK-N-MC, HCN-1A, HCN-2, NT2, SH-SY5y, hNSC11, or ReNcell VM. To ensure complete glycosylation, particularly sialylation and tyrosine sulfate formation, the cell line used for production can be enhanced by genetically engineering the host cells to co-express α-2,6-sialyltransferase (or both α-2,3- and α-2,6-sialyltransferases) and / or TPST-1 and TPST-2 enzymes, which are responsible for tyrosine-O-sulfation.

[0267] 5.5 Biomarkers / Sampling / Efficacy Monitoring Efficacy can be monitored by measuring cognitive function (e.g., prevention or reduction of neurocognitive decline), a decrease in disease biomarkers in CSF and / or serum (e.g., GAGs such as heparan sulfate and dermatan sulfate), and / or an increase in IDS enzyme activity in CSF and / or serum. Signs of inflammation and other safety events can also be monitored.

[0268] In one embodiment, the Specified provides a method for monitoring efficacy by determining whether a subject receiving or having received ERT treatment can discontinue ERT treatment after receiving the gene therapy of the Disclosure (e.g., rAAV encoding hIDS). For example, the Specified provides a method for treating and / or identifying a subject diagnosed with MPS II (e.g., a subject likely to discontinue ERT treatment), the method comprising: (a) administering a therapeutically effective dose of the gene therapy of the Disclosure (e.g., rAAV encoding hIDS) to the subject, the subject having received or receiving ERT treatment; (b) identifying the subject as likely to discontinue ERT treatment, i. obtaining or having obtained a biological sample from the subject; ii. determining the level of at least one biomarker in the biological sample; and iii. identifying the subject as likely to discontinue ERT treatment if the level of the biomarker in the biological sample is different from (e.g., higher or lower than) a reference level (e.g., a reference level for at least one biomarker); and (c) discontinuing ERT treatment for the subject. In some embodiments, the biomarkers are D2S6, HS, total GAG, and / or anti-IDS antibodies. In some embodiments, ERT is recombinant idursulfase. In some embodiments, subjects diagnosed with MPS II have hepatosplenomegaly.

[0269] In another embodiment, this specification provides a method for selectively treating a human subject having MPS II, comprising administering a therapeutically effective dose of rAAV encoding hIDS to the subject, wherein the subject has received or is undergoing treatment with ERT, and the subject is determined to be likely to discontinue 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 if the level of at least one biomarker is different from a standard (e.g., high or low), the subject is determined to be likely to discontinue ERT treatment. In some embodiments, the biomarkers are D2S6, HS, total GAG, and / or anti-IDS antibodies. In some embodiments, ERT is recombinant idursulfase. In some embodiments, the subject diagnosed with MPS II has hepatosplenomegaly.

[0270] In other embodiments, methods for identifying or diagnosing a subject as having neuropathic or non-neuropathic MPS II or MPS I are provided herein. In some embodiments, the method comprises determining the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, D2S6) in a biological sample derived from the subject. In some embodiments, the method comprises determining the level of undegraded glycosaminoglycans (GAGs) in a biological sample derived from the subject. In some embodiments, the subject is identified or diagnosed as having 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 as having neuropathic MPS II or MPS I if the level of GAG heparan 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 four disaccharides (D2S6, D0A0, D0S0, D0A6) in the cerebrospinal fluid (CSF) after enzymatic digestion (determined, for example, based on bioanalytic mass spectrometry). In some embodiments, for example, an increase in the level of D2S6 in the CSF of a subject (e.g., a pre-symptomatic subject) indicates neuropathic MPS II or MPS I. In some embodiments, the level of D2S6 indicates iduronate-2-sulfatase enzyme activity and can be used for therapeutic monitoring. In some embodiments, one or more disaccharides include one or more of D0A0, D0S0, D0A6, D2S6, or a combination thereof. In another embodiment, the Specified Method provides for identifying or diagnosing a subject having neuropathic or non-neuropathic MPS II or MPS I, wherein the subject is identified or diagnosed with neuropathic MPS II if the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample derived from the subject is elevated compared to a reference level. In some embodiments, the subject is pre-symptomatic or does not have visible or detectable MPS II or MPS I symptoms.In some embodiments, the subject has MPS I or MPS II, or has been diagnosed with it. In some embodiments, the reference level is the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in biological samples from one or more healthy individuals and / or one or more non-neuropathic subjects. In some embodiments, the reference level is the level of D2S6 in biological samples (e.g., CSF samples) from one or more healthy individuals and / or one or more non-neuropathic subjects. In some embodiments, the reference level is a predetermined level. In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, D2S6) is about or at least about 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, or greater than 40% of the total heparan sulfate disaccharides (HS) in a biological sample derived from the subject (e.g., a subject having MPS I or MPS II). In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 20% of the total heparan sulfate disaccharides (HS) in a biological sample derived from the subject (e.g., a subject having MPS I or MPS II). In some embodiments, the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample derived from a subject (e.g., a subject having MPS I or MPS II) is about or at least about 5%, 10%, 115%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more than 90% higher than the level of one or more disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in a biological sample derived from a reference (e.g., a healthy person).In some embodiments, the levels of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or 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, and 550 ng / mL. The levels are 575 ng / mL, 600 ng / mL, 625 ng / mL, 650 ng / mL, 675 ng / mL, 700 ng / mL, 725 ng / mL, 750 ng / mL, 775 ng / mL, 800 ng / mL, 825 ng / mL, 850 ng / mL, 875 ng / mL, 900 ng / mL, 925 ng / mL, 950 ng / mL, 975 ng / mL, 1000 ng / mL, or greater than 1000 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 500 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 600 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 700 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is about or at least about 800 ng / mL. In some embodiments, the level of total heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) is in the range of about 500 ng / mL to about 1000 ng / mL, about 300 ng / mL to about 1000 ng / mL, about 400 ng / mL to about 1000 ng / mL, about 550 ng / mL to about 1000 ng / mL, about 600 ng / mL to about 1000 ng / mL, about 700 ng / mL to about 1000 ng / mL, 500 ng / mL to about 900 ng / mL, about 300 ng / mL to about 900 ng / mL, about 400 ng / mL to about 900 ng / mL, about 550 ng / mL to about 900 ng / mL, about 600 ng / mL to about 900 ng / mL, or about 700 ng / mL to about 900 ng / mL.In some embodiments, the levels of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) are approximately or at least approximately 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 mL, 175ng / mL, 180ng / mL, 185ng / mL, 190ng / mL, 195ng / mL, 200ng / mL, 210ng / mL, 220ng / mL, 230ng / mL, 240ng / mL, 250ng / mL, 260ng / mL, 270ng / mL, 280ng / mL, 290ng / mL, 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, or greater than 400ng / mL. In some embodiments, the levels of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) are approximately or at least approximately 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, and greater than 200 ng / mL. In some embodiments, the levels of at least one heparin sulfate (e.g., D0A0, D0S0, D0A6, and / or D2S6) are approximately or at least approximately 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 embodiments, the level of one or more heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) or the level of D2S6 in a biological sample derived from a subject is approximately or at least approximately compared to a reference level (e.g., the level of one or more heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6)), or the level of D2S6 in a biological sample derived from one or more healthy individuals and / or one or more non-neuropathic subjects, or a predetermined value). 10ng / mL, 20ng / mL, 30ng / mL, 40ng / mL, 50ng / mL, 60ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 150ng / mL, 160ng / mL, 170ng / mL, 180ng / mL, 190ng / mL, 200ng / mL, 210ng / mL, 220ng / mL, 230ng / mL, 240ng / mL, 250ng / mL, 260ng / mL, 270ng / mL, 280ng / mL, 290ng / mL, 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, 410ng / mL, 420ng / mL, 430ng / mL, 440ng / mL, 450ng / mL, 460ng / mL, 470ng / mL, 480ng / mL, 490ng / mL, 500ng / mL, or higher than 500ng / mL.In some embodiments, the total level of heparan sulfate disaccharides (e.g., D0A0, D0S0, D0A6, and / or D2S6) in biological samples derived from the subjects is approximately or at least approximately 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 6 ng / mL, 6 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 5040 ng / mL, 50 ng / mL, 6 ng / mL, 40 ng / mL, 50 ng / mL, 6 ng / mL, 40 ng / mL, 50 ng / mL, 6 ng / mL, 40 ng / mL, 50 ng / mL, 6 ng / mL, 40 ng / mL, 50 ng / mL, 6 ng / mL, 40 ng / mL, 50 ng / mL, 40 ng / mL, 50 ng / mL, 40 ng / mL, 50 ng / mL, 40 ng / mL, 50 ng / mL, 40 ng / mL, 50 ng / mL, 40 ng / mL, 50 ng / mL, 50 ng / mL, 40 ng / mL, 50 ng / mL, 50 ng / mL, 0ng / mL, 70ng / mL, 80ng / mL, 90ng / mL, 100ng / mL, 150ng / mL, 160ng / mL, 170ng / mL, 180ng / mL, 190ng / mL, 200ng / mL, 210ng / mL, 220ng / mL, 23 0ng / mL, 240ng / mL, 250ng / mL, 260ng / mL, 270ng / mL, 280ng / mL, 290ng / mL, 300ng / mL, 310ng / mL, 320ng / mL, 330ng / mL, 340ng / mL, 350ng / mL, 360ng / mL, 370ng / mL, 380ng / mL, 390ng / mL, 400ng / mL, 410ng / mL, 420ng / mL, 430ng / mL, 440ng / mL, 450ng / mL, 460ng / mL, 470ng / mL, 480ng / mL, 490ng / mL, 500ng / mL, 510ng / mL, 520ng / mL, 530ng / mL, 540ng / mL, 550ng / mL, 560ng / mL, 570ng / mL, 580ng / mL, 590ng / mL, 600ng / mL, 610n g / mL, 620ng / mL, 630ng / mL, 640ng / mL, 650ng / mL, 660ng / mL, 670ng / mL, 680ng / mL, 690ng / mL, 700ng / mL, 710ng / mL, 720ng / mL, 730ng / mL, 7 40ng / mL, 750ng / mL, 760ng / mL, 770ng / mL, 780ng / mL, 790ng / mL, 800ng / mL, 850ng / mL, 900ng / mL, 950ng / mL, 1000ng / mL, or greater than 1000ng / mL.

[0271] In some embodiments, a subject is determined to be responsive to the treatment of the Disclosure (e.g., rAAV9 encoding an hIDUA for the treatment of MPS I; construct 2) based on the level of I0S6 in a biological sample derived from the subject. In some embodiments, an elevated level of I0S6 in a biological sample derived from a subject (e.g., a subject having MPS I) compared to a baseline indicates that the subject is responsive to the treatment of the Disclosure (or treatment with rAAV9 encoding an hIDUA for MPS I). In some embodiments, the baseline is the level of I0S6 in a biological sample derived from a healthy person or a population of healthy persons. In some embodiments, the baseline is the level of I0S6 in a biological sample derived from a subject having MPS I or a population of subjects having MPS I. In some embodiments, the baseline is the level of I0S6 in a biological sample derived from a subject not having MPS I or not diagnosed with MPS I, or a population of subjects not diagnosed with MPS I. In some embodiments, the baseline is the level of I0S6 in a biological sample derived from the same subject but obtained at different time points (e.g., at an earlier time point). In some embodiments, the criterion is a predetermined value.

[0272] 5.5.1. Disease Markers In certain embodiments, the effectiveness of therapy using recombinant nucleotide expression vectors is monitored by measuring the levels of the patient's disease biomarker. In certain embodiments, the levels of the disease biomarker are measured in the patient's CSF. In certain embodiments, the levels of the disease biomarker are measured in the patient's serum. In certain embodiments, the levels of the disease biomarker are measured in the patient's plasma. In certain embodiments, the levels of the disease biomarker are measured in the patient's urine. In certain embodiments, the disease biomarker is GAG. In some embodiments, the disease biomarker is I0S6. In preferred embodiments, the disease biomarker is heparan sulfate. In certain embodiments, the disease biomarker is D2S6. The I2S enzyme cleaves sulfate from HS in lysosomes, and the absence of I2S leads to the accumulation of long chains of fully sulfated D2S6. In some embodiments, quantitative measurement of D2S6 reflects the level of I2S enzyme activity, and elevated levels of HS and D2S6 are closely correlated with the neuropathic phenotype of MPS II. In some embodiments, D2S6 levels are inversely correlated with neurocognitive development. In some embodiments, the disease biomarker is an anti-AAV antibody (e.g., an anti-AAV9 antibody). In certain embodiments, the disease biomarker is dermatan sulfate. In certain embodiments, the disease biomarker is IDS enzyme activity. In certain embodiments, the disease biomarker is inflammation. In certain embodiments, the disease biomarker is a safety event.

[0273] In certain embodiments, the effectiveness of therapy using recombinant nucleotide expression vectors is monitored by measuring one or more of the following biomarkers in patient-derived samples: (a) levels of GAG in CSF, (b) levels of I2S in CSF, (c) levels of GAG in plasma, (d) levels of I2S in plasma, (e) levels of leukocyte I2S enzyme activity, (f) levels of GAG in urine, (g) levels of heparan sulfate in CSF, and (h) levels of dermatan sulfate in CSF. In certain embodiments, the effectiveness of therapy using recombinant nucleotide expression vectors is monitored by measuring I2S and / or GAG in CSF, urine, and / or plasma. In certain embodiments, the effectiveness of therapy using recombinant nucleotide expression vectors is monitored by measuring heparan sulfate in CSF, plasma, and / or urine. In certain embodiments, the effectiveness of therapy using recombinant nucleotide expression vectors is monitored by measuring non-reducing heparan sulfate. In some embodiments, heparan sulfate measured in CSF is the primary endpoint for determining the effectiveness of the therapy. In certain embodiments, the efficacy of therapy using recombinant nucleotide expression vectors is monitored by measuring D2S6 in CSF. In some embodiments, D2S6 is measured using any detectable / available assay or biological sample (e.g., CSF, urine, and / or plasma) used to detect D2S6. In certain embodiments, the efficacy of therapy using recombinant nucleotide expression vectors is monitored by measuring urinary total GAG, urinary HS, and / or plasma I2S enzyme activity. In some embodiments, urinary GAG indicates a systemic effect and / or is unrelated to ERT therapy. In some embodiments, the efficacy of the therapy of the Disclosure is determined based on the level of I2S protein concentration in a sample from the subject (e.g., an increase in the level of I2S protein concentration indicates efficacy). In some embodiments, heparan sulfate (HS) and D2S6 (glycosaminoglycan (GAG)) are measured in cerebrospinal fluid (CSF) at baseline and / or after administration of the recombinant vector of the Disclosure.In some embodiments, the determination or monitoring of the efficacy of MPS II treatment in a subject is performed by administering the rAAV of the Disclosure to the subject (for example, then) about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 35 The level of at least one biomarker (e.g., D2S6) is detected and determined in a biological sample derived from the subject obtained after 0, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days, or after 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, MPS in the subject is detected and determined. I. Determination or monitoring of the effectiveness of treatment by administering the rAAV of this disclosure to a subject (for example, then, compared to a baseline) about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 The level of at least one biomarker (e.g., IOS6) is detected and determined in a biological sample derived from the subject obtained after 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 days, or after 1, 2, 3, 4, 5, 6, 7, 8, 10, 16, 20, 24, 30, 35, 40, 45, 48, 50, 52, 56, 104 weeks, or after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0274] In some embodiments, the levels of HS and / or D2S6 are reduced in the subject after administration of the recombinant vector of the Disclosure compared to a reference (for example, the levels of HS and / or D2S6 in the subject before administration of the recombinant vector of the Disclosure, or baseline, or a predetermined value). In some embodiments, the level of HS decreases by approximately or at least approximately 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% after administration of the recombinant vector of the Disclosure, compared to a baseline before discontinuation of ERT (for example, compared to the level of HS in the subject before administration of the recombinant vector of the Disclosure). In some embodiments, after administration of the recombinant vector of the Disclosure, the level of D2S6 decreases by approximately or at least approximately 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% compared to a baseline before discontinuation of ERT (for example, compared to the level of D2S6 in the subject before administration of the recombinant vector of the Disclosure). In some embodiments, the level of I0S6 in the subject is reduced compared to a reference (for example, compared to the level of I0S6 in the subject before administration of the recombinant vector of this disclosure, or compared to a baseline or a predetermined value).In some embodiments, after administration of the recombinant vector of the Disclosure, the level of I0S6 decreases by approximately or at least approximately 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% compared to a baseline before discontinuation of ERT (for example, compared to the level of I0S6 in the subject before administration of the recombinant vector of the Disclosure). In some embodiments, biomarkers (e.g., HS, D2S6, I0S6, total GAG, and / or I2S enzyme) are administered before administration of the recombinant vector of the Disclosure, on the same day as administration of the recombinant vector of the Disclosure, one day after administration of the recombinant vector of the Disclosure, and / or approximately one, two, three, four, five, six, seven, eight, nine, ten, eleven, and twelve weeks after administration of the recombinant vector of the Disclosure. Measurements are taken after weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 30 weeks, 35 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 ​​weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 year, 2 years, or more than 2 years. In some embodiments, biomarkers (e.g., HS, D2S6, I0S6, total GAG, and / or I2S enzyme) are measured before administration of the recombinant vector of the Disclosure, on the same day as administration of the recombinant vector of the Disclosure, and 8 weeks, 16 weeks, 24 weeks, 32 weeks, 40 weeks, 48 ​​weeks, 56 weeks, 72 weeks, 104 weeks, 1 year, 2 years, or more than 2 years after administration of the recombinant vector of the Disclosure. In some embodiments, HS and / or D2S6 are measured in CSF in ng / ml.In some embodiments, biomarkers (e.g., HS, I0S6, and / or D2S6) are present in levels after administration of the recombinant vector of the Disclosure, compared to a baseline (e.g., baseline dose, or dose before treatment with the recombinant vector of the Disclosure), at approximately or at least approximately 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 mg / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, and 60 mg / ml. The levels decrease to ng / ml, 65ng / ml, 70ng / ml, 75ng / ml, 80ng / ml, 85ng / ml, 90ng / ml, 95ng / ml, 100ng / ml, 110ng / ml, 120ng / ml, 130ng / ml, 140ng / ml, 150ng / ml, 160ng / ml, 170ng / ml, 180ng / ml, 190ng / ml, 200ng / ml, 220ng / ml, 250ng / ml, 270ng / ml, 300ng / ml, or more than 300ng / ml. In some embodiments, total GAG (e.g., in urine) is measured in g / mol, CK. In some embodiments, a biomarker (e.g., total GAG) decreases by approximately or at least approximately 5 g / mol, 10 g / mol, 15 g / mol, 20 g / mol, 25 g / mol, 30 g / mol, 35 mg / ml, 40 mg / ml, 45 g / mol, 50 g / mol, 55 g / mol, 60 g / mol, 65 g / mol, 70 g / mol, 75 g / mol, 80 g / mol, 85 g / mol, 90 g / mol, 95 g / mol, 100 g / mol, 110 g / mol, 120 g / mol, 130 g / mol, 140 g / mol, 150 g / mol, 160 g / mol, 170 g / mol, 200 g / mol, or more than 200 g / mol after administration of the recombinant vector of the Disclosure, compared to a baseline (e.g., a baseline amount or an amount before treatment with the recombinant vector of the Disclosure).In some embodiments, biomarkers (e.g., HS, D2S6, I0S6, and / or total GAG) and / or hepatosplenomegaly are reduced after administration of the recombinant vector of the Disclosure by approximately or at least approximately 3%, 5%, 7%, 8%, 9%, 1% compared to, for example, the baseline dose or the dose before treatment with the recombinant vector of the Disclosure, compared to the baseline dose or the dose before treatment with the recombinant vector of the Disclosure. Decreases and / or mitigates by 0%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100%. In some embodiments, the determination of an increase or decrease in the biomarker in a subject is made before discontinuing ERT in the subject.In some embodiments, biomarkers (e.g., HS, IOS6, D2S6, and / or total GAG), and / or hepatosplenomegaly are observed after administration of the recombinant vector of the Disclosure, compared to a baseline (e.g., compared to the level of the biomarker in a subject before administration of the recombinant vector of the Disclosure, or compared to the level of the biomarker from a previously obtained subject-derived biological sample, or compared to baseline, or compared to the level of the biomarker in a healthy person), for approximately the following periods, 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, 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, 3 The period decreases and / or is mitigated for 0 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 longer than 10 years.For example, in some embodiments, biomarkers (e.g., HS, D2S6, I0S6, and / or total GAG) and / or hepatosplenomegaly show a reduction and / or easing of approximately 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% over approximately, at least approximately, or up to approximately 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, compared to, for example, baseline levels of the biomarker or hepatosplenomegaly or levels of the biomarker or hepatosplenomegaly before treatment using the recombinant vector of the Disclosure, or compared to levels of the biomarker or hepatosplenomegaly obtained from the subject at an earlier time. In some embodiments, a decrease in biomarkers (e.g., HS, D2S6, I0S6, and / or total GAG), alleviation of hepatosplenomegaly, and / or an increase in biomarkers (e.g., I2S) occur approximately or at least 2 weeks, 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 Weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, more than 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 ​​months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years are considered to be lasting.

[0275] In some embodiments, the level of a biomarker (e.g., I2S enzyme) increases by approximately or at least approximately 3%, 5%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 88%, 85%, 87%, 90%, 92%, 95%, 97%, 100%, or more than 100% after administration of the recombinant vector of the Disclosure, compared to a baseline (e.g., the level of the biomarker in the subject before administration of the recombinant vector of the Disclosure). In some embodiments, a biomarker (e.g., I2S enzyme) is elevated after administration of the recombinant vector of the Disclosure, compared to a baseline (e.g., compared to the level of the biomarker in a subject before administration of the recombinant vector of the Disclosure), for approximately the following periods: 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. 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 Increases in 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, a biomarker (e.g., I2S enzyme) increases by approximately or at least about 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% over approximately, at least approximately, or up to approximately 2 weeks, 4 weeks, 6 weeks, 8 weeks, 16 weeks, 24 weeks, 48 ​​weeks, 56 weeks, 104 weeks, 2 years, or more than 2 years after administration of the recombinant vector of the Disclosure (e.g., compared to the amount from baseline or the amount before treatment with the recombinant vector of the Disclosure). In some embodiments, a biomarker (e.g., I2S enzyme) increases by approximately or at least approximately 500 pg / ml, 1000 pg / ml, 1500 pg / ml, 10000 pg / ml, 15000 pg / ml, 20000 pg / ml, 25000 mg / ml, 30000 mg / ml, 35000 pg / ml, 40000 pg / ml, 45000 pg / ml, 50000 pg / ml, or more than 50000 pg / ml after administration of the recombinant vector of the Disclosure (e.g., compared to the amount from baseline or the pre-treatment amount using the recombinant vector of the Disclosure).

[0276] In some embodiments, heparan sulfate (HS) glycosaminoglycan (CAG) in CSF is measured using bioanalytical LC / MS. In some embodiments, the non-reducing end of heparan sulfate and total heparan sulfate in CNS are measured using bioanalytical LC / MS. In some embodiments, the non-reducing end of heparan sulfate and total heparan sulfate in plasma are measured using bioanalytical LC / MS. In some embodiments, HS CAG in urine is measured using bioanalytical LC / MS. In some embodiments, the total CAG concentration in urine is measured using a colorimetric assay. In some embodiments, a more detailed description of the available assays is provided in Section 6 of this disclosure.

[0277] In some embodiments, if, after administration of the recombinant vector of the Disclosure to a subject, biomarkers (e.g., D2S6, I0S6, HS, GAG (e.g., total GAG in urine)) increase and / or hepatosplenomegaly worsens and / or biomarker (e.g., I2S) decreases, another dose (e.g., the same dose as the previous dose, a lower dose, or a higher dose) of the recombinant vector of the Disclosure is administered to the subject. In some embodiments, if, after administration of the recombinant vector of the Disclosure to a subject, biomarkers (e.g., D2S6, HS, GAG (e.g., total GAG in urine)) increase and / or hepatosplenomegaly worsens and / or biomarker (e.g., I2S) increase, another therapy or treatment (e.g., a therapy or treatment used for the treatment of MPS II, a therapy or treatment used to treat the symptoms of an affected subject, a therapy or treatment for hepatosplenomegaly, and / or a therapy or treatment used to treat or prevent symptoms derived from MPS II) is administered to the subject. In some embodiments, if, after administering the recombinant vector of the present disclosure to a subject, a biomarker (e.g., I0S6) increases and / or hepatosplenomegaly worsens and / or a biomarker (e.g., I2S) decreases, another therapy or treatment (e.g., a therapy or treatment used for the treatment of MPS I, a therapy or treatment used to treat the symptoms of an affected subject, a therapy or treatment for hepatosplenomegaly, and / or a therapy or treatment used to treat or prevent symptoms derived from MPS I) is administered to the subject.For example, if a biomarker (e.g., D2S6, I0S6, HS, GAG (e.g., total GAG in urine)) increases by approximately or at least approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% compared to a baseline, and / or if hepatosplenomegaly worsens and / or if a biomarker (e.g., I2S) decreases, another dose of the recombinant vector of this disclosure (e.g., the same dose as the previous dose, a lower dose, or a higher dose) is administered to the subject and / or another therapy or treatment (e.g., ERT therapy) is administered to the subject. In some embodiments, the baseline is the control. In some embodiments, the criterion is a biomarker level and / or hepatosplenomegaly obtained from a prior biological sample or image of the subject (e.g., ultrasound). In some embodiments, the criterion is a biomarker level 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 criterion is a biomarker level and / or hepatosplenomegaly in a healthy subject or a subject that does not have MPS II or is undiagnosed. In some embodiments, the criterion is at least one biomarker level and / or hepatosplenomegaly in a biological sample obtained from a subject that has never undergone ERT or has not undergone ERT but is diagnosed with MPS II. In some embodiments, the criterion is a biomarker level and / or hepatosplenomegaly in a subject or population of subjects having MPS II.In some embodiments, after administration of the recombinant vector of the present disclosure, an increase in biomarkers (e.g., D2S6, I0S6, HS, GAG (e.g., total GAG in urine)) and / or exacerbation of hepatosplenomegaly and / or a decrease in biomarkers (e.g., I2S) occurred for approximately, at least approximately, or up to approximately 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13 weeks. 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, 8 0 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 Where permitted over periods of 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, another dose of the recombinant vector of this disclosure (e.g., the same dose as the previous dose, a lower dose, or a higher dose) may be administered for and / or for another therapy or treatment.

[0278] In some embodiments, subjects are receiving or have received enzyme replacement therapy (ERT) prior to and / or in conjunction with the administration of the recombinant vector of the Disclosure. In some embodiments, ERT treatment in subjects is discontinued before administration of the recombinant vector of the Disclosure. In some embodiments, ERT treatment in subjects is discontinued approximately 52 weeks after administration of the recombinant vector of the Disclosure. In some embodiments, ERT treatment in subjects is discontinued approximately 56 weeks after administration of the recombinant vector of the Disclosure. In some embodiments, ERT treatment in a subject is discontinued approximately 52 weeks after administration of the recombinant vector of the present disclosure to the subject, before or after that week (for example, 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 the present disclosure, before or after that period). In some embodiments, a patient may decide to discontinue enzyme replacement therapy (ERT) treatment after administration of the recombinant vector of the Disclosure, using biomarker levels and / or hepatosplenomegaly determination. In some embodiments, an increase in a biomarker (e.g., I2S) after administration of the recombinant vector of the Disclosure (e.g., compared to baseline) indicates that the subject can discontinue ERT treatment. In some embodiments, an increase in a biomarker (e.g., I2S) during the period after administration of the recombinant vector of the Disclosure indicates that the subject can discontinue ERT treatment. In some embodiments, if the subject is negative for anti-AAV antibodies (e.g., a biological sample derived from the subject does not show a detectable level of anti-AAV9 antibody), ERT treatment is discontinued before administration of the recombinant vector of the Disclosure to the subject.In some embodiments, ERT treatment is discontinued approximately or at least approximately 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 the recombinant vector of the Disclosure is administered to the subject. In some embodiments, a decrease in biomarkers (e.g., D2S6, HS, GAG (e.g., total urinary GAG)) and / or relief of hepatosplenomegaly after administration of the recombinant vector of the Disclosure to the subject (e.g., compared to baseline) indicates that treatment of the subject with ERT can be discontinued. In some embodiments, a decrease in biomarkers (e.g., D2S6, I0S6, HS, GAG (e.g., total urinary GAG)) and / or relief of hepatosplenomegaly over a period of time after administration of the recombinant vector of the Disclosure to the subject indicates that treatment of the subject with ERT can be discontinued.In some embodiments, approximately, at least approximately, or up to 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 after administration of the recombinant vector of the Disclosure. Between, 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, 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. A decrease in biomarkers (e.g., D2S6, I0S6, HS, GAG (e.g., total urinary GAG)) and / or relief of hepatosplenomegaly and / or an increase in biomarkers (e.g., I2S) over a period of 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or more than 10 years indicates that treatment of the target with ERT can be discontinued or interrupted. In some embodiments, ERT treatment is interrupted or discontinued if there is a decrease in biomarker levels (e.g., D2S6, I0S6, HS, GAG (e.g., total GAG in urine)) and / or relief of hepatosplenomegaly and / or an increase in a biomarker (e.g., I2S) of approximately 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% compared to baseline levels.In some embodiments, ERT treatment is interrupted or discontinued if biomarker levels (e.g., D2S6, I0S6, HS, GAG (e.g., total GAG in urine)) decrease by approximately or at least approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, or more than 100x compared to a baseline level (e.g., after administration of the recombinant vector of this disclosure). In some embodiments, ERT is discontinued indefinitely. In some embodiments, ERT is 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, Suspend or suspend the service for 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months, 40 months, 48 ​​months, 50 months, 55 months, 60 months, 65 months, 70 months, 75 months, 80 months, 85 months, 90 months, 95 months, 100 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, or more than 10 years. In some embodiments, ERT is interrupted approximately or at least approximately 52 weeks after administration of the recombinant vector of the present disclosure.

[0279] In some embodiments, biomarkers (e.g., I2S, I0S6, D2S6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly are monitored after discontinuation of ERT treatment. For example, in some embodiments, after interruption or discontinuation of ERT treatment and / or administration of the recombinant vector of this disclosure, biomarkers (e.g., I2S, I0S6, D2S6, HS, GAG (e.g., total GAG in urine)) and / or hepatosplenomegaly (e.g., liver or spleen size) are measured for approximately, at least approximately, or every 2, 5, 7, 10, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 weeks. Weeks, 52 weeks, 53 weeks, 54 weeks, 55 weeks, 56 weeks, 57 weeks, 58 weeks, 59 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, Measurements should be taken at 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 beyond 10 years.

[0280] In some embodiments, if biomarkers (e.g., D2S6, I0S6, HS, GAG (e.g., total urinary GAG)) increase and / or hepatosplenomegaly worsens and / or biomarkers (e.g., I2S) decrease after interruption or discontinuation of ERT treatment, ERT treatment is resumed for the subject. For example, if, compared to a baseline (e.g., after administration of the recombinant vector of this disclosure), there is an increase in a biomarker (e.g., D2S6, I0S6, HS, GAG (e.g., total GAG in urine)) and / or an exacerbation of hepatosplenomegaly and / or a decrease in a biomarker (e.g., I2S) of approximately or at least approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%, then the biomarker (e.g., D2S6, I0S6, HS, GAG (e.g., total GAG in urine)) is correct. Treatment with ERT is resumed for the subject for a period of time until G) is reduced or controlled, and / or hepatosplenomegaly is alleviated or controlled, and / or the biomarker (e.g., I2S) is increased or controlled. In some embodiments, the criterion is a control. In some embodiments, the criterion is biomarker levels and / or hepatosplenomegaly obtained from a prior biological sample or image of the subject (e.g., ultrasound). In some embodiments, the criterion is 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 criterion is a healthy person or an MPS The criteria are biomarker levels and / or hepatosplenomegaly in subjects who are not MPS II or are undiagnosed. In some embodiments, the criteria are biomarker levels and / or hepatosplenomegaly in subjects or populations of subjects who have MPS II. In some embodiments, the criteria are biomarker levels and / or hepatosplenomegaly in healthy individuals or subjects who are not MPS I or are undiagnosed. In some embodiments, the criteria are biomarker levels and / or hepatosplenomegaly in subjects or populations of subjects who have MPS I.In some embodiments, the criteria are biomarker levels and / or hepatosplenomegaly in subjects or populations of subjects who are receiving or have received ERT treatment.

[0281] In some embodiments, ERT therapy is discontinued after administration of the recombinant vector of the Disclosure. For example, ERT therapy is discontinued after administration of the recombinant vector of the Disclosure to a subject that has received ERT therapy and has anti-IDS antibody levels (e.g., anti-IDS antibody levels as a result of ERT therapy). In some embodiments, subjects with anti-IDS antibody levels stopped ERT therapy as soon as or immediately after administration of the recombinant vector of the Disclosure to the subject. In some embodiments, anti-IDS antibody levels are determined in a biological sample derived from the subject (e.g., a serum sample). In some embodiments, anti-IDS antibody levels are determined in a biological sample obtained from the subject before administration of the recombinant vector of the Disclosure to the subject. In some embodiments, anti-IDS antibody levels are monitored before and / or after administration of the recombinant vector of the Disclosure to the subject. In some embodiments, if the anti-IDS antibody level is higher than a reference level, ERT therapy is discontinued or stopped. In some embodiments, the reference level is a predetermined level. In some embodiments, the reference level is the anti-IDS antibody level in a prior 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., before 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 that 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 a population of subjects that have MPS II. In some embodiments, the reference level is the anti-IDS antibody level in subjects or a population of subjects that are receiving or have received ERT treatment. In some embodiments, ERT treatment is discontinued when anti-IDS antibody levels increase by approximately or at least approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 250%, 500%, 750%, 1000%, or more than 1000% compared to a baseline level.In some embodiments, ERT treatment is interrupted or discontinued when the anti-IDS antibody level becomes approximately or at least approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, 150x, 250x, 500x, 750x, 1000x, or more than 1000x greater than the reference level. In some embodiments, after ERT treatment, after interruption or discontinuation of ERT treatment, before administration of the recombinant vector of the Disclosure, and / or after administration of the recombinant vector of the Disclosure, the anti-IDS antibody level was reduced to 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, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months, 30 months, 36 months Measured in 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 over 10 years.

[0282] In some embodiments, the efficacy of treatment (e.g., using rAAV9 encoding hIDUA) or monitoring of the efficacy of treatment is based on the level of I0S6. In some embodiments, the level of I0S6 is determined from a biological sample derived from the subject. In some embodiments, the biological sample is plasma. In some embodiments, a decrease in the level of I0S6 after treatment with the recombinant vector of the Disclosure (e.g., rAAV9 encoding hIDUA) compared to a baseline indicates the efficacy of treatment. In some embodiments, the baseline is the level of I0S6 in a biological sample derived from the subject obtained before administration of the recombinant AAV of the Disclosure. In some embodiments, the baseline is a predetermined value. In some embodiments, the baseline is the level of I0S6 in a biological sample derived from another subject having MPS I, or from a population of subjects having MPS I. In some embodiments, the level of I0S6 decreases after administration of the recombinant AAV of the Disclosure compared to before administration of rAAV. In some embodiments, the subject had previously received ERT treatment before administration of rAAV and / or received ERT treatment after administration of rAAV. In some embodiments, a decrease in I0S6 level is a reduction of approximately or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to baseline. In some embodiments, the effectiveness of MPS I treatment is an improvement in at least one subtest of the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III) compared to baseline. In some embodiments, the at least one subtest is the Age Equivalence Score, Cognitive Development Quotient (DQ), Expressive Language DQ, Receptive Language DQ, Gross Motor DQ, and / or Fine Motor DQ. In some embodiments, the effectiveness of MPS I treatment is an improvement in WASI and / or VABS scores.In some embodiments, rAAV9, which encodes hIDUA, is administered to human subjects using a solution containing (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 at a concentration of approximately 0.206 g / L, (e) dextrose at a concentration of approximately 0.793 g / L, (f) poloxamer 188 at a concentration of approximately 0.010 g / L, (g) monobasic phosphate monohydrate at a concentration of approximately 0.0278 g / L, and (h) dibasic anhydride phosphate at a concentration of approximately 0.114 g / L.

[0283] 5.5.2. Hepatosplenomegaly In some embodiments, subjects diagnosed with MPS II have hepatosplenomegaly or experience symptoms associated with hepatosplenomegaly. In some embodiments, hepatosplenomegaly is diagnosed by physical examination. For example, a physician or healthcare worker may examine the area around the abdomen for signs of organ enlargement. In some embodiments, a physician or healthcare worker may feel or palpate the abdominal region of the subject for sensitivity or enlargement of the liver and / or spleen. In some embodiments, diagnostic tests are performed to detect hepatosplenomegaly. For example, hepatosplenomegaly can be detected using blood tests (e.g., liver function tests, hemocytometry, and / or coagulation factor tests). In some embodiments, hepatosplenomegaly is detected using imaging scans such as computed tomography (CT) scans or ultrasound. In some embodiments, hepatosplenomegaly is detected via biopsy.

[0284] In some embodiments, hepatosplenomegaly is measured before, during, and / or after treatment using the recombinant vector of the Disclosure. In some embodiments, hepatosplenomegaly is measured at different time points before, during, and / or after treatment using the recombinant vector of the Disclosure. In some embodiments, relief of hepatosplenomegaly indicates that the treatment is effective. In some embodiments, hepatosplenomegaly is evaluated or monitored to determine the dose, such as dose increase, dose decrease, and / or administration frequency.

[0285] In some embodiments, hepatosplenomegaly is determined before the subject initiates treatment with the Therapy or Therapy of the Disclosure and / or after the subject initiates treatment with the Therapy or Therapy of the Disclosure. In some embodiments, hepatosplenomegaly is determined during medication (for example, before the first dose (i.e., before initiating treatment using the recombinant vector of the Disclosure) and after the subject receives the first dose of the recombinant vector of the 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 treated by administration or initiation of treatment (e.g., by administration targeting the recombinant vector of this disclosure and / or by ERT treatment) over 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, The decision will be made after 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, and more than 10 years.

[0286] In some embodiments, hepatosplenomegaly (e.g., the size of the liver relative to the 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 more than 200% after administration of the recombinant vector of the present disclosure to the target. In some embodiments, hepatosplenomegaly is reduced by about or at least about 1.5, 2, 2.5, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 100, 200, or more than 200 times after administration of the recombinant vector of the Disclosure to the target. In some embodiments, the alleviation of hepatosplenomegaly corresponds to a reduction in the dimensions of the liver and / or spleen. In some embodiments, the alleviation of hepatosplenomegaly corresponds to a reduction in the mass or diameter of the liver and / or spleen. In some embodiments, the alleviation of hepatosplenomegaly corresponds to a reduction in the size of the liver and / or spleen. In some embodiments, the alleviation of hepatosplenomegaly corresponds to administration of the recombinant vector of the Disclosure to the target and / or administration during medication. In some embodiments, hepatosplenomegaly is alleviated after administration and / or ingestion of the recombinant vector of the present disclosure to a subject (for example, after administering a first dose, a second dose, a third dose, a fourth dose, a fifth dose, or several doses).In some embodiments, hepatosplenomegaly is diagnosed after administration of the recombinant vector of the present disclosure, at least about, or up to 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, Relax the restrictions for 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, the relief of hepatosplenomegaly is achieved by administering the recombinant vector of this disclosure to subjects for at least approximately 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, The relief lasts for 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, the relief of hepatosplenomegaly is greater in subjects receiving the treatments of the Disclosures compared to subjects receiving other treatments for hepatosplenomegaly or MPS II. In some embodiments, the relief of hepatosplenomegaly in subjects treated with the recombinant vector of this disclosure is greater than the relief of hepatosplenomegaly in comparable subjects having comparable hepatosplenomegaly who are receiving other treatments used for the treatment of hepatosplenomegaly or MPS II.In some embodiments, the relief of hepatosplenomegaly is about or at least about 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, 200%, or more than 200% greater (compared to the relief of hepatosplenomegaly in subjects receiving another treatment used for the treatment of hepatosplenomegaly or MPS II).

[0287] 5.5.3. Testing of neurocognitive and neurodegenerative functions In certain embodiments, the effectiveness of recombinant vector therapy is monitored by measuring the patient's level of cognitive function. Cognitive function can be measured by any method ...

Claims

1. A method for preventing hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring prevention of such hearing loss, comprising administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS), The method wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents the development of hearing loss in at least one ear.

2. The method according to claim 1, wherein the therapeutically effective dose of rAAV prevents the onset of hearing loss in 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 administration of the rAAV.

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

4. The method according to claim 3, wherein the hearing threshold is an estimated value of the behavioral threshold (eHL) obtained from the ABR threshold (nHL).

5. The method according to claim 3 or 4, further comprising performing an ABR test in at least one ear before and after administration of the rAAV to determine the hearing threshold of at least one ear before and after administration of the rAAV.

6. The method according to any one of claims 1 to 5, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic hearing test.

7. The method according to claim 6, further comprising performing the tympanic membrane audiometry test in at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

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

9. The method according to claim 8, wherein the therapeutically effective dose of rAAV stabilizes the hearing loss or prevents further progression of the hearing loss in 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 administration of the rAAV.

10. The method according to claim 9, wherein stabilization of the hearing loss or prevention of further progression of hearing loss is indicated by the hearing threshold measured by auditory brainstem response (ABR) testing in at least one ear after rAAV administration showing no change or only a slight change compared to the hearing threshold measured by ABR testing in at least one ear before rAAV administration.

11. The method according to claim 10, wherein the hearing threshold is an estimated value of the behavioral threshold (eHL) obtained from the ABR threshold (nHL).

12. The method according to claim 11, wherein stabilization of the hearing loss or prevention of further progression of hearing loss is indicated by the change in eHL in at least one ear after rAAV administration being about 10 dB or less compared to before rAAV administration.

13. The method according to any one of claims 10 to 12, further comprising performing an ABR test in at least one ear before and after administration of the rAAV to determine the hearing threshold of at least one ear before and after administration of the rAAV.

14. The method according to any one of claims 8 to 13, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic hearing test.

15. The method according to claim 14, further comprising performing the tympanic membrane audiometry test in at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

16. A method for improving hearing loss associated with mucopolysaccharidosis type II (MPS II) in a subject requiring improvement of such hearing loss, comprising administering to the subject a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS), The method wherein the subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV improves the hearing loss in at least one ear.

17. The method according to claim 16, wherein the therapeutically effective dose of rAAV improves the hearing of at least one ear about one week, about two weeks, about three weeks, about one month, about two months, about three months, about four months, about five months, about 24 weeks, about six months, about 48 weeks, about 52 weeks, or about one year after administration of the rAAV.

18. The method according to claim 16 or 17, wherein the improvement in hearing is indicated by a decrease in the hearing threshold measured by an auditory brainstem response (ABR) test in at least one ear after administration of rAAV compared with the hearing threshold measured by an ABR test in at least one ear before administration of rAAV.

19. The method according to claim 18, wherein the therapeutically effective dose of rAAV reduces the hearing threshold of 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 at least one ear before administration of the rAAV.

20. The method according to claim 18 or 19, wherein the hearing threshold is an estimated value of the behavioral threshold (eHL) obtained from the ABR threshold (nHL).

21. The method according to claim 20, wherein the improvement of hearing is indicated by a decrease of approximately 10 dB or more in eHL in at least one ear after rAAV administration compared to before rAAV administration.

22. The method according to any one of claims 18 to 21, further comprising performing an ABR test in at least one ear before and after administration of the rAAV to determine the hearing threshold of at least one ear before and after administration of the rAAV.

23. The method according to any one of claims 8 to 22, wherein the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural, as determined by tympanic hearing test.

24. The method according to claim 23, further comprising performing the tympanic membrane audiometry test in at least one ear before and after administration of the rAAV to determine whether the hearing loss is conductive, sensorineural, or a combination of conductive and sensorineural.

25. The method according to any one of claims 1 to 24, wherein the at least one ear includes the left ear or the right ear.

26. The method according to any one of claims 1 to 25, wherein the at least one ear includes a left ear and a right ear.

27. The method according to any one of claims 1 to 26, further comprising performing an additional treatment on the subject.

28. The method according to claim 27, wherein the additional treatment is enzyme replacement therapy comprising recombinant idursulfase.

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

30. The method according to claim 29, wherein the second administration of rAAV is in a larger dose than the first administration of rAAV.

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

32. The method according to any one of claims 1 to 31, wherein the rAAV is administered via intracision cell (IC) administration.

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

34. The method according to any one of claims 1 to 33, wherein the rAAV is administered in an amount not exceeding 10% of the total volume of cerebrospinal fluid of the subject.

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

36. The rAAV is administered to the subject as a solution, and the solution is (a) Sodium chloride at a concentration of approximately 8.77 g / L (b) Magnesium chloride hexahydrate at a concentration of approximately 0.244 g / L, (c) Potassium chloride at a concentration of approximately 0.224 g / L (d) Calcium chloride at a concentration of approximately 0.206 g / L (e) Dextrose at a concentration of approximately 0.793 g / L, (f) Poloxamer 188 at a concentration of approximately 0.010 g / L, (g) Sodium phosphate monobasic monohydrate at a concentration of approximately 0.0278 g / L, (h) The method according to any one of claims 1 to 35, comprising sodium dibasic anhydride at a concentration of approximately 0.114 g / L.

37. The aforementioned rAAV is approximately 1.3 × 10⁻⁶ １０ GC / brain weight in grams: approximately 6.5 x 10 １０ GC / brain weight in grams, or approximately 2 × 10⁻⁶ １１ The method according to any one of claims 1 to 36, wherein the drug is administered in GC / brain weight g, and the brain weight is measured by MRI.

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

39. The method according to any one of claims 1 to 38, wherein the subject is 5 years of age or older and under 18 years of age.

40. The method according to any one of claims 1 to 38, wherein the subject is 4 months of age or older and under 5 years of age.

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

42. The method according to claim 41, wherein the subject is not responding to the ERT.

43. The method according to claim 41 or 42, wherein the ERT comprises recombinant idursulfase.

44. The method according to any one of claims 41 to 43, wherein the ERT is discontinued 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 administration of the rAAV.

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

46. The method according to claim 45, wherein the rAAV comprises a human IDS expression cassette, and 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 according to claim 46, wherein the human IDS expression cassette comprises (i) an IDS transgene adjacent to an inverted terminal repeat sequence (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal.

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

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

45.

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

51. The method according to claim 50, wherein the immunosuppressive therapy comprises administering at least one corticosteroid, sirolimus, and / or tacrolimus.

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

53. The method according to any one of claims 1 to 52, further comprising administering at least one antibiotic to the subject before or simultaneously with the administration of rAAV.

54. The method according to claim 53, wherein the at least one antibiotic is trimethoprim, sulfamethoxazole, pentamidine, dapsone, and / or atobacuone.

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

56. A therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS), for use in subjects requiring prevention of hearing loss associated with mucopolysaccharidosis type II (MPS II), wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents the development of the hearing loss in at least one ear.

57. A therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS), used to stabilize hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring stabilization of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents further progression of the hearing loss in at least one ear.

58. A therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS), used to improve hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring improvement of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV is such that it improves hearing in at least one ear.

59. The use of a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS) for the prevention of hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring prevention of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subjects. The subject has not developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents the development of the hearing loss in at least one ear, as described above.

60. The use of a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS) to improve hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring improvement of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV improves the hearing in at least one ear, as described above.

61. The use of a therapeutically effective dose of recombinant adeno-associated virus vector (rAAV) encoding human iduronide-2-sulfatase (hIDS) to stabilize hearing loss associated with mucopolysaccharidosis type II (MPS II) in subjects requiring stabilization of such hearing loss, wherein the therapeutically effective dose of rAAV is administerable to the subject. The subject has developed hearing loss in at least one ear prior to the administration of the rAAV, and the therapeutically effective dose of the rAAV prevents further progression of hearing loss in at least one ear, as described above.

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

63. The formulation according to claim 62, wherein the rAAV comprises a human IDS expression cassette, and 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 according to claim 63, wherein the human IDS expression cassette comprises (i) an IDS transgene adjacent to an inverted terminal repeat sequence (ITR), (ii) a chicken β-actin intron, and (iii) a rabbit β-globin polyadenylation (polyA) signal.

65. The formulation according to claim 64, wherein the ITR is AAV2 ITR.

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

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