Gene therapy to treat mucopolysaccharidosis type I

Abstract

To provide a gene therapy for treating mucopolysaccharidosis type I.SOLUTION: Provided is a treatment regimen useful for the treatment of α-L-iduronidase deficiency in human patients, the regimen comprising administering to the patient: (a) a recombinant AAV having a nucleic acid, comprising an AAV9 capsid and a sequence encoding human α-L-iduronidase under the control of regulatory sequences that direct its expression in the patient; (b) a first immunosuppressant selected from glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides, or cytostatics; and (c) a second immunosuppressant selected from glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides, or cytostatics.SELECTED DRAWING: Figure 22

Description

[Technical field]

[0001] Declaration of Federally Funded Research This application contains work supported in part by grants from the United States Government, National Institutes of Health (NIH) Nos. R01DK54481, P40OD010939 and P30ES013508. The United States Government may have certain rights in the invention.

[0002] Technical Field The present invention relates to a gene therapy approach for treating Mucopolysaccharidosis Type I (MPS I), including patients diagnosed with Hurler Syndrome, Hurler-Scheie Syndrome and / or Scheie Syndrome. [Background technology]

[0003] Mucopolysaccharidoses are a group of inherited diseases caused by the deficiency of certain lysosomal enzymes involved in the breakdown of glycosaminoglycans (GAGs), also called mucopolysaccharides. The accumulation of partially degraded GAGs leads to disruption of cell, tissue and organ function. Over time, GAGs accumulate within cells, blood and connective tissues, resulting in increasing cell and organ damage.

[0004] MPS I, one of the most severe forms of mucopolysaccharidoses (MPS), is caused by a deficiency of α-L-iduronidase (IDUA). Specifically, α-L-iduronidase is reported to remove terminal iduronic acid residues from two GAGs called heparan sulfate and dermatan sulfate. α-L-iduronidase is found in lysosomes, compartments within cells that digest and recycle many types of molecules. The IDUA gene is reported to provide the instructions for producing α-L-iduronidase, which is essential for the breakdown of large sugar molecules called glycosaminoglycans (GAGs). Over 100 mutations in the IDUA gene have been found, causing mucopolysaccharidoses type I (MPS I). Mutations that change one DNA component (nucleotide), single nucleotide polymorphisms or "SNPs," are the most common.

[0005] Mutations causing MPS I reduce or completely eliminate the function of α-L-iduronidase, leading to three clinical syndromes: Hurler syndrome, Hurler-Scheie syndrome, and Scheie syndrome. Each is inherited in an autosomal recessive manner, with the degree of enzyme deficiency directly related to the severity of the clinical phenotype. Hurler syndrome represents the most severe manifestation of IDUA deficiency and usually occurs in the setting of a complete lack of enzyme activity due to two null mutations. Clinical diagnosis is established before the age of 2 years and is associated with multiple physical pathologies. In addition, natural history data confirm that patients with the Hurler syndrome genotype have CNS involvement leading to severe cognitive impairment and mental retardation. Hurler-Scheie syndrome is a more attenuated form that is usually diagnosed between the ages of 2 and 8 years. In contrast to Hurler syndrome, patients with Hurler-Scheie syndrome have a small amount of residual IDUA activity (theoretically) leading to a later onset of clinical manifestations and a more attenuated progression of the disease. Despite the more attenuated phenotype, some Hurler-Scheie patients experience multiple symptoms of CNS pathology associated with IDUA deficiency, including neurocognitive decline as evidenced by reduced IQ. Scheie syndrome is the mildest form of MPS I. Symptoms generally begin to appear after age 5, and the diagnosis is most commonly made after age 10. Children with Scheie syndrome may have normal intelligence or mild learning disabilities; some may have psychiatric problems. Glaucoma, retinal degeneration, and corneal opacities can significantly impair vision. Other problems include carpal tunnel syndrome or other nerve compression, joint stiffness, claw hands and deformed feet, short neck, and aortic valve disease. Some affected individuals also have obstructive airway disease and sleep apnea. Individuals with Scheie syndrome can live into adulthood.

[0006] In terms of clinical syndrome, the current standard of care for Hurler syndrome is hematopoietic stem cell transplantation (HSCT), e.g., bone marrow transplantation (BMT) or umbilical cord blood transplantation (UCBT). Treatment is performed as early as possible, before the age of 2 years, and affects both the physical and CNS aspects of the disease. However, HSCT for MPS I remains associated with a significant amount of morbidity and a mortality rate of 20%. If transplantation is not an option, then enzyme replacement therapy (ERT) may be initiated, which requires weekly infusions of enzymes to save the patient's life. ERT does not affect the progression of the CNS disease, but does partially improve the physical manifestations. Although disease aspects in the skeletal system, eyes, and heart are only partially improved, organomegaly is significantly improved. Patients may require surgery to stabilize the hips and knees and to treat carpal tunnel syndrome and finger retraction. Although surgery may ultimately be required, the heart disease is treated medically.

[0007] ERT for MPS I provides exogenous enzymes for uptake into lysosomes and increased catabolism of GAGs. Lysosomal enzymes function internally, but cell surface mannose-6-phosphate receptors can bind, internalize, and deliver these enzymes to lysosomes. Recombinant IDUA (Aldurazyme®, BioMarin) has been approved by the FDA for patients with the Hurler and Hurler-Scheie forms of MPS I, as well as for patients with the Scheie form who have moderate to severe symptoms and has been shown to improve lung function and walking ability. ERT has also been observed to reduce liver swelling and urinary GAG levels in patients with MPS I. However, because intravenous enzymes do not easily cross the brain, ERT does not currently address the neurological symptoms experienced by some MPS I patients.

[0008] The complexities of ERT revolve around lifelong complications of peripheral administration such as local and systemic inflammation along with immune responses to the recombinant enzyme that can range from mild to full-blown anaphylaxis. Up to 91% of patients receiving aldurazazidime develop antibodies to the enzyme, although it is unclear how much this affects efficacy. Furthermore, ERT requires weekly iv infusions administered over 3-8 hours in the hospital, which significantly impacts the patient's quality of life, is expensive, and is a major burden on the medical reimbursement system.

[0009] In view of these limitations, treatments that can more effectively correct the pathology associated with MPS I remain an unmet medical need. Summary of the Invention

[0010] Provided herein is a replication-deficient adeno-associated virus ("AAV") for delivery of the human alpha-L-iduronidase (hIDUA) gene to the CNS of a patient (human subject) diagnosed with Mucopolysaccharidosis Type I (MPS I). The recombinant AAV (rAAV) vector (rAAV.hIDUA) used to deliver the hIDUA gene should have tropism for the CNS (e.g., an rAAV carrying an AAV9 capsid), and the hIDUA transgene should be controlled by a specific expression control element, e.g., a hybrid of the cytomegalovirus (CMV) enhancer and chicken β-actin promoter (CB7). Pharmaceutical compositions suitable for intrathecal and intracapsular administration include a suspension of the rAAV.hIDUA vector in a formulation buffer that includes a physiologically compatible aqueous buffer, a surfactant, and optional excipients.

[0011] Provided is a therapeutic regimen useful for treating α-L-iduronidase deficiency in a human patient. In a particular embodiment, the regimen comprises: (a) a recombinant AAV (rAAV) having an AAV9 capsid and a human α-L under the control of regulatory sequences that direct its expression in the patient; - a nucleic acid comprising a sequence encoding iduronidase (hIDUA), wherein the human hIDUA coding sequence has a nucleotide sequence of SEQ ID NO: 1 encoding a functional hIDUA or a sequence that is at least about 80% identical to SEQ ID NO: 1; (b) at least a first immunosuppressant selected from at least one of a glucocorticoid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent; and (c) at least a second immunosuppressant selected from at least one of a glucocorticoid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent, wherein administration of at least one of the immunosuppressants begins prior to or on the same day as delivery of the AAV vector; and administration of at least one of the immunosuppressants continues for at least 8 weeks after vector administration. The patient may be initially administered intravenous steroids, followed by oral steroids. In certain embodiments, the immunosuppressant is one or more corticosteroids, and optionally, mycophenolate mofetil (MMF), and / or one or more macrolides. The one or more macrolides can be a calcineurin inhibitor (e.g., tacrolimus), an mTOR inhibitor (e.g., sirolimus, temsirolimus, everolimus, or another rapalog), or a combination thereof. In certain embodiments, administration of steroids to the patient is discontinued 12 weeks after vector administration. In certain embodiments, mycophenolate mofetil (MMF) and tacrolimus are delivered 0-15 days prior to vector administration. In certain embodiments, the immunosuppressant is mycophenolate mofetil (MMF) and sirolimus. In certain embodiments, when the immunosuppressant includes both tacrolimus and sirolimus, low doses of each are used to maintain blood trough levels of about 4 ng / mL to about 8 ng / mL, or a total of about 8 ng / mL to about 16 ng / mL. In certain embodiments where the adjuvant comprises only tacrolimus or sirolimus, the total dose ranges from about 16 ng / mL to about 24 ng / mL. In certain embodiments where only tacrolimus or sirolimus is used, the initial loading dose is about 3 mg / m 2 Specific In certain embodiments, immunosuppressive therapy is initiated from about -14 days to about -1 days prior to vector administration. In certain embodiments, the encoded hIDUA has a sequence selected from a synthetic human enzyme comprising a heterologous leader sequence fused to (a) amino acids from about 1 to about 653 of SEQ ID NO:2 (Genbank NP_000193); and (b) amino acids from about 27 to about 653 of SEQ ID NO:2. In certain embodiments, the nucleic acid sequence further comprises a 5' inverted terminal repeat (ITR) sequence, a chicken beta actin intron, a CB7 promoter, a polyA signal, and / or a 3' ITR sequence. In certain embodiments, the rAAV is in a suspension having a pH of 6 to 9. In certain embodiments, the rAAV is delivered via intrathecal injection. In certain embodiments, the rAAV comprising the hIDUA gene is administered intravenously. In certain embodiments, the efficacy of the treatment is assessed by measuring hearing ability changes, optionally by auditory brainstem testing. In certain embodiments, the rAAV is administered to a human subject (i) from about 1.2×10 12 ~Approx. 6.0×10 12 GC or approximately 6.0 x 10 12 ~Approx. 3.0×10 13 GC, (ii) about 2 × 10 in human subjects ≥ 9 months to < 18 months of age 12 ~Approx. 6.0×10 13 or about 1.0 x 10 13 ~Approx. 5.0×10 13 GC, (iii) about 2.2 × 10 in human subjects ≥ 9 months to < 18 months of age 12 ~Approx. 1.1×10 13 GC or approximately 1.1 x 10 13 ~Approx. 5.5×10 13 It is formulated for intrathecal injection into human subjects to administer a total plateau dose of GC. (a) Approximately 1.2 × 10 in human subjects aged ≥ 4 months to < 9 months 12 ~Approx. 6.0×10 12 GC or approximately 6.0 x 10 12 ~Approx. 3.0×10 13 GC; or (b) about 2 × 10 12 ~Approx. 6.0×10 13 GC or approximately 1.0 x 10 13~Approx. 5.0×10 13 GC; or (c) about 2.2 × 10 12 ~Approx. 1.1×10 13 GC or approximately 1.1 x 10 13 ~Approx. 5.5×10 13 Compositions are provided that include a recombinant AAV vector comprising a heterologous nucleic acid encoding human α-L-iduronidase (hIDUA) formulated for intrathecal injection into a human subject in need thereof to administer a total flat dose of GC. In certain embodiments, the human hIDUA coding sequence has the nucleotide sequence of SEQ ID NO: 1, which encodes a functional hIDUA, or a sequence that is at least about 80% identical to SEQ ID NO: 1. In certain embodiments, the composition comprises: (i) a glucocorticoid; and (ii) at least a first immunosuppressant selected from at least one of a steroid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent, and co-therapy with at least a second immunosuppressant selected from at least one of the foregoing, wherein administration of the immunosuppressant begins prior to or on the same day as delivery of the vector; and administration of at least one of the immunosuppressants continues for at least 8 weeks after administration of the vector.

[0012] Immunosuppressants are provided for use in combination therapy with a recombinant AAV vector comprising a heterologous nucleic acid encoding human alpha-L-iduronidase (hIDUA). In certain embodiments, the human hIDUA coding sequence has a nucleotide sequence of SEQ ID NO: 1 encoding a functional hIDUA, or a sequence at least about 80% identical to SEQ ID NO: 1. In certain embodiments, the immunosuppressant comprises (a) a composition comprising at least a first immunosuppressant selected from at least one of a glucocorticoid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent; and (b) a composition comprising at least a second immunosuppressant selected from at least one of a glucocorticoid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent, wherein administration of the immunosuppressant begins prior to or on the same day as delivery of the AAV vector, and administration of at least one of the immunosuppressants continues for at least 8 weeks after vector administration. In certain embodiments, the AAV vector is administered to a human subject (i) at least about 1.2×10 12 ~Approx. 6.0×10 12 GC or approximately 6.0 x 10 12 ~Approx. 3.0×10 13 GC, or (ii) about 2 × 10 in human subjects ≥ 9 months to < 18 months of age 12 ~Approx. 6.0×10 13 GC or approximately 1.0 x 10 13 ~Approx. 5.0×10 13 GC, or (iii) about 2.2 × 10 in human subjects ≥ 9 months to < 18 months of age 12 ~Approx. 1.1×10 13 GC or approximately 1.1 x 10 13 ~Approx. 5.5×10 13 To administer a total plateau dose of GC, it is formulated for intrathecal injection into a human subject in need thereof.

[0013] Such rAAV.hIDUA vector preparations can be administered to human subjects by intrathecal / intracascular injection to achieve therapeutic levels of hIDUA expression in the CNS. Patients who are candidates for treatment include pediatric and adult patients with MPS I and / or Hurler, Hurler-Scheie, and Scheie-related conditions.

[0014] Such rAAV.hIDUA vector preparations can be administered to human subjects by intrathecal / intracascular injection to achieve therapeutic levels of hIDUA expression in the CNS. Patients who are candidates for treatment include pediatric and adult patients with MPS I and / or Hurler, Hurler-Scheie, and Scheie-related conditions.

[0015] The therapeutically effective intrathecal / intracapsular dose of rAAV.hIDUA for MPS I patients is approximately 1 × 10 11 From 7.0×10 14 GC (flat dose) - 10 9 ~5×10 10 GC / Patient Alternatively, the following therapeutically effective plateau doses can be administered to patients in the indicated age groups: Newborn: Approx. 1 x 10 11 ~Approx. 3×10 14 G.C.; 3 to 9 months: approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; 9 months to 6 years: Approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; Ages 3 to 6: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.; 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0016] In another embodiment, the following therapeutically effective flat-line doses are administered to MPS patients in the following age groups: Newborn: Approx. 3.8 x 10 12 ~Approx. 1.9×10 14 G.C.; 3 to 9 months: approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; 9 months to 36 months: 10 13 ~Approx. 5×10 13 G.C.; Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 3-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.; 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0017] In certain embodiments, one or more of these ranges are used for patients of any age, and 1.2×10 12 Total genome copies (GC) (2.0 × 10 9 / g brain mass) or 6 × 10 12 Total GC (1×10 10 In certain embodiments, a dose of 2×10 GC / g brain mass is administered to patients aged ≧4 months and <9 months. 12 Total GC (2.0 × 10 9 GC / g brain mass) or 1×10 13 Total GC (1 × 10 10 In certain embodiments, a plateau dose of 2.2×10 GC / g brain mass is administered to patients ≥ 9 months and < 18 months of age. 12 Total GC (2.0 × 10 9 GC / g brain mass) or 1.1 × 10 13 Total GC (1 × 10 10GC / g brain matter In a particular embodiment, a plateau dose of 6.0×10 12 (1.0×10 10 GC / g brain mass) or 3 × 10 13 Total genome copies (GC) (5 × 10 10 In certain embodiments, a plateau dose of 1.0×10 GC / g brain mass is administered to patients ≥4 months and <9 months of age. 13 (1.0×10 10 GC / g brain mass) or 5.0 × 10 13 Total GC (5 × 10 10 In certain embodiments, a plateau dose of 1.1×10 GC / g brain mass is administered to patients ≥ 9 months and < 18 months of age. 13 (1.0×10 10 GC / g brain mass) or 5.5 × 10 13 Total GC (5 × 10 10 In certain embodiments, a plateau dose of 2.6×10 GC / g brain mass is administered to patients ≥ 18 months and < 3 years of age. 12 Genome copies (GC) (2.0 × 10 9 GC / g A plateau dose of 1.3×10 13 (GC)(1.0×10 10 A plateau dose of 1.4×10 GC / g brain mass is administered to patients aged 6 or older. In some embodiments, the dose administered to patients aged 12 years and older (including 18 years and older) is 1.4×10 13 Genome copies (GC) (1.1 × 10 10 In some embodiments, the dose administered to MPS I patients aged 12 years or older (including 18 years or older) is 7×10 13 GC(5.6×10 10 GC / g brain mass). Moreover, in a further embodiment, the dose administered to the MPS I patient is at least about 4×10 8 GC / g brain mass ~ approx. 4×10 11 GC / g brain mass. In certain embodiments, MP The dose administered to SI neonates is approximately 1.4 × 10 11 from about 1.4 × 10 14 The dose administered to infants aged 3 to 9 months is approximately 2.4 × 10 11 From about 2.4 × 10 14 The dose administered to children with MPS I aged 9 to 36 months is approximately 4 × 10 11 From about 4 × 10 14 The dose administered to children aged 3 to 12 years with MPS I is approximately 4.8 × 10 11 From about 4.8 x 10 14 The dose administered to children aged 12 years and older and adults is approximately 5.6 × 10 11 From about 5.6 × 10 14 This extends to GC.

[0018] The goal of treatment is to functionally replace the patient's defective α-L-iduronidase via rAAV-based CNS-directed gene therapy to treat the disease. The efficacy of the treatment can be measured by (a) prevention of neurocognitive decline in MPS I patients, and (b) by assessing biomarkers of disease, such as GAG levels and / or IDUA or hexosaminidase (Hex) enzyme activity in CSF, serum, and / or urine, and / or liver and spleen volumes. Neurocognition can be assessed by measuring intelligence quotient (IQ), for example, as measured by the Bayley Scales of Infant Development for Hurler subjects, or by the Wechsler Abbreviated Scale of Intelligence (WASI) for Hurler-Scheie subjects. Other suitable measures of neurocognitive development and function may be utilized, such as assessing developmental quotient (DQ) using the Bayley Scales of Infant Development (BSID-III), assessing memory using the Hopkins Verbal Learning Test, and / or using the Test of Variables of Attention (TOVA). Changes in hearing ability as measured by auditory brainstem response (ABR) testing.

[0019] Prior to treatment, MPS I patients can be assessed for neutralizing antibodies (Nab) against the capsid of the rAAV vector used to deliver the hIDUA gene. Such Nabs impede transduction efficiency and reduce the effectiveness of treatment. MPS I patients with baseline serum Nab titers of ≦1:5 are good candidates for treatment with rAAV.hIDUA gene therapy protocols. Treatment of MPS I patients with serum Nab titers >1:5 may require combination therapy, e.g., temporary co-treatment with immunosuppressants, prior to and / or during treatment with rAAV.hIDUA vector delivery. Optionally, immunosuppressant co-treatment may be used as a preventative measure without prior assessment of neutralizing antibodies against the AAV vector capsid and / or other components of the formulation. In certain embodiments, prior immunosuppressive therapy may be desirable to prevent adverse immune reactions to the product of the hIDUA transgene, especially in patients who do not actually have a level of IDUA activity, in which the product of the transgene may be viewed as "foreign." The results of preclinical studies in mice, dogs, and NHPs described in the Examples below are consistent with the development of immune responses and neuroinflammation to hIDUA. Although a similar response may not occur in human subjects, immunosuppressive therapy is recommended for all recipients of rAAV-hIDUA as a precautionary measure.

[0020] The combination of gene therapy delivery of rAAV.hIDUA to the CNS with systemic delivery of hIDUA is encompassed by the methods of the invention. Systemic delivery can be achieved using ERT (e.g., using Aldurazazime®) or by additional gene therapy with a liver-tropic rAAV.hIDUA (e.g., rAAV.hIDUA carrying an AAV8 capsid).

[0021] In certain embodiments, where the patient is an infant, child and / or adult who expresses therapeutic levels of hIDUA in the CNS, the patient is administered AAV.hIDUA via a liver-directed infusion to tolerize the patient to hIDUA, followed by administration of AAV.hIDUA via an intrathecal / intracascular infusion.

[0022] The benefits of the present invention are illustrated by the following examples, which demonstrate that IT administration of rAAV9.IDUA in animal studies resulted in widespread distribution of the vector within the CNS. Furthermore, a single dose of rAAV9 vector delivering fIDUA, cIDUA or hIDUA was successful in dose-dependently ameliorating or completely reversing histological and biochemical findings of CNS-associated MPS I in both 3-7 month old cat and 1 month old dog animal models. Similarly, a single IT dose of rAAV9.IDUA was clinically well tolerated in monkeys, including those injected as infants, for at least 2 years after injection. The only adverse effects associated with rAAV9.IDUA administration in animals were related to immune responses to the transgene across all species examined.

[0023] As shown in the Examples, the beneficial effects of rAAV9.IDUA treatment were limited by the development of anti-IDUA antibody responses. At the highest dose evaluated in non-tolerized MPS I dogs (Example 3) or rhesus monkeys (Example 7), adverse effects were observed. The variability in onset and time profile indicates that these effects are mediated by an immune response to the transgene product. In Example 3, both dogs at the highest dose had high CSF WBC and protein levels with hind limb weakness. These animals had histopathological lesions in the spinal cord and dorsal root ganglia due to an immune response to the transgene expressed in motor and sensory neurons that were not observed in animals at the two lower doses. In a toxicity study in rhesus monkeys (Example 7), both humoral and cell-mediated immune responses to hIDUA were observed, characterized by elevated nucleated cell counts and anti-IDUA antibodies in the CSF, and weak anti-IDUA T cell responses in the peripheral blood. Unlike dogs, no adverse clinical signs occurred, no neuronal necrosis was observed in the spinal cord, and monkeys appeared to tolerate the treatment. However, CNS lesions consisting of bilateral axonal degeneration in the dorsal columns of white matter were observed in the spinal cord at days 90 and 180. These axonal changes are thought to be secondary to immune-mediated effects on neurons in the dorsal root ganglion.

[0024] Acquired immune responses to human proteins observed in non-clinical species may not predict the nature or magnitude of the same response in humans. Nonetheless, in a preferred embodiment, human subjects should be prophylactically treated with immunosuppressants, particularly individuals who do not express hIDUA and are therefore not expected to be tolerant to the enzyme. The data in the Examples below show that sustained transduction and expression of IDUA was achieved in neonatal dogs and non-human primates tolerized to cIDUA or hIDUA prior to administration of the rAAV9.IDUA construct. In contrast, animals not pre-tolerized to IDUA generally mounted immune responses to both IDUA and AAV9 capsid antigens. In summary, the data in the Examples show that immunosuppressive treatment can also enhance the efficacy of rAAV9.IDUA.

[0025] In some embodiments, subjects receiving rAV9.IDUA are also treated with corticosteroids (methylprednisolone 10 mg / kg IV, starting with one predose on day 1 and oral prednisone 0.5 mg / kg / day on day 2, gradually tapered and discontinued by week 12), tacrolimus (1.0 mg PO twice daily, days 2-24), tapered over 8 weeks between weeks 24-32, and sirolimus (loading dose 1 mg / m 2 , 4 hours x 3 times, -2nd day, then -1st day: Shirori Mus 0.5mg / m 2 / day, divided twice a day, target blood concentration 4-8ng / ml at 48 weeks In some embodiments, the initial concomitant immunosuppressive therapy is tapered sequentially, first with discontinuation of prednisone, followed by tacrolimus and finally sirolimus.

[0026] Further aspects and advantages of the present invention will be apparent from the detailed description of the invention. [Brief description of the drawings]

[0027] [Figure 1] 1 is a schematic diagram of a vector genome packaged into an AAV as described herein, depicting the major components of the expression cassette flanked by the AAV 5' and 3' inverted terminal repeats (ITRs) of the vector genome, including the cytomegalovirus immediate early enhancer, CB7 promoter, chimeric intron, coding sequence (gene) for human α-L-iduronidase, and rabbit β-globin poly A signal. [Diagram 2] Figure 2A shows IDUA activity in CSF in native MPS I dogs (Figure 2A) or tolerized MPS I dogs (Figure 2B) treated with intramedullary injection with AAV9 expressing human IDUA. Dogs were treated at 1 month of age by intramedullary injection of vector into the cisterna magna. IDUA activity was measured in subsequent CSF samples. The vector dose (GC / kg) is indicated for each animal. The dashed line represents animals treated with intramedullary vector only. The solid line with black symbols represents animals pretreated on postnatal day 5 with intravenous AAV8 expressing human IDUA from a liver-specific promoter. The solid line with white symbols represents animals pretreated on postnatal days 7 and 14 with intravenous infusion of recombinant human IDUA. Animals I-665 and I-666 were euthanized on day 36 due to neurological signs. The horizontal dashed line represents the mean IDUA activity in CSF in normal dogs. The dotted line indicates the assay limit of quantification. [Diagram 3] Figure 1 shows CSF antibody titers against human IDUA. Antibody titers against human IDUA were measured by ELISA in CSF samples taken 50 days after vector administration. CSF samples examined from I-665 and I-666 were collected at necropsy (36 days after injection). Error bars = SEM. Antibody titers were significantly lower (Mann-Whitney test) in animals pretreated as neonates with AAV8 vectors (I-652, I-653, I-602, I-607, I-601, I-606) or recombinant human IDUA (I-663, I-664) compared to controls treated with IT vectors only (I-604, I-608, I-605, I-665, I-666). [Figure 4]Figure 4: CSF nucleated cell counts following intramedullary injection of AAV9. Total nucleated cell counts were measured in CSF samples from animals treated as neonates with systemic recombinant human IDUA (I-663 and I-664) or AAV8 vector expressing IDUA prior to receiving intramedullary AAV9 (Figure 4B) as well as naive dogs treated with intramedullary AAV9 (Figure 4A). Twenty-one days after vector injection, nucleated cell counts were significantly increased (Mann-Whitney test) in naive animals compared to those treated as neonates with AAV8 vector or recombinant human IDUA. [Diagram 5] Figure 1 shows normalization of brain hexosaminidase activity in MPS I dogs tolerant to human IDUA treated with intramedullary AAV9. Hex activity was measured in samples taken from six brain regions (frontal cortex, temporal cortex, occipital cortex, hippocampus, medulla and cerebellum). Average activity is shown for normal control dogs, untreated MPS I dogs and eight dogs tolerant to hIDUA treated with intramedullary injection of AAV9 expressing human IDUA. Open symbols indicate animals tolerized by infusion of recombinant human IDUA. Hex activity was significantly reduced in the high dose cohort compared to untreated controls (Kruskal-Wallis test followed by Dunn's multiple comparison test). [Figure 6] Figure 6 shows dose-dependent correction of brain accumulation pathology in dogs tolerant to human IDUA treated with intrathecal injection of AAV9 expressing human IDUA. Brains were sectioned and stained for LIMP2 and GM3. Meningeal GAG accumulation was visualized using Alcian blue staining. Automated quantification of GM3 (Figure 6A) and LIMP2 (Figure 6B) positive cells was performed on cortical brain images (n=10 per animal). Open symbols indicate animals tolerized by infusion of recombinant human IDUA. GM3 and LIMP2 were significantly reduced in the high dose cohort compared to untreated controls (Kruskal-Wallis test followed by Dunn's multiple comparison test). [Figure 7]Figure 1 shows partial normalization of brain hexosaminidase activity in naive dogs treated with intramedullary injection of AAV9 expressing human IDUA. Hexosaminidase activity was measured in samples taken from six brain regions (frontal cortex, temporal cortex, occipital cortex, hippocampus, medulla and cerebellum). Mean activity is shown for normal control dogs, untreated MPS I dogs, and dogs treated with intramedullary AAV9 expressing human IDUA at a dose of 1012GC / kg or 1011GC / kg at 1 month of age. [Figure 8] Figure 1 shows normalization of CSF hexosaminidase activity following IT AAV9 treatment in dogs tolerant to hIDUA. Hex activity was measured in the CSF of MPS I dogs tolerized to human IDUA at the end of the study. Open symbols indicate animals tolerized by infusion of recombinant human IDUA. CSF Hex activity was significantly reduced in all treated animals compared to untreated MPS I controls (Mann-Whitney test). [Figure 9] Figure 1. Resolution of cervical meningeal thickening in hIDUA-tolerant dogs treated with intramedullary AAV9 expressing human IDUA. Mean total meningeal thickness was measured in H&E stained sections of cervical spinal cord. Open symbols indicate animals tolerized by infusion of recombinant human IDUA. Meningeal thickness was significantly reduced in all treated animals compared to untreated MPS I controls (Mann-Whitney test). [Figure 10]Comparison of enzyme expression and correction of brain accumulation pathology in MPS I mice treated with AAV9 IT. MPS I mice were treated at 2-3 months of age by ICV injection of AAV9.CB.hIDUA at one of three doses: 3x108 GC (low), 3x109 GC (medium), or 3x1010 GC (high). (FIG. 10A) One cohort of animals sacrificed 3 weeks after vector injection, and brains were harvested for measurement of IDUA activity. (FIG. 10B) A second cohort of animals sacrificed 3 weeks after injection, and brains were stained for the lysosomal membrane protein LIMP2. Cells staining positive for LIMP2 were quantified in four cortical brain sections by a blinded reviewer. *p<0.05, one-way ANOVA followed by Dunnett's test. [Figure 11] 1 is a flowchart of a manufacturing process. [Figure 12] FIG. 1 illustrates a device (10) for intracisternal delivery of a pharmaceutical composition including a 10 cc vector syringe (12), a 10 cc pre-filled water flush syringe (14), a T-connector extension set (including tubing (20), clip (22) and connector (24) at the end of the tubing), a 22G x 5" subarachnoid puncture needle (26), and optional introducer needles for the coaxial insertion technique, including an optional 18G x 3.5" introducer needle (28). Also illustrated is a 4-way stopcock (16) with a swivel male luer lock. [Figure 13] FIG. 1 shows a schematic illustration of intracapsular injection. [Figure 14]FIG. 14 illustrates encephalitis and transgene-specific T cell responses in dogs treated with AAV9 ICV. One-year-old MPS I dogs were treated with ICV or IC injection of AAV9 expressing GFP. All animals were sacrificed 14 days after injection, except for I-567, which was found dead 12 days after injection. Brains were sectioned into coronal sections, which showed gross lesions proximal to the injection site (arrowheads) in ICV-treated animals. Histological sections of brain regions surrounding the gross lesions were stained with hematoxylin and eosin. Peripheral blood mononuclear cells were harvested from one ICV-treated dog (I-565) at the time of necropsy, and T cell responses against AAV9 capsid and GFP protein were measured by interferon-γ ELISPOT (FIG. 14). T cell responses against the GFP transgene product were measured using a single pool of overlapping 15 amino acid long peptides covering the complete GFP sequence. Peptides containing the AAV9 capsid protein were divided into three pools (designated pools A–C). * = positive reaction defined as >3-fold above background (unstimulated cells) and >55 spots per million cells. Ionomycin with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) served as positive controls for T cell activation. [Figure 15] 1 is a bar graph showing vector biodistribution in dogs treated with AAV9 ICV or IC. Dogs were sacrificed 14 days after injection with a single ICV or IC injection of AAV9 vector expressing GFP, except for animal I-567, which was necropsied 12 days after injection. Vector genome was detected in tissue samples by quantitative PCR. Values ​​are expressed as copies of vector genome per diploid cell (GC / diploid genome). Brain samples taken from the hippocampus or cerebral cortex are shown as injected or uninjected hemisphere for ICV-treated dogs, and for IC-treated animals, these are the right and left hemisphere, respectively. No samples were taken for PCR from the injected brain hemisphere of animal I-567. [Figure 16]Figure 1 is a bar graph showing vector biodistribution in NHPs treated with intramedullary AAV9. NHPs were sacrificed 14 days after intramedullary injection of AAV9 diluted in 5 mL of iohexol 180 via lumbar puncture. Two of the animals were placed in Trendelenburg position for 10 min after injection. Vector genome was detected in tissue samples by quantitative PCR. Values ​​are expressed as copies of vector genome per diploid cell (GC / diploid genome). [Figure 17] Figure 17. Elevated CSF spermine in MPS I. High-throughput LC / MS and GC / MS metabolite assessment was performed on CSF samples from MPS I dogs (n=15) and normal controls (n=15). (17A) Heatmap of the top 100 differentially detected metabolites (ANOVA). The youngest animal (28 days old) in the MPS I cohort is indicated by an asterisk. (17B) Graph showing spermine concentrations measured by quantitative isotope dilution LC / MS assay in CSF samples from six MPS I juveniles and two normal juveniles. [Figure 18]Figure 18. Spermine-dependent aberrant neurite outgrowth in MPS I neurons. Cortical neurons harvested from E18 wild-type or MPS I mouse embryos were plated 24 hours prior to treatment with spermine (50 ng / mL) or the spermine synthase inhibitor APCHA. Neurite number, length, and branching were quantified by a blinded reviewer for 45-65 randomly selected neurons from duplicate cultures per treatment condition. (Figure 18A) Bar graphs providing neurites for MPSI, MPSI+APCHA, or MPSI+APCHA+spermine compared to wild type. (Figure 18B) Bar graphs providing branch points for MPSI, MPSI+APCHA, or MPSI+APCHA+spermine compared to wild type. (Figure 18C) Bar graphs providing branch lengths for MPSI, MPSI+APCHA, or MPSI+APCHA+spermine compared to wild type. ***p<0.0001 (ANOVA followed by Dunnett's test). (FIG. 18D) Bar graph comparing neurites / cell for wild type treated with spermine compared to wild type. (FIG. 18E) Bar graph comparing branch points / cell for wild type treated with spermine compared to wild type. (FIG. 18F) Bar graph comparing branch length / cell for wild type treated with spermine compared to wild type. [Figure 19]Figure 19 shows normalization of CSF spermine levels and brain GAP43 expression in MPS I dogs following gene therapy. Five MPS I dogs were treated by intramedullary injection of AAV9 expressing canine IDUA at 1 month of age. Two of the dogs (I-549, I-552) were tolerized to IDUA by liver-directed gene therapy 1 day after birth to prevent antibody responses elicited against IDUA in some MPS I dogs. (Figure 19A) Bar graph showing results of IDUA activity measured in brain tissue 6 months after intramedullary vector injection. (Figures 19B and 19C) Graphs showing results following measurement of GAP43 in cortical brain samples quantified relative to β-actin by densitometry. CSF spermine was measured at the time of sacrifice by isotope dilution LC / MS (E). Untreated MPS I dogs (n=3) and normal dogs (n=2) served as controls. *p<0.05 (Kruskal-Wallis test followed by Dunn's test). [Figure 20] 20A-20B are graphs illustrating the use of spermine as a CSF biomarker for the evaluation of CNS-directed gene therapy in MPS I. Six MPS I dogs tolerized to human IDUA at birth were treated with intramedullary AAV9 expressing human IDUA at 1 month of age (10 GC / kg, n=2, 10 GC / kg, n=2, 10 GC / kg, n=2). (FIG. 20A) Results following measurement of CSF spermine levels measured 6 months after treatment are provided. Three MPS I cats were treated with intramedullary AAV9 expressing feline IDUA (10 GC / kg). (FIG. 20B) Results following quantification of CSF spermine 6 months after treatment are provided. Untreated MPS I dogs (n=3) and normal dogs (n=2) served as controls. [Figure 21] FIG. 1 shows the decrease in prediction accuracy for metabolites identified by random forest analysis. [Figure 22]

[0023] Figure 1 provides data from a non-human safety study using the AAV9.hIDUA vector described in Example 7. Serum and cerebrospinal fluid from non-human primates with no immunosuppression (no IS) and with immunosuppression (IS) from day 0 to day 90 are shown. Numbers in the legend reflect individual animals. [Figure 23]

[0023] Figure 1 provides data from a non-human safety study using the AAV9.hIDUA vector described in Example 7. The effect of immunosuppression on T cell immune responses at high dose (HD), with immunosuppression (IS) and without immunosuppression is shown. ElisSpots were performed for the vector capsid (AAV9) and transgene (hIDUA). Cells were stimulated as indicated. Results are provided in spot forming units (SFU) / million peripheral blood mononuclear cells (PBMC). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Provided herein is a replication-deficient adeno-associated virus ("AAV") for delivery of the human alpha-L-iduronidase (hIDUA) gene to the CNS of a patient (human subject) diagnosed with Mucopolysaccharidosis Type I (MPS I). The recombinant AAV (rAAV) vector (rAAV.hIDUA) used to deliver the hIDUA gene has tropism for the CNS (e.g., an rAAV carrying an AAV9 capsid), and the hIDUA transgene is expressed in a manner that incorporates specific expression control elements, e.g., the cytomegalovirus (CMV) enhancer and the chicken β enhancer. The rAAV.hIDUA vector is controlled by a hybrid with the actin promoter (CB7). In certain embodiments, a pharmaceutical composition suitable for intrathecal, intracapsular, and systemic administration is provided, which comprises a suspension of the rAAV.hIDUA vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optional excipients. The rAAV suspension further comprises: (i) the titer of rAAV genome copies (GC) is at least 1 × 10 9 GC / mL~1×1 0 14 GC / mL (+ / - 20%); (ii) the ratio of empty particles / full particles of the rAAV is between 0.01-0.05 (95%-99% free of empty capsids) as measured by SDS-PAGE analysis (see Example 6D), or in another embodiment, is at least about 50%, at least about 80%, at least about 85%, or at least about 90% free of empty capsids; and / or (iii) at least about 4 × 10 8 GC / g brain mass ~ approx. 4×10 11 rA of GC / g brain mass Administration of AV suspension is effective It is characterized by:

[0029] Potency can be measured by in vitro cell culture assays, for example, the in vitro potency assay described in Example 6G, where Huh7 or HEK293 cells are transduced with rAAV GC at a known multiplicity of infection per cell and supernatants are assayed for IDUA activity 72 hours post-transduction. hIDUA function (activity) and / or potency may be measured in a suitable in vitro assay, for example, by its ability to cleave the fluorogenic substrate 4-methylumbelliferyl α-L-iduronide. Specific activity is >7,500 pmol / min / μg when measured under the conditions described. See activity assay protocol at www.RnDSystems.com. Other suitable methods for measuring enzyme activity have been described, including those described herein [see, e.g., Kakkis, ED, et al (1994). Protein Expression Purif. 5:225-232; Rome, LH, et al (1979). Proc. Natl. Acad. Sci. USA, 76:2331-2334]. Activity may also be assessed using methods described, e.g., E. Oussoren, et al, Mol Genet Metab. 2013, Aug; 109(4):377-81. doi:10.1016 / j.ymgme.2013.05.016. Epub, 2013, Jun, 4.

[0030] Patients who are candidates for treatment include pediatric and adult patients with MPS I and / or Hurler, Hurler-Scheie, and Scheie-related conditions.

[0031] The therapeutically effective intrathecal / intracapsular dose of rAAV.hIDUA for patients with MPSI is 1 × 10 11 ~7.0×10 14 GC (flat dose) range, which is 10 9 ~5×10 10 GC / is equivalent to the patient's brain mass in g. Alternatively, the following therapeutically effective plateau doses can be administered to patients in the indicated age groups: Newborn: Approx. 1 x 10 11 ~Approx. 3×10 14 G.C.; 3 to 9 months: approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; 9 months to 6 years: Approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; Under 3 years old (newborn to 3 years old): Approx. 1 x 10 11 ~Approx. 1.2×10 13 GC Ages 3 to 6: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.; 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0032] In another embodiment, the following therapeutically effective flat-line doses are administered to MPS patients in the following age groups: Newborn: Approx. 3.8 x 10 12 ~Approx. 1.9×10 14 G.C.; 3 to 9 months: approx. 6 x 10 12 ~Approx. 3×1014 G.C.; 9~36 months: Approx. 10 13 ~Approx. 5×10 13 G.C.; Under 3 years old (newborn to 3 years old): Approx. 1 x 10 11 ~Approx. 1.2×10 13 GC Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 3-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.; 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0033] In certain embodiments, one or more of these ranges are used for patients of any age. 12 Total genome copies (GC) (2.0 × 10 9 GC / g brain matter amount) or 6×10 12 Total GC (1 × 10 10 A plateau dose of 2×10 GC / g brain mass is administered to patients ≥4 months and <9 months of age. 12 Total GC (2.0 × 10 9 GC / g brain mass) or 1×10 13 Total GC (1 × 10 10 GC / g brain mass) A plateau dose is administered to patients ≥ 9 months and < 18 months of age. In certain embodiments, a plateau dose of 2.2 x 10 12 Total GC (2.0 × 10 9 GC / g brain mass) or 1.1 × 10 13 Total GC(1×10 10 A plateau dose of 6×10 GC / g brain mass is administered to patients ≥ 18 months and < 3 years of age. 12 (1.0×10 10GC / g brain mass) or 3 × 10 13 Total genome copies (GC) (5 × 10 10 In certain embodiments, a plateau dose of 1.0×10 GC / g brain mass is administered to patients ≥4 months and <9 months of age. 13 (1.0×10 10 GC / g brain mass) or 5.0 × 10 13 Total GC (5 × 10 10 In certain embodiments, a plateau dose of 1.1×10 GC / g brain mass is administered to patients ≥ 9 months and < 18 months of age. 13 (1.0×10 10 GC / g brain mass) or 5.5 × 10 13 Total GC (5 × 10 10 In certain embodiments, a plateau dose of 2.6×10 GC / g brain mass is administered to patients ≥ 18 months and < 3 years of age. 12 Genome copies (GC) (2.0 × 10 9 A flat dose of 100 mg / g brain mass was administered to patients aged 6 years or older. In a particular embodiment, 1.3×10 13 (GC)(1.0×10 10 In some embodiments, a plateau dose of 1.4×10 GC / g brain mass is administered to patients at or above the age of 6. In some embodiments, a plateau dose of 1.4×10 GC / g brain mass is administered to patients aged 12 years or older (including 18 years or older) to patients with MPS I who are 12 years or older. 13 Genome copies (GC) (1.1 × 10 10 In some embodiments, the dose administered to MPS I patients aged 12 years or older (including 18 years or older) is 7×10 13 GC(5.6×10 10 GC / g brain mass). Moreover, in a further embodiment, the dose administered to the MPS I patient is at least about 4×10 8 GC / g brain mass ~ approx. 4×10 11 GC / g brain mass. In certain embodiments, the dose administered to a neonate with MPS I is about 1.4×10 11 from about 1.4 × 10 14 The dose administered to infants aged 3 to 9 months is approximately 2.4 × 10 11 From about 2.4 × 1014 The dose administered to children aged 9 to 36 months is approximately 4 × 10 11 From about 4 × 10 14 The dose administered to children aged 3 to 12 years is approximately 4.8 × 10 11 From about 4.8 x 10 14 The dose administered to children aged 12 years and older and adults is approximately 5.6 × 10 11 From about 5.6 × 10 14 This extends to GC.

[0034] The goal of treatment is to functionally replace the patient's deficient α-L-iduronidase via rAAV-based CNS-directed gene therapy as a viable approach to treat the disease. As expressed from the rAAV vectors described herein, expression levels of at least about 2% of normal levels as detected in CSF, serum, neurons or other tissues or fluids may provide therapeutic benefit. However, even higher expression levels may be achieved. Such expression levels may be from about 2% to about 100% of normal functional human IDUA levels. In certain embodiments, higher than normal expression levels may be detected in CSF, serum, or other tissues or fluids.

[0035] The present invention also provides for the preparation and characterization of rAAV.hIDUA pharmaceutical compositions (see below). Example 6).

[0036] As used herein, the term "intramedullary delivery" or "intramedullary administration" refers to the route of administration of an agent via injection into the spinal canal, more specifically into the subarachnoid space, so that it reaches the cerebrospinal fluid (CSF). Intramedullary delivery may include lumbar puncture, intraventricular, suboccipital / intramedical and / or C1-2 puncture. For example, a substance may be introduced for diffusion throughout the subarachnoid space by means of a lumbar puncture. In another example, injection may be into the cisterna magna.

[0037] As used herein, the term "intracascular delivery" or "intracascular administration" more specifically refers to routes of administration via suboccipital puncture, or by direct injection into the cisterna magna, or via a permanently placed tube directly into the cerebrospinal fluid of the ventricles, or into the cisterna magna-cerebellum-medulla. Figure 13 provides an explanation of how intracapsular injection is performed.

[0038] As used herein, a "therapeutically effective amount" refers to an amount of an AAV9.hIDUA composition that delivers to and expresses in a target cell an amount of an enzyme sufficient to ameliorate or treat one or more of the symptoms of Hurler syndrome, Hurler-Scheie syndrome, and / or Scheie syndrome of MPS I. "Treatment" may include preventing one symptom of MPS I syndrome from worsening, and perhaps reversing one or more of the symptoms. Methods for assessing therapeutic efficacy are described in detail below (see, e.g., Section 5.2.3, below).

[0039] A "therapeutically effective amount" for a human patient may be predicted based on animal models. Examples of suitable cat models and suitable dog models are described herein. See C. Hinderer, et al, Molecular Therapy, (2014); 22, 12, 2018-2027; A. Bradbury, et al, Human Gene Therapy Clinical Development. March, 2015, 26(1): 27-37, which are incorporated herein by reference. With regard to dog models, the models are usually immunosuppressed or tolerized animal models, since it has been observed that intravenous administration in dogs elicits a strong and sustained antibody response against human IDUA, whereas administration is well tolerated in human patients. In these models, reversal of certain symptoms may be observed, and / or prevention of progression of certain symptoms may be observed. For example, correction of corneal opacity may be observed, and / or correction of central nervous system (CNS) pathology is observed, and / or reversal of perivascular and / or meningeal GAG accumulation is observed.

[0040] As used herein, "functional human α-L-iduronidase" refers to a human α-L-iduronidase enzyme that functions normally in humans who lack MPS I or associated syndromes such as, for example, Hurler syndrome, Hurler-Scheie syndrome, and / or Scheie syndrome. Conversely, an α-L-iduronidase enzyme variant that causes MPS I or one of these syndromes is considered to be non-functional. In one embodiment, a functional human α-L-iduronidase has the amino acid sequence of wild-type human α-L-iduronidase as described by Bremer, et al, Mol. Genet. Metab. 104(3):289-294 (2011) and has the NCBI reference sequence NP_000194.2 reproduced in SEQ ID NO:2 (653 amino acids). However, several naturally occurring functional polymorphisms (variants) of this sequence have been described and may be encompassed within the scope of the present invention. Such variants have been described; see, for example, WO2014 / 151341, which is incorporated by reference herein, UniProtKB / Swiss-Prot; www.uniprot.org / uniprot / P35475, which is also incorporated by reference.

[0041] As used herein, the term "Nab titer" is a measure of how many neutralizing antibodies (e.g., anti-AAV Nabs) are produced that neutralize the physiological effect of the epitope (e.g., AAV) targeted thereto. Anti-AAV Nab titers are measured, for example, as described in Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of It may be measured as described in Infectious Diseases, 2009.199(3): p.381-390.

[0042] As used herein, "expression cassette" refers to a nucleic acid molecule that includes the IDUA gene and promoter, and optionally other regulatory sequences therefor, which may be delivered to a packaging host cell via a genetic element (e.g., capsid) or packaged into the capsid of a viral vector (e.g., viral particle). Typically, such expression cassettes for generating viral vectors contain an IDUA coding sequence flanked by packaging signals from the viral genome and other expression control sequences, such as those described herein.

[0043] The abbreviation "sc" refers to self-complementarity. "Self-complementary AAV" refers to constructs in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of a second strand, the two complementary halves of the scAAV associate to form a single double-stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See, e.g., DM McCarty, et al. al,“Self-complementary recombinant adeno-associated virus (scAAV) vectors promote transduction efficient independently of See, "AAV-specific self-complementary vectors for the development of AAV-specific ...

[0044] As used herein, the term "operably linked" refers both to expression control sequences adjacent to a gene of interest and to expression control sequences that act in trans or at some distance to regulate a gene of interest.

[0045] The term "heterologous," when used with reference to a protein or nucleic acid, indicates that the protein or nucleic acid comprises two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are usually produced recombinantly that have two or more sequences from unrelated genes arranged to create a new functional nucleic acid. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct expression of a coding sequence from a different gene. Thus, the promoter is heterologous with reference to the coding sequence.

[0046] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged into a viral capsid or envelope, where the sequences of the viral genome packaged within the viral capsid or envelope are also replication-defective, i.e., they are unable to produce progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication. (The genome can be engineered to be "underpowered" - containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome; these genes may be supplied during production. It is therefore considered safe for use in gene therapy, since no replication or infection of progeny virions will occur except in the presence of viral enzymes required for replication.

[0047] As used herein, "recombinant AAV9 viral particle" refers to a nuclease-resistant particle (NRP) having an AAV9 capsid, the capsid packaging a heterologous nucleic acid molecule therein, including an expression cassette for a desired gene product. Such an expression cassette usually contains 5' and / or 3' inverted terminal repeats of AAV flanking the gene sequence, which is operably linked to an expression control sequence. These and other suitable elements of the expression cassette are described in more detail below, or may be referred to herein as a transgene genome sequence. This may also be referred to as a "complete" AAV capsid. Such a rAAV viral particle is referred to as "pharmacologically active" if it delivers a transgene to a host cell capable of expressing the desired gene product carried by the expression cassette.

[0048] In many instances, rAAV particles are said to be DNase-resistant. However, in addition to this endonuclease (DNase), other endonucleases and exonucleases may also be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases may be selected to degrade single-stranded and / or double-stranded DNA and RNA. Such steps may contain a single nuclease or a mixture of nucleases directed to different targets, which may be endonucleases or exonucleases.

[0049] The term "nuclease resistant" indicates that the AAV capsid is fully assembled around an expression cassette that is designed to protect the packaged genomic sequence from degradation (digestion) during the nuclease incubation step that is designed to deliver the transgene to the host cell and to remove contaminating nucleic acids that may be present from the manufacturing process.

[0050] As used herein, "AAV9 capsid" refers to AAV9 having the amino acid sequence of GenBank accession AAS99264, incorporated herein by reference, with the AAVvp1 capsid protein reproduced in SEQ ID NO: 7. Some variation from this encoded sequence is encompassed by the present invention, which may include sequences having about 99% identity to the amino acid sequences referenced in GenBank accession AAS99264 and US7906111 (WO2005 / 033321) (i.e., less than about 1% variation from the referenced sequence). Such AAVs may include, for example, naturally occurring isolates (e.g., hu31 or hu32) or variants of AAV9 with amino acid substitutions, deletions or additions, including but not limited to amino acid substitutions selected from alternative residues "recruited" from corresponding positions in the capsids of other AAVs aligned with the capsid of AAV9, e.g., as described in US9,102,949, US8,927,514, US2015 / 349911; and WO2016 / 049230A1. However, in alternative embodiments, other variants of AAV9 or AAV9 capsids with at least about 95% identity to the above reference sequences may be selected. See, for example, U.S. Published Patent Application No. 2015 / 0079038. Methods for generating capsids, coding sequences therefor, and methods for producing rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. USA 100(10), 6081-6086 (2003) and US 2013 / 0045186 A1.

[0051] The term "AAV9 intermediate" or "AAV9 vector intermediate" refers to assembled rAAV capsids that lack the desired genomic sequence packaged therein. These may also be referred to as "empty" capsids. Such capsids may contain undetectable genomic sequences of an expression cassette, or only partially packaged genomic sequences that are insufficient to achieve expression of a gene product. These empty capsids are not functional to transfer a gene of interest to a host cell.

[0052] The terms "a" or "an" refer to one or more. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0053] The words "comprise," "comprises," and "comprising" should be interpreted inclusively and not exclusively. The words "consist," "consisting," and variations thereof should be interpreted exclusively and not inclusively. While various embodiments herein may be presented using "comprising" language under other circumstances, it is also intended that the related embodiments be construed and described using "consisting of" or "consisting essentially of" language.

[0054] The term "about" encompasses variations within and including ±10%, unless otherwise specified.

[0055] Unless defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published textbooks, which provide one of ordinary skill in the art with general guidance to many of the terms used in this application.

[0056] 5.1. AAV.hIDUA Constructs and Formulations 5.1.1. Expression Cassettes In a particular embodiment, an AAV vector is provided that includes an expression cassette containing a hIDUA gene characterized by having a nucleotide sequence of SEQ ID NO:1. This sequence, developed by the inventors, has about 83% identity to the published gene coding sequence of GenBank NP000194.2, which encodes SEQ ID NO:2. In another embodiment, the expression cassette contains a hIDUA gene characterized by having a nucleotide sequence that is at least about 80% identical to SEQ ID NO:1 and encodes a functional human α-L-iduronidase. In another embodiment, the sequence is at least about 85% identical to SEQ ID NO:1, or at least about 90% identical to SEQ ID NO:1 and encodes a functional human α-L-iduronidase. In one embodiment, the sequence is at least about 95% identical to SEQ ID NO:1, at least about 97% identical to SEQ ID NO:1, or at least about 99% identical to SEQ ID NO:1 and encodes a functional human α-L-iduronidase. In one embodiment, this encompasses the full length hIDUA gene including the leader peptide sequence of human α-L-iduronidase corresponding to about 1 to about 78 of SEQ ID NO:1 (i.e., encoding from about amino acid 26 or about amino acid 27 to about amino acid 653 of SEQ ID NO:2). In another embodiment, the hIDUA gene encodes a functional synthetic human α-L-iduronidase enzyme, i.e., a synthetic peptide including a heterologous leader sequence fused to a secreted portion of SEQ ID NO:2 from about amino acid 27 to about 653 of SEQ ID NO:2 or one of its functional variants identified herein. Yet further expression cassettes include those identified in SEQ ID NO:5 and SEQ ID NO:6. In each, each expression cassette is flanked by 5' and 3' ITRs of AAV2. Additionally, each contains a promoter, an enhancer, the hIDUA gene, and a polyA.

[0057] In another embodiment, the functional human α-L-iduronidase may comprise a synthetic amino acid sequence in which all or part of the first 26 amino acids of SEQ ID NO:2, which correspond to the leader (signal) peptide, are replaced with a heterologous leader peptide. This leader peptide, for example, a leader peptide derived from interleukin 2 (IL-2) or oncostatin, may improve the transport of the enzyme out of the cell and into the circulation via the secretory pathway. A suitable leader peptide is preferably, but not necessarily, of human origin. A suitable leader peptide may be selected from proline.bic.nus.edu.sg / spdb / zhang270.htm, which is incorporated herein by reference, or may be determined using various computer programs for determining leader (signal) peptides in selected proteins. Without limitation, such sequences may be about 15 to about 50 amino acids in length, or about 19 to about 28 amino acids in length, or may be longer or shorter as desired. In addition, at least one in vitro method has been described as useful for assessing the enzymatic activity of the IDUA enzyme [see, e.g., Kakkis, et al, Mol. Genet. Metabol, 2001, Mar;72(3):199-208].

[0058] Identity or similarity with respect to sequences is defined herein as the percentage of amino acid residues in a candidate sequence that are identical (i.e., identical residues) or similar (i.e., amino acid residues from the same group based on common side chain properties) to a region of the peptides and polypeptides provided herein, after aligning the sequences, introducing gaps as necessary, and achieving the maximum sequence identity percentage. Percentage (%) identity is a measure of the relatedness between two polynucleotides or two polypeptides as determined by comparing their nucleotide or amino acid sequences, respectively. In general, the two sequences being compared are aligned to obtain maximum correlation between the sequences. The alignment of the two sequences is examined, and the number of positions that give exact amino acid or nucleotide correspondences between the two sequences determined is divided by the entire length of the alignment and multiplied by 100 to obtain a % identity figure. This % identity figure may be determined over the entire length of the sequences being compared, which is particularly suitable for sequences of identical or very similar length and sequences that are highly homologous or over a shorter defined length, and is more suitable for sequences of unequal length or sequences with low levels of homology. There are numerous algorithms, and computer programs based thereon, available for use in the literature and / or publicly or commercially available for performing sequence comparisons and percent identity. The choice of algorithm or program is not a limitation of the present invention.

[0059] Examples of suitable sequence comparison programs include, for example, the software CLUSTALW under Unix (Hall, TA 1999, BioEdit: an easy to use biological sequence comparison editor and analysis program for Windows 95 / 98 / NT. Nucl. Acids. Symp. Ser. 41:95-98), which is then imported into the Bioedit program; the Wisconsin sequence analysis package, version 9.1 (Devereux, J. et al., Nucleic Acids Res., 12:387-395, 1984, available from Genetics Computer Group, Madison, Wis., USA). The programs BESTFIT and GAP may be used to determine the % identity between two polynucleotides and the % identity between two polypeptide sequences.

[0060] Other programs for determining identity and / or similarity between sequences include, for example, the BLAST family of programs available from the National Center for Biotechnology Information (NCB), Bethesda, Md., USA, accessible via the NCBI home page at www.ncbi.nlm.nih.gov, and the ALIGN program (version 2.0), which is part of the GCG sequence comparison software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4 can be used; and FASTA (Pearson, WR and Lipman, DJ, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988, available as part of the Wisconsin Sequence Analysis Package). SeqWeb software (a web-based interface to the GCG Wisconsin package: the Gap program).

[0061] In some embodiments, the cassette is designed to be expressed from a recombinant adeno-associated virus, and the vector genome also contains the inverted terminal repeats (ITRs) of AAV. In one embodiment, the rAAV is pseudotyped, i.e., the capsid of the AAV is derived from a different source of AAV than the AAV that provides the ITRs. In one embodiment, the ITRs of AAV serotype 2 are used. However, ITRs from other suitable sources may be selected. Optionally, the AAV may be a self-complementary AAV. The expression cassettes described herein utilized the 5' inverted terminal repeats (ITRs) of AAV and the 3' ITRs of AAV. However, other configurations of these elements may be suitable. A truncated version of the 5' ITR, called ΔITR, has been described, which lacks the D-sequence and the terminal separation site (trs). In another embodiment, the 5' ITR and / or 3' ITRs of full-length AAV are used. If pseudotyped AAV is to be made, the ITRs in the expression are selected from a source different from the AAV source of the capsid. For example, the ITRs of AAV2 may be selected for use with an AAV capsid that has a particular efficiency in targeting the CNS or tissues or cells within the CNS. In one embodiment, ITR sequences from AAV2 or deleted versions thereof (ΔITRs) are conveniently used to accelerate regulatory approval. However, ITRs from other AAV sources may be selected. If the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be called pseudotyped. However, other sources of the ITRs of AAV may be utilized.

[0062] In one embodiment, the expression cassette is designed for expression and secretion in the central nervous system (CNS), including the cerebrospinal fluid and the brain. In a particularly desirable embodiment, the expression cassette is useful for expression in both the CNS and liver, thereby allowing treatment of both the systemic and CNS-related effects of MPS I, Hurler syndrome, Hurler-Scheie syndrome, and / or Scheie syndrome. For example, the inventors have observed that certain constitutive promoters (e.g., CMV) do not promote expression at the desired level when delivered intramedullary, thereby providing suboptimal expression levels of hIDUA. However, the chicken β-actin promoter promotes expression sufficiently both intramedullary and systemic delivery. It is therefore a particularly desirable promoter. Other promoters may be selected, but the expression cassettes containing the same may not have all of the advantages of the chicken β-actin promoter. Various chicken β-actin promoters have been described, either alone or in combination with various enhancer elements (e.g., CB7 is a chicken β-actin promoter with a cytomegalovirus enhancer element, the CAG promoter which contains the first exon and first intron of chicken β-actin and the splice acceptor of the rabbit β-globin gene), the CBh promoter [SJ Gray, et al, Hu Gene Ther, 2011, Sep;22(9):1143-1153].

[0063] Examples of promoters that are tissue specific are well known, inter alia, for liver and other tissues (albumin, Miyatake, et al., (1997), J. Virol., 71:5124-32; Hepatitis B virus core promoter, Sandig, et al., (1996), Gene Ther., 3:1002-9; alpha-fetoprotein (AFP), Arbuthnot, et al., (1996), Hum. Gene Ther., 3:1002-9). her.,7:1503-14), bone osteocalcin (Stein,et al.,(1997),Mol.Biol.Rep.,24:185-96); bone sialoprotein (Chen,et al.,(1996),J.BoneMiner.Res.,11:654-64), lymphocyte (CD2,Hansal,et al.,(1998),J.Immunol.,161:1063-8; immunoglobulin heavy chain; T cell receptor chain), neuronal such as the neuron-specific enolase (NSE) promoter (Andersen,et al.,(1993),Cell.Mol.Neurobiol.,13:503-15), neurofilament light chain gene (Piccioli,et al., (1991), Proc. Natl. Acad. Sci. USA, 88:5611-5), and the neuron-specific vgf gene (Piccioli, et al., (1995), Neuron, 15:373-84). Alternatively, regulatable promoters may be selected. See, for example, WO2011 / 126808B2, incorporated herein by reference.

[0064] In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same or different. For example, the enhancers may include the alpha mic / bik enhancer or the CMV enhancer. The enhancer may be present in two copies located adjacent to each other. Alternatively, the duplicated copies of the enhancer may be separated by one or more sequences. In yet another embodiment, the expression cassette further contains an intron, for example, the chicken beta actin intron, the human beta globin intron, and / or the commercially available Promega® intron. Other suitable introns include those known in the art, for example, those described in WO2011 / 126808.

[0065] Furthermore, the expression cassette of the present invention is provided with a suitable polyadenylation signal. In one embodiment, the polyA sequence is rabbit globulin polyA. See, for example, WO2014 / 151341. Alternatively, another polyA, such as the human growth hormone (hGH) polyadenylation sequence, SV50 polyA, or synthetic polyA. Furthermore, other conventional regulatory elements may additionally or optionally be included in the expression cassette.

[0066] 5.1.2. Generation of rAAV.hIDUA viral particles In certain embodiments, recombinant adeno-associated virus (rAAV) particles are provided that have an AAV capsid, an AAV inverted terminal repeat packaged therein, a human alpha-L-iduronidase (hIDUA) gene under the control of regulatory sequences controlling its expression, wherein said hIDUA gene has a sequence as set forth in SEQ ID NO: 1 or a sequence at least about 95% identical thereto that encodes a functional human alpha-L-iduronidase. See also schematic diagram in FIG. 1. In one embodiment, the expression cassette for hIDUA is flanked by an AAV 5'ITR and an AAV 3'ITR. In another embodiment, the AAV may be a single-stranded AAV.

[0067] For intramedullary delivery, AAV9 is particularly desirable. Methods for generating vectors based on AAV9 sequences and AAV9 capsids are described in US7,906,111; US2015 / 0315612; WO2012 / 112832, which are incorporated herein by reference. Optionally, the rAAV9.hIDUA vector as described herein may be co-administered with a vector designed to specifically target the liver. Any of a number of rAAV vectors with liver tropism may be used. Examples of AAV that may be selected as a source for the capsid of rAAV include, for example, rh10, AAVrh64R1, AAVrh64R2, rh8 [see, e.g., U.S. Published Patent Application No. 2007-0036760-A1; U.S. Published Patent Application No. 2009-0197338]. -A1; EP1310571]. See also WO2003 / 042397 (AAV7 and other simian AAVs), US Patent 7790449 and US Patent 7282199 (AAV8), WO2005 / 033321 and US 7,906,111 (AAV9), and WO2006 / 110689], and rh10 [WO2003 / 042397], AAV3B; AAVdj [US2010 / 0047174]. One particularly preferred rAAV is AAV2 / 8.TBG.hIDUA.co.

[0068] In many instances, rAAV particles are referred to as DNase-resistant. However, in addition to this endonuclease (DNase), other endonucleases and exonucleases may also be used in the purification steps described herein to remove contaminating nucleic acids. Such nucleases may be selected to degrade single-stranded and / or double-stranded DNA and RNA. Such steps may contain a single nuclease or a mixture of nucleases directed against different targets, which may be endonucleases or exonucleases.

[0069] Methods for preparing vectors based on AAV are known. See, for example, U.S. Published Patent Application No. 2007 / 0036760 (February 15, 2007), which is incorporated herein by reference. The use of AAV capsids of AAV9 is particularly well suited for the compositions and methods described herein. Additionally, AAV8 sequences and methods for producing vectors based on AAV8 capsids are described in U.S. Patents 7,282,199 B2, 7,790,449, and 8,318,480, which are incorporated herein by reference. However, other AAV capsids may be selected or produced for use in the present invention. A number of such AAV sequences are provided in the above-cited U.S. Patents 7,282,199 B2, 7,790,449, 8,318,480, and 7,906,111, and are available from GenBank. Either sequence of the AAV capsid gene can be readily produced synthetically or using a variety of molecular biology and genetic engineering techniques. Suitable production methods are well known to those of skill in the art. See, e.g., Sambrook, et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY). Alternatively, oligonucleotides encoding peptides (e.g., CDRs) or the peptides themselves can be produced synthetically, e.g., by well-known solid phase peptide synthesis techniques (Merrifield, (1962), J. Am. Chem. Soc., 85:2149; Stewart and Young, Solid Phase Peptide Synthesis, (Freeman, San Francisco, 1969), pp. 27-62). These and other suitable production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention.

[0070] The recombinant adeno-associated virus (AAV) described herein may be produced using known techniques. See, for example, WO2003 / 042397; WO2005 / 033321, WO2006 / 110689; US7588772B2. Such methods involve culturing a host cell that contains an AAV capsid; a functional rep gene; an expression cassette consisting of, at a minimum, the inverted terminal repeats (ITRs) of AAV and a transgene; and nucleic acid sequences encoding sufficient helper functions to allow packaging of the expression cassette into AAV capsid proteins.

[0071] To calculate the hollow and full particle content, the VP3 band volume for a selected sample (herein sample, e.g., an iodixanol gradient purified preparation where GC number = number of particles) is plotted against the GC particles loaded. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test article peak. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL divided by GC / mL gives the particle to genome copy ratio (pt / GC). Pt / mL-GC / mL gives the empty Pt / mL. Empty Pt / mL divided by Pt / mL multiplied by 100 gives the percentage of empty particles.

[0072] In general, methods for assaying AAV vector particles with empty capsids and packaged genomes are known in the art. See, e.g., Grimm, et al., Gene Therapy, (1999), 6:1322-1330; Sommer, et al., Molec. Ther. (2003), 7:122-128. To test for denatured capsids, the method involves subjecting the processed AAV stock to SDS-polyacrylamide gel electrophoresis consisting of a gel capable of separating the three capsid proteins, e.g., a gradient gel containing 3-8% Tris-acetate in buffer, then running the gel until the sample material is separated, and blotting the gel against a nylon or nitrocellulose membrane, preferably nylon. Then, an anti-AAV capsid antibody, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV2 monoclonal antibody, is used as a primary antibody that binds to the denatured capsid protein (Wobus, et al., J. Virol. (2000) 74:9281-9293). Then, a secondary antibody that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound thereto, most preferably an anti-mouse IgG antibody covalently bound to horseradish peroxidase, is used. The binding between the primary antibody and the secondary antibody is determined semi-quantitatively using a method for detecting binding, preferably the detection method can detect the release of radioisotopes, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit. For example, for SDS-PAGE, samples from the column fractions were removed and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT) and capsid proteins were resolved in precast gradient polyacrylamide gels (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining methods, i.e., SYPROruby or Coomassie staining. In one embodiment, the concentration of AAV vector genomes (vg) in the column fractions can be measured by quantitative real-time PCR (Q-PCR).The samples are diluted and digested with DNase I (or other suitable nuclease) to remove extraneous DNA. After inactivation of the nuclease, the samples are further diluted and amplified with a TaqMan™ fluorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample in an Applied Biosystems Prism7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is employed to generate a standard curve in a Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the titer of the vector genome by normalizing it against the Ct value of the plasmid standard curve. An endpoint assay based on digital PCR can also be used.

[0073] In one embodiment, an optimized q-PCR is used that utilizes a broad spectrum serine protease, such as Proteinase K (as commercially available from Qiagen). More specifically, the optimized q-PCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with Proteinase K buffer, treated with Proteinase K, and then heat inactivated. Preferably, the sample is diluted with Proteinase K buffer in an amount equivalent to the sample size. The Proteinase K buffer may be concentrated two-fold or more. Typically, Proteinase K treatment is about 0.2 mg / mL, but may vary from 0.1 mg / mL to about 1 mg / mL. The treatment step is generally performed at about 55° C. for about 15 minutes, but may also be performed at lower temperatures (e.g., about 37° C. to about 50° C.) for longer times (e.g., about 20 minutes to about 30 minutes), or at higher temperatures (e.g., up to about 60° C.). Heat inactivation may be performed at about 95° C. for about 15 minutes, but the temperature may be lowered (e.g., from about 70° C. to about 90° C.) and the time may be increased (e.g., from about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described for standard assays.

[0074] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, a method for measuring the titer of single-stranded and self-complementary AAV vector genomes by ddPCR has been described. See, for example, M. Lock, et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014, Apr; 25(2): 115-25. doi: 10.1089 / hgtb.2013.131. Epub, 2014, Feb, 14.

[0075] Briefly, a method for separating rAAV9 particles with packaged genome sequences from genome-defective AAV9 intermediates involves subjecting a suspension containing recombinant AAV9 viral particles and AAV9 capsid intermediates to high performance liquid chromatography, where the AAV9 viral particles and AAV9 intermediates are bound to a strong anion exchange resin equilibrated at a pH of 10.2 and subjected to a salt gradient while the eluate is monitored for UV absorbance at about 260 and about 280. Although less optimal for rAAV9, the pH may range from about 10.0 to 10.4. In this method, AAV9 complete capsids are recovered from the fraction that is eluted when the ratio of A260 / A280 reaches an inflection point. In one example, for the affinity chromatography step, the diafiltered product may be applied to Capture Select™ Poros- AAV2 / 9 affinity resin (Life Technologies), which efficiently captures AAV2 / 9 serotypes. Under these ionic conditions, AAV particles are efficiently captured, while a significant fraction of the remaining cellular DNA and proteins pass through the column.

[0076] The rAAV.hIDUA vector can be prepared as shown in the flow chart provided in Figure 11, which is described in further detail in Section 5.4 below and in Example 5.

[0077] 5.1.3. Pharmaceutical Formulations of rAAV.hIDUA The rAAV9.hIDUA formulation is a suspension containing an effective amount of the AAV.hIDUA vector suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6-8, or pH 6.5-7.5, pH 7.0-7.7, or pH 7.2-7.8. The pH of cerebrospinal fluid is about 7.28 to about 7.32, so a pH within this range may be desired for intrathecal delivery, whereas for intravenous delivery, a pH of 6.8 to about 7.2 may be desired. However, other pH's within the broadest range and subranges of these may be selected for other routes of delivery.

[0078] A suitable surfactant or combination of surfactants may be selected among non-toxic non-ionic surfactants. In one embodiment, a bifunctional block copolymer surfactant terminated with a primary hydroxyl group is selected, for example Pluronic® F68 [BASF], also known as Poloxamer 188, having a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers, i.e. non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxycaprylic Acid Glyceride), Polyoxy 10 Oleyl Acetonitrile, etc. Copolymers may be selected from the group consisting of tert-butyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are commonly named by the letter "P" (for poloxamer) followed by three numbers, the first two numbers times 100 giving the approximate molecular weight of the polyoxypropylene core, and the last number times 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount of from about 0.0005% to about 0.001% of the suspension.

[0079] In one embodiment, the formulation has a concentration of at least about 1×10 IgG as measured by oqPCR or digital droplet PCR (ddPCR) as described, for example, in M. Lock, et al, Hu Gene Therapy Methods, Hum Gene Ther. Methods. 2014, Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131. Epub, 2014, Feb. 14, which is incorporated herein by reference. 9 GC / mL ~ approx. 3×10 13 Concentration in GC / mL may also contain

[0080] In one embodiment, a frozen composition is provided that contains rAAV in a frozen form in a buffer solution as described herein. Optionally, one or more surfactants (e.g., Pluronic F68), stabilizers or preservatives are present in the composition. Suitably, for use, the composition is thawed and titrated to the desired dose with a suitable diluent, e.g., sterile saline or buffered saline.

[0081] In one example, the formulation may contain, for example, sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate·7H2O), magnesium chloride, in water. potassium chloride, calcium chloride (for example, calcium chloride·2H2O), sodium phosphate The formulation may also contain a buffered saline solution containing one or more of: sodium phosphate dibasic, ...

[0082] In another embodiment, the formulation may contain one or more permeation enhancers. Suitable permeation enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.

[0083] In certain embodiments, a kit is provided that includes the concentrated vector (optionally frozen) suspended in a formulation, an optional dilution buffer, and an implement, and other components required for intrathecal administration are provided. In other embodiments, the kit may additionally or instead include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow for infusion. Such buffer may allow for dilution of the concentrated vector 1:1 to 1:5 or more. In other embodiments, greater or lesser amounts of buffer or sterile water are included to allow for dose titration and other adjustments by the treating physician. In yet other embodiments, one or more components of the implement are included in the kit.

[0084] In certain embodiments, the dilution is performed in a clinical pharmacy using aseptic techniques, such as The procedure may be performed in a fast air flow cabinet. A syringe and needleless bottle adapter are used to withdraw a predefined volume of suspension containing AAV9.hIDUA (which may have been previously frozen), then the syringe is capped with a sterile stainless steel blunt needle covered with a plastic needle sheath. A syringe and needleless bottle adapter are used to withdraw a predefined volume of diluent, then the syringe is capped with a sterile stainless steel blunt needle covered with a plastic needle sheath. The diluent and then the suspension containing AAV9.hIDUA are delivered to a third "dosing" syringe using aseptic technique, ensuring that the exterior sterility of the syringe is maintained. The syringe is capped with a tip cap and mixed by inversion. The syringe is uncapped, primed to the desired delivery volume, capped, labeled, and packaged in a sterile bag, which can be transported to the operating room for use within 6 hours.

[0085] 5.2. Gene Therapy Protocols 5.2.1. Target patient population Provided herein is a method of treating mucopolysaccharidosis type I comprising delivering a therapeutically effective amount of an expression cassette of a modified hIDUA as described herein. In particular, provided herein is a method of preventing, treating and / or ameliorating neurocognitive decline in a patient diagnosed with MPS I comprising delivering to a patient in need thereof a therapeutically effective amount of rAAV.hIDUA as described herein. A "therapeutically effective amount" of a rAAV.hIDUA vector as described herein may ameliorate one or more of the symptoms identified in any one of the following paragraphs.

[0086] Patients who are candidates for treatment are pediatric and adult patients with MPS I and / or Hurler, Hurler-Scheie and Scheie-related conditions. MPS I disease is a spectrum of disease ranging from early severe forms (Hurler) to late onset forms (Scheie). Hurler syndrome is usually characterized by no IDUA enzyme activity (0%), is diagnosed early, and is characterized by developmental delay, hepatosplenomegaly, skeletal lesions, corneal opacities, joint lesions, hearing loss, cardiac involvement and death by age 10. Hurler-Scheie patients are observed to have some IDUA enzyme activity (greater than 0%, but usually less than 2%) and have variable intellectual outcomes, respiratory disease, obstructive airway disease, cardiovascular disease, joint stiffness / spasticity, skeletal abnormalities, decreased vision, and death in the teens or twenties. Patients with Scheie syndrome usually have at least 2% "normal" IDUA enzyme activity and are diagnosed late; such patients usually have normal intelligence but hepatosplenomegaly, joint involvement, nerve entrapment, hearing loss, cardiac involvement, and a normal life span. Newborn screening for Mucopolysaccharidosis Type I (MPS I): A Systematic Review of Evidence Report Final Findings, Final Version 1.1, prepared for the Maternal and Child Health Administration. See www.hrsa.gov / advisorycommittees / mchbadvisory / -heritabledisorders / nominatecondition / reviews / mps1finalreport.pdf.

[0087] The compositions of the invention avoid the complications of long-term enzyme replacement therapy (ERT) associated with immune responses to the recombinant enzyme, which can range from mild to full-blown anaphylaxis, as well as the complications of long-term peripheral administration, such as local and systemic infections. In contrast to ERT, the compositions of the invention do not require long-term repeated weekly injections. Without wishing to be bound by theory, it is believed that the therapeutic methods described herein are useful in correcting at least the central nervous system phenotype associated with MPS I disease by providing efficient, long-term gene transfer obtained by a vector with high transduction efficiency that provides continuous high circulating IDUA levels that provide therapeutic impact outside the CNS compartment. Additionally, provided herein are methods of providing active tolerance to the enzyme by various routes, including by direct systemic delivery of the enzyme in the form of a protein or in the form of rAAV.hIDUA, prior to AAV-mediated delivery to the CNS, and preventing the formation of antibodies.

[0088] In some embodiments, a patient diagnosed with Hurler Syndrome is treated according to the methods described herein. In some embodiments, a patient diagnosed with Hurler-Scheie Syndrome is treated according to the methods described herein. In some embodiments, a patient diagnosed with Scheie Syndrome is treated according to the methods described herein. In some embodiments, a pediatric subject with MPS I having neurocognitive deficits is treated according to the methods described herein.

[0089] In certain embodiments, newborns (under 3 months of age) are treated according to the methods described herein. In certain embodiments, infants between 3 months and 9 months of age are treated according to the methods described herein. In certain embodiments, toddlers between 9 months and 36 months of age are treated according to the methods described herein. In certain embodiments, children between 3 and 12 years of age are treated according to the methods described herein. In certain embodiments, children between 12 and 18 years of age are treated according to the methods described herein. In certain embodiments, adults 18 years of age or older are treated according to the methods described herein.

[0090] In one embodiment, the patient may have Hurler syndrome and may be a male or female at least 3 months to less than 12 months of age. In another embodiment, the patient may have an MPS I condition and may be 49 months (over 4 years) to about 72 months (6 years) of age. In another embodiment, the patient may have Hurler-Scheie syndrome, male or female, and may be at least about 6 to 18 years of age. In another embodiment, the subject may be older or younger, male or female.

[0091] Suitably, patients selected for treatment may include those with one or more of the following characteristics: a documented diagnosis of MPS I confirmed by absent or reduced IDUA activity as measured in plasma, fibroblasts or leukocytes; documented evidence of early neurocognitive deficits due to MPS I, defined as either an IQ test score one standard deviation below the mean or a score in one domain of neuropsychological function (language, memory, attention or non-verbal abilities), unless explained by other neurological or psychiatric factors; or documented historical evidence of greater than one standard deviation decline on sequential tests. Alternatively, elevated GAG in urine or genetic testing may be used.

[0092] Prior to treatment, subjects, e.g., infants, are preferably genotyped to identify MPS I patients, i.e., patients with a mutation in the gene encoding hIDUA. In certain populations, subjects may be older, e.g., from under 3 years of age to 72 months (6 years) or even older. Prior to treatment, MPS I patients may be evaluated for neutralizing antibodies (Nab) against the AAV serotype used to deliver the hIDUA gene. In certain embodiments, MPS I patients with neutralizing antibody titers against AAV that are 5 or less are treated according to one or more of the methods described herein.

[0093] Prior to treatment, MPS I patients can be assessed for neutralizing antibodies (Nab) against the capsid of the AAV vector used to deliver the hIDUA gene. Such Nabs can interfere with transduction efficiency and reduce therapeutic efficacy. MPS I patients with baseline serum Nab titers of ≦1.5 are good candidates for treatment with rAAV.hIDUA gene therapy protocols. Treatment of MPS I patients with serum Nab titers >1:5 may require concomitant therapy, such as temporary co-treatment with an immunosuppressant, prior to and / or during treatment with delivery of the rAAV.hIDUA vector. Optionally, immunosuppressant co-treatment can be used as a preventative measure without prior assessment of neutralizing antibodies against the AAV vector capsid and / or other components of the formulation. hIDUA may be used as a prophylactic against hIDUA transgene product. Prior immunosuppressive therapy may be desirable to prevent a potential adverse immune response to the hIDUA transgene product, especially in patients who do not actually have the level of IDUA activity and in whom the transgene product may be viewed as "foreign." The results of preclinical studies in mice, dogs, and NHPs described below are consistent with the development of an immune response and neuroinflammation to hIDUA. Although a similar response may not occur in human subjects, prophylactic immunosuppressive therapy is recommended for all recipients of rAAV.hIDUA.

[0094] Immunosuppressants for such co-therapy include, but are not limited to, cytostatics including glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides, and alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins. One or more macrolides selected are immunosuppressants. In certain embodiments, the macrolide is a non-antibiotic immunosuppressant. These non-antibiotic immunosuppressants may have different mechanisms of action. In certain embodiments, a calcineurin inhibitor (e.g., tacrolimus), an mTOR inhibitor (e.g., sirolimus, temsirolimus, everolimus, or another rapalog), or a combination thereof. Another suitable non-biological immunosuppressant may be pimecrolimus.

[0095] Immunosuppressants include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mycophenolate mofetil, methotrexate, leflunomide (Arava), cyclophosphamide, chlorambucil (Leukeran), chloroquine (e.g., hydroxychloroquine), quinine sulfate, mefloquine, combinations of atovaquone and proguanil, sulfasalazine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, antibodies directed against the IL-2 receptor (CD25) or CD3, anti-IL-2 receptor (IL-2 receptor) ... 2 antibodies, abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), rituximab (Rituxan), tocilizumab (Actemra) and tofacitinib (Xeljanz), cyclosporine, tacrolimus, mTOR inhibitors (such as sirolimus (i.e. rapamycin), temsirolimus or rapalogs), IFN-β, IFN-γ, opioids or TNF-α (tumor necrosis factor-alpha) binders, and combinations of these drugs. In certain embodiments, the immunosuppressive therapy may be initiated 0, 1, 2, 7 or more days prior to administration of the gene therapy. Such therapy may involve co-administration of two or more agents on the same day. In one embodiment, the two or more drugs can be, for example, one or more corticosteroids (e.g., prednelisone or prednisone) and optionally, MMF, and / or a calcineurin inhibitor (e.g., tacrolimus), and / or an mTOR inhibitor (temsirolimus or sirolimus (i.e., rapamycin)). In one embodiment, the two or more drugs are mycophenolate mofetil (MMF) and / or sirolimus. In another embodiment, the two or more drugs can be, for example, methylprednisolone, prednisone, tacrolimus, and / or sirolimus. In certain embodiments, the immunosuppressive regimen consists of a corticosteroid, tacrolimus, and sirolimus.In certain embodiments, the medications are MMF and tacrolimus for 0-15 days prior to vector delivery, about 8 weeks with MMF, and / or maintained throughout follow-up appointments with tacrolimus. One or more of these drugs may continue after administration of gene therapy at the same or adjusted doses. In certain embodiments, the patient is first dose-loaded with IV steroids (e.g., methylprednisolone), followed by oral steroids (e.g., prednisolone), which are gradually tapered so that the patient is off by week 12. Corticosteroid treatment may be supplemented with tacrolimus (24 weeks) and / or sirolimus (12 weeks), and further supplemented with MMF. When both tacrolimus and sirolimus are used, the dose of each is between 4 ng / mL and about 8 ng / mL, or a total of about 8 ng / mL to about 16 ng / mL of blood tonicity. It is better to have a low dose adjusted to maintain rough levels. In certain embodiments, when only one of these agents is used, the total dose for tacrolimus and / or sirolimus can range from about 16 ng / mL to about 24 ng / mL. When only one of the agents is used, the labeled dose (higher dose); for example, 0.15 to 0.20 mg / kg / day of tacrolimus given as two divided doses every 12 hours; and 1 mg / m 2 Sirolimus should be used per day; the loading dose is 3 mg / m 2 should be. If MMF is added to the regimen, the doses of tacrolimus and / or sirolimus may be maintained due to different mechanisms of action. These and other therapies may be initiated on about day -14 to -1 (e.g., day -2, day 0, etc.) and continued for up to about 1 week (7 days), or up to about 60 days, or up to about 12 weeks, or up to about 16 weeks, or up to about 24 weeks, or up to about 48 weeks, or longer as needed. In certain embodiments, a dosing regimen that does not include tacrolimus is selected.

[0096] That said, in one embodiment, patients with one or more of the following characteristics may be excluded from treatment at the discretion of the attending physician: Have a contraindication to IC injection, including any of the following: * Review of baseline MRI scan indicates contraindication to IC injection. - History of head / neck surgery that contraindicates IC injection. - There is a contraindication to CT (or contrast agent) or general anesthesia. -There is a contraindication to MRI (or gadolinium). 〇<30mL / min / 1.73m 2 have an estimated glomerular filtration rate (eGFR) of Have a neurocognitive deficit not attributable to MPS I or have a diagnosis of a neuropsychiatric condition. -Historic hypersensitivity reaction to one or more of the following: tacrolimus, sirolimus, prednisolone, methylprednisolone, or prednisolone. Inappropriate immunosuppressive therapy (e.g., <1.3 × 10 3 Neutrophils / μL Absolute number of <100 × 10 3 / μL and platelet count <12g / dL (male) or <10g / dL (female) hemoglobin. -There is a contraindication to lumbar puncture. Undergoing HSCT. -Has received laronidase via IT administration within 6 months prior to treatment. Have received IT laronidase at any time and experienced a serious adverse event that is considered to be related to IT treatment that places the patient at undue risk. History of lymphoma or another cancer other than squamous cell or basal cell carcinoma of the skin that has not been in complete remission for at least 3 months prior to treatment. · Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) of >3× upper limit of normal (ULN) or total bilirubin of >1.5×ULN, unless the patient has a previously known history of Gilbert's syndrome and fractionated bilirubin showing bound bilirubin of <35% of total bilirubin. ·History of a positive test for Human Immunodeficiency Virus (HIV), active or recurrent Hepatitis B or C, or a positive screening test for Hepatitis B, Hepatitis C, or HIV. ·Pregnant, <6 weeks postpartum, breastfeeding, or planning pregnancy (autologous or partnered). ·History of alcohol or drug abuse within 1 year prior to treatment. ·Has a serious or unstable medical or psychological condition that jeopardizes patient safety. · Poorly controlled seizures.

[0097] In another embodiment, the attending physician may determine that the presence of one or more of these physical characteristics (medical history) should not preclude treatment as provided herein.

[0098] Given that HSCT is the standard of care for children with MPS I in the United States, Elephants may be adults aged 18 years or older. Enrollment of children aged 6 years or older will begin only if no safety concerns are identified in these subjects 8 weeks after gene therapy.

[0099] In other countries, e.g., Brazil, HSCT and / or ERT are limited and the unmet medical need in patients with Hurler syndrome is significant. Therefore, enrolling patients under 3 years of age is justified as these patients have the greatest prospect of benefiting from treatment with rAAV9.hIDUA. Eligible patients include those with severe forms of MPS I and at risk for early-stage neurocognitive impairment. The pharmacodynamic effects of AAV9.hIDUA can be measured using biomarkers of disease, and the efficacy of AAV9.hIDUA in subjects with Hurler syndrome can be measured using cognitive function.

[0100] In such a population, patients may meet the following criteria: In certain embodiments, subjects must meet all of the following inclusion criteria: 1) Male or female under the age of 3. 2) The subject's legal guardian is willing and able to provide signed informed consent after the nature of the clinical trial has been explained to them and before any procedures related to the clinical trial are performed. 3) Proven diagnosis of severe MPS I-Hurler: 4) Presence of clinical signs and symptoms compatible with MPS I-Hurler, and / or 5) Homozygosity or compound heterozygosity for mutations exclusively associated with a severe phenotype. 6) Have an IQ score of 55 or higher 7) Have sufficient hearing and vision to complete required protocol testing with or without assistive devices and are willing to comply with wearing assistive devices on testing days, if applicable.

[0101] In certain embodiments, subjects who meet any of the following exclusion criteria are ineligible for treatment. 8) Contraindicated for IC infusions containing any of the following: a) Review of baseline magnetic resonance imaging (MRI) studies by a neuroradiologist / neurosurgeon adjudication committee indicates contraindication to IC injection. b) History of previous head / neck surgery that contraindicated IC injection based on review of available information by a panel of neuroradiologists / neurosurgeons. c) Contraindicated to computed tomography (CT) (or contrast) or general anesthesia. d) MRI (or gadolinium) is contraindicated. e) Estimated glomerular filtration rate (eGFR) < 30 mL / min / 1.73 m 2 It is. 9) Have a neurocognitive disorder not attributable to MPS I or a diagnosis of a neuropsychiatric condition that, in the opinion of the PI, may confound the interpretation of the study results. 10) Lumbar puncture is contraindicated. 11) Having undergone hematopoietic stem cell transplantation (HSCT) 12) Previous treatment with AAV-based gene therapy products 13) Received intrathecal (IT) laronidase at any time and experienced a significant AE considered to be related to IT administration that, in the opinion of the PI, placed the subject at undue risk. 14) History of lymphoma that has not been in complete remission for at least 3 months prior to screening or a history of another cancer other than squamous cell or basal cell carcinoma of the skin. 15) Uncontrolled hypertension (systolic blood pressure [BP] >180mmHg, diastolic blood pressure >100mmHg) despite maximal medical therapy. 16) Platelet count <100,000 / microliter (μL) 17) Alanine aminotransferase (ALA) was not detected at screening unless the subject had a history of Gilbert's syndrome. have amyotrophic lateral laminase (ALT) or aspartate aminotransferase (AST) >3×upper limit of normal (ULN) or total bilirubin >1.5×ULN. 18) History of human immunodeficiency virus (HIV) or hepatitis B or C virus infection, or positive screening test for hepatitis B surface antigen, hepatitis B core antibody, hepatitis C or HIV antibody. 19) Received an investigational drug within 30 days or 5 half-lives (whichever is longer) prior to signing the informed consent form (ICF) 20) Being a first-degree family member of an employee of a medical institution or other individual involved in the conduct of the clinical trial, or being an employee of a medical institution or other individual involved in the conduct of the clinical trial. 21) There is a clinically significant ECG abnormality that, in the opinion of the PI, compromises the safety of the subject. 22)Has a serious or unstable medical or psychological condition that, in the opinion of the PI, compromises the subject's safety or successful participation in the trial or interpretation of the trial results. 23) Ventricular shunts which, in the opinion of the institution's neuroradiologist / neurosurgeon and in discussion with the medical monitor, may affect the subject's dosing and proper dosing.

[0102] In certain embodiments, subjects may be excluded from treatment based on the following criteria related to immunosuppressive therapy: 21) History of hypersensitivity reactions to tacrolimus, sirolimus, or prednisone; 22) History of primary immunodeficiency (e.g., common variable immunodeficiency syndrome), splenectomy, or any underlying disease that predisposes the subject to infection. 23) Herpes zoster, cytomegalovirus, or Epstein-Barr virus (EBV) infection that had not completely resolved at least 12 weeks prior to screening. 24) Any infection requiring hospitalization or treatment with parenteral anti-infectives that did not resolve at least 8 weeks prior to Visit 2. 25) Any active infection requiring oral anti-infectives (including antivirals) within 10 days prior to Visit 2. 26) History of active tuberculosis (TB) or positive QuantiFERON TB Gold test during screening. 27) Any live vaccine within 8 weeks prior to signing the ICF. 28) Major surgery within 8 weeks prior to signing the ICF or planned during the study period. 29) Anticipate the need for adenoidectomy or tonsillectomy within 6 months of enrollment. If adenoidectomy or tonsillectomy is anticipated, it should be performed prior to screening. 30) Absolute neutrophil count < 1.3 x 10 3 / μL. 31) Any condition or laboratory abnormality that the clinician considers to be inappropriate for immunosuppressive therapy.

[0103] 5.2.2. Dosage and route of administration A pharmaceutical composition suitable for administration to a patient comprises a suspension of the rAAV.hIDUA vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In certain embodiments, the pharmaceutical compositions described herein are administered intrathecally. In another embodiment, the pharmaceutical compositions described herein are administered intracapsularly. In another embodiment, the pharmaceutical compositions described herein are administered intravenously. In certain embodiments, the pharmaceutical compositions are delivered via a peripheral vein by infusion over a period of 20 minutes (±5 minutes). However, this time may be adjusted as necessary or desired. However, still other routes of administration may be selected. Alternatively or additionally, routes of administration may be combined as desired.

[0104] While a single dose of rAAV is expected to be effective, administration may be repeated (e.g., four times a year, particularly in the treatment of neonates, twice a year, annually, or otherwise as needed). Optionally, the initial dose of a therapeutically effective amount may be delivered over a period of divided infusions / infusions, taking into account the age and ability of the subject to tolerate the infusion / infusion. However, repeated weekly infusions of the full therapeutic dose are not required, providing advantages to the patient in terms of both comfort and therapeutic outcome.

[0105] In some embodiments, the rAAV suspension comprises at least 1×10 9 rAA in GC / mL In certain embodiments, the rAAV suspension has a titer of at least about 4 × 10 genome copies (GC) of rAAV. In certain embodiments, the ratio of empty / full particles of rAAV in the rAAV suspension is between 0.01 and 0.05 (95% to 99% empty capsids free). In some embodiments, an MPS I patient in need thereof has a titer of at least about 4 × 10 genome copies (GC) of rAAV. 8 GC / g brain mass ~ approx. 4×10 11 Use of GC / g brain mass The amount is administered.

[0106] The therapeutically effective intrathecal / intracapsular dose of rAAV.hIDUA for patients with MPSI is approximately 1 × 10 11~7.0×10 14 GC (flat dose) (10 9 ~5×10 10 Brain mass of GC / g patients and Alternatively, the following therapeutically effective flat-line doses can be administered to patients in the indicated age groups: Newborn: Approx. 1 x 10 11 ~Approx. 3×10 14 G.C.; 3 to 9 months: approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; 4 months to under 9 months: approx. 1.2 x 10 12 ~Approx. 6×10 12 ; 9 months to 18 months: Approx. 2 x 10 12 ~Approx. 1.0×10 13 ; 18 months to under 3 years: approx. 2.2 x 10 12 ~Approx. 1.1×10 13 ; 9 months to 6 years: Approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; Under 3 years old (newborn to 3 years old): Approx. 1 x 10 11 ~Approx. 1.2×10 13 G.C.; Ages 3 to 6: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.; 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0107] In another embodiment, the following therapeutically effective flat-line doses are administered to MPS patients in the following age groups: Newborn: Approx. 3.8 x 10 12 ~Approx. 1.9×10 14 G.C.; 3 to 9 months: approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; 4 months to under 9 months: approx. 6.0 x 10 12 ~Approx. 3.0×10 13 ; 9 months to 18 months: Approx. 1.0 x 10 13 ~Approx. 5.0×10 13 ; 18 months to under 3 years: approx. 1.1 x 10 13 ~Approx. 5.5×10 13 ; 9~36 months: Approx. 10 13 ~Approx. 5×10 13 G.C.; Under 3 years old (newborn to 3 years old): Approx. 1 x 10 11 ~Approx. 1.2×10 13 G.C.; Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 3-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.; 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0108] In certain embodiments, one or more of these ranges are used for patients of any age. 12 Total genome copies (GC) (2.0 × 10 9 GC / g brain matter amount) or 6×10 12 Total GC (1 × 10 10 A plateau dose of 2×10 GC / g brain mass is administered to patients ≥4 months and <9 months of age. 12 Total GC (2.0 × 10 9 GC / g brain mass) or 1×10 13 Total GC (1 × 10 10 GC / g brain mass) A plateau dose is administered to patients aged ≥ 9 months to < 18 months. In certain embodiments, a plateau dose of 2.2 x 10 12 Total GC (2.0 × 10 9 GC / g brain mass) or 1.1 × 10 13 Total GC(1×10 10 In certain embodiments, a plateau dose of 6.0×10 GC / g brain mass is administered to patients ≥ 18 months and < 3 years of age. 12 (1.0×10 10 GC / g brain matter amount) or 3×10 13 Total genome copies (GC) (5 × 10 10 In certain embodiments, a plateau dose of 1.0×10 GC / g brain mass is administered to patients ≥4 months and <9 months of age. 13 (1.0×10 10 GC / g brain mass) or 5.0 × 10 13 Total GC (5 × 10 10 In certain embodiments, a plateau dose of 1.1×10 GC / g brain mass is administered to patients ≥ 9 months and < 18 months of age. 13 (1.0×10 10 GC / g brain mass) or 5.5 × 10 13 Total GC (5 × 10 10 In certain embodiments, a plateau dose of 2.6×10 GC / g brain mass is administered to patients ≥ 18 months and < 3 years of age. 12 Genome copies (GC) (2.0 × 10 9 A flat-line dose of 1000 mg / g brain mass is administered to patients aged 6 years and older. In the embodiment, 1.3 x 10 13 (GC)(1.0×10 10 A plateau dose of 1.4×10 GC / g brain mass is administered to patients aged 6 years and older. In some embodiments, the dose administered to patients aged 12 years and older (including 18 years and older) with MPS I is 1.4×10 13 Genome copies (GC) (1.1 × 10 10 In some embodiments, the dose administered to patients aged 12 years or older (including those aged 18 years or older) with an MPS I is 7×10 13 Genome copies (GC) (5.6 × 1010 GC / g brain mass). In yet a further embodiment, the dose administered to an MPS I patient is about 4×10 8 GC / g brain mass ~ approx. 4×10 11 GC / g brain mass. In certain embodiments, The dose administered to an MPS I neonate is approximately 1.4 × 10 11 from about 1.4 × 10 14 GC; the dose administered to a 3- to 9-month-old infant is approximately 2.4 × 10 11 From about 2.4 × 10 14 GC; the dose administered to children 9 to 36 months of age is approximately 4 × 10 11 From about 4 × 10 14 GC; the dose administered to children aged 3 to 12 years is approximately 4.8 × 10 11 From about 4.8 x 10 14 GC; the dose administered to children aged 12 years and older and adults is approximately 5.6 × 10 11 From about 5.6 × 10 14 This extends to GC.

[0109] Suitable volumes for delivery of these doses and concentrations may be determined by one of skill in the art. For example, a volume of about 1 μL to 150 mL may be selected, with higher volumes being selected for adults. Typically, for neonates, a suitable volume is about 0.5 mL to about 10 mL, and for older infants, about 0.5 mL to about 15 mL may be selected. For infants, a volume of about 0.5 mL to about 20 mL may be selected. For children, a volume of up to about 30 mL may be selected. For prepubertal children and teenagers, a volume of up to about 50 mL may be selected. In yet another embodiment, patients may receive intrathecal administration at a selected volume of about 5 mL to about 15 mL, or about 7.5 mL to about 10 mL. Other suitable volumes and dosages may be determined. Dosages are adjusted to balance therapeutic benefit against side effects, and such dosages may vary depending on the therapeutic application in which the recombinant vector is employed.

[0110] In one embodiment for intramedullary delivery, the patient is an adult subject and the dose is about 1×10 8 GC ~5×10 14 In another embodiment, the dose is about 3.8×10 12 ~Approx. 1.9×10 14 In further embodiments, the patient is an infant subject with Hurler syndrome at least about 3 months to 12 months of age, at least about 3 months to 24 months of age, or at least about 3 months to 36 months of age, at least about 3 months to 6 years of age, at least 6 months to 6 years of age, at least 12 months to 6 years of age, and the dose is 4×10 8 rAA of GC V9.hIDUA / g brain mass ~3×10 12 In another example, patients are children up to 3 years of age with Hurler-Scheie syndrome and the dose is at least 4 x 10 8 GC rAAV9.hIDUA / g brain mass ~ 5×10 10 In another example, the patient is a child at least about 6 to 18 years of age with Hurler-Scheie syndrome and the dose is at least 4 x 10 8 GC rAAV9.hIDUA / g brain mass ~3 x 10 12 G.C. Contains the equivalent of rAAV9.hIDUA / g brain mass.

[0111] 5.2.3. Monitoring Effectiveness The efficacy of the treatment will be demonstrated by (a) preventing neurocognitive decline in patients with MPS I and (b) reducing disease progression. Biomarkers of disease can be measured, for example, by assessing the levels and / or enzyme activity of GAGs in CSF, serum and / or urine, and / or liver and spleen volumes. Neurocognition can be assessed by measuring intelligence quotient (IQ), for example, as measured by the Bayley Scales of Infant Development for Hurler subjects, or by the Wechsler Abbreviated Scale of Intelligence (WASI) for Hurler-Scheie subjects. Other suitable measures of neurocognitive development and function may be utilized, for example, assessing developmental quotient (DQ) using the Bayley Scales of Infant Development (BSID-III), assessing memory using the Hopkins Verbal Learning Test, and / or using the Test of Variables of Attention (TOVA). Changes in hearing ability as measured by auditory brainstem response (ABR) testing. Other neuropsychological functions, for example, Vineland Adaptive Behavior Scales (e.g., Vineland II), visual processing, fine motor, communication, socialization, daily living skills, and emotional and behavioral health, may be monitored. Magnetic resonance imaging (MRI) of the brain to obtain volumetric diffusion tensor imaging (DTI) as well as resting state data, mean nerve cross-sectional area by ultrasound, improvement in spinal cord compression, safety, liver size and spleen size will also be managed.

[0112] Optionally, other measures of efficacy may include assessment of biomarkers (e.g., spermine and other polyamines as described herein) and clinical outcomes. Urine is assessed for total GAG content, concentration of GAG relative to creatinine, as well as pGAG specific for MPS I. Serum and / or plasma is assessed for IDUA activity, anti-IDUA antibodies, pGAG, and concentrations of heparin cofactor II-thrombin complexes and markers of inflammation. CSF is assessed for IDUA activity, anti-IDUA antibodies, hexosaminidase (hex) activity, and pGAG (e.g., heparan sulfate and dermatan sulfate). The presence of neutralizing antibodies against the vector (e.g., AAV9) and binding antibodies to anti-IDUA antibodies may be assessed in CSF and serum. T cell responses against the vector capsid (e.g., AAV9) or hIDUA transgene product may be assessed by ELISPOT assay. Vector concentrations (PCR for AAV9 DNA) as well as the pharmacokinetics of IDUA expression in serum and urine may also be monitored.

[0113] The combination of gene therapy delivery of rAAV.hIDUA to the CNS with systemic delivery of hIDUA is encompassed by the methods of the invention. Systemic delivery can be achieved using ERT (e.g., using Aldurazyme™) or with additional gene therapy using liver-tropic rAAV.hIDUA (rAAV.hIDUA carrying the AAV8 capsid).

[0114] Additional measures of clinical efficacy associated with systemic delivery include, for example, orthopedic measurements, such as bone mineral density, bone mineral mass, bone geometry and strength, bone density measured by dual energy x-ray absorptiometry (DXA); height (Z-score for height / supine length for age); markers of bone turnover; measurements of serum osteocalcin (OCN) and bone specific alkaline phosphatase (BSAP), carboxy-terminal telopeptide of collagen type I (ICTP) and carboxy-terminal telopeptide alpha 1 chain of collagen type I (CTX); flexibility and muscle strength. ; Biodex and physical therapy including a 6-minute walk test (assessing muscle strength at the knee and elbow for each participant using the Biodex III isokinetic muscle testing system); active range of motion (ROM); Children's Health Questionnaire / Health Status Questionnaire (CHAQ / HAQ) Disability Index score; electromyography (EMG) and / or oxygen utilisation to monitor individual cardiopulmonary fitness; peak oxygen uptake during exercise testing (VO2 peak); apnea-hypopnea index (AHI); forced vital capacity (FVC); left ventricular mass (LVM) may be included.

[0115] In certain embodiments, methods are provided for diagnosing and / or treating or monitoring treatment of MPS I in a patient. The methods include administering to a patient suspected of having MPS I. The method involves obtaining a sample of cerebrospinal fluid or plasma from a patient, detecting a concentration level of spermine in the sample, diagnosing the patient with a mucopolysaccharidosis selected from MPS I in patients with spermine concentrations greater than 1 ng / mL, and delivering an effective amount of human α-L-iduronidase (hIDUA) to the patient diagnosed as provided herein, e.g., using a device as described herein.

[0116] In another embodiment, the method involves monitoring and adjusting MPS I therapy. Such a method involves obtaining a sample of cerebrospinal fluid or plasma from a human patient being treated for MPS I, detecting the concentration level of spermine in the sample by performing mass spectrometry analysis, and adjusting the administration level of the MPS I therapeutic agent. For example, a "normal" human spermine concentration is 1 ng / mL or less in cerebrospinal fluid. However, a patient with untreated MPS I may have spermine concentration levels of more than 2 ng / mL up to about 100 ng / mL. If the patient has levels approaching normal levels, the administration of concomitant ERT may be reduced. Conversely, if the patient has higher than desired spermine levels, a higher dose or additional therapy, such as ERT, may be provided to the patient.

[0117] Spermine concentration may be determined using a suitable assay. See, for example, J. Sanchez-Lopez, et al., "Underivatives of polyamines." "A novel method for the analysis of plant samples by ion pair liquid chromatography coupled with electrospray tandem mass spectrometry," Plant Physiology and Biochemistry, 47(2009):592-598, available online, February 28, 2009; M.R. Hakkinen, et al, "Analysis of underivatized polyamines by reversed phase liquid chromatography with electrospray tandem mass spectrometry", J. Pharm. Biomec. Analysis, 44 (2007): 625-634, a quantitative isotope dilution liquid chromatography (LC) / mass spectrometry (MS) assay. Other suitable assays may be used.

[0118] In some embodiments, the efficacy of the therapeutic agents described herein is determined by assessing neurocognition after 52 weeks of administration in pediatric subjects with MPS I who have early neurocognitive deficits. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the relationship of CSF glycosaminoglycans (GAGs) to neurocognition in MPS I patients. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the effect of the therapeutic agent on physical changes to the CNS in MPS I patients as measured by magnetic resonance imaging (MRI), for example, by volumetric analysis of gray and white matter and CFS ventricles. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the pharmacokinetic effect of the therapeutic agent on biomarkers (e.g., GAGs, HS) in the cerebrospinal fluid (CSF), serum, and urine of MPS I patients. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the effect of the therapeutic agent on quality of life (QOL) in MPS I patients. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the effect of the therapeutic agent on motor function in MPS I patients. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the effect of the therapeutic agent on growth and development outcomes in MPS I patients.

[0119] As expressed from the rAAV vectors described herein, CSF, serum or When detected in other tissues, an expression level of at least about 2% may provide a therapeutic effect. However, even higher expression levels may be achieved. Such expression levels may be from 2% to about 100% of normal, functional human IDUA levels. In certain embodiments, higher than normal expression levels may be detected in CSF, serum, or other tissues.

[0120] In certain embodiments, the methods of treating, preventing and / or ameliorating MPS I and / or symptoms thereof described herein result in a significant increase in the Intelligence Quotient (IQ) in the treated patient as assessed using the Bayley Infant Development Scales for Hurler Subjects. In certain embodiments, the methods of treating, preventing and / or ameliorating MPS I and / or symptoms thereof described herein result in a significant increase in the neurocognitive IQ in the treated patient as measured by the Wechsler Abbreviated Intelligence Scale for Hurler-Scheie Subjects (WASI). In certain embodiments, the methods of treating, preventing and / or ameliorating MPS I and / or symptoms thereof described herein result in a significant increase in the neurocognitive DQ in the treated patient as assessed using the Bayley Infant Development Scales.

[0121] In certain embodiments, the methods of treating, preventing and / or ameliorating MPS I and / or symptoms thereof described herein result in a significant increase in functional human IDUA levels. In certain embodiments, the methods of treating, preventing and / or ameliorating MPS I and / or symptoms thereof described herein result in a significant increase in levels of GAG as measured in a patient's serum, urine and / or cerebrospinal fluid (CSF) samples.

[0122] Combination Therapy The combination of gene therapy delivery of rAAV.hIDUA to the CNS with systemic delivery of hIDUA is encompassed by the methods of the invention. Systemic delivery may be accompanied by additional gene therapy with ERT (e.g., using Aldurazyme®) or liver-tropic rAAV.hIDUA (rAAV.hIDUA with an AAV8 capsid).

[0123] In certain embodiments, the intramedullary administration of rAAV9.hIDUA is co-administered with a second AAV.hIDUA injection, for example, directed to the liver. In such instances, the vectors may be identical. For example, the vectors may have the same capsid and / or the same vector genome sequence. Alternatively, the vectors may be different. For example, each of the vector stocks may be designed with different regulatory sequences (e.g., each with a different tissue-specific promoter), for example, a liver-specific promoter and a CNS-specific promoter. Additionally or alternatively, each of the vector stocks may have a different capsid. For example, the vector stocks directed to the liver may have a capsid selected from AAV8, AAVrh64R1, AAVrh64R2, rh8, rh10, AAV3B, or AAVdj, among others. In such a dosing regimen, the dose of each vector stock may be adjusted so that the total of the vectors delivered intramedullarily is about 1×10 8 GC~1×10 14 GC Scope in another embodiment, the combined vector delivered by both routes is within 1×10 11 ~1×10 16 Alternatively, each vector is in the range of about 10 8 GC~about 10 12 The amount of GC / vector may be delivered in a dose of 100 mg / kg / day. The administration of the active ingredient(s) may be simultaneous or at different times, for example, from about 1 day to about 12 weeks apart, or from about 3 days to about 30 days apart, or at other suitable times.

[0124] In some embodiments, the patient is co-administered AAV.hIDUA via liver-directed infusion and intramedullary infusion. In some embodiments, the method of treatment includes: (a) treating a patient with MPS I and / or symptoms associated with Hurler syndrome, Hurler-Scheie syndrome, and Scheie syndrome; (b) administering a sufficient amount of hIDUA enzyme or liver-directed rAAV.hIDUA to the CNS of the patient to induce transgene-specific tolerance; and (b) administering rAAV9.hIDUA to the CNS of the patient, wherein rAAV.hIDUA directs expression of therapeutic levels of hIDUA in the patient.

[0125] In further embodiments, methods are provided for treating human patients with MPS I and / or Hurler, Hurler-Scheie and Scheie syndrome-associated conditions that involve tolerizing the patient with a sufficient amount of hIDUA enzyme or liver-directed rAAV.hIDUA to induce transgene-specific tolerance, followed by CNS-directed rAAV-mediated delivery of hIDUA to the patient. In certain embodiments, for example, if the patient is under 4 weeks of age (neonatal stage) or an infant, the patient is administered rAAV.hIDUA via liver-directed infusion to tolerize the patient to hIDUA, and if the patient is an infant, child and / or adult, the patient is subsequently administered rAAV.hIDUA via intramedullary infusion to express therapeutic concentrations of hIDUA in the CNS.

[0126] In one example, an MPS I patient is tolerized by delivering hIDUA to the patient within about 2 weeks of birth, e.g., within about 0 to about 14 days, or within about 1 to 12 days, or within about 3 to about 10 days, or within about 5 to about 8 days, i.e., the patient is a newborn. In another embodiment, an older infant may be selected. The tolerizing dose of hIDUA may be delivered via rAAV. However, in another embodiment, the dose may be delivered by direct delivery of the enzyme (enzyme replacement therapy). Methods for producing recombinant hIDUA in Chinese hamster ovary (CHO) cells and soluble hIDUA in tobacco cells [L H Fu, et al, Plant Science, (Impact Factor:3.61). 12 / 2009; 177(6): 668-675] or in plant seeds [X He, et al, Plant Biotechnol J. 2013, Dec; 11(9): 1034-1043] have been described in the literature.

[0127] Additionally, recombinant hIDUA is commercially produced as Aldurazyme® (Laronidase), and a fusion protein of anti-human insulin receptor monoclonal antibody and α-L-iduronidase [AGT-181; ArmaGen, Inc] may be useful. Although currently less preferred, the enzyme may be delivered via "naked" DNA, RNA, or another suitable vector. In one embodiment, the enzyme is delivered intravenously and / or intramedullarily to the patient. In another embodiment, another route of administration is used (e.g., intramuscular, subcutaneous, etc.). In one embodiment, the MPS I patient selected for tolerization is unable to express detectable amounts of hIDUA prior to the initiation of tolerization administration. When recombinant human IDUA enzyme is delivered, the intramedullary rhIDUA infusion may consist of about 0.58 mg / kg body weight or about 0.25 mg to about 2 mg total rhIDUA per infusion (e.g., intravenous or intramedullary). For example, 3 cc of enzyme (e.g., approximately 1.74 mg Aldurazyme® (Laronidase)) is diluted with 6 cc of Elliot B® for a total injection of 9 cc. Alternatively, larger or smaller doses may be selected. Similarly, when expressed from a vector, low levels of expressed protein may be delivered. In one embodiment, the amount of hIDUA delivered for tolerization is less than the therapeutically effective amount. However, other doses may be selected.

[0128] Typically, following administration of the tolerizing dose, the therapeutic dose is delivered to the subject, for example, within about 3 days to about 6 months after the tolerizing dose, more preferably within about 7 days to about 1 month after the tolerizing dose, however, other time points within these ranges may be selected, as may longer or shorter waiting periods.

[0129] In certain embodiments, the immunosuppressive co-therapy may be delivered in combination with the sole gene therapy vector or a combination of gene therapy vectors described herein. Alternatively, immunosuppressive therapy may be given in addition to the vector before, during, and / or following vector administration. The immunosuppressive therapy may include prednisolone, mycophenolate mofetil (MMF) and tacrolimus or sirolimus, as described above. In another embodiment, the immunosuppressive therapy may be initiated about 2 days before vector administration and may include a single intravenous dose of methylprednisolone about 2 days before the vector, an oral dose of prednisone, an oral dose of tacrolimus, and an oral dose of sirolimus. Prednisone is administered orally daily from about 2 days before vector administration until about 16 weeks after treatment. Tacrolimus is administered orally daily from about 2 days before vector administration until about 24 weeks after treatment. In certain embodiments, a tacrolimus-free dosing regimen described below may be preferred. Sirolimus may be administered orally daily starting about 2 days prior to vector administration until about 48 weeks after treatment.

[0130] In certain embodiments, a therapeutic regimen useful for treating alpha-L-iduronidase deficiency in a human patient comprises: (a) a recombinant AAV (rAAV) comprising a nucleic acid comprising an AAV9 capsid and a sequence encoding human alpha-L-iduronidase (hIDUA) under the control of regulatory sequences that direct its expression in the patient, wherein the human hIDUA coding sequence has the nucleotide sequence of SEQ ID NO:1 that encodes a functional hIDUA, or a sequence that is at least about 80% identical to SEQ ID NO:1; (b) a glucocorticoid, steroid, or antimetabolite; and (c) at least a first immunosuppressant selected from at least one of an anti-cancer drug, a T-cell inhibitor, a macrolide, or a cytostatic; and (c) at least a second immunosuppressant selected from at least one of a glucocorticoid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic, wherein administration of the at least one immunosuppressant begins prior to or on the same day as delivery of the AAV vector; and administration of the at least one immunosuppressant continues for at least 8 weeks after vector administration. The patient may be initially administered intravenous steroids, followed by oral steroids. In certain embodiments, the immunosuppressant combination includes one or more corticosteroids, and optionally, mycophenolate mofetil (MMF), and / or a calcineurin inhibitor, and / or an mTOR inhibitor. The one or more calcineurin inhibitors may be tacrolimus. The one or more mTOR inhibitors may be temsirolimus or sirolimus, or another rapalog (e.g., everolimus). In certain embodiments, administration of steroids to the patient is discontinued 12 weeks after vector administration. In certain embodiments, mycophenolate mofetil (MMF) and tacrolimus are delivered 0-15 days prior to vector administration. In certain embodiments, the immunosuppressant is mycophenolate mofetil (MMF) and sirolimus.

[0131] In embodiments in which the immunosuppressant includes both tacrolimus and sirolimus, low doses of each can be used to maintain blood trough levels of about 4 ng / mL to about 8 ng / mL, or a total of about 8 ng / mL to about 16 ng / mL. In embodiments in which the immune support includes only tacrolimus or sirolimus, the total dose ranges from about 16 ng / mL to about 24 ng / mL. In certain embodiments in which only tacrolimus or sirolimus is used, the initial loading dose is about 3 mg / m 2 It could be.

[0132] In certain embodiments, immunosuppressive therapy is initiated from about -14 days to about -1 day prior to vector administration.

[0133] In certain embodiments, the encoded hIDUA is (a) SEQ ID NO:2 (Genbank NP_000193); or (b) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids 1 to 653 of SEQ ID NO:2. do.

[0134] In certain embodiments, the nucleic acid sequence packaged into the AAV capsid further comprises a 5' inverted terminal repeat (ITR) sequence, a chicken beta-actin intron, a CB7 promoter, a polyA signal, and / or a 3' ITR sequence.

[0135] In certain embodiments, the rAAV is in a suspension having a pH of 6-9.

[0136] In certain embodiments, the rAAV is delivered via intrathecal injection.

[0137] In certain embodiments, the regimen further comprises intravenously co-administering a rAAV containing the hIDUA gene.

[0138] In certain embodiments, the efficacy of treatment includes measuring changes in hearing ability, optionally by auditory brainstem testing.

[0139] In certain embodiments, a composition is provided comprising a recombinant AAV vector comprising a heterologous nucleic acid encoding human alpha-L-iduronidase (hIDUA) for use in a therapeutic regimen, wherein the human hIDUA coding sequence has a nucleotide sequence of SEQ ID NO:1 encoding a functional hIDUA or a sequence that is at least about 80% identical to SEQ ID NO:1, and the composition further comprises at least a first immunosuppressant selected from at least one of (b) and (c) at least a second immunosuppressant selected from at least one of a glucocorticoid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent, wherein administration of the immunosuppressant begins prior to or on the same day as delivery of the AAV vector; and administration of at least one of the immunosuppressants continues for at least 8 weeks after vector administration. In certain embodiments, the macrolide is one or more anti-calcineurin inhibitors, one or more mTOR inhibitors, or a combination thereof.

[0140] In certain embodiments, one or more compositions comprising at least one immunosuppressant for use in combination therapy with a recombinant AAV vector comprising a heterologous nucleic acid encoding human alpha-L-iduronidase (hIDUA) are provided, where the human hIDUA coding sequence has a nucleotide sequence of SEQ ID NO:1 encoding a functional hIDUA, or a sequence that is at least about 80% identical to SEQ ID NO:1, and the immunosuppressant comprises (a) a composition comprising at least a first immunosuppressant selected from at least one of a glucocorticoid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent, and (b) a composition comprising at least a second immunosuppressant selected from at least one of a glucocorticoid, a steroid, an antimetabolite, a T-cell inhibitor, a macrolide, or a cytostatic agent, where administration of at least one of the immunosuppressants begins prior to or on the same day as delivery of the AAV vector; and administration of at least one of the immunosuppressants continues for at least 8 weeks after vector administration. In certain embodiments, the macrolide is one or more anti-calcineurin inhibitors, one or more mTOR inhibitors, or a combination thereof.

[0141] 5.4.Manufacturing The present invention provides for the preparation of the rAAV.hIDUA pharmaceutical composition described herein (Example 5 below). An illustrative manufacturing process is provided in FIG. 11. The rAAV.hIDUA vector can be prepared as shown in the flow chart shown in FIG. 11. Briefly, cells are prepared by suitable cell culture (e.g., HEK293 cells). Methods for producing the gene therapy vectors described herein include methods well known in the art, such as the production of plasmid DNA used in the generation of the gene therapy vector, the production of the vector, and the purification of the vector. In some embodiments, the gene therapy vector is AAV.hIDUA. The vectors are AAV cis-plasmids encoding the AAV genome and the gene of interest, AAV trans-plasmids containing the AAV rep and cap genes, and adenovirus helper plasmids. The process of vector production can include method steps such as initiating cell culture, passaging cells, seeding cells, transfecting cells with plasmid DNA, changing the medium to serum-free medium after transfection, and harvesting vector-containing cells and culture medium. The harvested vector-containing cells and culture medium are referred to herein as crude cell harvest.

[0142] The crude cell harvest may then be subjected to process steps such as, for example, concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the fluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare large quantities of vector.

[0143] The vector drug product is purified and empty capsids are removed using a two-step affinity chromatography purification at high salt concentration followed by anion exchange resin chromatography. These methods are described in further detail in International Patent Application No. PCT / US2016 / 065970, filed December 9, 2016, and its priority documents, U.S. Patent Application Nos. 62 / 322,071, filed April 13, 2016, and 62 / 226,357, filed December 11, 2015, entitled "Scalable Purification Method for AAV9," which are incorporated herein by reference. and "AAV8 purification methods," International Patent Application No. PCT / US2016 / 065976, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,098, filed April 13, 2016, and U.S. Patent Application No. 62 / 266,341, filed December 11, 2015, and rh10, International Patent Application No. PCT / US16 / 66013, filed December 9, 2016, and its priority documents, U.S. Patent Application No. 62 / 322,055, filed April 13, 2016, and "AAV No. 62 / 266,347, filed December 11, 2015, entitled "Scalable Purification Method for rh10," and for AAV1, International Patent Application No. PCT / US2016 / 065974, filed December 9, 2016, and its priority document, U.S. Patent Application No. 62 / 322,083, filed April 13, 2016, and U.S. Patent Application No. 62 / 26,351, filed December 11, 2015, entitled "Scalable Purification Method for AAV1," are all incorporated herein by reference.

[0144] 5.5. DEVICES AND METHODS FOR DELIVERY OF PHARMACEUTICAL COMPOSITIONS TO THE CEREBROSPINAL FLUID In one embodiment, the vectors provided herein may be administered intrathecally via the methods and / or devices provided in this section and further described in the Examples and FIG. 12. Alternatively, other devices and methods may be selected. The method includes advancing a subarachnoid needle into the patient's cisterna magna, connecting a length of flexible tubing to the proximal hub of the subarachnoid needle and connecting an output port of a valve to the proximal end of the flexible tubing, and allowing the tubing to become self-sufficient with the patient's cerebrospinal fluid after the advancing and connecting steps, then connecting a first container containing a quantity of an isotonic solution to the flush inlet port of the valve, and then connecting a second container containing a quantity of a pharmaceutical composition to the vector inlet port of the valve. After connecting the first and second containers to the valve, a path for fluid flow is opened between the vector inlet and outlet ports of the valve, and the pharmaceutical composition is injected into the patient through the subarachnoid puncture needle; after injecting the pharmaceutical composition, a path for fluid flow is opened between the water flush inlet and outlet ports of the valve, and an isotonic solution is injected through the subarachnoid puncture needle to flush the pharmaceutical composition into the patient.

[0145] In another aspect, a device for intracisternal delivery of a pharmaceutical composition is provided, the device comprising a first container containing a quantity of the pharmaceutical composition, a second container containing an isotonic solution, and a subarachnoid puncture needle through which the pharmaceutical composition may be ejected from the device directly into the cerebrospinal fluid in the cisterna magna of a patient. The device further includes a valve having a first inlet port interconnected to the first container, a second inlet port interconnected to the second container, an outlet port interconnected to the intrathecal needle, and a luer lock that controls the flow of the pharmaceutical composition and the isotonic solution through the intrathecal needle.

[0146] As used herein, the term computed tomography (CT) refers to an x-ray in which a three-dimensional image of a body structure is constructed by a computer from a series of planar cross-sectional images made along an axis.

[0147] The apparatus or medical device 10 as shown in Figure 12 includes one or more containers 12 and 14 interconnected via a valve 16. The containers 12 and 14 provide a fresh source of a pharmaceutical composition, drug, vector or similar substance and a fresh source of an isotonic solution such as saline, respectively. The containers 12 and 14 may be any form of medical device that allows for the infusion of fluids into a patient.

[0148] By way of example, each container 12 and 14 may be provided in the form of a syringe, cannula, etc. For example, in the illustrated embodiment, container 12 is provided as a separate syringe containing a quantity of the pharmaceutical composition, referred to herein as a "vector syringe." For purposes of example only, container 12 may contain approximately 10 cc of the pharmaceutical composition, or the like.

[0149] Similarly, container 14 may be provided in the form of a separate syringe, cannula, or the like, containing a quantity of saline solution, and may be referred to as a "rinse syringe." By way of example only, container 14 may contain approximately 10 cc of saline solution.

[0150] Alternatively, containers 12 and 14 may be provided in forms other than syringes and may be integrated into a single device, such that the integrated medical injection device has a pair of separate chambers, one for the pharmaceutical composition and one for the saline solution, and the size of the chambers or containers may be provided as needed to contain the desired amount of fluid.

[0151] In the described embodiment, the valve 16 is provided as a four-way stopcock with a swivel male luer lock 18. The valve 16 interconnects the containers 12 and 14 (i.e., the vector syringe and the water rinse syringe in the described embodiment), and the swivel male luer lock allows the path through the valve 16 to be opened or closed to the containers 12 and 14, respectively. In this manner, the path through the valve 16 may be closed to both the vector syringe and the water rinse syringe, or may be open to a selected one of the vector syringe and the water rinse syringe. As an alternative to a four-way stopcock, the valve may be a three-way stopcock or a fluid control device.

[0152] In the illustrated embodiment, the valve 16 is connected to one end of a length of extension tubing 20 or similar conduit for the fluid. The tubing 20 may be selected based on the desired length or internal volume. By way of example only, the tubing may be approximately 6-7 inches in length.

[0153] In the illustrated embodiment, the opposite end 22 of tubing 12 is connected to a T-connector extension set 24 which is in turn connected to a subarachnoid needle 26. By way of example, needle 26 may be a 5 inch 22 or 25 gauge subarachnoid needle. Additionally, as an option, subarachnoid needle 26 may be connected to an introducer needle 28, such as, for example, a 3.5 inch 18 gauge introducer needle.

[0154] In use, the intrathecal needle 26 and / or optional introducer needle 28 may be advanced into the patient toward the cisterna magna. After needle advancement, the needle 26 and / or 28 and associated tissue (e.g. A computed tomography (CT) image may be obtained that allows visualization of the intrathecal structures (e.g., lumbar paraspinal muscles, bones, brain stem, and spinal cord). Correct needle placement is confirmed by observation of cerebrospinal fluid (CSF) at the needle hub and visualization of the needle tip within the cisterna magna. A relatively short length of tubing 20 may then be connected to the inserted subarachnoid puncture needle 26, and a four-way stopcock 16 may then be connected to the opposite end of tubing 20.

[0155] The above assembly allows for the patient's CSF to become "self-supplied". A pre-filled saline flush syringe 14 is then connected to the flush inlet of the four-way stopcock 16, and then a vector syringe containing the pharmaceutical composition is connected to the vector inlet of the four-way stopcock 16. The output port of the stopcock 16 is then opened to the vector syringe 12, and the contents of the syringe may be slowly infused into the patient over a period of time via the vector valve and assembly device. For purposes of example only, this period of time may be approximately 1-2 minutes, and / or any other period of time desired.

[0156] After injecting the contents of the vector syringe 12, the swing lock 18 on the stopcock 16 is turned to a second position so that the connected pre-filled water flush syringe 14 can be used to flush the stopcock 16 and needle assembly with a desired amount of saline. By way of example only, 1-2 cc of saline may be used, although more or less may be used as needed. The saline ensures that all or most of the pharmaceutical composition is forced through the assembly tool and into the patient, such that little or none of the pharmaceutical composition remains on the assembly tool.

[0157] After the construction tool has been flushed with saline, the entire construction tool including the needle(s), extension tubing, stopcock and syringe is slowly removed from the subject and placed on a surgical tray for disposal in a biohazard waste container or hard container (for needle(s)).

[0158] The screening process that may ultimately lead to an intracisternal (IC) procedure may be undertaken by the investigator. The investigator may describe the process, procedure, administration procedure itself, and all possible safety risks so that the subject (or designated caregiver) is fully informed. Medical history, concomitant medications, physical examination, vital signs, electrocardiogram (ECG), and laboratory test results are obtained or performed and provided to the neuroradiologist, neurosurgeon, and anesthesiologist for use in screening evaluation of the subject's eligibility for IC procedure.

[0159] To allow adequate time to reconsider eligibility, the following procedure may be performed any time between the initial screening visit and up to 1 week prior to the study visit. For example, gadolinium (i.e., eGFR > 30 mL / min / 1.73 m) should be administered on "Day 0"; 2 ) with or without head Magnetic resonance imaging (MRI) of the neck may be obtained. In addition to the head and neck MRI, the investigator may determine the need for further evaluation of the neck via flexion / extension testing. The MRI protocol may include T1, T2, DTI, FLAIR, and CINE protocol images.

[0160] In addition, a head / neck MRA / MRV may be obtained per institutional protocol to allow proper assessment of CSF flow and identification of possible disconnections or lack of communication between CSF spaces (i.e., subjects with a history of intradural / transdural surgery may be excluded or may require further testing (e.g., radionucleotide cisternography)).

[0161] Neuroradiologists, neurosurgeons, and anesthesiologists will ultimately discuss and decide on each subject's eligibility for IC procedure based on all available information (scans, medical history, physical exam, laboratory tests, etc.) Airway, neck (shortened / thickened), and head range of motion (degree of neck flexion). A pre-operative evaluation of anesthesia may also be obtained on "Day -28" to "Day 1" providing a detailed assessment of anesthesia, taking note of the specific physiological needs of the MPS subject.

[0162] Prior to the IC procedure, the CT lab will verify that the following equipment and medications are present: adult lumbar puncture (LP) kit (supplied by facility); BD (Becton Dickinson) 22 or 25 gauge × 3-7” subarachnoid needle (Quincke bevel); Coaxial introducer needle (for introduction of subarachnoid puncture needle) used at the direction of the interventional physician; 4-way small diameter stopcock with swivel (rotating) male luer lock; T-connector extension set with female luer lock adapter (plumbing), 6.7 inch approximate length; Omnipaque 180 (iohexol) for intramedullary administration; Iodinated contrast media for intravenous (IV) administration; 1% lidocaine solution for injection (if not supplied in adult LP kit); A pre-filled 10cc flush syringe of saline (sterile); radiopaque marker(s); Surgical preparation instruments / shaving razor blades; pillows / supports to allow proper positioning of the intubated subject; Endotracheal intubation equipment, general anesthesia machines and ventilators; Intraoperative neurophysiological monitoring (IONM) equipment (and necessary personnel); and A 10 cc syringe containing the vector; prepared according to a separate pharmacy manual, is brought to the CT / operating room (OR).

[0163] Informed consent for the procedure is confirmed and documented in the chart and / or study file. Separate consent for the procedure from radiology and anesthesiology staff is obtained per institutional requirements. Subjects have intravenous access (e.g., two IV access sites) placed in an appropriate hospital nursing unit per institutional guidelines. Intravenous fluids are administered at the discretion of the anesthesiologist. At the discretion of the anesthesiologist and per institutional guidelines, subjects are induced and receive endotracheal intubation with administration of general anesthesia in an appropriate patient care unit, holding area, or surgical / CT procedure room.

[0164] A lumbar puncture is performed and 5 cc of cerebrospinal fluid (CSF) is first removed, followed by intramedullary injection of contrast agent (Omnipaque 180) to aid in visualization of the cisterna magna. Appropriate positioning of the subject is performed to facilitate diffusion of the contrast agent into the cisterna magna.

[0165] The subject is fitted with intraoperative neurophysiological monitoring (IONM) equipment. The subject is placed on the CT scanner table in prone or lateral position. Appropriate staff must be present to ensure the subject's safety during transportation and positioning. If deemed appropriate, the subject may be positioned in a manner that provides for a degree of cervical flexion determined to be safe during preoperative evaluation, and with normal neurophysiological monitor signals documented following positioning.

[0166] It may be confirmed that the following staff are present and identified on-site: the interventionalist / neurosurgeon who will perform the procedure; the anesthesiologist and respiratory technologist(s); nurses and physician assistants; CT (or OR) technologists; neurophysiology technologists; and the site coordinator. A "time out" may be completed per Joint Commission / hospital protocol to verify the correct subject, procedure, site, positioning, and presence of all necessary equipment in the room. The lead site investigator may then confirm with staff that he / she may proceed with preparing the subject.

[0167] The subject's skin below the base of the skull is shaved appropriately. A CT scout image is taken, followed by a pre-procedure planning CT with IV contrast by the interventionalist if deemed necessary to localize the target location and image the vasculature. After the target site (cisterna) is identified and the needle trajectory is planned, the skin is prepared and draped using sterile technique as per institutional guidelines. A radiopaque marker is placed at the target skin location as indicated by the interventionalist. The skin below the marker is anesthetized via infiltration with 1% lidocaine. A 22G or 25G intrathecal puncture needle is then advanced toward the cisterna magna, with the option of using a coaxial introducer needle.

[0168] After needle advancement, CT images are obtained using the thinnest CT slice thickness feasible with the facility's equipment (ideally ≦2.5 mm). Sequential CT images are obtained using the lowest radiation dose possible that allows adequate visualization of the needle and associated soft tissues (e.g., lumbar paraspinal muscles, bone, brain stem, and spinal cord). Correct needle placement is confirmed by observation of CSF at the needle hub and visualization of the needle tip within the cisterna magna.

[0169] The interventional physician ensures that the vector syringe is located close to, but outside, the sterile field. Prior to handling or administering the pharmaceutical composition in the vector syringe, gloves, masks and eye protection are donned by staff assisting with the procedure within the sterile field.

[0170] An extension tubing is attached to the inserted subarachnoid needle, which is then attached to the 4-way stopcock. Once the device is "self-primed" with the subject's CSF, a 10 cc pre-filled saline flush syringe is attached to the flush inlet port of the 4-way stopcock. A vector syringe is then provided to the interventionalist and attached to the vector inlet port of the 4-way stopcock.

[0171] After the stopcock outlet has been opened to the vector syringe by placing the stopcock swivel lock in the first position, slowly inject (over approximately 1-2 minutes) the contents of the vector syringe, taking care not to apply excessive force to the syringe plunger during injection. After the vector syringe contents have been injected, turn the stopcock swivel lock to the second position so that the stopcock and needle assembly can be flushed with 1-2 cc of saline using a pre-filled water flush syringe attached.

[0172] When ready, the interventionalist then alerts the staff that he / she will remove the equipment from the subject. In a single motion, slowly remove the needle, extension tubing, stopcock, and syringe from the subject and place them on a surgical tray for disposal in a biohazard waste container or hard container (for needles).

[0173] The needle insertion site will be inspected for signs of bleeding or CSF leakage and treated as directed by the investigator. The site will be dressed with gauze, surgical tape and / or Tegaderm dressing as directed. The subject will then exit the CT scanner and be placed on a stretcher in a supine position. Appropriate staff will be present to ensure the subject's safety during transport and positioning.

[0174] Anesthesia is discontinued and the subject is cared for per facility guidelines for post-anesthesia care. Neurophysiologic monitors are removed from the subject. The head of the gurney on which the subject lies should be slightly elevated (approximately 30 degrees) during recovery. The subject is transferred to a suitable post-anesthesia care unit per facility guidelines. After the subject has adequately regained consciousness and is in a stable condition, he / she will be admitted to the appropriate floor / unit for evaluation as mandated by protocol. Neurological evaluations will follow as per protocol, and the Principal Investigator will oversee the subject's care in collaboration with hospital and study staff.

[0175] In one embodiment, a method of delivery of a composition provided herein comprises advancing a subarachnoid needle into the cisterna magna of a patient; connecting a length of flexible tubing to the proximal hub of the subarachnoid needle and connecting an output port of a valve to the proximal end of the flexible tubing; after said advancing and connecting steps and after allowing the tubing to be self-sufficient with the patient's cerebrospinal fluid, connecting a first container containing a quantity of isotonic solution to the flush inlet port of the valve and thereafter connecting a second container containing a quantity of a pharmaceutical composition to the vector inlet port of the valve; after connecting said first and second containers to the valve, opening a pathway for fluid flow between the vector inlet and outlet ports of the valve and injecting the pharmaceutical composition into the patient via the subarachnoid needle; after injecting the pharmaceutical composition, opening a pathway for fluid flow through the flush inlet and outlet ports of the valve and injecting an isotonic solution into the subarachnoid needle to flush the pharmaceutical composition into the patient. In certain embodiments, the method further includes verifying correct placement of the distal tip of the intrathecal needle within the cisterna magna prior to connecting the tubing and valve to the hub of the intrathecal needle. In certain embodiments, the verifying step includes visualizing the distal tip of the intrathecal needle within the cisterna magna by computed tomography (CT). In certain embodiments, the verifying step includes observing the presence of cerebrospinal fluid of the patient at the hub of the intrathecal needle.

[0176] In the method described above, the valve may be a stopcock with a swivel luer lock adapted to be rotated to a first position that allows flow from the vector inlet to the outlet while simultaneously blocking flow through the water flush inlet, and to a second position that allows flow from the water flush inlet to the outlet while simultaneously blocking flow through the vector inlet, where the swivel luer lock is placed in the first position when the pharmaceutical composition is injected into the patient, and is placed in the second position when the pharmaceutical composition is flushed into the patient by an isotonic solution. In certain embodiments, after injecting the isotonic solution into the intrathecal needle to flush the pharmaceutical composition into the patient, the intrathecal needle is withdrawn from the patient along with the tubing, valve and container connected thereto as a structure. In certain embodiments, the valve is a four-way stopcock with a swivel male luer lock. In certain embodiments, the first and second containers are separate syringes. In certain embodiments, a T-connector is placed on the hub of the intrathecal needle to interconnect the tubing to the intrathecal needle. Optionally, the intrathecal needle includes an introducer needle at the distal end of the intrathecal needle. The intrathecal needle is a 5 inch, 22 or 24 gauge intrathecal needle. In a particular embodiment, the introducer needle is a 3.5 inch, 18 gauge introducer needle.

[0177] In certain aspects, the method utilizes a device that is comprised of at least a first container containing a quantity of a pharmaceutical composition; a second container containing an isotonic solution; a subarachnoid needle through which the pharmaceutical composition may be extruded from the device directly into the cerebrospinal fluid in the cisterna magna of the patient; a first inlet interconnected to the first container, a second inlet interconnected to the second container, an outlet interconnected to the subarachnoid needle, and a luer lock for controlling the flow of the pharmaceutical composition and the isotonic solution through the subarachnoid needle. In certain embodiments, the valve is a stopcock with a swivel luer lock adapted to be rotated to a first position that allows flow from the first inlet to the outlet while simultaneously blocking flow through the second inlet, and to a second position that allows flow from the second inlet to the outlet while simultaneously blocking flow through the first inlet. Optionally, the valve is a four-way stopcock with a swivel male luer lock. In certain embodiments, the first and second containers are separate syringes. In certain embodiments, the intrathecal needle is interconnected to the valve via a length of flexible tubing. A T-connector may interconnect the tubing to the intrathecal needle. In certain embodiments, the intrathecal needle is a 5 inch, 22 or 24 gauge intrathecal needle. In certain embodiments, the device further includes an introducer needle connected to the distal end of the intrathecal needle. Optionally, the introducer needle is a 3.5 inch, 18 gauge introducer needle.

[0178] This method and this device may each optionally be used for intramedullary delivery of the compositions provided herein, although alternatively, other methods and delivery methods may be used for such intramedullary delivery.

[0179] The following examples are illustrative only and are not limiting of the invention described herein. EXAMPLES

[0180] Example 1: Protocol for Treatment of Human Subjects This example relates to a gene therapy treatment for a patient with MPS I. In this example, a gene therapy vector, AAV9.CB7.hIDUA, a replication-deficient adeno-associated virus vector 9 expressing a modified hIDUA gene encoding a wild-type hIDUA enzyme, is administered to the central nervous system (CNS) of a patient with MPS I. A dose of AAV vector is injected directly into the CNS under general anesthesia. Clinical measurements of neurocognitive development and / or surrogate markers, including biomarkers, as described herein, for example, reduction in the concentration of pathogenic GAGs and / or hexosaminidases in the CSF or serum of the subject, are used to assess efficacy of the treatment.

[0181] A. Gene Therapy Vectors An illustrative gene therapy vector, AAV9.CB.hIDUA, is described in Example 3. Expression from the transgene cassette is driven by the CB7 promoter, a hybrid of the CMV immediate early enhancer (C4) and chicken β-actin promoter, while transcription from this promoter is enhanced by the presence of the chicken β-actin intron (CI). The polyA signal for the expression cassette is RBG polyA. The vector is suspended in formulation buffer (Elliot's B solution, 0.001% Pluronic F68). The manufacturing process is described in detail in Example 5 below.

[0182] B. Administration and Route of Administration Patients aged 6 years or older had a 2.6 × 10 12 GC(2.0×10 9 GG / g brain mass) (low use amount) or 1.3 x 10 13 GC(1.0×10 10 Patients will receive a single intrathecal / intracascular injection of 1000 GC / g brain mass (high dose) of rAAV9.CB7.hIDUA. For vector administration, patients will be placed under general anesthesia. A lumbar puncture will be performed to first remove 5cc of CSF, followed by IT injection of contrast to aid in visualization of the cisterna magna. CT (with contrast) will be used to guide needle insertion and administration of rAAV9.CB7.hIDUA into the suboccipital space.

[0183] In another embodiment, subjects under the age of three (<3) with a severe MPIS phenotype (Hurler Syndrome), which may be identified by a known mutation resulting in Hurler Syndrome, may be treated with rAAV9.CB7.hIDUA in a modified Elliot B® solution containing 0.001% Pluronic® F68. In a specific embodiment, the composition is administered at two dose levels: 1×10 10 GC / g brain mass and 5×10 10 The vector will be administered as a single dose via intracisternal administration at 1 of GC / g brain mass. No subject will receive more than one dose IP. Vector administration will be as described above. The proposed starting clinical dose is 1 x 10 10 The starting dose was the dose at which toxicity was observed in untreated (non-tolerized) dogs with MPS I (1×10 11 GC / g brain mass) and was 100-fold lower than the lowest dose (1.1 × 10 10 GC / g brain mass). In summary, a starting dose of 1 × 10 10 The choice of GC / g brain mass is justified because a) it is the lowest dose with a reasonable likelihood of clinical benefit, b) it maintains a reasonable margin of safety relative to the dose at which toxicity was observed in the canine model of MPS I, and c) while a NOAEL was not identified in NHPs, lowering the dose would not reduce this potential toxicity in the absence of a dose response associated with histopathological findings. Importantly, none of the NHPs showed clinical symptoms associated with the histopathological findings. 10 The higher dose of GC / g brain mass was greater than the dose at which toxicity was observed in MPS I dogs (1 × 10 12 GC / g brain mass) and the highest dose (1.1 × 10 11 GC / g brain mass), approximately two-fold lower.

[0184] Total dosage by age

[0185] [Table 1]

[0186] AAV9.hIDUA is aseptically diluted with a diluent (similar in composition to the formulation buffer) and the pH adjusted to near-physiological conditions as part of the dose preparation. The total volume of the delivered product for both the lower and higher doses is 10 mL or less after appropriate dilution prior to administration.

[0187] The following therapeutically effective flat-line doses are administered to patients in the indicated age groups: Newborn: Approx. 1 x 10 11 ~Approx. 3×10 14 G.C.; 3 to 9 months: approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; 9 months to 6 years: Approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; Ages 3 to 6: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.; 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0188] In another embodiment, the following therapeutically effective flat-line doses are administered to MPS patients in the following age groups: Newborn: Approx. 3.8 x 10 12 ~Approx. 1.9×10 14 G.C.; 3 to 9 months: approx. 6 x 10 12 ~Approx. 3×10 14 G.C.; 9 months to 36 months: Approx. 10 13 ~Approx. 5×1013 G.C.; Ages 6-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 3-12: Approx. 1.2 x 10 13 ~Approx. 6×10 14 G.C.; Ages 12 and up: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.; 18 years old and over (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0189] To ensure that empty capsids are removed from the dose of rAAV9.CB7.hIDUA administered to patients, the empty capsids are separated from the vector particles by cesium chloride gradient ultracentrifugation or by ion exchange chromatography during the vector purification process, as discussed in Example 5 herein.

[0190] Immunosuppressive therapy may be given in addition to the vector. Immunosuppressive therapy includes corticosteroids (methylprednisolone 10 mg / kg intravenous [IV] once on day -2 and oral prednisone 0.5 mg / kg / day starting on day -1, gradually tapered and discontinued by week 16), tacrolimus (0.2 mg / kg / day oral [PO] from day -2 to week 24), and sirolimus (once daily [QD] from day -2 to week 48 visit). Exemplary sirolimus doses include (6 mg PO on day -2, then 2 mg QD from day -1 to week 48 visit). Sirolimus dose adjustments can be made to maintain whole blood trough concentrations within 16-24 ng / mL. Adjustments can also be made to other drugs in the regimen, including shorter or longer delivery of the drug. For most subjects, dose administration can be based on the equation: new dose = current dose x (target concentration / current concentration). Subjects can continue the new maintenance dose for at least 7-14 days before further dose adjustments by monitoring concentrations. Optionally, patients can be allowed to remain on a stable regimen of intravenous enzyme replacement therapy (ERT, e.g., Aldurazyme™ [laronidase]), as well as symptomatic treatment (e.g., physical therapy). Serious adverse events may include possible drug-induced liver injury, with hyperbilirubinemia defined as an ALT of approximately 3 x ULN and bilirubin of approximately 2 x ULN (>35% direct), referred to as "Hy's Law."

[0191] In some embodiments, the immunosuppressive therapy regimen is as follows:

[0192] Corticosteroids On the morning of vector administration (pre-dose day 1), patients will receive methylprednisolone 10 mg / kg IV (maximum 500 mg) over at least 30 minutes. Methylprednisolone will be administered prior to lumbar puncture and intrathecal (IC) injection of rAAV9.CB7.hIDS. Premedication with acetaminophen and antihistamines is optional.

[0193] On day 2, oral prednisone is initiated with the goal of discontinuing prednisone by week 12. Prednisone doses are as follows: Day 2 through the end of week 2: 0.5 mg / kg / day. Weeks 3 and 4: 0.35 mg / kg / day. Weeks 5-8: 0.2 mg / kg / day. Weeks 9-12: 0.1 mg / kg.

[0194] Prednisone is discontinued after 12 weeks. The exact dose of prednisone can be adjusted to the next higher clinically practical dose.

[0195] Sirolimus: 2 days before vector administration (day -2): 1 mg / m every 4 hours 2 Sirolim Sirolimus 0.5 mg / m2 loading dose x 3 doses. From day -1: Sirolimus 0.5 mg / m2 2 / day, Sirolimus was administered twice daily in divided doses with a target blood concentration of 4-8 ng / ml. Sirolimus was discontinued after the 48-week visit.

[0196] Tacrolimus: Tacrolimus is initiated on day 2 (the day after rAAV9.CB7.hIDUA administration) at a dose of 1 mg twice daily and titrated to achieve blood levels of 4-8 ng / mL for 24 weeks. Beginning at the week 24 visit, tacrolimus is tapered over 8 weeks. At week 24, the dose is reduced by approximately 50%. At week 28, the dose is reduced again by approximately 50%. Tacrolimus is discontinued at week 32.

[0197] In another embodiment, the immunosuppressive therapy for patients under 3 years of age is as follows:

[0198] Corticosteroids On the morning of vector administration (pre-dose day 1), patients will receive methylprednisolone 10 mg / kg IV (maximum 500 mg) over at least 30 minutes. Methylprednisolone should be administered prior to lumbar puncture and IC injection of rAAV9.CB7.hIDS. Premedication with acetaminophen and antihistamines is optional at the investigator's discretion.

[0199] On day 2, oral prednisone is initiated with the goal of discontinuing prednisone by week 12. Prednisone doses are as follows: Day 2 through the end of week 2: 0.5 mg / kg / day Weeks 3 and 4: 0.35 mg / kg / day Weeks 5-8: 0.2 mg / kg / day Weeks 9-12: 0.1 mg / kg Prednisone is discontinued after 12 weeks. The exact dose of prednisone can be adjusted to the next higher clinically practical dose.

[0200] Sirolimus Two days before vector administration (day -2): 1 mg / m every 4 hours 2 For Sirolimus loading Administer 3 doses of the same amount. From day 1: Sirolimus 0.5 mg / m 2 / day to a target blood concentration of 1-3ng / ml The dose was divided into two doses per day. Sirolimus will be discontinued after the Week 48 visit.

[0201] Tacrolimus Tacrolimus will be initiated on day 2 (the day after rAAV9.CB7.hIDUA administration) at a dose of 0.05 mg / kg twice daily and titrated to achieve a plasma concentration of 2-4 ng / mL for 24 weeks. Beginning at the week 24 visit, tacrolimus will be tapered over 8 weeks. At week 24, the dose will be reduced by approximately 50%. At week 28, the dose will be reduced again by approximately 50%. Tacrolimus will be discontinued at week 32. - Monitoring of tacrolimus and sirolimus blood levels

[0202] C. Patient Subpopulations Suitable patients include: MPS confirmed by enzyme activity when measured in plasma, fibroblasts or leukocytes Those with a documented diagnosis of I; Documented evidence (medical records) of early neurocognitive deficit due to MPS I, defined as either an IQ test score 2:1 standard deviations below the mean or a score in one domain of neuropsychological functioning (verbal comprehension, memory, attention, or perceptual reasoning), unless explained by other neurological or psychiatric factors; those with documented historical evidence (medical charts) of greater than one standard deviation decline in sequential testing; may include those who have sufficient hearing and vision abilities to comply with completing required protocol testing and willingly wear assistive devices, with or without assistive devices, if applicable on the test date; Optionally, on a stable regimen of ERT (eg, Aldurazyme® [laronidase] IV) for at least 6 months.

[0203] Prior to treatment, patients are screened and one or more of the following criteria may indicate that this treatment is not suitable for the patient. Have a contraindication to IC injection, including any of the following: Review of baseline MRI indicates contraindication to IC injection History of previous head / neck surgery contraindicating IC injection Contraindications to CT (or contrast medium) or general anesthesia Contraindications to MRI (or gadolinium) <30mL / min / 1.73m 2 have an estimated glomerular filtration rate (eGFR) of Have a neurocognitive deficit or neuropsychiatric condition not attributable to MPS I - History of hypersensitivity reaction to sirolimus, MMF or prednisolone Inappropriate immunosuppressive therapy (e.g., <1.3 × 10 3 Neutrophils / μL Absolute number of <100 × 10 3 / μL and platelet count <12g / dL [male] or <10g / dL [female] -Contraindications to lumbar puncture -Having undergone HSCT -Has received laronidase via IT administration within 6 months prior to treatment Received IT laronidase at any time and experienced a significant adverse event that was considered to be related to IT administration that placed the patient at undue risk History of lymphoma or another cancer other than squamous cell or basal cell carcinoma of the skin that has not been in complete remission for at least 3 months prior to treatment Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) of >3× the upper limit of normal (ULN) or total bilirubin of >1.5× the ULN, unless the patient has a previously known history of Gilbert's syndrome and fractionated bilirubin showing bound bilirubin of <35% of total bilirubin History of a positive test for human immunodeficiency virus (HIV), active or recurrent hepatitis B or C, or a positive screening test for hepatitis B, hepatitis C, or HIV Pregnant, <6 weeks postpartum, breastfeeding, or planning a pregnancy (yourself or your partner's) History of alcohol or drug abuse within one year prior to treatment ·Has a serious or unstable medical or psychological condition that jeopardizes patient safety. · Poorly controlled seizures.

[0204] Suitable patients include · Newborns; 3-9 months old; ·4 months to under 9 months of age; Ages 9 months to under 18 months; 9-36 months of age; 18 months to under 3 years old; 3-12 years old; 12 years or older; The program is aimed at both men and women aged 18 and above.

[0205] In certain embodiments, suitable patients include males or females under the age of 3 and one or more or all of the following: 1) The subject's legal guardian is willing and able to provide signed informed consent after the nature of the clinical trial has been explained to him or her and before any procedures related to the clinical trial are performed. 2) Proven diagnosis of severe MPS I-Hurler: a) the presence of clinical signs and symptoms compatible with MPS I-Hurler, and / or b) Homozygosity or compound heterozygosity for mutations exclusively associated with a severe phenotype. 3) Have an IQ score of 55 or higher 4) Have sufficient hearing and vision to complete required protocol testing with or without assistive devices and are willing to comply with wearing the assistive device on testing days, if applicable.

[0206] Subjects who meet any of the following exclusion criteria are not eligible to participate in the study. 5) Contraindicated for IC infusions containing any of the following: a) Review of baseline magnetic resonance imaging (MRI) studies by a neuroradiologist / neurosurgeon adjudication committee indicates contraindication to IC injection. b) History of previous head / neck surgery that contraindicated IC injection based on review of available information by a panel of neuroradiologists / neurosurgeons. c) Contraindicated to computed tomography (CT) (or contrast) or general anesthesia. d) MRI (or gadolinium) is contraindicated. e) Estimated glomerular filtration rate (eGFR) < 30 mL / min / 1.73 m 2 It is. 6) Have a neurocognitive disorder not attributable to MPS I or have a diagnosis of a neuropsychiatric condition that, in the physician's opinion, may confound the interpretation of the study results. 7) Lumbar puncture is contraindicated. 8) Having undergone hematopoietic stem cell transplantation (HSCT) 9) Previously treated with AAV-based gene therapy products 10) Received intrathecal (IT) laronidase at any time and experienced a significant AE considered to be related to IT administration that, in the opinion of the investigator, placed the subject at undue risk. 11) History of lymphoma that has not been in complete remission for at least 3 months prior to screening or a history of another cancer other than squamous cell or basal cell carcinoma of the skin. 12) Uncontrolled hypertension (systolic blood pressure [BP] >180mmHg, diastolic blood pressure >100mmHg) despite maximal medical therapy. 13) Platelet count <100,000 / microliter (μL) 14) Have alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >3× upper limit of normal (ULN) or total bilirubin >1.5×ULN at screening, unless the subject has a history of Gilbert's syndrome. 15) History of human immunodeficiency virus (HIV) or hepatitis B or C virus infection, or positive screening test for hepatitis B surface antigen, hepatitis B core antibody, hepatitis C or HIV antibody. 16) Within 30 days prior to signing the Informed Consent Form (ICF) received the investigational drug within 5 half-lives (whichever is longer) 17) Being a first-degree family member of an employee of a medical institution or other individual involved in the conduct of the clinical trial, or being an employee of a medical institution or other individual involved in the conduct of the clinical trial. 18) There is a clinically significant ECG abnormality that, in the opinion of the PI, compromises the safety of the subject. 19)Has a serious or unstable medical or psychological condition that, in the opinion of the PI, compromises the subject's safety or successful participation in the trial or interpretation of the trial results. 20) Ventricular shunts which, in the opinion of the institution's neuroradiologist / neurosurgeon and in discussion with the medical monitor, may affect the subject's dosing and proper dosing.

[0207] Exclusion criteria related to immunosuppressive therapy: 21) History of hypersensitivity reactions to tacrolimus, sirolimus, or prednisone; 22) History of primary immunodeficiency (e.g., common variable immunodeficiency syndrome), splenectomy, or any underlying disease that predisposes the subject to infection. 23) Herpes zoster, cytomegalovirus, or Epstein-Barr virus (EBV) infection that had not completely resolved at least 12 weeks prior to screening. 24) Any infection requiring hospitalization or treatment with parenteral anti-infectives that did not resolve at least 8 weeks prior to Visit 2. 25) Any active infection requiring oral anti-infectives (including antivirals) within 10 days prior to Visit 2. 26) History of active tuberculosis (TB) or positive QuantiFERON TB Gold test during screening. 27) Any live vaccine within 8 weeks prior to signing the ICF. 28) Major surgery within 8 weeks prior to signing the ICF or planned during the study period. 29) Anticipate the need for adenoidectomy or tonsillectomy within 6 months of enrollment. If adenoidectomy or tonsillectomy is anticipated, it should be performed prior to screening. 30) Absolute neutrophil count < 1.3 x 10 3 / μL. 31) Any condition or laboratory abnormality that the clinician considers to be inappropriate for immunosuppressive therapy.

[0208] D. Assessing clinical objectives Primary clinical objectives include preventing and / or optionally reversing neurocognitive decline associated with MPS I deficiency and / or slowing or halting neurodevelopmental decline, as determined, for example, by measuring intelligence quotient (IQ) as measured by the Bayley Scale of Infant and Toddler Development, Third Edition (Bayley III) and / or the Wechsler Preschool and Primary Scales of Intelligence, Fourth Edition (WPPSI-IV) and Adaptive Behavior (Vineland-2) for Hurler subjects, or by the WASI for Hurler-Scheie subjects. Other suitable measures of neurocognitive development and function may be utilized, such as assessing the Developmental Quotient (DQ) using the Bayley Scales of Infant Development (BSID-III), assessing memory using the Hopkins Verbal Learning Test, and / or the WASI-I and / or the Bayler-III, and / or the Test of Variables of Attention (TOVA).

[0209] Secondary endpoints include assessment of biomarkers and clinical outcomes. Urine will be assessed for MPS I-specific pGAG as well as total glycosaminoglycan (GAG) content. Serum will be assessed for IDUA activity, anti-IDUA antibodies, pGAG, and heparin cofactor II-thrombin complex concentrations. Animal data indicate that systemic effects may be possible. and plasma is monitored for biomarkers (GAG and IDUA). CSF is assessed for IDUA activity, anti-IDUA antibodies, hexosaminidase (hex) activity, and pGAG. The presence of neutralizing antibodies to the vector (e.g., AAV9) and binding antibodies to IDUA may be assessed in CSF and serum, T cell responses to the vector capsid (e.g., AAV9) may be assessed by ELISPOT assay, and vector concentrations (quantitative PCR (qPCR) for AAV9 DNA) as well as the pharmacokinetics of IDUA expression in CSF, serum, and urine may be monitored. Vector shedding in CSF, plasma, and urine may be monitored.

[0210] Exploratory endpoints include one or more of the following: immunogenicity measures, e.g., neutralizing antibodies to AAV9 and binding antibodies to IDUA in CSF and serum, enzyme-linked immunospot (ELISPOT) assays: T cell responses to AAV9 and IDUA, and flow cytometry: AAV- and IDUA-specific regulatory T cells. Other exploratory endpoints may include CNS structural abnormalities assessed by brain magnetic resonance imaging (MRI); liver and spleen volumes assessed by abdominal ultrasound; hearing ability changes measured by auditory brainstem response (ABR) testing; biomarkers in plasma (GAG and IDUA), CSF (GAG, IDUA and spermine) and urine (GAG); viral shedding: vector concentrations in CSF, serum and urine (quantitative polymerase chain reaction [qPCR] for AAV9.hIDUA deoxyribonucleic acid [DNA]); effects on systemic symptoms of disease (compared to CSF) and quality of life.

[0211] Example 2: Neonatal systemic AAV induces tolerance to CNS gene therapy in MPS I dogs and non-human primates This example demonstrates that liver-directed gene transfer using adeno-associated virus (AAV) vectors in neonates induces a sustained state of immune tolerance to the transgene and substantially improves the efficacy of subsequent vector administrations targeting the central nervous system (CNS) in both dogs and non-human primates. This approach was applied to a canine model of mucopolysaccharidosis type I (MPS I), a lysosomal storage disease characterized by progressive CNS disease due to a deficiency in the activity of the enzyme α-L-iduronidase (IDUA). CNS-targeted gene transfer using intramedullary AAV delivery in 1-month-old dogs resulted in the induction of antibodies against canine IDUA, which partially attenuated the amelioration of brain pathology. MPS I dogs systemically treated with vectors expressing canine IDUA at 1 week of age did not develop antibodies against the enzyme and showed robust expression in the CNS upon intramedullary AAV delivery at 1 month of age, resulting in complete correction of brain storage disease. Neonatal rhesus macaques systemically treated with an AAV vector expressing human IDUA similarly developed tolerance to the transgene, resulting in dramatically increased incidence of IDUA in the CSF and the absence of subsequent antibody induction in the CNS following gene therapy. These findings suggest the possibility of improving the efficacy and safety of gene therapy by inducing tolerance to the transgene during a critical period of immunological development.

[0212] A. Materials and Methods 1. Vector Construction The test article consisted of an AAV9 capsid packaging an expression cassette consisting of a chicken β-actin promoter (CB7), a chimeric intron (CI), a codon-optimized canine IDUA transgene (cIDUA) and a polyadenylation signal (RBG). The expression construct was flanked by AAV serotype 2 inverted terminal repeats. The vector was designated AAV2 / 9.CB7.CI.cIDUA.RBG or AAV9.CB7.CI.cIDUA.RBG. Some animals were treated intravenously as neonates with a different vector to induce tolerance to the canine IDUA protein. The vector contained a liver-specific thyroid hormone-binding globulin promoter (TBG), an artificial intron (PI), a codon-optimized canine IDUA transgene (cIDUA) and a polyadenylation signal (RBG). The vector consisted of an AAV8 capsid that packages an expression cassette consisting of the nucleotide sequence ...

[0213] 2.Animals A colony of MPS I dogs was maintained at the University of Pennsylvania School of Veterinary Medicine under NIH and USDA guidelines for the care and use of animals in research. All MPS I dog study protocols were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. For vector injections in neonatal MPS I dogs, AAV8 vectors were diluted in 0.5–1 mL of sterile saline and injected via the jugular vein. Intramedullary injections of AAV9 vectors and collection of CSF were performed via a suboccipital approach as previously described [C.Hinderer,et al,Intrathecal Gene Therapy Corrects CNS Pathology in a Feline Model of Mucopolysaccharidosis I.Molecular therapy:the journal of the American Society of Gene Therapy,(2014);published online EpubJul,16]. A total of nine MPS I dogs were included in this study. Genotypes were confirmed at birth by PCR and serum enzyme assays. Six dogs were transfected with AAV serotype 8 vectors (5 × 10 12 One died 3 days after birth. The remaining five treated animals as well as three untreated MPS I dogs received intramedullary AAV9 (10 12 MPS I mice were treated with 100 mg / kg GC. Blood was collected from a peripheral vein weekly for the first 7 weeks of age and monthly thereafter. CSF (1 mL) was collected at the time of intramedullary injection (1 month of age), on days 7 and 21 of injection, and monthly thereafter. Euthanasia was performed by administration of sodium pentobarbital 80 mg / kg, IV). Five animals were euthanized at 9 months of age, the remainder at 11 months of age. Untreated MPS I and controls were euthanized between 6 and 26 months of age. Tissues were collected and processed as previously described [Hinderer, et al, 2014].

[0214] All animal procedures that conformed to the Animal Welfare Act were approved by the University of California, Davis, Laboratory Animal Care and Use Committee prior to their implementation. Animal care activities were performed in accordance with the operating procedures of the California National Primate Research Center. Adult normally cycling female rhesus macaques (Macaca mulatta; N=4) were bred and identified as pregnant using established methods [AF Tarantal, in The Laboratory Primate. (2005), pp. 317-352]. All mothers selected for the study were prescreened to ensure that they were seronegative for AAV antibodies. Fetuses were monitored by ultrasound during pregnancy to ensure normal growth and development [AF Tarantal (2005)], and neonates were delivered by Caesarean section at term (160±2 days of gestation) according to established protocols [AF Tarantal, et al, Mol. Ther. 12, 87-98 (2005); published online EpubJul]. Neonates were placed in an incubator after delivery and artificially reared for the study. Infant health, food intake and weight were recorded daily or weekly (depending on age) in the neonatal room according to established protocols. All animals were administered the selected AVV vector IV at birth. At 1 month postnatal and monthly baselines thereafter (until 2 months post-transduction, until now), infants were sedated with ketamine (10 mg / kg intramuscularly, IM) and dexmedetomidine (0.015-0.075 mg / kg, IM) in preparation for collection of CSF (approximately 0.5 mL; pre-infusion, then weekly or monthly) and intrathecal infusion (approximately 0.5 mL, 1 month after CSF collection and immediately) all under sterile conditions. CBC and clinical laboratory panels were monitored to monitor and Peripheral blood samples (approximately 3-6 mL) were collected at birth and monthly for serum and plasma collection. Upon completion of sampling, reversible atipamezole was given IM at a dose comparable to dexmedetomidine.

[0215] DNA was isolated from tissues and vector genomes were quantified by TaqMan PCR as described [L. Wang, 2011]. Assays for IDUA and Hex activity were performed as described [Hinderer, et al., 2014].

[0216] Measurement of pGAG in CSF was performed by the Glycotechnology Core at the University of California, San Diego using a method previously described [R. Lawrence, et al, Nature chemical biology, 8, 197-204 (2012); published online EpubFeb]. Briefly, GAGs were extracted from CSF samples and digested with heparinases I, II, and III to disaccharides. Aniline was synthesized by reductive coupling. 12 The disaccharides were tagged with C and dried in a SpeedVac. The dried samples were reconstituted in LC-MS grade water and analyzed for known concentrations of aniline. 12 C-tagged standards were spiked in. Samples were analyzed on a LTQ Orbitrap Discovery electrospray ionization mass spectrometer (Thermo Scientific) equipped with a Thermo Scientific Ultimate 3000 HPLC system.

[0217] ELISA for antibodies to canine IDUA was performed as described [Hinderer, et al, 2014], except that an expression construct containing a canine cDNA under the control of the thyroid hormone-binding globulin promoter and cIDUA protein was produced in Huh7 cells. The detection antibody used was HRP-conjugated sheep anti-dog (Pierce, Rockford, IL). Assays for antibodies to human IDUA in rhesus monkeys were identical, except that 10 μg / mL of aldurazyme (Genzyme, Cambridge, MA) was used as the coating antigen and the detection antibody was a polyclonal goat anti-human (Jackson ImmunoResearch Laboratories, West Grove, PA).

[0218] Histological analysis of brains from MPS I dogs was performed as previously described [C. Hinderer, 2014] with the following modifications to quantify accumulation of neurons positive for monosialodihexosylganglioside (GM3), cholesterol and lysosomal membrane protein (LIMP2); images of LIMP2 and filipin stained cortical sections were taken with a 10x objective such that the border between layer I (molecular layer) and layer II formed the upper border of the image. A total of 10 images were acquired from each animal. Images of GM3 stained brain sections were taken with a 4x objective from the area immediately below the cortical surface including the molecular layer of the brain. Seven images from each animal were analyzed. All images were processed with ImageJ software (Rasband, WS, National Institutes of Health, USA; rsb.info.nih.gov / ij / ) using the “Threshold” and “Analyze particles” modules as previously described [M. Aldenboven, et al, Biology of Blood and Marrow Transplantation, 14, 485-498 (2008); published online Epub May].

[0219] B. Results 1. Induction of antibodies against canine IDUA following intramedullary AAV9-mediated gene transfer in MPS I dogs The MPS I canine model faithfully recapitulates many of the symptoms of the human disease [E. Kakkis, et al, Molecular genetics and metabolism, 83, 163-174 (2004); published online Epub Sep-Oct; RMShull, et al, Am. J. Pathol. 114, 487-495 (1984)]. These animals have no detectable IDUA activity due to a splice site mutation resulting in retention of the first intron of IDUA [K. Menon, et al, (Genomics, 14, 763-768 (1992)]. Given the lack of detectable IDUA expression in these animals, and because these individuals generally carry alleles that produce non-full-length IDUA and remain immunologically naive to the protein, we anticipated that they would model the immune response to intramedullary gene therapy that occurs in patients with a severe form of MPS I [NJ Terlato, GF Cox, Can mucopolysaccharidosis type I, 1999]. MPS I disease severity be predicted based on a patient's genotype? A comprehensive review of the literature.Genetics in medicine: official journal of the American College of Medical Genetics 5,286-294(2003);published online EpubJul-Aug]. The brains of MPS I dogs show a wide range of MPS I-related abnormalities, including widespread accumulation of gangliosides such as GM3 in neurons, as well as abnormal accumulation of cholesterol and lysosomal membrane proteins, including LIMP2. MPS I dogs also show marked accumulation of glycosaminoglycans (GAGs) in the meninges, resulting in significant meningeal thickening, a process that contributes to spinal cord compression in some MPS I patients [E. Kachur, et al, Neurosurgery, 47, 223-228 (2000); published online EpubJul;A.Taccone,et al,Pediatric Radiology,23,349-352(1993);published online EpubSep;S.Vijay,JEWraith,Clinical presentation and follow-up of patients with the attenuated phenotype of mucopolysaccharidosis type I. Acta Paediatrica, 94, 872-877 (2005)].

[0220] Three dogs were initially treated at 1 month of age by intrathecal injection of an AAV9 vector carrying a canine IDUA sequence under the control of a ubiquitous promoter. Injections were well tolerated in all animals, and no clinical signs were observed throughout the study. CSF analysis was generally unremarkable, with only mild, transient increases in CSF lymphocytes occurring in two animals. A single CSF sample in one animal showed marked CSF pleocytosis consisting primarily of monocyte-like cells. Subsequent punctures showed no evidence of CSF pleocytosis, and at the time of euthanasia, there was no histological evidence of inflammation in the brain or spinal cord in any of the treated animals. The vector distributed throughout the CNS, transducing cells in all analyzed regions of the brain and spinal cord. All animals showed supraphysiological expression of IDUA in the CSF, which declined into the normal range in one animal and below normal levels in two over the course of three months, after which CSF enzyme levels remained essentially stable for five months until the animals were euthanized. The absence of clinical signs, loss of vector genome, or histological evidence of encephalitis indicated that the decline in CSF IDUA activity was not due to killing of transduced cells by cytotoxic T lymphocytes, which was also supported by the persistent residual CSF IDUA activity. Instead, the decline in CSF IDUA activity was associated with the induction of high titers of antibodies against canine IDUA.

[0221] B. Induction of tolerance to IDUA by neonatal gene transfer To determine whether neonatal expression of canine IDUA could induce immune tolerance to the enzyme in MPS I dogs, we administered hepatic IDUA to dogs on postnatal day 1 (N=3) or day 7 (N=3). Six dogs were treated by IV injection of an AAV serotype 8 vector expressing canine IDUA from a liver-selective promoter. One of the dogs treated on the day of birth died 2 days after treatment. The surviving newborn dogs had an approximately 20% mortality rate in the first 2 weeks of age, overall similar to historical data for untreated MPS I dogs [Vite, R. et al, Molecular therapy: the journal of the American Society of Gene Therapy 15, 1423-1431 (2007); published online EpubAug]. The cause of this early mortality in MPS I dogs has not been determined; in this treated animal, necropsy showed possible evidence of systemic bacterial infection as well as systemic lesions typical of MPS I. Treated animals demonstrated elevated serum IDUA followed by a rapid decline. This is consistent with observations of transient expression due to loss of the vector genome during hepatocyte division in a previous study using non-integrating vectors for hepatic gene transfer in neonates [L. Wang, et al, Human gene therapy, 23, 533-539 (2012); published online Epub May].

[0222] At 1 month of age, the five surviving dogs that had received IV AAV8 at 1 week of age were injected with AAV9 vectors using an intramedullary approach. All five animals demonstrated peak levels of IDUA greater than 30-fold higher than normal levels in the CSF following intramedullary vector injection, with prolonged CSF enzyme levels 3-100-fold higher than those achieved in untreated animals. Antibodies to canine IDUA were undetectable in the CSF of dogs treated on postnatal day 1 and were only slightly above the limit of detection in animals treated on postnatal day 7, suggesting a state of immune tolerance to the enzyme in both groups. 2. Correction of biochemical and histological abnormalities in the CNS of dogs with MPS I The lysosomal enzyme hexosaminidase (Hex) is upregulated in tissues of MPS I animals, and elevated Hex activity in both brain tissue and CSF serves as a useful marker for abnormal cellular processes occurring downstream of IDUA deficiency [Hinderer, et al. (2014)]. Measurement of CSF Hex activity at the time of intramedullary delivery of vector (approximately 1 month of age) demonstrated abnormally elevated Hex activity in all MPS I dogs. Animals treated with intramedullary AAV9 alone showed a moderate decrease in Hex activity, and only animals with the highest residual IDUA expression reached normal range Hex activity. All five animals treated neonatally by systemic gene transfer and subsequently receiving intramedullary administration of vector demonstrated complete normalization of CSF Hex. Although the effect was somewhat reduced in the two animals with the lowest CSF IDUA levels that received intramedullary treatment only, Hex activity in brain tissue samples showed a greater response to treatment than CSF Hex, accompanied by a substantial decrease in brain Hex activity in all treated animals.

[0223] GAG concentrations in the CSF were measured using an assay specific for the non-reducing end of pathological GAG (pGAG) that accumulates due to IDUA deficiency [R. Lawrence, et al; Nature chemical biology, 8, 197-204 (2012); published online EpubFeb]. Three weeks after intramedullary AAV injection, all animals showed a significant reduction in CSF pGAG concentrations. Dogs that were not immune-tolerant to IDUA maintained higher residual pGAG in the CSF than immune-tolerant dogs, but this reduction persisted at day 112. Histological analysis revealed severe storage lesions throughout the brain of untreated MPS I dogs, accompanied by widespread neuronal accumulation of GM3, cholesterol, and LIMP2. Only the animal with the highest IDUA in the CSF experienced complete resolution of neuronal accumulation, whereas animals treated with intramedullary AAV9 alone demonstrated substantial amelioration of storage lesions. The other two intramedullary treated dogs had residual storage lesions. CNS storage lesions were completely reversed in all five dogs that were neonatally treated with AAV8 systemic gene transfer and subsequently received intramedullary AAV9. In addition to storage lesions in the brain parenchyma, untreated MPS I dogs showed GAG accumulation in the meninges visible by Alcian blue staining. This meningeal GAG accumulation and resulting meningeal thickening did not require surgical intervention. It is involved in many cases of spinal cord compression leading to CSF ​​resorption and may also contribute to the development of communicating hydrocephalus in some MPS I patients by interfering with normal pathways of CSF reabsorption. All treated animals showed evidence of improvement in meningeal GAG accumulation. While the meninges appeared almost completely normal in all tolerant dogs and one intolerant dog, the two intolerant animals with the lowest CSF IDUA activity retained some GAG ​​accumulation.

[0224] 3. Induction of tolerance to human IDUA in neonatal rhesus monkeys To assess whether the neonatal window for immune tolerance induction observed in MPS I dogs could be found in primates, a similar study was performed in neonatal rhesus macaques (N=4). Because these animals are not IDUA deficient, a human IDUA transgene was used to model the immune response that might be expected to a species-specific transgene in patients lacking an active endogenous gene. Two neonatal rhesus macaques were administered IV at birth with an AAV8 vector expressing human IDUA from a liver-specific promoter. Both demonstrated a transient increase in serum IDUA activity. Two additional neonates were administered IV at birth with an AAV8 vector expressing an unrelated transgene (human factor IX). All four animals were administered an AAV9 vector expressing human IDUA by intrathecal injection at 1 month of age. Similar to MPS I dogs, IDUA-naive animals showed a decline in CSF IDUA activity 3 weeks after infusion, returning to near baseline levels by 2 months post-treatment. These animals also developed transgene-specific antibodies in the CSF. Two animals administered the IDUA gene transduction IV at birth did not develop antibodies to human IDUA in the CSF and maintained CSF enzyme activity >10-fold higher than normal 2 months after intramedullary AAV9 administration. All animals remained robust and healthy for the duration of the study, with no evidence of adverse events, normal growth trajectories based on age and compared to historical controls, and complete blood counts (CBC) and biochemistry within normal limits.

[0225] C. Consideration Immune activation against wild-type therapeutic proteins is a potential concern for recessive diseases. Antibody responses to protein replacement therapy are particularly challenging for some LSDs, as they can interfere with the distribution and uptake of intravenously delivered enzymes [EJ Langereis,et al,Molecular genetics and metabolism,(2014);published online EpubOct,29]. Antibodies are equally problematic for gene therapy targeting these diseases, as they can interfere with the reciprocal correction mediated by enzymes secreted from transduced cells.

[0226] This study demonstrates that intramedullary delivery of AAV9 can effectively target cells throughout the canine CNS and achieve sufficient expression to correct biochemical and histological abnormalities associated with MPS I in the large animal brain. Vector biodistribution data indicate that there is less than one vector genome per cell in the brain, indicating that the widespread reduction in storage pathology observed is due to cross-correction by secreted enzymes. However, of three animals treated with intramedullary vector alone, two developed sufficiently robust anti-transgene antibody responses to prevent complete resolution of storage lesions in the CNS. Only animals that maintained near-normal IDUA activity in the CSF after antibody induction against the transgene demonstrated a complete response to CNS gene therapy. From this result, we conclude that IDUA activity in the CSF is a reasonable predictor of efficacy following intramedullary gene transfer and that near-normal levels are required for full therapeutic benefit. This is consistent with our findings with intramedullary gene therapy in cats with MPS I [Hinderer (2014)]. MPS I cats generally display weaker antibody responses to intramedullary gene transfer than MPS I dogs and more stable IDUA activity in the CSF. This may be related to the underlying mutations in the two models, as MPS I cats express an inactive mutant IDUA, potentially rendering them partially immune tolerant to the enzyme. Importantly, the MPS I canine counterparts in MPS I dogs are more likely to be involved in the pathogenesis of MPS I than in MPS I dogs. Current data indicate that even for severely diseased MPS I patients who, like dogs, have no residual IDUA activity, the anti-transgene antibody response that may arise after intramedullary gene transfer does not result in adverse clinical events, and substantial efficacy is maintained despite the antibody response. However, these data also suggest that preventing antibody responses to IDUA in the CNS would improve the efficacy of intramedullary gene therapy for MPS I.

[0227] Using liver-directed gene transfer, we investigated the effect of early exposure to IDUA on the subsequent immune response after intramedullary gene therapy. Neonatal IDUA expression induced tolerance to the enzyme in MPS I dogs, where very high CSF enzyme levels were achieved by intramedullary gene therapy at 1 month of age. The high CSF IDUA levels in the immune-tolerant group consistently resulted in complete reversal of neuropathology, providing a strong example of the efficacy possible with intramedullary gene therapy of LSD once interfering antibody responses are overcome. The finding that a neonatal window for induction of immune tolerance to a transgene also exists in nonhuman primates seems promising for translation to the clinic. There are several important limitations to this study. Due to the high risks associated with performing intramedullary injections of vectors in neonatal MPS I patients, systemic gene transfer rather than CNS-directed gene therapy has been used in neonates as a means of inducing tolerance.

[0228] The approach used in this example had the advantage that intramedullary gene therapy was performed in the same manner and at the same age in all experimental groups, allowing direct comparison of CSF IDUA levels between animals without confounding effects of differences in transduction efficiency in animals of different ages. This study did not exclude the possibility that previous liver-directed gene therapy contributed to improved correction of brain pathology in immune-tolerant animals, although this seems unlikely given that IDUA was not detectable in the CSF in these animals at the time of intramedullary injection of the vector, and CSF Hex activity and pGAG concentrations showed no evidence of correction prior to intramedullary gene transfer. This is consistent with a previous study in cats with MPS I in which extremely high serum IDUA activity had no effect on brain pathology [C.Hinderer,PNAS,111:14894-14899(2014)]. Based on the observation that detectable antibodies began to appear in treated MPS I dogs on postnatal day 7, this period was estimated to last only 1-2 weeks, which could serve as a useful starting point for human studies.

[0229] If human neonates have the same potential for transgene-specific immune tolerance as demonstrated herein in dogs and non-human primates, neonatal gene transfer may have enormous potential to treat numerous genetic disorders in which immune responses limit the safety or efficacy of treatment. To make clinical trials feasible, prenatal and neonatal screening will be essential to identify patients early enough for this approach to be effective. For MPS I, neonatal screening is now being performed, offering the potential opportunity to perform first-in-human trials [PV Hopkins, et al, J. Pediatr, (2014); published online EpubOct.18].

[0230] Example 3: Induction of transgene-specific immune tolerance allows accurate evaluation of human gene therapy in canine disease models. A. Materials and Methods The vector is a non-replicating recombinant adeno-associated virus (AAV) of serotype 9 expressing human iduronidase (hIDUA). The AAV9 serotype allows efficient expression of the hIDUA product in the CNS following IC administration.

[0231] 1. Vector Construction The AAV-hIDUA vector genome plasmid, pAAV.CB7.CI.hI DUAco.RBG(p3032) is 7,165 bp in size. The vector genome derived from this plasmid is a single-stranded DNA genome with ITRs from AAV2 flanking the hIDUA expression cassette. Expression from the transgene cassette is driven by the CB7 promoter, a hybrid between the CMV immediate early enhancer (C4) and the chicken β-actin promoter, while transcription from this promoter is enhanced by the presence of an intron (CI) from chicken β-actin. The polyA signal for the expression cassette is RBG polyA. The plasmid was constructed by codon optimizing and synthesizing the hIDUA sequence, and the resulting construct was then cloned into the expression elements of the plasmid, pENN.AAV.CB7.CI.RBG(p1044), an expression cassette flanked by the ITRs of AAV2 containing CB7, CI, and RBG to obtain pAAV.CB7.CI.hIDUAco.RBG(p3032).

[0232] Describing array elements Inverted Terminal Repeats (ITR): The AAV ITRs (GenBank #NC001401) are sequences that are identical at both ends but in inverted orientation. The AAV2 ITR sequences both function as origins of replication of vector DNA and as packaging signals for the vector genome when AAV and adenovirus helper functions are provided in trans. As such, the ITR sequences represent the only cis sequences required for replication and packaging of the vector genome.

[0233] CMV immediate early enhancer (382 bp, C4; GenBank #K03104.1): This element is present in the plasmid of the vector genome.

[0234] Chicken β-actin promoter (282 bp; CB; GenBank #X00182.1): A promoter and is used to drive high level expression of hIDUA.

[0235] Chicken β-actin intron: A 973 bp intron derived from the chicken β-actin gene (GenBank #X00182.1) is present in the expression cassette of the vector. The intron is transcribed but is removed from the mature messenger RNA (mRNA) by splicing and joined to sequences on either side of it. The presence of an intron in the expression cassette has been shown to facilitate transport of mRNA from the nucleus to the cytoplasm, thus improving the accumulation of steady-state levels of mRNA for translation. This is a common feature of genetic vectors aimed at high levels of gene expression. This element is present in both vector genome plasmids.

[0236] The coding sequence of α-L-iduronidase: hIDUA (Genbank NP_000194) was codon-optimized and synthesized [SEQ ID NO:1]. The encoded protein is 653 amino acids with a predicted molecular weight of 73 kD and an apparent molecular weight of 83 kD by SDS-PAGE [SEQ ID NO:2].

[0237] Polyadenylation signal: The 127 bp rabbit beta globin polyadenylation signal (GenBank #V00882.1) provides a cis sequence for efficient polyadenylation of the antibody mRNA. This element functions as a signal for transcription termination, a specific cleavage event at the 3' end of the nascent transcript, and the addition of a long polyadenylation tail. This element is present in both vector genome plasmids.

[0238] Inverted terminal repeats (ITRs): AAV ITRs (GenBank #NC001401) are sequences that are identical at both ends but in the opposite orientation. The AAV2 ITR sequences are involved in the replication of vector DNA when AAV and adenovirus helper functions are provided in trans. The ITR sequences function as the start of replication and packaging signal for the vector genome. As such, the ITR sequences represent the only cis sequences required for replication and packaging of the vector genome.

[0239] The constructs were packaged into AAV9 capsids and purified and titered as previously described in M. Lock, et al., Human Gene Ther, 21:1259-1271 (2010).

[0240] 2. Animal Treatment A colony of MPS I dogs was maintained at the University of Pennsylvania School of Veterinary Medicine under NIH and USDA guidelines for the care and use of animals in research. All study protocols were approved by the University of Pennsylvania Institutional Animal Care and Use Committee. For infusion of recombinant human IDUA, laronidase (Genzyme) was diluted 5-fold in saline immediately prior to use. Infusion was administered over 2 hours via a peripheral venous catheter. Intramedullary injection of AAV9 vectors and collection of CSF were performed via a suboccipital approach as previously described [C. Hinderer, et al, (Mol. Ther. J. Am. Soc. Gene Ther. 22, 2018-2027 (2014)]. Euthanasia was performed by administration of sodium pentobarbital (80 mg / kg, IV). Tissues were harvested and processed as previously described [Hinderer (2014)].

[0241] 3. Enzyme Assay IDUA and Hex activities were measured in tissue lysates and CSF as previously described [C. Hinderer, et al., (Mol. Ther. J. Am. Soc. Gene Ther. 22, 2018-2027 (2014))].

[0242] 4. Anti-IDUA ELISA Polystyrene ELISA plates were coated overnight at 4°C with recombinant human IDUA (Genzyme) diluted to 5 μg / mL in phosphate buffer, pH 5.8. Plates were washed and blocked with 2% BSA in phosphate buffer, pH 5.8. Plates were incubated with CSF samples diluted 1:50 in PBS for 1 h at room temperature. Plates were washed and bound antibodies were detected with HRP-conjugated anti-dog IgG (Pierce, Rockford, IL) diluted 1:10,000 in phosphate buffer with 2% BSA. ELISA was developed with tetramethylbenzidine substrate for 15 min, then the reaction was stopped with 2N sulfuric acid, and absorbance was measured at 450 nm. Titers were calculated from a standard curve of serially diluted positive samples.

[0243] 5. Histology, Biodistribution and Statistics Histological analysis of brains was performed as previously described [C. Hinderer, 2014] with the following modifications to quantify neurons positive for GM3, cholesterol and LIMP2 accumulation; images of LIMP2 and filipin stained cortical sections were taken with a 10x objective such that the border between layer I (molecular layer) and layer II formed the upper edge of the image. A total of 10 images were acquired from each animal. Images of GM3 stained brain sections were taken with a 4x objective from the area immediately below the cortical surface including the molecular layer of the brain. Seven images from each animal were analyzed. All images were processed with ImageJ software (Rasband, WS, National Institutes of Health, USA; rsb.info.nih.gov / ij / ) using the "Threshold" and "Analyze particles" modules as previously described [M. Aldenboven, et al., Biology of Blood and Marrow Transplantation, 14, 485-498 (2008); published online Epub May]. Quantification of cervical meningeal thickening was performed on H&E stained sections of cervical spinal cord. Fifteen measurements of total meningeal thickness were made per slide at 300 μm intervals.

[0244] Vector biodistribution was assessed as follows. DNA was isolated from tissues and purified as previously described [LL Wang, et al., Impact of Pre-Existing Immunity on Gene Transfer to Nonhuman Primate Liver with Adeno-Associated Virus]. Vector genomes were quantified by TaqMan PCR as described in [8 Vectors. Human gene therapy, 22, 1389-1401, (2011); published online EpubNov]. Data were evaluated using the Kruskal-Wallis test followed by Dunn's test or Mann-Whitney test, as appropriate. p < 0.05 was considered statistically significant. All statistical analyses were performed using Prism 6.0 (GraphPad Software).

[0245] B. Results 1. Intramedullary AAV9 expressing human IDUA elicits strong transgene-specific immunity in MPS I dogs.

[0246] MPS I dogs carry a mutation in IDUA that results in the inclusion of the first intron in the mature mRNA, creating an immediate stop codon. The mutation in MPS I dogs results in undetectable IDUA activity [KP. Menon, et al, Genomics, 14:763-768 (1992); NJ. Terlato, et al, Genet Med, 5:286-294 (2003); XX. He, et al, Mol. Genet Metab, 67:106-112 (1999)]. In the absence of lysosomal IDUA activity, undegraded GAGs accumulate in cells [GN. Sando, et al, Cell, 12:619-627 (2011)]. This major GAG deposit in affected tissues can be detected histologically directly by Alcian blue staining [C. Hinderer, et al, Mol. Ther. J. Am. Soc. Gene Ther. 22, 2018-2027 (2014); NJ Terloato, et al. (2003); RM Shull, et al, Am. J. Pathol. 114, 487-95 (1984); RM Shull, et al, Proc. Natl. Acad. Sci. USA 91, 12937-12941 (1994); LA Clarke, et al., Pediatrics, 123, 229-40 (2009); NM Ellinwood, et al., Mol. Genet. Metab. 91, 239-250 (2007); ME Haskins, et al. al, Am. J. Pathol. 112, 27 (1983); A. Chen, et al, Apmis, 119, 513-521 (2011)]. In addition to the primary GAG storage pathology, lysosomal GAG accumulation leads to a characteristic cascade of cellular abnormalities. Upregulated GAGs cause lysosomal enlargement, which is visible histologically by intense staining for lysosomal membrane proteins such as LIMP2. Neurons also show secondary accumulation of substances such as gangliosides (e.g., GM3) and unesterified cholesterol. Lysosomal accumulation also induces aberrant overexpression of lysosomal enzymes such as hexosaminidase (Hex).

[0247] MPS I dogs were treated at 1 month of age with a single intramedullary injection into the cisterna magna of a clinical candidate AAV9 vector expressing human IDUA. The vector dose was 10 11 Genome copies / kg (GC / kg) (n=2) to 10 12 GC / kg (n=1). The procedure was well tolerated by all subjects. IDUA activity in the CSF rose rapidly following vector administration and exceeded that of normal controls by day 7 (Figure 2A, untreated). However, by day 21 post-vector administration, CSF IDUA levels had declined to baseline, accompanied by an increase in CSF anti-hIDUA antibody titers (Figure 2A, untreated). Day 21 CSF samples All animals also showed lymphocytic CSF pleocytosis (Figure 4A, untreated). In this cohort, elevated CSF antibodies and cell counts were not associated with clinical signs or other laboratory abnormalities, and CSF pleocytosis resolved spontaneously. At necropsy 6 months after injection, histological evaluation showed no evidence of pathology in the brain or spinal cord. Vector biodistribution demonstrated widespread CNS transduction and persistence of the vector genome.

[0248] Although it was not normalized at any dose, brain hexosaminidase overexpression was reduced compared to untreated MPS I dogs (Figure 7). Histology also showed partial resolution of brain storage lesions by immunostaining for LIMP2 and GM3, which did not appear to be dose-dependent.

[0249] Based on the favorable safety profile observed in these dogs, a 10-fold higher dose (10 13Two additional MPS I dogs were treated with vector at 100 mg / kg (100 mg / kg). These dogs developed CSF pleocytosis with kinetics similar to animals treated with the two lower doses (Figure 4A, untreated); however, in these two subjects, the response was more pronounced and the pleocytosis was temporally associated with the onset of neurological signs. Beginning 21 days after vector administration, the animals exhibited hyporeflexia and weakness of the hind limbs, and pain on neck flexion. The pain and CSF pleocytosis began to resolve following treatment with analgesics and corticosteroids; however, hind limb weakness persisted, and 2 weeks after symptom onset, the animals were euthanized. Histopathology demonstrated robust transduction of spinal motor neurons, especially in the lumbar spinal cord, and lymphocytic infiltration around the transduced neurons. Systematic evaluation of sections throughout the brain and spinal cord confirmed that the pathology was primarily localized to the lumbar spinal cord, although occasional infiltration was observed in the brain.

[0250] 2. Neonatal exposure to human IDUA via hepatic gene transfer induces tolerance to subsequent intramedullary gene transfer To evaluate the AAV9 vector expressing human IDUA in the absence of an exaggerated immune response to the transgene, we attempted to induce immune tolerance to the human protein via neonatal exposure. Five days after birth, six MPS I dogs were treated with a single intravenous injection of an AAV serotype 8 vector (AAV8) expressing human IDUA from a liver-specific promoter. At 1 month of age, the following were administered: 10 , 10 11 and 10 12Animals were treated by intrathecal injection into the cisterna magna of different doses of AAV9 vector expressing human IDUA in three cohorts (n=2 animals per cohort) as GC / kg. All animals showed a dose-dependent increase in CSF IDUA activity similar to non-tolerized animals (Figure 2A), but in this cohort CSF enzyme expression persisted beyond day 21 and remained detectable for the duration of the experiment (Figure 2B, tolerized). CSF antibody responses were blunted compared to those seen when treatment-naive (i.e., non-tolerized) animals received intrathecal vector; only two animals in the tolerized cohort (I-602 and I-606) showed detectable titers, which were approximately 20-fold lower than treatment-naive animals treated with equivalent vector doses (Figure 3). Only the dog with the highest antibody titer in this cohort (I-606) showed high lymphocyte counts in the CSF at day 21, albeit at lower levels than treatment-naïve animals (Figures 4A and 4B). There were no clinical adverse events in these cohorts.

[0251] 3. Intramedullary AAV9-mediated expression of hIDUA achieves dose-dependent correction of brain biochemical abnormalities and accumulation lesions Six MPS I dogs tolerized to human IDUA via neonatal gene transfer were sacrificed 6 months after intramedullary injection of AAV9. Brain lysates demonstrated complete normalization of hexosaminidase activity at the highest dose of vector, with partial correction at the lowest dose (Figure 5). Hexosaminidase activity was normalized in CSF at all vector doses (Figure 8). Thickening of cervical meninges, which may contribute to spinal cord compression in MPS I patients, was not observed at all doses of treatment. The effects of LIMP2 and GM3 were reversed in animals treated with hIDUA (Figure 9). Histological evaluation of the brain showed a dose-dependent decrease in LIMP2 and GM3 accumulation in hIDUA-tolerant dogs (Figures 6A, 6B). Animals treated with the highest vector dose showed staining for LIMP2 and GM2 similar to normal controls; at the lowest dose, there was no measurable improvement in some markers (LIMP2 and Hex), whereas GM2 accumulation was not appreciably reduced. Thus, 10 The low dose of GC / kg appeared to be the minimal effective dose (MED).

[0252] The MED of AAV9.CB7.hIDUA IT was established in eight MPS I dogs previously tolerized to human IDUA to assess efficacy in the absence of a confounding immune response to human proteins. Dogs were treated with AAV9.CB7.hIDUA IT at 1 month of age and euthanized 6 months later for evaluation of brain storage lesions. Establishment of the MED utilized well-characterized histological criteria of MPS I disease in CNS tissues, including LIMP2 and GM3. All lysosomal storage lesion criteria were assessed at the highest dose (10 12 Although animals in this cohort did not reach the normal range for GM3 or LIMP2 accumulation, consistent improvements were observed at a 10-fold lower dose (10 11 GC / kg body weight. 10 In the 10-mg / kg body weight group, histological evidence of lysosomal storage showed moderate improvement by LIMP2 staining and minimal improvement in GM3 storage. 10 GC / kg body weight was estimated to be the MED of AAV9.CB7.hIDUA IT. Dose-dependent resolution of brain accumulation lesions correlated with CSF IDUA activity and inversely correlated with CSF spermine concentrations, indicating that CSF IDUA activity and CSF spermine may be useful biomarkers for evaluation of AAV9.CB7.hIDUA pharmacokinetics in clinical trials.

[0253] These data suggest that in MPS I dogs, the MED of AAV9.CB7.hIDUA is 10 10 It indicates that the concentration is GC / kg.

[0254] Administration of AAV9.CB7.hIDUA was also evaluated in treatment-naïve MPS I dogs. MPS I dogs (n=5) received intramedullary injections of the AAV9.MPS I test vector at 1 month of age. 11 GC / kg body weight and 10 12All animals treated with GC / kg body weight showed mild, self-limited lymphocytic cerebrospinal fluid pleocytosis. The animals appeared healthy throughout the study, and at necropsy 6 months after injection, there was no evidence of inflammation in the brain, spinal cord, or meninges.10 13 Two dogs treated with the GC / kg body weight dose appeared to do well initially, but 3 months after infusion developed more severe cerebrospinal fluid pleocytosis and neurological signs consistent with histological evidence of a T cell response to the transduced cells, with mononuclear cells surrounding dead motor neurons in the lumbar spinal cord. These results are consistent with dose-dependent immunotoxicity mediated by lymphocytes targeting transduced spinal motor neurons. Given the sequence differences between the human and canine IDUA proteins, it is not surprising that human IDUA is immunogenic in dogs.

[0255] In non-tolerized MPS I dogs, the MTD is 10 12 The MTD was 9 × 10 GC / kg. This is a conservative estimate of the MTD since it is based on the dog's immune response to human proteins. Based on a brain mass of 45 g for 1-month-old dogs, and with an average body weight of 2 kg, this dose is approximately 9 × 10 GC / kg brain mass in adults (approximately 5 × canine MED on a GC / kg brain mass basis). 10 Sum or 2×10 9 GC / g brain mass, or approximately 1.4 × 10 13 GC total (1. 1×10 10 Equivalent to GC / g brain mass).

[0256] 4. Infusion of recombinant hIDUA in neonatal MPS I dogs is sufficient to induce tolerance to intramedullary AAV9-mediated expression of hIDUA To determine whether hepatic expression of human IDUA is required for the induction of tolerance, we administered recombinant human IDUA ( Two MPS I dogs (I-663 and I-664) were treated with 0.58 mg / kg intravenous infusion of IDUA. Similar to dogs treated as neonates with a vector expressing human IDUA, the enzyme-treated dogs continued to show high levels of CSF IDUA activity (Fig. 2A, 2B) and minimal antibody responses to human IDUA (Fig. 2) or CSF pleocytosis (Fig. 4A-4B3). Brain hexosaminidase activity was reduced in both animals (Fig. 5), and storage lesions were effectively cleared (Fig. 6A, 6B).

[0257] C. Consideration Evaluating the efficacy of intramedullary AAV9 delivery for the treatment of MPS I required the evaluation of both the distribution of vectors that can be achieved via injection into the CSF and the impact of that degree of transduction on disease-specific markers. These studies required the use of an animal model that could accurately reflect the pathophysiology of the disease while also exhibiting sufficiently similar size and anatomy to allow for a meaningful evaluation of the clinical delivery method and the resulting vector distribution. The MPS I canine model faithfully replicates the human phenotype, displaying the same biochemical and histological lesions as well as the same clinical findings [KP. Menon, et al, Genomics, 14:763-8 (1992); RM. Shull, et al, (1984); RM. Shull, et al, (1994); C. Ciron, et al, Ann Neurol, 60:204-213 (2006); P. Dickson, et al, Ann. Neurol, 60:204-213 (2006)]. Due to their phenotypic similarity to MPS I in humans, MPS I dogs have been used extensively in the development of enzyme replacement therapy for the treatment of systemic disease [RM. Shull, et al, PNAS, 91:12937-12941 (1994); P. Dickson, et al, J. Clin. Invest, 118:2868-2876 (2008)]. MPS I dogs also mimic the CNS findings of disease that sporadically develop spinal cord compression and hydrocephalus [P. Dickson, et al, Mol. Genet. Metab. 99, S15-S15 (2010); PI Dickson, et al, Mol. Genet. Metab. 98, 70-70 (2009); CH Vite, et al, Comp. Med. 63, 163-173 (2013)]. Although no cognitive testing has been reported in MPS I dogs, the histological and biochemical findings in the brain have been well characterized and closely recapitulate findings in humans with severe forms of the disease [RM Shull (1984); C. Ciron (2006); SU Walkley, et al, Acta Neuropathol. (Berl.) 75, 611-620 (1988)].The brains of MPS I dogs demonstrate accumulation of lysosomal membrane proteins (LIMP2) and gangliosides (GM3) and upregulation of lysosomal enzymes such as hexosaminidase (Hex). Ganglioside accumulation correlates with cognitive function in MPS I and other lysosomal storage diseases and is therefore an important marker for assessing disease severity and treatment outcome [SU Walkley, MT Vanier, Secondary lipid accumulation in lysosomal disease, Biochim. Biophys. Acta BBA-Mol. Cell Res. 1793, 726-736 (2009); G. Constantopoulos, et al, J. Neurochem. 34, 1399-1411 (1980)]. MPS I dogs also show changes in neuronal morphology similar to those identified in patients [SU Walkley, (1988)]. These striking similarities make this a compelling model for evaluation of intramedullary delivery of AAV as a novel therapeutic approach to the CNS manifestations of MPS I in humans. The ability of this large animal model to recapitulate the route of administration used clinically for IT delivery of AAV9 as well as the distribution of the resulting vector in the CNS further supported the relevance of MPS I dogs for these studies.

[0258] Although MPS I dogs appeared to be an excellent model for the evaluation of clinical vectors, the immune response to human IDUA presented a critical obstacle. It is clear that the immune response to human IDUA in dogs is much more extreme than that observed in patients. Intravenous delivery of the protein in both dogs and MPS I patients often induces antibodies, but in dogs these responses are much stronger, are less likely to subside upon successive administration, and are often associated with anaphylactic reactions to subsequent infusions [RM. Shull, et al, 1994; E. Kakkis, et al, Proc. Natl. Acad. Sci. UA. 101, 829-34 (2004)]. The difference in the immune response to human IDUA in the CNS is even more striking; MPS I dogs treated with intrathecal infusions of the enzyme show evidence of detectable antibody responses in the CSF as well as meningitis. In contrast, children and adults with MPS I treated with repeated IT infusions of the protein show no evidence of detectable antibody responses in the CSF as well as meningitis. For both patients, there were no similar adverse effects, and only one of five patients treated for CSF antibodies against IDUA was positive [C. Ciron (2006); P. Dickson, et al, (2010); PI Dickson, et al, Mol. Genet. Metab. 98, 70-70 (2009); PI Dickson, et al, Mol. Genet. Metab. 101, 115-122 (2010); PI Dickson, et al, Mol. Genet. Metab. 93, 247-247 (2008); E. Kakkis, et al, Mol. Genet. Metab. 83, 163-174 (2004); T. C. Rund, et al, Mol. Genet. Metab. 111, S74 (2); M. Vera, et al, Pediatr. Res. 74, 712-720 (2013)]. Interestingly, MPS MPS I dogs also develop CSF antibodies against canine IDUA, albeit at lower levels than against the human enzyme, suggesting that this model has a greater overall tendency toward immunity against IDUA, which is exacerbated by the use of a protein that is not species-specific. These striking differences in the outcomes of both intravenous and intramedullary delivery of human IDUA in MPS I dogs and patients suggest a consistently exacerbated immune response to human IDUA in MPS I dogs and that preventing this response is necessary to recapitulate vector activity expected in humans. Inducing tolerance to the protein via neonatal exposure allowed the evaluation of human vector efficacy in this model without the intervention of an exacerbated immune response. This provided important information that allowed for the accurate determination of the minimum effective dose, an essential factor in the design of first-in-human gene therapy trials in the most relevant animal models. More specifically, extensive dose-ranging studies were performed in MPS I dogs. The minimum effective dose was determined in immune-tolerant animals, but was greater than 2 × 10 as determined by the oqPCR method described herein. 9 The dose is estimated to be 100 GC / g brain mass. The study was conducted in immunocompetent dogs (i.e., naive to IDUA and AAV) and toxicity was 10 12 GC / g. 12 Based on the findings of the dose-limiting toxicity study (DLT) in GC / g, a 10-fold lower dose will be administered in the formal Good Laboratory Practice (GLP) toxicity study in rhesus monkeys. The dose evaluated in the formal GLP non-human primate (NHP) toxicity study is 1.1 × 10 11 GC / g brain mass. If no toxicity is encountered, the clinical starting dose would be 1.1×10 10GC / g brain mass. This starting dose is approximately 5-fold higher than the minimal effective dose (MED) in the canine MPS I model and approximately the same dose that demonstrated reliable histological responses in studies of MPS I dogs and cats, supporting a reasonable expectation of clinical efficacy at this dose. The starting dose is also approximately 90-fold lower than the dose at which toxicity was observed in MPS I dogs and 10-fold lower than the dose examined in non-human primates, providing an acceptable safety margin to account for the potential for human subjects to exhibit greater susceptibility to vector- or transgene-related toxicity. Based on these data, the starting dose represents an acceptable benefit:risk profile, where the dose is expected to be within the therapeutic range (and thus may provide clinical benefit), but below the toxic vector dose (and thus should be reasonably safe). The following calculations show how the dose in dogs can be extrapolated to the starting dose in humans: 1-month-old dog brain = 45 grams; naive dogs: MED9 x 10 10 GC total (2×10 9 GC / g brain mass);Adult brain = 1300 grams; human starting dose (5 x canine MED), 1.4 x 10 13 GC total (1.1×10 10 GC / g brain mass). Without this approach, the only option would be to extrapolate efficacy data from vectors with species-specific transgenes, which may have important differences in efficacy, or to move studies to less representative animal models that are more immune-tolerant to human proteins. Pharmacological immunosuppression could also be employed in this setting, although induction protocols of neonatal tolerance have the distinct advantage of avoiding secondary consequences of immunosuppressive drugs.

[0259] Although efficacy evaluation was confounded by immune responses and loss of circulating IDUA in non-tolerant dogs treated with human vector, some useful data can be derived from these animals. While the robust immune response is unlikely to represent an immune response in humans, it may be aligned with monitoring plans for first-in-human trials by revealing key features of immune-mediated toxicity. In this case, we observed that immune-mediated toxicity was dose-dependent, with a peak of the immune response occurring 3 weeks after vector administration, with focal motor symptoms likely due to a high degree of transduction of spinal motor neurons, and with CSF pleocytosis. These findings may be directly integrated into a Phase I trial protocol with extensive monitoring of immune-mediated toxicity and neurological symptoms that extend for several weeks after vector administration, and CSF analysis of CSF pleocytosis that occurs 2-4 weeks after injection. If neurological symptoms accompanied by CSF pleocytosis manifest with similar kinetics in human study subjects, the findings in treatment-naive dogs suggest that toxicity is due to an immune response (as opposed to, e.g., overexpression toxicity) and may guide treatment decisions.

[0260] In MPS I dogs tolerized to human IDUA, a strong correlation emerged between vector dose, CSF enzyme levels, and correction of brain accumulation lesions. The relationship between IDUA activity in the CSF and correction of brain lesions may be a useful observation when this approach progresses to human trials, where IDUA activity detected in the CSF may be a useful predictor of clinical response. Even more useful would be the identification of CSF markers that directly reflect the severity of CNS accumulation lesions. CSF biomarkers are valuable tools to assess the correction of underlying CNS lesions in MPS I patients, and the dog model may be an ideal system for the identification of such markers. In this study, we evaluated one potential CSF biomarker, the enzyme hexosaminidase. While Hex activity is substantially elevated in brain tissue from MPS I dogs, it is only moderately elevated in the CSF. Regardless of the extent of tissue reaction, CSF Hex normalized in all treated animals. Thus, CSF Hex may be useful to confirm vector activity in clinical trials, but is unlikely to predict treatment response. Future studies with MPS I canine models may allow for the evaluation of additional CSF markers and their correlation with brain accumulation lesions, which may ultimately yield powerful new tools to non-invasively assess the severity of CNS lesions in MPS I and the efficacy of novel therapeutic approaches.

[0261] The present findings indicate that neonatal exposure to human IDUA can induce tolerance using two different sources of enzyme. While Example 3 shows that AAV-mediated expression may induce transgene-specific tolerance in neonates, this example shows that infusion of recombinant enzyme may also induce tolerance. If this approach can be generalized to other proteins, it may be useful for more accurate preclinical evaluation of many human therapeutics in animal models. Furthermore, if a similar approach can induce tolerance to foreign proteins in human neonates, it may have enormous potential to improve the efficacy of protein replacement therapy for diseases in which antibody responses to normal proteins limit efficacy. While most MPS I patients appear to tolerate intrathecal infusion of IDUA, the majority develop serum antibodies against intravenous enzyme replacement, and these antibodies can reduce response to therapy. Induction of tolerance in neonates as a gene replacement therapy or protein replacement therapy may be useful. Combination use may substantially improve patient outcomes. The availability of an approved recombinant enzyme makes MPS I an excellent candidate for the first human trials of this approach. If human newborns exhibit a similar time window of 1-2 weeks for induction of tolerance, neonatal screening will be essential to identify patients early enough for successful intervention. Thus, MPS I is a promising candidate for the first human trials of this approach. The ongoing implementation of newborn screening for lysosomal storage disorders will be crucial to the clinical evaluation of protocols for the induction of neonatal tolerance.

[0262] Example 4: Intramedullary AAV-mediated human IDUA gene transfer in young rhesus monkeys A. Intramedullary delivery The objective of this study was to evaluate the safety of intrathecal (IT) administration of AAV2 / 9.CB7.CI.hIDUAco.RBG, an AAV9 vector expressing human IDUA, in 1-month-old rhesus macaques, a model that is developmentally similar to 6- to 9-month-old human infants. In addition, the study assessed whether antibodies to the transgene product in serum or cerebrospinal fluid (CSF) affected the safety of vector administration and the activity of human IDUA.

[0263] Four rhesus macaques were included in the study. Preliminary studies have shown that rhesus macaques can make antibodies against human α-L-iduronidase (IDUA). Because an antibody response to the human IDUA transgene product was expected in rhesus macaques, two of the animals were tolerized at birth by intravenous (IV) administration of an AAV8 vector expressing human IDUA from a liver-specific promoter (AAV2 / 8.TBG.PI.COhIDUA.nRBG). To control for procedural effects and exposure to AAV8 vectors, the other two rhesus macaques were administered IV at birth with an AAV8 vector expressing an unrelated transgene (human clotting factor IX) (AAV2 / 8.LSP.IVS2.hFIXco.WPRE.BGH). At 1 month of age, the animals were tolerized at 3 × 10 12 All four animals were dosed by IT infusion at a dose of 100 GC / kg. Animals were observed for 16 months after dosing at the time of publication of this report and will remain on study for at least another year. Endpoints evaluated throughout the study included general observations, body weight, and global clinical pathology (blood counts with differential and serum laboratory values). In addition, IDUA enzyme activity and antibody responses to the transgene were measured in CSF.

[0264] The study revealed no vector-associated pathologies and clinical sequelae (0). All animals showed normal growth trajectories. Serum laboratory values ​​and blood counts were within the normal ranges for historical control rhesus macaques of comparable age and housing conditions. Antibodies to the transgene were detected in the CSF of two non-tolerized animals but not in two animals tolerized to human IDUA as neonates. In both animals tolerized to IDUA, IDUA activity at least 15% higher than baseline levels was detectable in the CSF throughout the study. In non-tolerized animals, CSF IDUA activity rapidly increased after administration of the AAV9.MSPI test vector but declined to baseline following the induction of antibodies. The presence of transgene-specific antibodies in the CSF did not affect the safety of IT administration of AAV9.CB7.hIDUA but did affect the ability to detect levels of human IDUA in the CSF.

[0265] In conclusion, a single dose of AAV9.CB7.hIDUA administered intrathecally resulted in 3 × 10 12 The dose of 1000 mg / kg GC was well tolerated in 1-month-old rhesus monkeys. At this dose, levels of hIDUA at least 15% above baseline were detectable in the CSF of animals tolerized to human IDUA at birth; non-tolerized animals developed an antibody response to hIDUA that was detectable in the CSF and negatively correlated with hIDUA expression. No treatment-related effects on growth, behavior, or clinical laboratory or hematological parameters were observed in either animals positive for anti-IDUA antibodies or those not positive for antibodies. There wasn't.

[0266] A. Materials and Methods Vectors used in this study included rAAV9.hIDUA, rAAV8.hIDUA and an AAV8 vector carrying an unrelated transgene (hFIX).

[0267] Intrathecal (IT) administration via suboccipital puncture was chosen because it is the proposed route for clinical use. This study evaluated a single vector dose adapted to the animal's weight. To induce immune tolerance to human IDUA, two animals were administered 100 mg of rAAV8.hIDUA (100 mg / kg) on ​​postnatal day 1 (day 0 in the study). 12 Animals were administered an IV infusion of 1000 mg / kg of AAV8.hFIX (100 mg / kg GC) at postnatal day 1. Control animals received a control vector (rAAV8.hFIX) expressing an unrelated transgene (human factor IX) from a liver-specific promoter. All animals then received the AAV9.MPSI test vector IT via suboccipital puncture at 1 month of age (study day 30).

[0268] B. Results and Conclusions Four 1-month-old rhesus monkeys (M. mulatta) were given 3 × 10 12 GC / kg of rAAV9.hIDUA was administered IT and monitored for >1 year after vector administration. Two of these animals were tolerized to the human IDUA protein at birth.

[0269] There were no treatment-related effects on body weight or weight gain, and no treatment-related clinical signs. There were no treatment-related effects on clinical laboratory or hematological parameters. Antibodies to the transgene product were detectable in the CSF of two animals that had not been pretreated to induce tolerance to the human protein. There were no differences in the endpoints assessed (clinical signs, body weight, hematology, and clinical laboratory) between tolerized and non-tolerized animals, indicating that CSF antibodies to the protein were not associated with overt toxicity. In animals tolerant to IDUA, one animal had sustained expression of IDUA in the CSF at more than twice baseline levels, and the other animal was approximately 15% above baseline.

[0270] In conclusion, intrathecal administration of a single dose of rAAV9.CB7.hIDUA reduced the number of immunizations by 3 × 10 12A dose of 1000 GC / kg was well tolerated in 1-month-old rhesus monkeys. At this dose, levels of hIDUA that were 115-200% of baseline were detectable in the CSF of animals tolerized to human IDUA at birth; non-tolerized animals developed antibody responses to hIDUA that were detectable in the CSF and negatively correlated with hIDUA expression. No treatment-related effects on growth, behavior, or clinical laboratory or hematological parameters were observed in either animals positive or non-seropositive for anti-IDUA antibodies.

[0271] Example 5: Intramedullary AAV-mediated transfer of the human IDUA gene in cynomolgus monkeys The vectors consisted of an AAV9 capsid packaging an expression cassette consisting of a cytomegalovirus promoter (CMV), a chimeric intron (PI), a codon-optimized human IDUA transgene (hIDUA), and a polyadenylation signal (SV40). The expression cassette was flanked by inverted terminal repeats of AAV serotype 2. A single vector was used in this study and is designated as either AAV2 / 9.CMV.PI.hIDUA.SV40, AAV2 / 9.CMV.PI.hIDUAco.SV40, AAV2 / 9.CMV.PI.hIDUAco.SV40PA, or AAV9.CMV.PI.hIDUA.SV40.

[0272] A. Materials and Methods This study included two female cynomolgus monkeys (ID06-09 and 07-19). 12 Patients received 1000 genome copies / kg body weight (GC / kg) of AAV2 / 9.CMV.PI.hIDUAco.SV40PA. Intrathecal (IT) administration via suboccipital puncture was chosen because it is the proposed route for clinical use.

[0273] [Table 2]

[0274] 1. Dose per gram of brain mass is based on a 90g brain.

[0275] B. Results and Conclusions Two adult female cynomolgus monkeys were treated by intrathecal injection of an AAV9 vector expressing human IDUA from a CNV promoter. Body weight, physical examination, and blood counts and serum laboratory values ​​were assessed on study days 1, 7, 14, 28, 91, 118, 147, 182, 208, 239, 261, 294, 322, 350, 378, 413, 434, 462, 490, 518, 561, 589, 624, and 636 after vector administration, and animals were subsequently necropsied for histopathology and vector biodistribution analysis. There were no vector-related clinical adverse events. One animal developed a femoral artery aneurysm 600 days after vector administration. This was likely secondary to repeated blood draws and is unlikely to be treatment-related. There were no treatment-related effects on clinical pathology parameters, including final CSF parameters. Histopathology showed no evidence of CNS lesions and no obvious vector-related abnormalities in peripheral organs. Biodistribution analysis showed vector deposition in the brain and spinal cord of both NHPS, 1-2 orders of magnitude greater than in peripheral organs, with one exception. Significant liver distribution occurred in one animal with no preexisting neutralizing antibodies to the AAV9 capsid, while animals with preexisting serum antibodies to the vector showed minimal liver transduction. Immunostaining of brain sections from both animals revealed expression of human IDUA.

[0276] This study provided evidence that IT AAV9-mediated gene transfer allows long-term expression of IDUA in the brain. This study also provided preliminary evidence of the safety of this approach.

[0277] Example 6: Intracerebroventricular (ICV) delivery of AAV9.hIDUA in mice in the context of pre-existing immunity to hIDUA A pilot study was designed to evaluate evidence of toxicity following intracerebroventricular (ICV) administration of AAV9.hIDUA in treatment-naive mice as well as mice with pre-existing antibodies to the transgene product, human iduronidase (IDUA).

[0278] The test article consisted of an AAV9 capsid packaging an expression construct consisting of a chicken β-actin promoter (CB7), a chimeric intron (CI), a codon-optimized human IDUA transgene (hIDUAco) and a polyadenylation signal (RBG). The expression construct was flanked by inverted terminal repeats of AAV serotype 2. This vector is designated AAV9.CB7.CI.hIDUA.RBG. The final product was diluted in Elliot's formulation buffer (EFB).

[0279] This non-GLP study was originally planned as an aid to designing a GLP toxicity study in pre-immunized mice, but the GLP study was not performed based on FDA feedback that experiments based on immunization against a non-species specific protein in normal mice were unlikely to be representative of patients previously treated with enzyme replacement therapy (ERT). At the time the decision was made to conduct a GLP toxicity study in pre-immunized mice, the first pilot study was already underway. The results of the pilot study are included in this report.

[0280] The study included 100 adult C57BL / 6 mice (50 / sex). Half of the animals were immunized against human IDUA by intramuscular (IM) injection of recombinant human IDUA (Aldurazyme®) in adjuvant (TiterMax). Six months after immunization, both immunized and naïve animals received two doses (5×10 10 GC or 2.5×10 11 Animals were treated with AAV9.hIDUA ICV injection into one of the 100-mL mice (GC). Animals from each treatment group were sacrificed at one of five time points (7, 14, 30, 60, or 90 days) after vector administration. Brain, spinal cord, heart, lungs, liver, spleen, kidneys, and gonads were harvested for histopathology.

[0281] In the naive (non-immune) cohort (n=50, 25 / sex), no animals died prior to scheduled necropsy and showed no clinical abnormalities. Brain histopathology showed dilatation of the lateral ventricles and visible needle track in some animals, consistent with an ICV route of administration. Minimal to mild lymphocytic infiltration of the meninges and / or brain parenchyma occurred in 12 of 50 animals, which showed no clear correlation with vector dose or time after injection. Hepatitis occurred in a dose-dependent manner and in correlation with time after vector administration. Minimal to moderate hepatitis occurred in all five animals in the high-dose cohort sacrificed 14 days after vector administration. Only minimal hepatitis was observed in high-dose animals sacrificed at 7, 30, 60, or 90 days. Only minimal hepatitis was observed in the low-dose cohort, which occurred in eight animals and showed no clear correlation with time after vector administration. One animal in the high-dose cohort developed moderate myocarditis 60 days after vector administration; three additional animals in the high-dose cohort showed minimal myocarditis 30 or 90 days after vector administration. Two animals in the low-dose cohort showed minimal myocarditis and one showed mild myocarditis, all occurring 60 days after vector administration. No other potentially vector-related abnormalities were observed in the naive (non-immune) cohort.

[0282] In the cohort (n=50, 25 / sex) immunized against human IDUA prior to vector administration, 3 animals (2 males, 1 female); high dose (2.5 × 10 11 GC) and two mice that received a low dose (5 × 10 10One animal received 100 mg / kg / day GC and 1 died. All three died on days 18 or 19 of the study. Histopathology in the immunized groups was consistent with an intense cell-mediated immune response to the transduced cells in peripheral organs, with moderate to severe myocarditis occurring in 8 of 50 animals and moderate to severe hepatitis occurring in 8 of 50 animals. Both findings correlated with vector dose and timing of vector administration, with the most severe findings occurring 14 days after vector administration. Findings in the brain were less severe; moderate meningitis or encephalitis occurred in 3 animals treated with the high vector dose and 1 animal treated with the low vector dose. These findings did not correlate with time of vector administration. Other findings in the brain were minimal or mild.

[0283] Overall, results in the immune naive (non-immune) cohort were consistent with the induction of an immune response to the human transgene, as evidenced by lymphocytic infiltration in the liver and, to a lesser extent, the heart, although both organs were transduced by AAV9 that escaped into the peripheral circulation following IT injection. 1,2 In this situation, the highest dose evaluated (2.5 × 10 11 The toxicity was evident in GC.

[0284] In pre-immunized cohorts, the immunization strategy appeared to induce a strong cell-mediated immune response against the transgene and resulted in moderate to severe myocarditis and hepatitis in some vector-treated animals. Toxicity correlated with vector dose. Because the experimental design was based on immunization against a non-species-specific protein in normal mice, its applicability to patients previously treated with ERT is unclear.

[0285] Example 7: AAV9.CB7.hIDUA Vector Injected Intramedullarily (IT) in Non-Human Primates The following non-human primate (NHP) safety study involved fluoroscopically guided suboccipital injection (cistem magna) using two doses: the low dose (LD) was 1.1 × 10 10 GC / g, and the high dose (HD) is 1.1 × 10 11The dose was GC / g (equivalent to the canine maximum tolerated dose (MTD)). For the arm with HD with immunosuppression (IS), the protocol included co-administration of MMF and sirolimus as follows: MMF from days 21 to 60, and sirolimus from days -21 to 90. In the NHPs studied, there were no clinical findings and no clinically significant abnormalities in serum chemistry or hematological parameters. There was evidence of anti-AAV and anti-hIDUA immune responses and immune-mediated axonopathy. These data are provided in Figures 22-23 and the tables below.

[0286] Histological findings are not shown. Axonopathy in the dorsal columns (ascending sensory tracts) at high dose was found with an axonopthy score of 2 (0-4 scale) (vehicle control, normal). High dose ganglia gangioneuritis was observed with perineuronal inflammatory cells, neuronal degeneration, satellite cell activation and proliferation (vehicle control, normal).

[0287] For AAV9.hIDUA, neuronal degeneration is limited to the DRG (data not shown). For high-dose cervical spinal cord (ventral horn) without immunosuppression, no signs of inflammation around motor neurons expressing hIDUA are observed. For high-dose cervical (DRG) without immunosuppression, inflammation is observed around DRG neurons expressing hIDUA. A prevalence of about 1-2 neurons per 100 is observed. It has been shown that the inflammatory infiltrate is mainly CD 20-positive B lymphocytes and CD3-positive T lymphocytes, with few CD68-positive macrophages. Lymphocytes cluster around hIDUA-positive transduced neurons. Small inflammatory nodules replacing missing neurons.

[0288] Immunosuppression improved but did not uniformly prevent immune-mediated DRG ganglioneurite outgrowth (data not shown). Illustrated results are four images from three different animals, all receiving the same vector, but with variation in their immune response. For high dose RA1404 cervical with immunosuppression (DRG), MMR ceases at day 36, with an axonopathy score of 0 and cumulative of 0. No inflammation is observed around hIDUA-expressing DRG neurons. For high dose RA0747 cervical with immunosuppression (DRG), with an axonopathy score (cervical 1, cumulative of 4), inflammation is observed around hIDUA-expressing DRG neurons. CD3+ T lymphocytes are neither organized nor clustered. CD20+ positive B lymphocytes are not observed in the infiltrate. For the cervix (DRG) of the third animal (RA1528) at the high dose with immunosuppression, the axonapthy score was cervical 1 and cumulative 4. Inflammation is observed around the DRG neurons expressing hIDUA. CD3+ T lymphocytes and CD20+ positive B lymphocytes are observed in clusters.

[0289] Immunosuppression improved but did not uniformly impede immune-mediated DRG ganglioneurite apoptosis. Data are presented in tabular format and include control, low dose, high dose, and high dose with immunosuppression.

[0290] [Table 3]

[0291] A. Safety and Biodistribution This study evaluated the safety and biodistribution of AAV9.CB7.hIDUA up to 180 days post-injection by image-guided suboccipital puncture in rhesus monkeys. Adult rhesus monkeys (n=9, 6 females, 3 males; Groups 1A, B, and C) were administered 1.1 × 10 11 Approximately equivalent to a dose of 10 GC / g brain mass 13GCs were administered a single dose of AAV9.CB7.hIDUA. An additional three animals (2 females, 1 male, Groups 2A and B) were administered a single dose of vehicle (Elliot B® + 0.001% Pluronic® F68) by image-guided suboccipital puncture. Animals were euthanized and necropsied on Day 14 (Groups 1A and 2A), Day 90 (Groups 1B and 2B), or Day 180 (Group 1C) following administration of the test or control article. Toxicity was assessed by daily observations on Days 0, 3, 7, 14, 21, 30, 45, 60, and 90 of the study, as well as by physical examination, CBC and serum chemistry panel, coagulation panel, and CSF cell count analysis, protein, and glucose concentrations. At necropsy, a pathologist evaluated tissues for gross lesions and examined microscopically. T cell responses to the vector capsid and transgene product were assessed by ELISPOT, antibody responses to the transgene product were measured in serum and CSF by ELISA, and vector biodistribution was assessed by qPCR.

[0292] There were no treatment-related adverse events (AEs). From day 14 of the study, the first time point evaluated, the vector genome could be detected throughout the brain and spinal cord of all treated animals (10 4 Levels around GC / μgDNA), euthanasia at 90 and 180 days The cytotoxicity was observed in the brain and spinal cord of the animals at necropsy and persisted at the same level. 5 ~10 6 GC / μgDNA), reticuloendothelial tissue (lymph nodes and bone marrow 10 3 ~10 4 GC / μgDNA) and heart (10 3 ~10 4 There was also significant distribution of vector to the peripheral and hepatic GC / μg DNA. These data suggest that vector spread peripherally and hepatic GC / μg DNA follows intramedullary delivery of vector. These results suggest that liver transduction is possible.

[0293] Both humoral and T cell-mediated immune responses were elicited against the human IDUA protein. This response appeared to correlate with the histological findings of a transient increase in mononuclear cells in the CSF and axonal degeneration in the dorsal white matter of the spinal cord, observed throughout the spinal cord at 90 and 180 days. These findings were not associated with clinical abnormalities or histological evidence of damage to tissues other than the dorsal funiculus of the spinal cord. Based on these findings, the no observed adverse effect level (NOAEL) was determined to be 10 in cynomolgus monkeys. 13 GC (approximately 1.1 × 10 11 The effect of GC / g brain mass on the toxicity of nicotinamide adenine diphosphate ...

[0294] 1. Materials and Methods The AAV9.hIDUA test vector (in Elliot B®+0.001% Pluronic® F68) was evaluated. Twelve rhesus monkeys were included in the study. Animals were randomly assigned to five groups. Test groups 1A, 1B, and 1C, consisting of one male and two females, were treated with the test vector and were euthanized and necropsied on days 14±2, 90±2, or 180±2 of the study, respectively. Animals in groups 2A and 2B were treated with vehicle (Elliot formulation buffer) and were euthanized and necropsied on days 14±2 or 90±2, respectively.

[0295] The IT route via image-guided suboccipital puncture was chosen because it is the proposed route for clinical use, and because this dose is similar to the maximum tolerated dose in MPS I dogs (on a dose per gram brain mass basis) and is 10-fold higher than the starting dose proposed for the first-in-human study. 13 The dose of GC was selected.

[0296] On day 0 of the study, animals were anesthetized and placed on an X-ray table in a lateral decubitus position with the head bent for collection and administration of CSF into the cisterna magna. The site of injection was aseptically prepared. Using aseptic technique, a 21-27 gauge, 1-1.5 inch Quincke needle (Becton Dickinson) was advanced into the suboccipital space until CSF flow was observed. Up to 1.0 mL of CSF was collected for baseline analysis. Correct placement of the needle was verified via myelography using a fluorescent microscope (OEC9800 C-Arm, GE). After CSF collection, a Luer access extension catheter was connected to the intrathecal needle to facilitate administration of iohexol (Omnipack 180 mg / mL, General Electric Healthcare) contrast agent and test or control substance. Up to 1 mL of iohexol was administered via the catheter and intrathecal needle. After confirming correct needle placement by observation of contrast agent in the cisterna magna, a syringe containing the test substance or solvent (1.4 mL volume, equal to 1 mL plus the volume of the syringe and the dead space of the linker) was attached to the flexible linker and injected slowly over 20-60 seconds. The needle was removed and direct pressure was applied to the puncture site.

[0297] 2.Results There were no adverse events related to vector administration treatment. AAV vector genomes were detected throughout the brain and spinal cord of all animals treated with the AAV9.hIDUA test vector at all time points measured, and levels were comparable in these tissues over time. There was also significant vector distribution in peripheral organs, particularly the liver, and vector genome levels in peripheral tissues were comparable over time. There were no clinical, gross, or histological findings in either vehicle controls or test vector animals euthanized at day 14. Mild, transient CSF mononucleosis was observed in 5 / 6 AAV9.hIDUA-treated animals, peaking around day 30 post-administration.

[0298] Serum and CSF antibodies against hIDUA (transgene product) were 6 / 6 on day 21. Peripheral T cell responses to the hIDUA peptide were detected in AAV9.hIDUA test vector-treated animals and observed in 4 / 6 test vector-treated 90 day animals and 1 / 3 treated 180 day animals. Microscopically, there was minimal to moderate axonal degeneration in the dorsal sensory white matter tracts of the spinal cord suggesting cell body damage within sensory neurons of the dorsal root ganglion (DRG not available for histological evaluation) in 6 / 6 AAV9.hIDUA test vector-treated animals. The anatomical location of axonal degeneration in the ascending dorsal sensory tracts suggests specific involvement of sensory neurons from the dorsal root ganglion. The fact that these neurons were usually significantly transduced after intrathecal administration of AAV and the time course of CSF antibodies (from day 21) and CSF cerebrospinal fluid cellularity (peaking at day 30), as well as the presence of transgene-specific T cell responses in the majority of animals at day 90, suggest that a cell-mediated cytotoxic immune response against hIDUA was generated in the dorsal root ganglion.

[0299] B.NHP toxicity The purpose of this exploratory GLP study is to 12 GC total (1.1×10 10 GC / g brain mass) and 1×10 13 GC total (1.1×10 11 The objectives of this study were to evaluate the safety of IC AAV9.hIDUA test vector at two dose levels (1000 mg / g brain mass) and to evaluate the effect of immunosuppressive therapy on the safety of IC R AAV9.hIDUA test vector at a higher dose. The immunosuppressive regimen consisted of sirolimus (rapamycin) and mycophenolate mofetil (MMF) administered at least 2 weeks prior to AAV9.hIDUA test vector administration and continued through days 60 (MMF) and 90 (sirolimus) at doses that maintained plasma trough levels as close as possible to 10-15 μg / L for sirolimus and 2-3.5 μg / mL for mycophenolate (the active metabolite of MMF).

[0300] Adult rhesus monkeys (N = 9, 6 males and 3 females, groups 2–4) were administered 1 × 10 12 or 1013 Male animals received a single dose of either GC whole AAV9.hIDUA or GC whole AAV9.hIDUA. Additional male animals (Group 1) received a single dose of vehicle (Elliot B® + 0.001% Pluronic® F68) via image-guided suboccipital puncture. Consistent with the studies in Part A of this Example, there were no AEs related to the administration procedure and no treatment-related effects on clinical performance, weight changes, CBC, serum chemistry, or coagulation parameters. IS had the expected effects primarily on weight and CBC.

[0301] AAV vector genomes were detected throughout the brain, spinal cord, and dorsal root ganglia of all AAV9.hIDUA test vector-treated animals. All but one AAV9.hIDUA test vector-treated animal developed both humoral and T cell immune responses to hIDUA that were not dose-dependent. IS inhibited only the humoral immune response to hIDUA. IS does not prevent the cellular immune response to hIDUA or AAV9 capsid that occurred between days 60 and 90 in 2 / 3 of IS animals. Treatment-related findings were observed in histological analysis and consisted of minimal to mild dorsal column axonopathy that was not dose-dependent. In the dorsal root ganglia, which contain neuronal cell bodies projecting to the dorsal columns, there was minimal to moderate neuronal cell body degeneration with mononuclear cell infiltration. Dose level of AAV9.hIDUA did not affect the presence and intensity of findings in the spinal cord and dorsal root ganglia. IS did not eliminate axonopathy in the majority of animals that received IS. These data support the potential efficacy of IC administration of AAV9.hIDUA to reverse CNS symptoms of MPS I, but also suggest that IS may be necessary in the clinical setting to achieve optimal efficacy and safety.

[0302] Based on the observed findings of DRG neuron degeneration, T cell and B lymphocyte infiltration around hIDUA-expressing sensory neurons, and humoral and T cell immune responses to hIDUA, it is possible that immune-mediated destruction of a small portion of the transduced sensory neurons in the dorsal root ganglion may be involved. It is concluded that degeneration of axons belonging to injured DRG neurons (dieback phenomenon) leads to minimal to mild dorsal column axonopathy.

[0303] Low dose group (1×10 12 GC) and the high-dose group (1 × 10 13 In rhesus monkeys receiving IT injections of the AAV9.hIDUA test vector, a NOAEL could not be defined. 13 The incidence and severity of findings was reduced in the GCIS group, suggesting that the cause of these findings was immune-related.

[0304] C. Study Summary and Conclusions These data indicate that immune-mediated destruction of DRG neurons can cause axonopathy. Perineuronal inflammatory infiltrates were observed around hIDUA-expressing DRG neurons and neuronal death. Mild to moderate axonopathy was observed in the dorsal funiculus, including ascending axons from the DRG. All animals with abnormal findings had both humoral and T cell responses to the transgene product. Animals were clinically normal throughout the study. The immunosuppressive regimen used (MMF + sirolimus) attenuated but did not consistently prevent the immune response to the transgene. No correlation was found between axonopathy and anti-AAV9 responses. From this, it was concluded that in the first study with AAV9.hIDUA, immunosuppression is necessary to minimize the risk of immune-mediated damage to neuronal cells. In conclusion, preclinical studies support the development of IT AAV9-based gene therapy to address neurocognitive symptoms in MPS I (AAV9.hIDUA). Preclinical results call for the inclusion of immunosuppression along with treatment with AAV9.hIDUA to minimize the risk of immune-mediated toxicity.

[0305] Example 8: Production of rAAV9.CB7.hIDUA Vector rAAV9.CB7.hIDUA is generated by triple transfection of human HEK293MCB cells with (i) the hIDUA vector genome plasmid, (ii) an AAV helper plasmid called pAAV29, which contains the wild-type AAV rep2 and cap9 genes, and (iii) a helper adenovirus plasmid called pAdΔF6(Kan). The size of the packaged vector genome is 4344 nt.

[0306] Cloning of the above plasmid pAAV.CV7.CI.hIDUAco.RGB: The plasmid is 7,165 bp in size. The vector genome derived from this plasmid is a single-stranded DNA genome with ITRs from AAV2 flanking the hIDUA expression cassette. Expression from the transgene cassette is driven by the CB7 promoter, a hybrid between the cytomegalovirus (CMV) immediate early enhancer (C4) and the chicken β-actin promoter, while transcription from this promoter is enhanced by the presence of the chicken β-actin intron (CI). The polyA signal for the expression cassette is the rabbit β-globin (RBG) polyA. A plasmid was constructed by codon optimizing and synthesizing the hIDUA sequence [SEQ ID NO:1] and the resulting construct was then cloned into the expression elements of the plasmid, pENN.AAV.CB7.CI.RBG (p1044), an expression cassette flanked by the ITRs of AAV2 containing CB7, CI and RBG to obtain pAAV.CB7.CI.hIDUAco.RBG (p3032).

[0307] Cloning of cis-plasmid pAAV.CB7.CI.hIDUA.RBG.KanR: The vector genome was excised from p3032 using PacI restriction enzyme and cloned into a pKSS-based plasmid backbone (p2017) containing the kanamycin resistance gene. The final vector genome plasmid is pAAV.CB7.CI.hIDUAco.RBG.KanR.

[0308] AAV2 / 9 helper plasmid pAAV29KanRRep2 The AAV2 / 9 helper plasmid pAAV29KanRRep encodes the four wild-type AAV2 rep proteins and the three wild-type AAV VP capsid proteins derived from AAV9. To create the chimeric packaging construct, the AAV2 cap gene was first removed from plasmid p5E18, which contains the wild-type AAV2 rep and cap genes, and replaced with a PCR fragment of the AAV9 cap gene amplified from liver DNA. The resulting plasmid was given the identifier pAAV2-9(p0008). Note that the AAV p5 promoter, which normally drives expression of rep, is moved from the 5' end of rep to the 3' end of cap in this construct. This arrangement serves to introduce a spacer between the promoter and the rep gene (i.e., the backbone of the plasmid), downregulating expression of rep and enhancing the ability to support vector production. The backbone of the plasmid in p5E18 is derived from pBluescriptKS. The AAV2 / 9 helper plasmid pAAV29KanRRep2 encodes the four wild-type AAV2 rep proteins, the three wild-type AAV VP capsid proteins, and kanamycin resistance.

[0309] The pAdDeltaF6(Kan) adenovirus helper plasmid is 15,770 bp in size. The plasmid contains the regions of the adenovirus genome that are important for AAV replication, namely E2A, E4, and VA RNA (the adenovirus E1 function is provided by 293 cells), but does not contain other adenovirus replicative or structural genes. Since the plasmid does not contain cis elements important for replication such as the adenovirus inverted terminal repeats, infectious adenovirus is not expected to be generated. It was derived from an Ad5 E1, E3 deleted molecular clone (pBHG10, a pBR322-based plasmid). Deletions were introduced into the Ad5 DNA to eliminate expression of unnecessary adenovirus genes and to reduce the amount of adenovirus DNA from 32 Kb to 12 kb. Finally, the amplicon resistance gene was replaced with a kanamycin resistance gene to obtain pAdΔF6(Kan). The functional elements of the adenoviral genes E2, E4 and VAI required for the construction of AAV vectors remain in this plasmid. The function of the adenoviral E1 essential gene is provided by HEK293 cells. The DNA plasmid was sequenced by Qiagen Genomic Services and showed 100% homology with the following important functional elements of the reference sequence pAdDeltaF6(Kan)p1707FH-Q: E4 ORF6 3692-2808bp; E2A DNA binding protein 11784-10194bp; VA RNA region 12426-13378bp.

[0310] A flow chart summarizing the manufacturing process is provided in FIG.

[0311] Cell seeding: A qualified human embryonic kidney 293 cell line will be used in the production process. Cells will be seeded at 5 x 10 using Corning T flasks and CS-10 to generate sufficient cell mass to seed up to 50 HS-36 for vector production per BDS lot. 9 ~5× 10 10The number of cells will be expanded to 10000. The cells will be cultured in a medium consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) sourced from gamma-irradiated US. The cells are anchorage dependent and cell dissociation will be achieved using TrypLE Select, an animal product-free dissociation reagent. Cell seeding will be achieved using sterile, disposable bioprocess bags and tubing sets. The cells will be maintained in an atmosphere of 5% (±0.5%) CO2 at 37°C (±2°C). Cell Culture The nutrient medium will be replaced with fresh serum-free DMEM medium and transfected with the three production plasmids using an optimized PEI-based transduction method. All plasmids used in the production process will be produced in the context of a CMO quality system and infrastructure that utilizes the most prominent features of cGMP manufacturing: traceability, document control, and material segregation.

[0312] Sufficient DNA plasmid transfection complexes will be prepared in the BSC to transfect up to 50 HS-36 (per BDS batch). First, a DNA / PEI mix will be prepared containing 7.5 mg of pAAV.CB7.CI.hIDUAco.RBG.KanR vector genome plasmid, 150 mg of pAdDeltaF6 (Kan), 75 mg of pAAV29KanRRep2 AAV helper plasmid, and GMP grade PEI (PEIPro, PolyPlus Transfection SA). This plasmid ratio was determined to be optimal for AAV production in small-scale optimization studies. After thorough mixing, the solution is allowed to sit at room temperature for 25 minutes, and then serum-free medium is added to stop the reaction, and then added to the HS-36. The transfection mix is ​​balanced between all 36 layers of the HS-36, and the cells are incubated at 37°C (±2°C) in an atmosphere of 5% (±0.5%) CO2. .

[0313] Harvesting cell media: Transfected cells and media will be harvested from each HS-36 by aseptically drawing the media out of the unit using a disposable bioprocess bag. Following media harvest, an approximately 80 liter volume will be supplemented with MgCl2 (a cofactor for benzonase) to a final concentration of 2 mM and Benzonase Nuclease (Cat. No: 1.016797.0001, Merck Group) will be added to a final concentration of 25 units / ml. The product (in the disposable bioprocess bag) will be incubated for 2 hours at 37°C in an incubator to provide sufficient time for enzymatic digestion of residual cellular and plasmid DNA present in the harvest as a result of the transfection procedure. This step is performed to minimize the amount of residual DNA in the final vector. After the incubation period, NaCl will be added to a final concentration of 500 mM to aid in product recovery during filtration and downstream tangential flow filtration (see steps 4 and 5 below).

[0314] Clarification: Cells and cell debris will be removed from the product using depth filter capsules (1.2 μm / 0.22 μm) connected in series as a closed tubing and bag set driven by a peristaltic pump. Clarification ensures that downstream filters and chromatography columns are protected from contamination, while bioburden reduction filtration, at the end of the filter circuit, ensures that any bioburden that may have been introduced during the upstream production process is removed prior to downstream purification. The recovered material will pass through a Sartorius Sartoguard PES capsule filter (1.2 / 0.22 μm) (Sartorius Stedim Biotech Inc.).

[0315] Large-scale tangential flow filtration: Volume reduction (10-fold) of the clarified product may be achieved by tangential flow filtration (TFF) using custom-made sterile closed bioprocess tubing, bags, and membranes. The principle of TFF is to flow a solution under parallel pressure through a membrane of suitable porosity (100 kDa). The pressure difference carries smaller molecules through the membrane and into a waste stream, while retaining molecules larger than the membrane pores. By recirculating the solution, the parallel flow sweeps the membrane surface, preventing contamination of the membrane pores. By selecting the appropriate membrane pore size and surface area, the liquid sample may be rapidly reduced in volume while retaining and concentrating the desired molecules. Diafiltration in TFF applications involves the addition of fresh buffer to the recirculating sample and to the waste stream at the same rate that the liquid passes through the membrane. With increasing diafiltration volume, an increasing amount of small molecules are removed from the recirculating sample. This results in modest purification of the clarified product, but also achieves a buffer exchange that is compatible with the subsequent affinity chromatography step. Therefore, we utilize a 100 kDa PES membrane for concentration that is then diafiltered with 4 volumes of a buffer composed of 20 mM Tris pH 7.5 and 400 mM NaCl. The diafiltered product will be stored overnight at 4°C and then further clarified by a 1.2 μm / 0.22 um depth filter capsule to remove precipitated material.

[0316] Affinity chromatography: The diafiltered product efficiently isolated AAV2 / 9 serotypes. The resulting solution will be applied to Capture Select™ Poros-AAV2 / 9 affinity resin (Life Technologies), which selectively captures AAV particles. Under these ionic conditions, AAV particles are efficiently captured while a significant proportion of residual cellular DNA and proteins flow through the column. Application is followed by washing the column to remove additional feed impurities, followed by a low pH step elution (400 mM NaCl, 20 mM sodium citrate, pH 2.5) that is immediately neutralized by recovery into one-tenth volume of neutralization buffer (Bis Tris Propane, 200 mM, pH 10.2).

[0317] Anion exchange chromatography: To achieve further reduction of in-process impurities including empty AAV particles, the Poros-AAV2 / 9 elution pool is diluted 50-fold (20 mM Bis-Tris propane, 0.001% Pluronic F68; pH 10.2) to reduce ionic strength and allow binding to a CIMultus Q monolith matrix (BIA Separations). Following a low-salt wash, the vector product is eluted using a 60 CV NaCl linear salt gradient (10-180 mM NaCl). This shallow salt gradient efficiently separates vector genome-free capsid particles (hollow particles) from particles containing vector genomes (full particles), resulting in a preparation enriched for full capsids. Fractions are collected in tubes containing 1 / 100th volume of 0.1% Pluronic F68 and 1 / 27th volume of Bis-Tris pH 6.3 to minimize non-specific binding to the tube and length of exposure to high pH, ​​respectively. Appropriate peak fractions will be collected and the peak areas assessed to determine approximate vector yields and compared with previous data.

[0318] Final formulation and sterile filtration to obtain BDS: Final formulation will be achieved on the pooled AEX fractions using TFF through a 100 kDa membrane. This will be achieved by diafiltration with 4 volumes of formulation buffer (Elliot's B solution, 0.001% Pluronic F68), thereby obtaining a ≥ 5 x 10 13 Peak areas derived from anion exchange chromatography will be compared to previous data to estimate concentration factors to achieve a titer in GC / ml. Samples will be removed for BDS testing (described in the following section). Filtered purified bulk will be stored in sterile polypropylene tubes and frozen at ≦-60°C in quarantine until release for final filling. Preliminary stability studies indicate that DP does not lose activity following freezing and thawing in our proposed formulation buffer. Additional studies are underway to evaluate stability following long-term storage at -80°C.

[0319] Final Fill: Frozen BDS will be thawed, pooled, diluted to target titer with final formulation buffer, and filled through a 0.22 um filter (Millipore, Billerica, MA) into West Pharmaceutical "ready to use" (pre-sterilized) 2 ml glass vials with 13 mm stoppers and seals with a fill volume of ≥ 0.6 ml to ≤ 2.0 ml per vial. Individually labeled vials will be labeled according to the following specifications. Labeled vials will be stored at ≤ -60°C.

[0320] The vector (drug product) will be packaged in a vial at a single fixed concentration, with the only variable being the volume per vial. To achieve low dose concentrations, the drug product will be diluted with Elliot's B solution, 0.001% Pluronic F68. High dose vectors will be used directly without dilution, while low dose vectors will require a 1:5 dilution with formulation buffer, which will be performed by the pharmacy at the time of administration.

[0321] Example 9: Vector testing Characterization assays are performed, including serotype identity, hollow particle content, and transgene product identity. Descriptions of the assays appear below.

[0322] A. Uniqueness of the Vector Genome: DNA Sequencing Genomic DNA of the viral vector will be isolated and sequenced by 2-fold sequence coverage using primer walking. Sequence comparison will be performed and compared to the expected sequence.

[0323] B. Vector Capsid Identity: Mass Spectroscopic Analysis of VP3 AAV Capsids Confirmation of the AAV2 / 9 serotype of the vector is achieved by an assay based on the analysis of peptides of the VP3 capsid protein by mass spectrometry (MS). The method involves multiple enzymatic digestion (trypsin, chymotrypsin, and endoproteinase Glu-C) of the VP3 protein band excised from an SDS-PAGE gel, followed by characterization by UPLC-MS / MS on a Q-Exactive Orbitrap mass spectrometer to sequence the capsid protein. A tandem mass spectrometry (MS) method was developed that allows subtraction of host protein products and deduced sequence of the capsid protein from the mass spectrum.

[0324] C. Genome copy (GC) titer Genome copy titers based on oqPCR will be determined over a range of serial dilutions and compared to a cognate plasmid standard (pAAV.CB7.CI.hIDUAco.RBG.KanR). oqPCR utilizes sequential digestion with DNase I and Proteinase K followed by qPCR analysis to measure encapsidated vector genome copies. Detection of DNA will be achieved using sequence-specific primers targeting the RBG polyA region in combination with a fluorescently tagged probe that hybridizes to this same region. Comparison to a standard curve of plasmid DNA allows titer determination without the need for post-PCR sample manipulation. A number of standards, validation samples and controls (for background and DNA contamination) are being introduced into the assay. The assay is not currently limited, but will be limited by the CMO. The assay will be limited by establishing and defining the parameters of the assay, including sensitivity, limit of detection, range of limits, and intra- and inter-assay precision. An internal AAV9 reference lot will be established and used for qualification testing. Of note, our previous experience suggests that titers obtained by the optimized qPCR described here are generally 2.5-fold higher than those achieved by our standard qPCR used in the generation of preclinical data.

[0325] D. Ratio of hollow particles to whole particles The total particle content of the drug product will be determined by SDS-PAGE analysis. A preparation of reference vector purified on an iodixanol gradient will be analyzed by various methods (analytical ultracentrifugation, electron microscopy, and absorbance at 260 / 280 nm) to verify that the preparation contains >95% genome-containing (intact) particles. This reference material will be serially diluted to known genome copy numbers (and therefore by extension particle numbers) and run on an SDS-PAGE gel along with a similar dilution series of the drug product. The volumes of the peak areas of both the reference material and the drug product VP3 protein bands will be measured by densitometry, and the volume of the reference material will be plotted against particle number. The total particle concentration of the drug substance will be determined by extrapolation from this curve, and the genome copy (GC) titer will then be subtracted to obtain the titer of the hollow particles. The ratio of hollow particles to intact particles is the ratio of the GC titer to the hollow particle titer.

[0326] E. Infectious titer Infectious titers (IU) are used to determine productive uptake and replication of vector in RC32 cells (HeLa cells expressing rep2). A 96-well end-point format similar to that previously published is employed. Briefly, RC32 cells are simultaneously infected with serial dilutions of rAAV9.CB.hIDUA and a single dilution of Ad5 with 12 copies of rAAV at each dilution. 72 hours post-infection, cells are lysed and qPCR is performed to determine the input of rAAV vector. The amplification is detected relative to the total volume of the virus. End-point dilutions (Spearman-Karber) for TCID50 calculations are performed to determine the replication titer expressed as IU / ml. Since "infectivity" values ​​depend on the particles contacting the cells, receptor binding, internalization, transport to the nucleus, and genome replication, they are influenced by the assay structure and the presence of appropriate receptors and post-binding pathways in the cell line used. Since receptors and post-binding pathways are not usually maintained in immortalized cell lines, the titer of the infectivity assay is not an absolute measure of the number of "infectious" particles present. However, the ratio of encapsidated GC to "infectious units" (described as the GC / IU ratio) can be used as a measure of product consistency from lot to lot. The GC / IU ratio is a measure of product consistency. The calculated GC / IU ratio is obtained by dividing the oqPCR titer (GC / ml) by the "infectious units" (IU / ml).

[0327] F. Assay for replication-competent AAV (rcAAV) Samples will be analyzed for the presence of replication-competent AAV2 / 9 (rcAAV) that may have arisen during production. A three-passage assay has been developed consisting of cell-based amplification and passaging followed by detection of rcAAV DNA (cap9 target) by real-time qPCR. The cell-based component consists of inoculated monolayers (P1) of HEK293 cells with dilutions of the test sample and wild-type human adenovirus type 5 (Ad5). 10 10The GC vector product will be the largest amount of product examined. Due to the presence of adenovirus, replication-competent AAV will replicate in cell culture. After 2 days, cell lysates are generated and Ad5 is heat inactivated. The clarified lysates are then placed in second round cells (P2) to increase sensitivity (again in the presence of Ad5). After 2 days, cell lysates are generated and Ad5 is heat inactivated. The clarified lysates are then placed in third round cells (P3) to maximize sensitivity (again in the presence of Ad5). After 2 days, cells are lysed, DNA is released and then subjected to qPCR to detect AAV9 cap sequences. Amplification of the AAV9 cap sequence in an Ad5-dependent manner indicates the presence of rcAAV. The use of an AAV2 / 9 surrogate positive control containing the AAV2 rep gene and the AAV9 cap gene allowed the limit of detection (LOD) of the assay to be determined (0.1, 1, 10, and 100 IU) and serial dilutions (1 × 10 10 , 1×10 9 , 1×10 8 , 1×10 7 GC) to determine the presence of The approximate levels of rcAAV expressed can be quantified.

[0328] G. In Vitro Efficacy To relate qPCR GC titers to gene expression, an in vitro bioassay will be performed by transducing Huh7 or HEK293 cells with GC per cell of known infection efficiency and assaying the supernatant for IDUA activity 72 hours after transduction. IDUA activity will be measured by incubating samples diluted in 0.1 ml water with 0.1 ml of 100 mmol / L 4MU-iduronide for 1-3 hours at 37 degrees. The reaction will be stopped by the addition of 2 ml of 290 mmol / L glycine, 180 mmol / L sodium citrate pH 10.9, and liberated 4MU will be quantified by comparing the fluorescence against a standard dilution of 4MU. Highly active preclinical and tox vector preparations will allow interpretation of product activity.

[0329] H. Determination of total protein, capsid protein, protein purity and capsid protein ratio Vector samples are first quantified for total protein against a standard curve of bovine serum albumin (BSA) using the bicinchoninic acid assay. Measurements are performed by mixing an equal volume of sample with the Micro-BCA reagent provided in the kit. The same procedure is applied for dilutions of the BSA standard. The mixture is incubated at 60°C and the absorbance is measured at 562 nm. A four-parameter fit is used to generate a standard curve from the absorbance of standards of known concentrations. Unknown samples are quantified according to the four-parameter regression.

[0330] To provide a semi-quantitative determination of AAV purity, samples were then normalized for genomic titer and run on SDS-polyacrylamide (SDS-PAGE) gels under reducing conditions to yield 5 × 10 9 GC is separated. The gel is then stained with SYPRO Ruby dye. Impurity bands are quantified by densitometry by comparison with simultaneously electrophoresed BSA standards of 25, 50, and 100 ng protein. These amounts represent 1%, 2%, and 4% of the total AAV protein sample. Stained bands appearing upon addition of the three AAV-specific proteins VP1, VP2, and VP3 are considered protein impurities. All impurity bands are compared to reference proteins and approximate molecular weights are reported as well as the mass percentage of the impurities. SDS-PAGE gels will be used to quantitate VP1, VP2, and VP3 proteins and their ratios will also be determined.

[0331] Example 10: Biodistribution and Brain Enzymes 1 × 10 in adult cynomolgus monkeys 12 GC / kg AAV9.CMV.hIDUA was injected suboccipitally and 636 days later tissues were harvested and immediately frozen at -80°C. QIAamp DNA Total cellular DNA is extracted from tissues using the Qiagen Mini Kit (Qiagen, Valencia, CA, USA). Detection and quantification of vector genomes in extracted DNA is performed by real-time PCR (TaqMan Universal Master Mix, Applied Biosystems, Foster City, CA, USA) using a set of primers and probes targeting sequences within the SV40 polyA. PCR conditions are set with 100 ng of total cellular DNA as template, 300 nM primers, and 200 nM probe, respectively. The cycles were 95.8°C for 10 min, 40 cycles of 95.8°C for 15 s and 60.8°C for 1 min.

[0332] 3 × 10 8 , 3×10 9 , or 3 × 10 10 GC / mice are injected with AAV9.CB7.hIDUA in the right lateral ventricle. After 21 days, whole brains are harvested and immediately frozen at -80°C. Tissue samples are homogenized in lysis buffer (0.2% Triton-X100, 0.9% NaCl, pH 4.0) and sonicated briefly. Samples are then freeze-thawed and clarified by centrifugation. Protein concentration is determined by BCA assay. IDUA activity is measured by incubating 0.1 mL of water-diluted samples with 0.1 mL of 100 mmol / L 4MU-iduronide (Toronto Research Chemicals, Toronto, Canada; Glycosynth, Warrington, England) in IDUA buffer (0.15 mol / L NaCl, 0.05% Triton-X100, 0.1 mol / L sodium acetate, pH 3.58) for 1-3 h at 37°C. The reaction is stopped by the addition of 2 ml of 290 mmol / L glycine, 180 mmol / L sodium citrate pH 10.9. The released 4MU is quantified by comparing the fluorescence to a standard dilution of 4MU. Units are given as nmoles of 4MU released per mg of protein per hour.

[0333] Example 11: Biomarkers of MPS I In this study, metabolite profiling of CSF samples from dogs with MPS I was performed, which revealed substantial disease-related changes in the CSF metabolome. The most striking difference was a 30-fold increase in spermine levels compared to normal controls. This finding was confirmed in a feline model of MPS I as well as in samples from MPS I patients. Spermine binds to HS and the uptake of spermine into cells depends on this interaction [M. Belting, S. Persson, L.-A. Fransson, Proteoglycan involvement in polyamine uptake. Biochemical Journal, 338, 317-323 (1999); JE Welch, P. Bengtson, K. Svensson, A. Wittrup, GJ Je nniskens, GBTen Dam, THVan Kuppevelt, M. Belting, Single chain fragment anti-heparan sulfate antibody targets the polyamine transport system and attenuates polyamine-dependent cell proliferation. International journal of oncology, 32, 749-756 (2008); published online EpubApr]. Cell surface proteoglycans such as glypican-1 can bind spermine via their HS moieties, and after endocytosis of glypican proteins, intracellular cleavage of the HS chains releases the bound spermine intracellularly (K. Ding, S. et al, The Journal of biological chemistry, 276, 46779-46791, (2001); published online EpubDec, 14). Thus, recycling of intact HS is essential for spermine uptake. In MPS I, accumulation of extracellular spermine may occur either through inhibition of this uptake mechanism due to insufficient HS recycling or through simple binding of spermine to extracellular GAGs that accumulate in MPS, shifting the equilibrium of spermine binding to more favor extracellular distribution. Further studies should address the relative contributions of these mechanisms to spermine accumulation in the CSF of MPS I.

[0334] We found that inhibitors of spermine synthesis blocked excessive neurite outgrowth in MPS neurons, and that neurite outgrowth could be induced in WT neurons with spermine concentrations similar to those found in the CSF of patients. Gene therapy in the MPS I canine model reversed spermine accumulation and normalized GAP43 expression, suggesting that the same pathway is affected in vivo. We could not directly assess the effect of spermine synthesis inhibition in vivo because the available inhibitors do not cross the blood-brain barrier, and chronic direct CNS administration from birth is not feasible in our animal model. While our in vitro findings support a role for spermine in abnormal neurite outgrowth in MPS I, it is important to mention that inhibiting spermine synthesis did not completely reverse the phenotype, and addition of spermine to normal neurons did not increase neurite outgrowth to the level of MPS I neurons. The effect of spermine modulation may have been limited by the relatively short duration of treatment. It is also possible that spermine accumulation is not the only mediator contributing to neurite outgrowth in MPS I. In particular, many neurotrophic factors bind via HS-modified receptors, and interactions with HS in the extracellular matrix can affect neurite outgrowth [D. Van Vactor, et al, Heparan sulfate proteoglycans and the emergence of neuronal connectivity. Current opinion in neurobiology, 16, 40-51 (2006); published online Epu...

Claims

1. A combination preparation for a therapeutic regimen useful for treating α-L-iduronidase deficiency in a human patient, the preparation comprising: (a) a recombinant AAV (rAAV) comprising an AAV9 capsid and a nucleic acid comprising an expression cassette comprising a sequence encoding human alpha-L-iduronidase (hIDUA) under the control of regulatory sequences directing expression of said hIDUA in said patient, said human hIDUA coding sequence having the nucleotide sequence of nucleotides 1-1962 of SEQ ID NO:1 or having a sequence at least 95%-99% identical to nucleotides 1-1962 of SEQ ID NO:1 encoding a hIDUA having the amino acid sequence of SEQ ID NO:2, said expression cassette further comprising a chicken β-actin promoter, a cytomegalovirus immediate early (CMV IE enhancer), a chicken β-actin intron, and a polyadenylation (poly A) signal sequence; (b) at least one first immunosuppressant agent and at least one second immunosuppressant agent, (i) the first immunosuppressant comprises an intravenous corticosteroid; (ii) the at least one first immunosuppressant agent and the at least one second immunosuppressant agent, wherein the second immunosuppressant agent comprises an oral corticosteroid; Including, The combination formulation, wherein administration of at least one of the immunosuppressants is initiated prior to or on the same day as delivery of the rAAV, and at least one of the immunosuppressants is administered continuously for at least 8 weeks after administration of the rAAV.

2. The rAAV is formulated for intrathecal administration to a human subject, (i) about 1.2×10 12 to about 6.0×10 12 genome copies (GC) or about 6.0×10 12 to about 3.0×10 13 GC for human subjects ≧4 months to <9 months of age; (ii) about 2×10 12 to about 6.0×10 13 or about 1.0×10 13 to about 5.0×10 13 GC for human subjects ≧9 months to <18 months of age; or (iii) about 2.2×10 12 to about 1.1×10 13 GC or about 1.1×10 13 to about 5.5×10 13 GC for human subjects ≧9 months to <18 months of age. The formulation of claim 1, wherein a total flat dose of

3. A composition comprising a recombinant AAV (rAAV) for use in a method for treating alpha-L-iduronidase deficiency in a human patient, the method comprising administering the composition to the patient, the rAAV comprising an expression cassette comprising a heterologous nucleic acid encoding human alpha-L-iduronidase (hIDUA), the human hIDUA coding sequence having the nucleotide sequence of nucleotides 1-1962 of SEQ ID NO:1 or having a sequence at least 95% to 99% identical to nucleotides 1-1962 of SEQ ID NO:1 encoding a hIDUA having the amino acid sequence of SEQ ID NO:2, the expression cassette further comprising a chicken beta-actin promoter, a cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta-actin intron, and a polyadenylation (polyA) signal sequence, the rAAV vector being formulated for intrathecal injection into a human subject in need thereof, (a) from about 1.2×10 12 to about 6.0×10 12 GC or from about 6.0×10 12 to about 3.0×10 13 GC for human subjects ≧4 months to <9 months of age; (b) from about 2×10 12 to about 6.0×10 13 GC or from about 1.0×10 13 to about 5.0×10 13 GC for human subjects ≧9 months to <18 months of age; or (c) about 2.2×10 12 to about 1.1×10 13 GC or about 1.1×10 13 to about 5.5×10 13 GC for human subjects ≧9 months to <18 months of age. is administered, the method further comprising administering to the patient at least one first immunosuppressant agent and at least one second immunosuppressant agent; (i) the first immunosuppressant comprises an intravenous corticosteroid; (ii) the second immunosuppressant comprises an intravenous corticosteroid; The composition, wherein administration of at least one of the immunosuppressants is initiated prior to or on the same day as administration of the rAAV, and administration of at least one of the immunosuppressants is continued for at least 8 weeks after administration of the rAAV.

4. An immunosuppressant for use in combination therapy with a recombinant AAV (rAAV) comprising an expression cassette comprising a heterologous nucleic acid encoding human alpha-L-iduronidase (hIDUA), wherein the human hIDUA coding sequence has the nucleotide sequence of nucleotides 1-1962 of SEQ ID NO:1 or has a sequence at least 95% to 99% identical to nucleotides 1-1962 of SEQ ID NO:1 encoding a hIDUA having the amino acid sequence of SEQ ID NO:2, the expression cassette further comprising a chicken beta actin promoter, a cytomegalovirus immediate early (CMV IE) enhancer, a chicken beta actin intron, and a polyadenylation (polyA) signal sequence, and the immunosuppressant comprises at least one first immunosuppressant and at least one second immunosuppressant, (a) the first immunosuppressant comprises an intravenous corticosteroid; (b) the second immunosuppressant comprises an oral corticosteroid; The administration of the immunosuppressants is initiated prior to or on the same day as administration of the rAAV, and administration of at least one of the immunosuppressants is continued for at least 8 weeks after administration of the rAAV.

5. The AAV vector is formulated for intrathecal injection into a human subject in need thereof, (i) from about 1.2×10 12 to about 6.0×10 12 GC or from about 6.0×10 12 to about 3.0×10 13 GC for human subjects ≧4 months to <9 months of age; (ii) from about 2×10 12 to about 6.0×10 13 GC or from about 1.0×10 13 to about 5.0×10 13 GC for human subjects ≧9 months to <18 months of age; or (iii) about 2.2×10 12 to about 1.1×10 13 GC or about 1.1×10 13 to about 5.5×10 13 GC for human subjects ≧9 months to <18 months of age. The immunosuppressant for use according to claim 4, wherein a total flat dose of 6. The formulation of claim 1, the composition for use of claim 3, or the immunosuppressant for use of claim 4 or 5, wherein the rAAV comprises a vector genome comprising an AAV 5' ITR, an expression cassette, and a nucleic acid molecule comprising an AAV 3' ITR.

7. A formulation described in any one of claims 1 to 2 or 6, a composition for use described in claim 3 or claim 6, or an immunosuppressant for use described in any one of claims 4 to 6, wherein the expression cassette comprises a nucleic acid sequence having the nucleic acid sequence of nucleotides 1 to 1962 of SEQ ID NO:

1.

8. A formulation described in any one of claims 1 to 2 or 6 to 7, a composition for use described in any one of claims 3 or 6 to 7, or an immunosuppressant for use described in any one of claims 4 to 7, wherein the expression cassette comprises a nucleic acid sequence of nucleotides 198 to 4126 of SEQ ID NO:

3.

9. The formulation of claim 6, the composition for use of claim 6, or the immunosuppressant for use of claim 6, wherein the rAAV comprises a vector genome comprising the nucleic acid sequence of nucleotides 1 to 4344 of SEQ ID NO:

3.

10. The rAAV is formulated for intrathecal administration to a human in need thereof, and 9 ~10 11 The formulation according to any one of claims 1 to 2 or 6 to 9, the composition for use according to any one of claims 3 or 6 to 9, or the immunosuppressant for use according to claim 4 or 9, administered in a dose of GC / g brain mass.

11. The formulation of any one of claims 1-2 or 6-10, the composition for use of any one of claims 3 or 6-10, or the immunosuppressant for use of any one of claims 4-10, wherein the intravenous corticosteroid is administrable to the patient prior to rAAV administration and the oral corticosteroid is administrable to the patient after rAAV administration.

12. The formulation of any one of claims 1-2 or 6-11, the composition for use of any one of claims 3 or 6-11, or the immunosuppressant for use of any one of claims 4-11, wherein the intravenous corticosteroid is methylprednisolone and can be administered once at 10 mg / kg one day prior to rAAV administration.

13. The formulation of any one of claims 1-2 or 6-12, the composition for use of any one of claims 3 or 6-12, or the immunosuppressant for use of any one of claims 4-12, wherein the oral corticosteroid is prednisone and can be administered at 0.5 mg / kg / day starting on day 2 after rAAV administration.

14. A formulation according to any one of claims 1 to 2 or 6 to 13, a composition for use according to any one of claims 3 or 6 to 13, or an immunosuppressant for use according to any one of claims 4 to 13, wherein the immunosuppressant further comprises one or more macrolides.

15. The formulation of claim 14, the composition for use of claim 14, or the immunosuppressant for use of claim 14, wherein the one or more macrolides comprise tacrolimus, sirolimus, or a combination of tacrolimus and sirolimus.

16. A formulation according to any one of claims 1 to 2, 6 or 13, or a composition for use according to any one of claims 3 or 6 to 13, or an immunosuppressant for use according to any one of claims 6 to 13, wherein administration of corticosteroids is discontinued 12 weeks after rAAV administration.

17. A formulation according to any one of claims 14 to 15, or a composition for use according to any one of claims 14 to 15, or an immunosuppressant for use according to any one of claims 14 to 15, wherein tacrolimus is administrable on the second day after rAAV administration.

18. A formulation according to any one of claims 14 to 15, or a composition for use according to any one of claims 14 to 15, or an immunosuppressant for use according to any one of claims 14 to 15, wherein sirolimus can be administered on day -2 prior to rAAV administration.

19. A formulation according to any one of claims 14 to 15 or 17, or a composition for use according to any one of claims 14 to 15 or 17, or an immunosuppressant for use according to any one of claims 14 to 15 or 17, wherein administration of tacrolimus is discontinued 32 weeks after rAAV administration.

20. A formulation according to any one of claims 14-15 or 18, or a composition for use according to any one of claims 14-15 or 18, or an immunosuppressant for use according to any one of claims 14-15 or 18, wherein administration of sirolimus is discontinued 48 weeks after rAAV administration.

21. A formulation according to any one of claims 14-15 or 17-20, or a composition for use according to any one of claims 14-15 or 17-20, or an immunosuppressant for use according to any one of claims 14-15 or 17-20, wherein when the immunosuppressant comprises both tacrolimus and sirolimus, low doses of tacrolimus and sirolimus are used to maintain blood trough levels of (i) for tacrolimus, 2 ng / mL to 4 ng / mL, or about 4 ng / mL to about 8 ng / ml, or about 8 ng / mL to about 16 ng / mL in total, and (ii) for sirolimus, 1 ng / mL to 3 ng / mL.

22. A formulation according to any one of claims 14-15 or 17-21, or a composition for use according to any one of claims 14-15 or 17-21, or an immunosuppressant for use according to any one of claims 14-15 or 17-21, wherein the immunosuppressant comprises only one of tacrolimus or sirolimus, and the total dose is within the range of about 16 ng / mL to about 24 ng / mL.

23. The formulation of any one of claims 14-15 or 17-21, or the composition for use of any one of claims 14-15 or 17-21, or the immunosuppressant for use of any one of claims 14-15 or 17-21, wherein when the combination treatment regimen includes both tacrolimus and sirolimus, the initial dose of tacrolimus is 0.05 mg / kg twice daily.

24. A formulation described in any one of claims 14 to 15 or 17 to 21, or a composition for use described in any one of claims 14 to 15 or 17 to 21, or an immunosuppressant for use described in any one of claims 14 to 15 or 17 to 21, wherein only tacrolimus or sirolimus is used and the initial loading dose is about 1 mg / m2.

25. The formulation of any one of claims 1 to 2 or 6 to 24, the composition for use of any one of claims 3 or 6 to 24, or the immunosuppressant for use of any one of claims 4 to 24, wherein the immunosuppressive therapy is initiated about -14 days to -1 days prior to administration of rAAV.

26. The formulation of any one of claims 1-2 or 6-25, the composition for use of any one of claims 3 or 6-25, or the immunosuppressant for use of any one of claims 4-25, wherein the rAAV is in a suspension having a pH of 6 to 9, and optionally the rAAV is administered via intrathecal injection.

27. A formulation according to any one of claims 1 to 2 or 6 to 26, a composition for use according to any one of claims 3 or 6 to 26, or an immunosuppressant for use according to any one of claims 4 to 26, wherein the rAAV is for administration by intraventricular or intracapsular delivery.

28. The formulation described in claim 27 (when the rAAV is delivered via intrathecal injection), a composition for use described in any one of claims 3 or 6 to 27, or an immunosuppressant for use described in any one of claims 4 to 27, further comprising intravenous co-administration of a rAAV containing a hIDUA gene.

29. A formulation according to any one of claims 1 to 2 or 6 to 28, a composition for use according to any one of claims 3 or 6 to 28, or an immunosuppressant for use according to any one of claims 4 to 28, wherein the effectiveness of the treatment is assessed by measuring changes in hearing ability, optionally by auditory brainstem testing.

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