Gene therapy for treating mucopolysaccharidosis type ii

Abstract

To provide treatments for Hunter syndrome and the symptoms associated with Hunter syndrome.SOLUTION: A suspension useful for AAV9-mediated intrathecal and / or systemic delivery of an expression cassette containing a hlDS gene is provided herein. Also provided are methods and kits containing these vectors and compositions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Statement Regarding Federally Sponsored Research This invention was made with government support under grant numbers R01DK54481, P40OD010939, and P30ES013508 from the National Institutes of Health. The federal government may have certain rights in this invention.

[0002] Incorporation by reference of electronic material Applicants incorporate by reference the sequence listing submitted electronically herewith under File No. "UPN-16-7771PCT_ST.25."

[0003] 1. Introduction The present invention relates to a gene therapy approach for treating mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome. [Background technology]

[0004] 2. Background of the invention MPS II, also known as Hunter syndrome, is a rare X-linked recessive genetic disorder affecting primarily males, affecting 1 in 100,000 to 1 in 170,000 individuals. This progressive and devastating disease is caused by mutations in the IDS gene, leading to a deficiency of the lysosomal enzyme iduronate-2-sulfatase, an enzyme required for lysosomal catabolism of heparan sulfate and dermatan sulfate. These ubiquitous polysaccharides, called GAGs (glycosaminoglycans), accumulate in the tissues and organs of MPS II patients, resulting in characteristic storage lesions and diverse disease sequelae. Morbidity and mortality are high in this patient population—patients with a severe phenotype (characterized by neurocognitive decline) have reported deaths at a mean age of 11.7 years; patients with a mild or attenuated phenotype have reported deaths at a mean age of 21.7 years.

[0005] Although patients with MPS II appear normal at birth, signs and symptoms of the disease typically present in a severe form between 18 months and 4 years of age and in a weakened form between 4 and 8 years of age. Signs and symptoms common to all affected patients include short stature, coarse facies, macrocephaly, macroglossia, hearing loss, hepatomegaly and splenomegaly, dysostosis multiplex, joint contractures, spinal stenosis, and carpal tunnel syndrome. Frequent upper respiratory tract and ear infections occur in most patients, and progressive airway obstruction is common, leading to sleep apnea and often death. Heart disease is the leading cause of death in this population and is characterized by valvular dysfunction resulting in left and right ventricular hypertrophy and heart failure. Death is generally due to obstructive airway disease or heart failure.

[0006] In severe forms of the disease, developmental delays are readily apparent by 18 to 24 months, although early developmental milestones may be achieved. Some patients do not meet screening tests in the first year, and other milestones, including the ability to sit unsupported, walk, and speak, are delayed. Developmental progress begins to plateau for approximately 6.5 years. Half of children with MPS II are toilet trained, but most, if not all, children lose this ability as the disease progresses.

[0007] Patients with significant neurological involvement exhibit severe behavioral disturbances, including hyperactivity, stubbornness, and aggression, beginning in the second year of life and continuing until age 8–9 years, when neurodegeneration diminishes this behavior.

[0008] Epileptic seizures are reported in more than half of severely affected patients who reach the age of 10 years, and by the time of death, most patients with CNS involvement have severe mental disabilities and require constant care. Although patients with weakened disease exhibit normal intellectual function, MRI images reveal global brain abnormalities in all patients with MPS II, including white matter lesions, enlarged ventricles, and brain atrophy.

[0009] Enzyme replacement therapy ("ERT") using recombinant idursulfase (Elaprase®, Shire Human Genetic Therapies) is the only approved treatment for Hunter syndrome and is administered as a weekly infusion. However, currently administered ERT does not cross the blood-brain barrier ("BBB") and therefore does not address the unmet needs of patients with severe disease, i.e., MPS II with CNS / neurocognitive and behavioral involvement. Current efforts to address this problem aim to modify the enzyme so that it can cross the BBB. Summary of the Invention [Means for solving the problem]

[0010] 3. Summary of the Invention Provided herein is the use of a replication-deficient adeno-associated virus ("AAV") to deliver the human iduronate-2-sulfatase ("hIDS") gene to the CNS of a patient (human subject) diagnosed with MPS II, also known as Hunter syndrome. The recombinant AAV ("rAAV") vector ("rAAV.hIDS") used to deliver the hIDS gene should have tropism for the CNS (e.g., an rAAV carrying an AAV9 capsid), and the hIDS transgene should be controlled by specific expression control elements, such as a hybrid of the cytomegalovirus (CMV) enhancer and the chicken beta-actin promoter (CB7). A pharmaceutical composition suitable for intrathecal / intracisternal administration comprises a suspension of the rAAV.hIDS vector in a formulation buffer containing a physiologically compatible aqueous buffer, surfactant, and optional excipients. The rAAV suspension is further characterized by: (i) an rAAV genome copy (GC) titer of at least 1.0 × 10 13 GC / ml (+ / -20%); (ii) the rAAV has an empty particle / total particle ratio of 0.01 to 0.05 (95% to 99% free of empty capsids) as determined by SDS-PAGE analysis (see Example 5D), or in other embodiments, 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 2.5 × 10 10 GC / g brain mass ~ approx. 3.6 x 10 11 The dose of rAAV suspension in GC / g brain mass has a titer.

[0011] Also provided herein are pharmaceutical compositions provided herein that can be administered by intrathecal injection to a human subject in need thereof. In certain embodiments, a pharmaceutical composition comprising the rAAV.hIDS described herein is used to prepare a medicament that can be administered by intrathecal injection to a human subject in need thereof. The human subject (patient) may have previously been diagnosed with mucopolysaccharidosis II (MPS II) or severe Hunter syndrome.

[0012] Titers can be measured in an in vitro cell culture assay, such as the in vitro titer assay described in Example 5G herein, in which HEK293 or Huh7 cells are transduced with a known multiplicity of rAAVGC per cell and the supernatants are assayed for IDS activity 72 hours post-transduction using a 4MU-iduronide enzyme assay.

[0013] Such rAAV.hIDS vector preparations may be administered to pediatric or adult human subjects by intrathecal / intracisternal injection to achieve therapeutic levels of hIDS expression in the CNS. Candidates for treatment include pediatric and adult patients with severe or attenuated MPS II disease. Severe disease is defined as early neurocognitive deficits with a Developmental Quotient (DQ) (BSID-III) at least one standard deviation below the mean or documented historical evidence of a decline of more than one standard deviation on consecutive tests.

[0014] The therapeutically effective intrathecal / intracisternal dose of rAAV.hIDS in patients with Hunter syndrome is 10 / g of patient brain mass. 10 ~5×10 10 GC equals 1.4 x 10 13 ~7.0×10 13 GC (flat dose), or 10 per gram of patient brain mass 10 ~5×10 10 GC equals 3.8 x 10 12 ~7.0×10 13 Alternatively, the following therapeutically effective flat doses may be administered to patients in the indicated age groups: Newborn: Approx. 3.8 x 10 12 ~Approx. 1.9×10 14 GC; 3 to 9 months: Approx. 6 x 10 12 ~Approx. 3×10 14 GC; 9-36 months: Approx. 10 13 ~Approx. 5×10 14 GC; ·3~12 years: Approx. 1.2×10 13 ~about 6×10 14 GC; Ages 12+: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 GC; 18+ (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0015] In some embodiments, the dose administered to MPS II patients aged 12+ years (including 18+ years) is 1.4 x 10 13 genome copies (GC) (1.1 × 10 10 In some embodiments, the dose administered to MPS II patients aged 12+ (including those aged 18+) is 7×10 13 GC(5.6×10 10 In yet a further embodiment, the dose administered to an MPS II patient is at least about 4 x 10 8 GC / g brain mass ~ approx. 4×10 11GC / g brain mass. In certain embodiments, the dose administered to an MPS II newborn is about 1.4 x 10 11 ~Approx. 1.4×10 14 The dose administered to infants 3 to 9 months of age is approximately 2.4 x 10 11 ~Approx. 2.4×10 14 The dose administered to children 9 to 36 months of age with MPS II is approximately 4 × 10 11 ~Approx. 4×10 14 GC ranges from 3 to 12 years old; MPS The dose given to children is approximately 4.8 x 10 11 ~Approx. 4.8×10 14 The dose administered to children and adults aged 12+ is approximately 5.6 x 10 11 ~Approx. 5.6×10 14 This is the scope of GC.

[0016] The goal of this treatment is to functionally replace the patient's defective iduronate-2-sulfatase via rAAV-based CNS-directed gene therapy as a viable approach to treating the disease. The efficacy of the treatment can be measured by (a) preventing neurocognitive decline in MPS II (Hunter syndrome) patients; and (b) assessing disease biomarkers, such as GAG levels and / or enzyme activity (IDS or hexosaminidase) in CSF, serum, and / or urine, and / or liver and spleen volume. Infant neurocognition can be measured using the Bayley Scales of Infant and Toddler Development, 3rd Edition, BSID-III. Neurocognitive and adaptive behavioral assessments (e.g., using the Bayley Scale of Infant Development and Vineland Adaptive Behavior Scales, respectively) may also be performed.

[0017] Prior to treatment, MPS II patients may be evaluated for neutralizing antibodies (Nab) against the capsid of the rAAV vector used to deliver the hIDS gene. Such Nabs may interfere with transduction efficiency and reduce treatment titers. MPS II patients with baseline serum Nab titers ≤ 1:5 are at increased risk of treatment with the rAAV-hIDS gene therapy protocol. These patients are good candidates for treatment. Treatment of MPS II patients with serum Nab titers >1:5 may require combination therapy, such as transient cotreatment with immunosuppressants, before and / or during treatment with rAAV-hIDS vector delivery. If necessary, combination immunosuppressant therapy may be used as a preventative measure without prior assessment of neutralizing antibodies to the AAV vector capsid and / or other components of the formulation. In certain embodiments, prior immunosuppressive therapy may be desirable to prevent a potentially adverse immune response to the hIDS transgene product, which may be considered "foreign," particularly in patients with substantially no IDS activity. While reactions similar to those observed in animals may not occur in human subjects, preventative immunosuppressive therapy is recommended for all recipients of rAAV-hIDS.

[0018] The combination of gene therapy delivery of rAAV.hIDS to the CNS with systemic delivery of hIDS is encompassed by the methods of the invention. Systemic delivery may be achieved using ERT (e.g., using Elaprase® (idursulfase)) or additional gene therapy using a rAAV.hIDS with liver tropism (e.g., rAAV.hIDS with an AAV8 capsid).

[0019] In certain embodiments, patients are administered AAV.hIDS by liver-specific injection to tolerize the patient to hIDS, and the patient subsequently receives AAV.hIDS by intrathecal injection if the patient is an infant, child, and / or adult to express therapeutic concentrations of hIDS in the CNS.

[0020] Certain embodiments are based, in part, on (i) encouraging data generated in a mouse model of MPS II (described in Example 2, below), which shows that treatment with the rAAV.hIDS vectors of the invention normalized IDS expression; reduced disease biomarkers; and improved behavioral (CNS) symptoms; and (ii) safety and biodistribution in non-human primates (NHPs), as described in Example 3, below. Certain embodiments of the invention are also illustrated by examples describing the preparation and characterization of rAAV.hIDS pharmaceutical compositions (Examples 4 and 5, below).

[0021] As used herein, the terms "intrathecal delivery" or "intrathecal administration" refer to the route of administration of a drug by injection into the spinal canal, more specifically into the subarachnoid space, such that the drug reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular, suboccipital / intracisternal, and / or C1-2 puncture. For example, material may be introduced for diffusion throughout the subarachnoid space via lumbar puncture. In another example, injection may be intracisternal.

[0022] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to the route of administration of a drug directly into the cerebrospinal fluid of the cisterna magna, more specifically via suboccipital puncture, or by direct injection into the cisterna magna, or via a permanently placed tube.

[0023] As used herein, a "therapeutically effective amount" refers to an amount of an AAV.hIDS composition that delivers and expresses an enzyme in a target cell in an amount sufficient to ameliorate or treat one or more symptoms of MPS II. "Treatment" can include preventing the worsening of one symptom of MPS II syndrome, and possibly reversing one or more of its symptoms. For example, a therapeutically effective amount of rAAV.hIDS is an amount that improves neurocognitive function in patients with MPS II. Such improvement in neurocognitive function can be measured by assessing the subject's neurocognitive development quotient (DQ) using the Bayley Scales of Infant and Toddler Development. Improvement in neurocognitive function can also be measured, for example, but not limited to, the Wechsler Abbreviated Scale of Development. Intelligence quotient (IQ) of a subject can be measured using methods known in the art, including the use of the Weekly Assessment of Intelligence (WASI) (IQ), Bayley's Infantile Development Scale, the Hopkins Verbal Learning Test (memory), and / or the Tests of Variables of Attention (TOVA). In another embodiment, the therapeutically effective amount of rAAV.hIDS is an amount that reduces the concentration of pathogenic GAGs, heparan sulfate, and / or hexosaminidase in urine and / or cerebrospinal fluid, and / or serum and / or other tissues. In yet other embodiments, correction of corneal opacity can be observed, correction of lesions in the central nervous system (CNS) is observed, and / or reversal of perivascular and / or meningeal space accumulation is observed.

[0024] A "therapeutically effective amount" may be determined based on animal models rather than human patients. An example of a suitable mouse model is described herein.

[0025] As used herein, "functional human iduronate-2-sulfatase" refers to a human iduronate-2-sulfatase enzyme that normally functions in humans without MPS II or a related syndrome. Conversely, a human iduronate-2-sulfatase enzyme variant that causes MPS II or a related syndrome is considered non-functional. In one embodiment, a functional human iduronate-2-sulfatase is a human iduronate-2-sulfatase enzyme that is functional as described by Wilson et al. The amino acid sequence of wild-type human iduronate-2-sulfatase is described in [PubMed] al., Proc. Natl. Acad. Sci. USA 87(21):8531-8535 (1990), with NCBI reference sequence NP_000193.1 reproduced in SEQ ID NO:2 (550 amino acids); this preproprotein contains a signal peptide (amino acids 1-25), a propeptide (amino acids 26-33), and mature peptides consisting of amino acids 34-455 (42 kDa chain) and amino acids 456-550 (14 kDa chain). See also UniProtKB / Swiss-Prot (P22304.1).

[0026] As used herein, the term "NAb titer" refers to a measure of the production of neutralizing antibodies (e.g., anti-AAV Nabs) that neutralize the physiological effect of the targeted epitope (e.g., AAV). Anti-AAV NAb titers can be measured, for example, as described in Calcedo, R. et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses (Global Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses). Journal of Infectious Diseases, 2009. 199(3): p381-390.

[0027] As used herein, "expression cassette" refers to a nucleic acid molecule that includes an IDS gene, a promoter, and may include other regulatory sequences therefor; this cassette may be delivered to a packaging host cell via a genetic element (e.g., a plasmid) and packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such expression cassettes for generating viral vectors include the IDS coding sequence described herein adjacent to a packaging signal of the viral genome and other expression control sequences, such as those described herein.

[0028] The abbreviation "sc" stands for self-complementary. "Self-complementary AAV" refers to a construct designed such that the coding region carried by the recombinant AAV nucleic acid sequence forms an intramolecular double-stranded DNA template. After 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 ready for immediate replication and transcription. See, e.g., D.M. McCart y et al., “Self-complementary recombinant See "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," Gene Therapy (August 2001), Vol. 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0029] As used herein, the term "operably linked" refers both to expression control sequences that are contiguous with a gene of interest and to expression control sequences that act in trans or at a distance to regulate the gene of interest.

[0030] The term "heterologous," when used with respect to a protein or nucleic acid, indicates that the protein or nucleic acid contains two or more sequences or subsequences that are not naturally found in the same relationship to each other. For example, nucleic acids are typically produced recombinantly, with 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, when referring to a coding sequence, the promoter is heterologous.

[0031] "Replication-deficient 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. Any viral genomic sequences that are also packaged within the viral capsid or envelope are replication-deficient; that is, they cannot produce progeny virions, but may 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 may be engineered to be "gutless," containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes may be supplied during production. Thus, it is considered safe for use in gene therapy, as replication and infection by progeny virions cannot occur without the presence of viral enzymes required for replication.

[0032] As used herein, "recombinant AAV9 viral particle" refers to a nuclease-resistant particle (NRP) having an AAV9 capsid, which internally packages a heterologous nucleic acid molecule containing an expression cassette for a desired gene product. Such expression cassettes typically contain AAV 5' and / or 3' inverted terminal repeat sequences flanking the gene sequence, which is operably linked to expression control sequences. These and other suitable elements of the expression cassette are described in more detail below and may alternatively be referred to herein as the genomic sequence of the transgene. This may also be referred to as a "complete" AAV capsid. Such rAAV viral particles are said to be "pharmacologically active" when they deliver a transgene to a host cell capable of expressing the desired gene product carried by the expression cassette.

[0033] rAAV particles are often 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 be performed using a single nuclease, or multiple nucleases directed against different targets. The enzyme may comprise a mixture of nucleases and may be either endonucleases or exonucleases.

[0034] The term "nuclease resistant" indicates that the AAV capsid is fully assembled around an expression cassette designed to deliver a transgene into a host cell and protects these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove any contaminating nucleic acids from the production process.

[0035] 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: 13. Some variations from this encoded sequence are encompassed by the present invention, which may include sequences with about 99% identity to the reference amino acid sequence in GenBank Accession AAS99264, SEQ ID NO: 13, and US7906111 (also WO2005 / 033321) (i.e., less than about 1% variation from the reference sequence). Such AAVs include, for example, naturally occurring isolates (e.g., hu31 or hu32), or variants of AAV9 with amino acid substitutions, deletions, or additions, such as amino acid substitutions selected from surrogate residues "supplemented" from corresponding positions in any other AAV capsid aligned with the AAV9 capsid; e.g., substitutions described in U.S. Patent No. 9,102,949, U.S. Patent No. 8,927,514, U.S. Patent Application Publication No. 2015 / 349911, and WO2016 / 049230A1. However, in other embodiments, other variants of AAV9 or AAV9 capsids with at least about 95% identity to the above sequences may be selected. See, e.g., U.S. Patent Application Publication No. 2015 / 0079038. Accordingly, methods for generating capsids, coding sequences therefor, and methods for producing rAAV viral vectors have been described. For example, Gao et al. See, e.g., et al., Proc. Natl. Acad. Sci. USA, 100(10), 6081-6086 (2003) and U.S. Patent Application Publication No. 2013 / 0045186A1.

[0036] The term "AAV9 intermediate" or "AAV9 vector intermediate" refers to an assembled rAAV capsid that lacks the desired genomic sequence packaged therein. These may also be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of the expression cassette or may contain only partially packaged genomic sequence that is insufficient to achieve expression of the gene product. These empty capsids do not function to transfer a gene of interest into a host cell.

[0037] The terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0038] The terms "comprise," "comprises," and "comprising" are to be interpreted inclusively and not exclusively. The terms "consist," "consisting," and variations thereof are to be interpreted exclusively and not inclusive. Although various embodiments herein are presented using the word "comprising," under other circumstances, the relevant embodiment shall also be construed and described using the words "consisting of" or "consisting essentially of."

[0039] The term "about" encompasses variations within ±10% unless otherwise specified.

[0040] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art and with reference to published texts, which generally guide one of ordinary skill in the art to many of the terms used in this application. [Brief explanation of the drawings]

[0041] [Figure 1] Schematic diagram of the AAV9.CB.hIDS vector genome. The IDS expression cassette is flanked by inverted terminal repeats (ITRs), and expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and chicken beta-actin promoter (CB7). The transgene contains a chicken beta-actin intron and a rabbit beta-globin polyadenylation (polyA) signal. [Figure 2] Figures 2A-2C provide IDS expression in the CNS and serum of MPS II mice after IT vector treatment. MPS II mice were treated at 2-3 months of age with ICV injection of AAV9.CB.hIDS at one of three doses: 3 x 10 GC (low), 3 x 10 GC (medium), or 3 x 10 GC (high). Animals were sacrificed 3 weeks after injection. IDS activity was measured in CSF (Figure 2A), whole brain homogenate (Figure 2B), and serum (Figure 2C). Wild-type and untreated MPS II mice were used as controls. [Figure 3] Figure 1 shows the biodistribution of vector DNA in MPS II mice administered AAV9.CB.hIDS. MPS II mice were treated with an ICV injection of 3x1010GC AAV9 vector. Three weeks after injection, the animals were sacrificed, and vector genomes were quantified in tissue DNA by Taqman PCR. [Figure 4] Figures 4A-4D show correction of peripheral GAG accumulation after IT vector delivery in MPS II mice. MPS II mice were treated at 2-3 months of age by ICV injection of AAV9.CB.hIDS at one of three doses: 3 × 10 GC (low), 3 × 10 GC (medium), or 3 × 10 GC (high). Animals were sacrificed 3 months after injection. Hexosaminidase activity was measured in the liver (Figure 4A) and heart (Figure 4B). Correction of accumulation was seen in the liver at all doses and in the heart at the medium and high doses. GAG content was measured in the liver (Figure 4C) and heart (Figure 4D). Wild-type and untreated MPS II mice served as controls. *p<0.05, one-way ANOVA followed by Dunnett's test. [Figure 5]Figures 5A-5L show the dose-dependent resolution of brain accumulation lesions in MPS II mice. MPS II mice were treated at 2–3 months of age by intravenous injection of AAV9.CB.hIDS at one of three doses: 3 × 10 GC (low), 3 × 10 GC (medium), or 3 × 10 GC (high). Three months after injection, animals were sacrificed, and brains were stained for the lysosomal membrane protein LIMP2 and ganglioside GM3. Cells staining positive for GM3 (Figures 5A, 5C, 5E, 5G, and 5I) and LIMP2 (Figures 5B, 5D, 5F, 5H, and 5J) were quantified by blinded reviewers in four cortical brain sections from each animal. Representative cortical brain sections are shown (GM3, Figure 5K; LIMP2, Figure 5L). Wild-type and untreated MPS II mice were used as controls. *p<0.05, one-way ANOVA followed by Dunnett's test. [Figure 6] Figures 6A-6C show improved object discrimination in vector-treated MPS II mice. Untreated MPS II mice and WT male littermates underwent behavioral testing at 4-5 months of age. MPS II mice were treated at 2-3 months of age with ICV injections of AAV9.CB.hIDS at one of three doses: 3 x 10 GC (low), 3 x 10 GC (medium), or 3 x 10 GC (high). Two months after injection, animals underwent behavioral testing. Figure 6A shows Y-maze behavior, and Figure 6B shows contextual fear conditioning, assessed 2 months after injection by a blinded reviewer. "Pre" indicates freezing time when re-exposed to the enclosure 24 hours after receiving a 1.5 mA foot shock without a signal. *p<0.05, two-way ANOVA followed by Sidak's multiple comparison test. Figure 6C shows the percentage of time spent exploring novel or familiar objects in a novel object recognition task, used to assess long-term memory. Wild-type and untreated MPS II mice were used as controls. *p<0.05, t-test with Bonferroni correction for multiple comparisons. [Figure 7]Figures 7A-7B show a comparison of enzyme expression and correction of brain accumulation lesions in MPS I mice treated with IT AAV9. MPS I mice were treated at 2-3 months of age with ICV injection of AAV9.CB.hIDUA at one of three doses: 3 x 10 GC (low), 3 x 10 GC (medium), or 3 x 10 GC (high). One cohort of animals was sacrificed 3 weeks after vector injection, and brains were harvested for measurement of IDUA activity, as shown in Figure 7A. Figure 7B shows that a second cohort of animals was sacrificed 3 months after injection, and brains were stained for the lysosomal membrane protein LIMP2. Cells stained positive for LIMP2 were quantified by a blinded reviewer in four cortical brain sections. Wild-type and untreated MPS II mice served as controls. *p<0.05, one-way ANOVA followed by Dunnett's test. [Figure 8] Figure 1 shows antibody responses to human IDS in MPS II mice treated with ICV AAV9.CB.hIDS. MPS II mice were treated at 2–3 months of age with ICV injection of AAV9.CB.hIDS at one of three doses: 3 × 10 GC (low), 3 × 10 GC (medium), or 3 × 10 GC (high). Serum was collected at necropsy from mice sacrificed 21 or 90 days after vector administration. Antibodies to human IDS were assessed by indirect ELISA. The dashed line indicates 2 SD above the mean titer in naive serum from untreated animals. Most animals had no detectable antibodies (at background levels in naive serum). [Figure 9] Figures 9A-9D show normal open-field activity and Y-maze performance in MPS II mice. Untreated MPS II mice and WT male littermates underwent behavioral testing at 4-5 months of age. Open-field activity was measured by XY-axis beam breaks for horizontal activity (Figure 9A), Z-axis beam breaks for vertical activity (Figure 9B), and percent center beam breaks for center activity (Figure 9C). Total arm entries (Figure 9D) were recorded during an 8-min Y-maze testing session. [Figure 10] 10A-10B show a flow diagram of the manufacturing process. [Figure 11] Image of device (10) for intracisternal delivery of pharmaceutical compositions, including an optional introducer needle for a coaxial insertion method (28), with a 10 cc vector syringe (12), a 10 cc prefilled flush syringe (14), a T-connector extension set (including tubing (20), clips on the ends of tubing (22) and connector (24)), a 22G x 5 inch spinal needle (26) and an optional 18G x 3.5 inch introducer needle (28). Also shown is a 4-way stopcock with a swivel male luer lock (16). [Figure 12] Figures 12A-12I show encephalitis and transgene-specific T cell responses in dogs treated with ICV AAV9. One-year-old MPS I dogs were treated with a single ICV or IC injection of an AAV9 vector expressing GFP. All animals were sacrificed 14 days after injection, except for I-567, which died 12 days after injection. Brains were sectioned coronally to reveal gross lesions (arrowheads) near the injection site in ICV-treated animals (Figures 12A-12F). Tissue sections from brain regions surrounding the gross lesions were stained with hematoxylin and eosin (Figures 12G and 12H). Original magnification = 4x (left panel) and 20x (right panel). Peripheral blood mononuclear cells were collected from one ICV-treated dog (I-565) at necropsy, and T cell responses to the AAV9 capsid and human IDUA protein were measured by interferon-γ ELISPOT (Figure 12I). T cell responses to the GFP transgene product were measured using a single pool of overlapping 15-amino acid peptides covering the entire GFP sequence. Peptides containing the AAV9 capsid protein were divided into three pools (referred to as pools A–C). * = positive response, defined as >3x background (unstimulated cells) and >55 spots per million cells. Phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) with ionomycin served as positive controls for T cell activation. [Figure 13]1 is a bar graph showing vector biodistribution in dogs treated with ICV or IC AAV9. Dogs were sacrificed 14 days after a single ICV or IC injection of a GFP-expressing AAV9 vector, except for animal I-567, which was necropsied 12 days after injection. Vector genomes were detected in tissue samples by quantitative PCR. Values ​​are expressed as vector genome copies per diploid cell (GC / diploid genome). Brain samples taken from the hippocampus or cerebral cortex are shown as injected or uninjected hemispheres for ICV-treated dogs; for IC-treated animals, these are the right and left hemispheres, respectively. No samples were taken for PCR from the injected cerebral hemisphere of animal I-567. [Figure 14] Figures 14A-14H show GFP expression in the brain and spinal cord of dogs treated with ICV or IC AAV9. GFP expression was assessed by direct fluorescence microscopy of brain and spinal cord samples collected from dogs treated with ICV or IC injection of an AAV9 vector expressing GFP. Representative sections are shown for frontal cortex samples and from the anterior horn of the spinal cord collected at the cervical, thoracic, and lumbar levels. Original magnification is 10x. [Figure 15]Figures 15A-15Q show stable transgene expression and the absence of encephalitis in MPS VII dogs treated with ICV AAV9 expressing the lysosomal enzyme β-glucuronidase (GUSB). Six-week-old dogs with a genetic deficiency of GUSB (a model of MPS VII) were treated with a single ICV injection of an AAV9 vector expressing GUSB. GUSB enzyme activity was measured in CSF samples collected at the time of injection and on days 7 and 21 post-injection (Figure 15A). Dogs were sacrificed 3 weeks post-injection. Gross and microscopic evaluation of the brain region surrounding the injection site was performed (Figures 15B-15D). Original magnification = 4x (middle panel) and 10x (right panel). GUSB activity was detected in brain and spinal cord sections using a substrate that produces a red product when cleaved by active GUSB (Figures 15E-15N). Representative sections are shown for samples of cerebral cortex, cerebellum, and anterior horn of the spinal cord collected at the cervical, thoracic, and lumbar levels. Original magnification = 4x (cortex and cerebellum) and 10x (spinal cord). Sections of cerebral cortex taken from untreated MPS VII dogs, normal dogs, and MPS VII dogs treated with ICV AAV9 were stained for ganglioside GM3, which pathologically accumulates in the brains of MPS VII dogs (Figures 15O-Q). Original magnification = 4x. [Figure 16] Figures 16A-16B show the distribution of contrast agent after lumbar intrathecal injection in non-human primates (NHPs). Adult cynomolgus monkeys received intrathecal injection of AAV9 vector diluted in 5 mL of Iohexol 180 via lumbar puncture. The distribution of contrast agent along the spinal cord was assessed by fluoroscopy. Representative images of the thoracic and cervical regions are shown. Contrast material (arrowheads) was visible along the entire length of the spinal cord within 10 minutes of injection in all animals. [Figure 17]Figure 1 is a bar graph showing vector biodistribution in NHPs treated with intrathecal AAV9. NHPs were sacrificed 14 days after intrathecal injection via lumbar puncture of AAV9 vector diluted in 5 mL of iohexol 180. Two animals were placed in Trendelenburg position for 10 minutes after injection. Vector genomes were detected in tissue samples by quantitative PCR. Values ​​are expressed as vector genome copies per diploid cell (GC / diploid genome). [Figure 18] Figures 18A-18H show GFP expression in the brain and spinal cord of NHPs treated with intrathecal AAV9. GFP expression was assessed by direct fluorescence microscopy of brain and spinal cord samples taken from NHPs treated with intrathecal injection of AAV9 vectors. The vector was administered by lumbar puncture. Two animals were placed in Trendelenburg position for 10 minutes after injection. Representative sections are shown for frontal cortex samples and from the anterior horn of the spinal cord collected at the cervical, thoracic, and lumbar levels. Due to the presence of autofluorescent material in some NHP tissues, red blood cell images were captured to distinguish between autofluorescence and GFP signal. The autofluorescence image is overlaid on magenta. Original magnification = 4x (cortex) and 10x (spinal cord). [Figure 19] Figures 19A-19B show elevated CSF spermine during MPS I. High-throughput LC / MS and GC / MS metabolite screening was performed on CSF samples from MPS I dogs (n=15) and normal controls (n=15). A heat map of the top 100 differentially detected metabolites (ANOVA) is shown (Figure 19A). The youngest animal in the MPS I cohort (28 days old) is indicated by an asterisk. Spermine concentrations were measured by quantitative isotope dilution LC / MS assay in CSF samples from six infants with MPS I and two normal infants (Figure 19B). [Figure 20]Figures 20A-20J show spermine-dependent aberrant neurite outgrowth in MPS I neurons. Cortical neurons harvested from E18 wild-type or MPS I mouse embryos were treated with spermine (50 ng / mL) or the spermine synthase inhibitor APCHA 24 hours after plating. Phase-contrast images were acquired 96 hours after plating (Figures 20A-20D). A blinded reviewer quantified the number, length, and branching of neurites in 45-65 randomly selected neurons from duplicate cultures per treatment condition (Figures 20E and 20H, 20G and 20J, and 20F and 20I). ***p<0.0001 (ANOVA followed by Dunnett's test). [Figure 21] Figures 21A-21K show normalization of CSF spermine levels and brain GAP43 expression in MPS I dogs after gene therapy. Five MPS I dogs were treated by intrathecal injection of an AAV9 vector expressing canine IDUA at 1 month of age. To prevent IDUA-induced antibody responses in some MPS I dogs, two of the dogs (I-549 and I-552) were tolerized to IDUA DNA by liver-specific gene therapy at postnatal day 1. Six months after intrathecal vector injection, IDUA activity was measured in brain tissue (Figure 21A). Brain accumulation lesions were assessed by staining for the lysosomal membrane protein LIMP2 (Figures 21B-21H). GAP43 was measured in cortical brain samples by Western blot (Figure 21I) and quantified relative to β-actin by densitometry (Figure 21J). CSF spermine was measured at sacrifice by isotope dilution LC / MS (Figure 21K). Untreated MPS1 dogs (n=3) and normal dogs (n=2) served as controls. *p<0.05 (Kruskal-Wallis test followed by Dunn's test). [Figure 22]Figures 22A-B illustrate the use of spermine as a CSF biomarker for evaluation of CNS-directed gene therapy in MPS I. Six MPS I dogs tolerized to human IDUA at birth were treated at 1 month of age with intrathecal AAV9 expressing human IDUA (10 GC / kg, n = 2; 10 GC / kg, n = 2; 10 GC / kg, n = 2). CSF spermine levels were measured 6 months after treatment (Figure 22A). Three MPS I cats were treated with intrathecal AAV9 expressing feline IDUA (10 GC / kg). CSF spermine was quantified 6 months after treatment (Figure 22B). Untreated MPS I dogs (n = 3) and normal dogs (n = 2) served as controls. [Figure 23] The mean decreasing accuracy of metabolites identified by random forest analysis is shown. [Figure 24] 1 shows the expression of enzymes in the polyamine synthesis pathway in MPS I dog brain samples. [Figure 25] 1 shows spermine concentrations in MPS VII canine CSF. [Figure 26] Figures 26A-26C show no effect of APCHA treatment on WT neuronal proliferation. [Figure 27] Figure 1 shows a dose-dependent reduction in MPS II-associated pathology in MPS II mice (IDSγ / -) treated with ICV AAV9.CB7.CI.hIDS.rBG at various doses (3e8, 3x10 GC; 3e9, 3x10 GC; 3e10, 3x10 GC). Cumulative pathology scores were assessed and plotted on the y-axis. Heterozygous mice (IDSγ / +) served as controls. Results showed a dose-dependent reduction in MPS II-associated pathology in MPS II mice treated with ICV AAV9.CB7.CI.hIDS.rBG. [Figure 28]Figure 1 shows CSF pleocytosis in rhesus monkeys as described in Example 9. White blood cells (leukocytes, y-axis) were counted in CSF samples collected on various days (x-axis, days 0, 7, 14, 21, 30, 45, 60, and 90). Diamonds represent data from RA 2198, which was not treated with AAV9.CB7.hIDS. Squares, triangles, and x represent data from RA1399, RA2203, and RA2231, which were treated with 5 x 10 GC of AAV9.CB7.hIDS, respectively. *, circle, and + represent data from RA 1358, RA 1356, and RA 2197, which were treated with 1.7 x 10 GC of AAV9.CB7.hIDS, respectively. [Figure 29] Figure 1 shows the results of an ELISA measuring anti-hIDS antibody development in the serum of NHPs at day 60, as described in Example 9. Dilution is plotted as the x-axis, while optical density (OD) is plotted as the y-axis. Diamonds represent data from RA2198 without treatment with AAV9.CB7.hIDS. Squares represent data from RA2197 treated with 1.7 x 10 GC of AAV9.CB7.hIDS, while triangles and x represent data from RA 2203 and RA 2231, respectively, treated with 5 x 10 GC of AAV9.CB7.hIDS. DETAILED DESCRIPTION OF THE INVENTION

[0042] 5. Detailed Description of the Invention The use of replication-deficient AAV to deliver a hIDS gene to the CNS of a patient (human subject) diagnosed with MPS II is provided. The recombinant AAV ("rAAV") vector ("rAAV.hIDS") used to deliver the hIDS gene has tropism for the CNS (e.g., rAAV with an AAV9 capsid), and the hIDS transgene is controlled by specific expression control elements, such as a hybrid of the cytomegalovirus (CMV) enhancer and the chicken beta-actin promoter (CB7). In certain embodiments, a pharmaceutical composition suitable for intrathecal, intracisternal, and systemic administration is provided, comprising a suspension of the rAAV.hIDS vector in a formulation buffer containing a physiologically compatible aqueous buffer, a surfactant, and optional excipients. The rAAV suspension is further characterized by: (i) an rAAV genome copy (GC) titer of at least 1.0 × 10 13 is GC / mL; (ii) the rAAV has an empty particle / total particle ratio of 0.01 to 0.05 (95% to 99% free of empty capsids) as measured by SDS-PAGE analysis (see Example 5D); in other embodiments, the rAAV9.hIDS provided herein is 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 rAAV suspension 8 GC / g brain mass ~ approx. 4×10 11 The dose is titrated in GC / g brain mass.

[0043] Potency may be measured in an in vitro cell culture assay, such as the in vitro potency assay described in Example 5G, in which HEK293 cells are transduced with a known multiplicity of rAAVGC per cell and supernatants are assayed for IDS activity 72 hours post-transduction. For example, the function (activity) and / or potency of hIDS may be measured in a suitable in vitro assay using the 4MU-iduronide enzyme assay, which measures the ability of hIDS to cleave the fluorogenic substrate 4-methylumbelliferyl alpha-L-iduronide-2 sulfate. The specific activity measured under the described conditions is >7,500 pmol / min / μg. See the Activity Assay Protocol at www.RnDSystems.com. Other suitable methods for measuring enzyme activity have been described (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), including the methods described herein. Activity can also be measured by 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. Patients who are candidates for treatment include pediatric and adult patients with MPS II (Hunter syndrome) and / or symptoms associated with MPS II.

[0044] MPS II patients in the indicated age groups may receive the following therapeutically effective flat doses of rAAV9.hIDS: Newborn: Approx. 3.8 x 10 12 ~Approx. 1.9×10 14 GC; 3 to 9 months: Approx. 6 x 10 12 ~Approx. 3×10 14 GC; 9-36 months: Approx. 10 13 ~Approx. 5×10 14 GC; ·3~12 years: Approx. 1.2×10 13 ~about 6×10 14 GC; Ages 12+: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 GC; 18+ (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0045] In some embodiments, the dose administered to MPS II patients aged 12+ years (including 18+ years) is 1.4 x 10 13 genome copies (GC) (1.1 × 10 10 In some embodiments, the dose administered to MPS II patients aged 12+ years (including 18+ years) is 7×10 13 GC(5.6×10 10 In yet a further embodiment, the dose administered to an MPS II patient is at least about 4 x 10 8 GC / g brain mass ~ approx. 4×10 11 GC / g brain mass. In certain embodiments, the dose administered to an MPS II newborn is about 1.4 x 10 11 ~Approx. 1.4×10 14 The dose administered to infants 3 to 9 months of age is approximately 2.4 x 10 11 ~Approx. 2.4×10 14 The dose administered to children 9 to 36 months of age with MPS II is approximately 4 × 10 11 ~Approx. 4×10 14 The dose administered to children with MPS II aged 3 to 12 years is approximately 4.8 × 10 11 ~Approx. 4.8×10 14 The dose administered to children and adults aged 12+ is approximately 5.6 x 10 11 ~Approx. 5.6×10 14 This is the scope of GC.

[0046] The goal of this treatment is to functionally replace a patient's defective iduronate-2-sulfatase via rAAV-based CNS-directed gene therapy as a viable approach to treating the disease. When expressed from the rAAV vectors described herein, an expression level of at least about 2% detected in CSF, serum, neurons, or other tissues can provide therapeutic benefit. However, higher expression levels may be achieved. Such expression levels may be 2% to about 100% of normal functional human IDS levels. In certain embodiments, higher-than-normal expression levels may be detected in CSF, serum, or other tissues.

[0047] The efficacy of treatment can be measured by (a) preventing neurocognitive decline in MPS II (Hunter syndrome) patients; and (b) assessing disease biomarkers, such as GAG levels and / or enzyme activity reductions in CSF, serum, and / or urine, and / or liver and spleen volume. Neurocognition in infants can be measured using the Bayley Scales of Infant and Toddler Development, 3rd Edition, BSID-III. Neurocognitive and adaptive behavioral assessments (e.g., using the Bayley Scales of Infant Development and Vineland Adaptive Behavior Scales, respectively) may also be performed.

[0048] The combination of gene therapy delivery of rAAV.hIDS to the CNS with systemic delivery of the hIDS is encompassed by certain embodiments. Systemic delivery can be achieved by ERT (e.g., using Elaprase®) or delivery of a rAAV.hIDS with liver tropism (e.g., A This may be achieved using additional gene therapy using rAAV.hIDS) carrying the AV8 capsid.

[0049] Certain embodiments also provide for the preparation and characterization of an rAAV.hIDS pharmaceutical composition (Example 4 below).

[0050] 5.1 AAV.hIDS Constructs and Formulations 5.1.1. Expression Cassette An AAV vector is provided that includes an expression cassette containing a hIDS gene characterized by having the nucleotide sequence of SEQ ID NO:1 (CCDS14685.1). This sequence is a published gene sequence encoding Genbank NP000193.1 and is also included herein as SEQ ID NO:2. In another embodiment, the expression cassette contains a hIDS gene characterized by having a nucleotide sequence at least about 75% identical to SEQ ID NO:1 and encodes a functional human iduronate-2-sulfatase. In another embodiment, the expression cassette contains a hIDS gene characterized by having a nucleotide sequence at least about 80% identical to SEQ ID NO:1 and encodes a functional human iduronate-2-sulfatase. 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 iduronate-2-sulfatase. In one embodiment, the sequence is at least about 95% identical to SEQ ID NO: 1, or 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 iduronate-2-sulfatase. In one embodiment, the sequence is at least about 77% identical to SEQ ID NO: 1. In another embodiment, the expression cassette comprises a hIDS coding sequence from nt 1177 to nt 2829 of SEQ ID NO: 8, also designated as nt 1937 to nt 3589 of SEQ ID NO: 11. In another embodiment, the expression cassette comprises a hIDS coding sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to nt 1177 to nt 2829 of SEQ ID NO: 8 (also designated as nt 1937 to nt 3589 of SEQ ID NO: 11).

[0051] In another embodiment, the functional human iduronate-2-sulfatase may comprise a synthetic amino acid sequence in which all or part of the first 25 amino acids of the preproprotein, SEQ ID NO: 2, corresponding to the leader (signal) peptide, have been replaced with a heterologous leader peptide. This leader peptide, such as that derived from interleukin-2 (IL-2) or oncostatin, may improve transport of the enzyme out of the cell through its secretory pathway into the circulatory system. Suitable leader peptides are preferably, but not necessarily, of human origin. Suitable leader peptides may be selected from proline.bic.nus.edu.sg / spdb / zhang270.htm, 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. Furthermore, at least one in vitro assay has been described as useful for assessing the enzymatic activity of IDS enzymes [see, e.g., Dean et al., Clinical Chemistry, 2006 Apr;52(4):643-649]. In addition to removing all or a portion of the preproprotein (amino acids 1-25 of SEQ ID NO:2), all or a portion of the proprotein (amino acids 26-33 of SEQ ID NO:2) may be removed and, if desired, replaced with a heterologous mature peptide.

[0052] In another embodiment, an AAV vector comprising an expression cassette further comprising the SUMF1 gene, characterized in that the SUMF1 gene has the nucleotide sequence from nt 3423 to nt 4547 of SEQ ID NO:5. (CDS of GenBank: AB448737.1) is provided. This sequence is a published gene sequence encoding NCBI Reference Sequence: NP_877437.2, also incorporated herein as SEQ ID NO:7 and SEQ ID NO:10. Some studies have suggested that expression of sulfatases such as IDS may be limited by the availability of the sulfatase modifier SUMF1, which is required for post-translational modification of IDS (Fraldi et al., Biochemical J, 2007:403:305-312). In another embodiment, the expression cassette comprises a SUMF1 gene characterized by having a nucleotide sequence at least about 80% identical to nt 3423 to nt 4547 of SEQ ID NO:5, and encodes functional human SUMF1. In another embodiment, the sequence is at least about 85% identical to nt 3423 to nt 4547 of SEQ ID NO:5, or at least about 90% identical to nt 3423 to nt 4547 of SEQ ID NO:5, and encodes functional human SUMF1. In one embodiment, the sequence is at least about 95%, about 97%, or about 99% identical to nt 3423 to nt 4547 of SEQ ID NO: 5 and encodes functional human SUMF1. In one embodiment, the sequence is at least about 76.6% identical to nt 3423 to nt 4547 of SEQ ID NO: 5. In another embodiment, the expression cassette comprises an hSUMF1 coding sequence from nt 3423 to nt 4553 of SEQ ID NO: 8. In another embodiment, the expression cassette comprises an hSUMF1 coding sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to nt 3423 to nt 4553 of SEQ ID NO: 8.

[0053] In one embodiment, an AAV vector is provided that includes an expression cassette comprising a hIDS coding sequence and a hSUMF1 coding sequence. In another embodiment, the hIDS coding sequence and the hSUMF1 coding sequence are linked by an internal ribosome entry site (IRES). In a further embodiment, the IRES has the sequence from nt 2830 to nt 3422 of SEQ ID NO:5 or from nt 2830 to nt 3422 of SEQ ID NO:8. In another embodiment, the expression cassette comprises from nt 1177 to nt 4547 of SEQ ID NO:5 or from nt 1177 to nt 4553 of SEQ ID NO:8, which comprises the hIDS coding sequence, the IRES, and the hSUMF1 coding sequence.

[0054] Sequence identity or similarity is defined herein as the percentage of amino acid residues in a candidate sequence that are identical (i.e., the same residues) or similar (i.e., amino acid residues from the same group based on common side chain properties, see below) to the peptide and polypeptide regions provided herein, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Percent (%) identity is a measure of the relationship between two polynucleotides or two polypeptides, determined by comparing their nucleotide or amino acid sequences, respectively. Generally, the two sequences being compared are aligned to maximize the 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 is divided by the total length of the alignment and multiplied by 100 to obtain a percent identity figure. This percent identity figure can be determined over the entire length of the sequences being compared, which is particularly appropriate for sequences of the same or very similar length and highly homologous, or over a shorter, defined length, and is more appropriate for sequences of unequal length or with a lower level 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 alignments and percent identity. The choice of algorithm or program is not a limitation of the present invention.

[0055] Examples of suitable alignment programs include, for example, the software CLUSTALW on Unix, and then the Bioedit program (Hall, TA 1999, BioEdit: a user-friendly biological sequence alignment program). Clustal Omega, available from EMBL-EBI (Sievers, Fabian et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega," Molecular Systems Biology 7.1 (2011):539 and Goujon, Mickael et al., "A new bioinformatics analysis tools framework at EMBL-EBI," Nucleic Acids Research 38.suppl 2 (2010):W695-W699); 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 can be used to determine the percent identity between two polynucleotide and two polypeptide sequences. 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 from the NCBI homepage (www.ncbi.nlm.nih.gov http: / / www.ncbi.nlm.nih.gov / ), and the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may 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) may be used. SeqWeb Software (a web-based interface to the GCG Wisconsin Package: Gap program) may also be used.

[0056] In some embodiments, the cassette is designed to be expressed from a recombinant adeno-associated virus, and the vector genome also contains AAV inverted terminal repeats (ITRs). In one embodiment, the rAAV is pseudotyped, i.e., the AAV capsid is derived from an AAV source different from the AAV that provides the ITRs. In one embodiment, the ITRs of AAV serotype 2 are used. However, ITRs from other suitable sources may also be selected. Optionally, the AAV may be a self-complementary AAV.

[0057] The expression cassettes described herein utilized AAV 5' inverted terminal repeats (ITRs) and AAV 3' ITRs. However, other configurations of these elements may also be suitable. A shortened version of the 5' ITR, called ΔITR, has been described in which the D-sequence and terminal release sites (trs) are deleted. In other embodiments, full-length AAV 5' and / or 3' ITRs are used. When pseudotyped AAVs are produced, the ITRs being expressed are selected from a source different from the AAV source of the capsid. For example, AAV2 ITRs may be selected for use with AAV capsids with particular efficiency for targeting the CNS or tissues or cells within the CNS. In one embodiment, ITR sequences from AAV2 or deleted versions thereof (ΔITRs) are used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources may also 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 referred to as pseudotyped. However, other sources of AAV ITRs may also be utilized.

[0058] 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 enabling treatment of both systemic and CNS-related effects of MPS II. For example, the inventors have observed that certain constitutive promoters (e.g., CMV) do not drive expression at desirable levels when delivered intrathecally, thereby providing suboptimal hIDS expression levels. However, the chicken beta-actin promoter drives expression well both intrathecally and systemically. Thus, it is a particularly desirable promoter. Other promoters may be selected, but expression cassettes containing the same will not have all of the advantages of those with the chicken beta-actin promoter. Various chicken beta-actin promoters have been described, either alone or in combination with various enhancer elements (e.g., CB7 is a chicken beta-actin promoter with a cytomegalovirus enhancer element, the CAG promoter, which contains the promoter, the first exon and first intron of chicken beta-actin, and the splice acceptor of the rabbit beta-globin gene), or the CBh promoter [SJ Gray et al., Hu Gene Ther, 2011 Sep;22(9):1143-1153.

[0059] Examples of promoters that are tissue-specific include promoters specific 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., 7:1503-14), bone osteocalcin (Stein et al., (1997) Mol. Biol. Rep., 24:185-96); bone sialoprotein (Chen et al., (1996) J. Bone Miner. Res., 11:654-64), lymphocytes (CD2, Hansal et al., (1996) J. Bone Miner. Res., 11:654-64), among others. al., (1998) J. Immunol., 161:1063-8; immunoglobulin heavy chain; T cell receptor chain), neuronal, e.g., 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 neuron-specific vgf gene (Piccioli et al., (1995) Neuron, 15:373-84). Alternatively, a regulatable promoter may be selected. See, e.g., WO 2011 / 126808B2, incorporated herein by reference.

[0060] In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette comprises two or more expression enhancers. These enhancers may be the same or different. For example, the enhancer may comprise 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 comprises an intron, such as a chicken beta-actin intron, a human β-globin intron, and / or a commercially available Promega® intron. Other suitable introns include those known in the art, for example, those described in WO 2011 / 126808.

[0061] Furthermore, an expression cassette having an appropriate polyadenylation signal is provided. In one embodiment, the polyA sequence is rabbit globulin polyA. For example, see WO2014 / 151341. Alternatively, another polyA, such as human growth hormone (hGH) polyadenylation sequence, SV40 polyA, SV50 polyA, or synthetic polyA, may be used. Furthermore, other conventional regulatory elements may additionally or optionally be included in the expression cassette.

[0062] Exemplary rAAV.hIDS vector genomes are set forth in nt 2 to nt 3967 of SEQ ID NO:3, nt 11 to nt 4964 of SEQ ID NO:5, nt 11 to nt 4964 of SEQ ID NO:8, nt 2 to nt 3967 of SEQ ID NO:11, or nt 2 to nt 3965 of SEQ ID NO:14.

[0063] 5.1.2. Production of rAAV-hIDS viral particles In one embodiment, a recombinant adeno-associated virus (rAAV) particle is provided, comprising an AAV capsid and packaging therein an AAV inverted terminal repeat sequence, a human iduronate-2-sulfatase (hIDS) gene under the control of regulatory sequences controlling its expression, wherein the hIDS gene has the sequence set forth in SEQ ID NO: 1 ( FIG. 1 ), or a sequence at least about 95% identical thereto, encoding functional human iduronate-2-sulfatase. In one embodiment, the hIDS expression cassette is flanked by the AAV 5′ ITR and the AAV 3′ ITR. In another embodiment, the AAV is a single-stranded AAV.

[0064] AAV9 is particularly desirable for intrathecal and / or intracisternal delivery. Optionally, the rAAV9.hIDS vector 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 AAVs that can be selected as the source of rAAV capsid include, for example, rh10, AAVrh64R1, AAVrh64R2, and rh8 (see, for example, U.S. Patent Application Publication No. 2007-0036760-A1; U.S. Patent Application Publication No. 2009-0197338-A1; European Patent No. 1310571). See also WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7,790,449 and U.S. Patent No. 7,282,199 (AAV8), WO2005 / 033321 and U.S. Patent No. 7,906,111 (AAV9) and WO2006 / 110689], and rh10 [WO2003 / 042397], AAV3B; AAVdj [US2010 / 0047174]. One particularly desirable rAAV is AAV2 / 8.TBG.hIDS.co.

[0065] rAAV particles are often 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 involve a single nuclease or a mixture of nucleases directed against different targets, and may be endonucleases or exonucleases.

[0066] Methods for preparing AAV-based vectors are known. See, for example, U.S. Patent Application Publication No. 2007 / 0036760 (February 15, 2007), which is incorporated herein by reference. The use of the AAV capsid of AAV9 is particularly well suited for the compositions and methods described herein. The sequence of AAV9 and methods for producing vectors based on the AAV9 capsid are described in U.S. Patent No. 7,906,111; U.S. Patent Application Publication No. 2015 / 0315612; and WO2012 / 112832, which are incorporated herein by reference. However, other AAV capsids may be selected or produced. For example, the sequence of AAV8 and methods for producing vectors based on the AAV8 capsid are described in U.S. Patent Nos. 7,282,199B2, 7,790,449, and 8,318,480, which are incorporated herein by reference. Numerous such AAV sequences are set forth in the above-referenced U.S. Patent Nos. 7,282,199B2, 7,790,449, 8,318,480, and 7,906,111 and / or available from GenBank. Any AAV capsid sequence can be readily produced synthetically or using various molecular biology and genetic engineering techniques. Suitable production techniques are well known to those skilled in the art. See, for example, 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, for example, 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 skilled in the art and are not a limitation of the present invention.

[0067] The recombinant adeno-associated viruses (AAVs) described herein can be produced using known techniques. See, for example, WO2003 / 042397; WO2005 / 033321; WO2006 / 110689; and U.S. Patent No. 7,588,772 B2. Such methods include culturing a host cell containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette composed of at least the AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to allow packaging of the expression cassette into AAV capsid proteins.

[0068] To calculate the empty total particle content, the VP3 band volume of a selected sample (e.g., an iodixanol gradient purified preparation, where in the examples herein, the number of GCs = the number of particles) is plotted against the number of GC particles loaded. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the peak of the specimen. The number of particles (pt) per 20 μL loaded is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the particle to genome copy ratio (pt / GC). Pt / mL - GC / mL gives empty pt / mL. Empty pt / mL is divided by pt / mL and multiplied by 100 to obtain the percentage of empty particles.

[0069] Generally, methods for assaying for empty capsids and AAV vector particles with packaged genomes are known in the art (see, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer See Wobus et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsids, the method involves subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis using any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% Tris-acetate in buffer, running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane, preferably nylon. An anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. 2000) 74:9281-9293). A means for detecting binding of a secondary antibody to the primary antibody and more preferably an anti-IgG antibody having a detection molecule covalently attached thereto, most preferably a sheep anti-mouse IgG antibody covalently attached to horseradish peroxidase, is then added. A secondary antibody containing the antibody is used. The binding between the primary and secondary antibodies is determined semiquantitatively using a method for detecting binding, preferably a detection method that can detect radioisotope radiation, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescent detection kit. For example, in SDS-PAGE, samples from the column fractions are collected and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and the capsid proteins are separated on a precast gradient polyacrylamide gel (e.g., Novex). Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other appropriate staining methods, i.e., SYPRO Ruby or Coomassie staining. In one embodiment, the concentration of AAV vector genome (vg) in the column fractions may be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another appropriate nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using TaqMan™ fluorogenic probes specific to the primers and the DNA sequence between them. The number of cycles required to reach a defined fluorescence level (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in a Q-PCR reaction. The cycle threshold (Ct) value obtained from the sample is used to determine the vector genome titer by normalizing it to the Ct value of the plasmid standard curve. Digital PCR-based endpoint assays may also be used.

[0070] In one embodiment, an optimized q-PCR method utilizing a broad-spectrum serine protease, such as proteinase K (such as that commercially available from Qiagen), is used. More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and heat-inactivated after proteinase K treatment. Suitably, the sample is diluted with a volume of proteinase K buffer equal to the sample size. The proteinase K buffer may be concentrated two-fold or more. Typically, proteinase K treatment is at 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 periods (e.g., about 20 minutes to about 30 minutes) or at higher temperatures (e.g., up to about 60°C) for shorter periods (e.g., about 5 to 10 minutes). Similarly, heat inactivation is generally at about 95°C for about 15 minutes, although the temperature may be lowered (e.g., from about 70°C to about 90°C) and the time may be extended (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 in standard assays.

[0071] Additionally or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al., Hu Gene See Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0072] In summary, a method for separating rAAV9 particles with packaged genome sequences from genome-deleted AAV9 intermediates involves subjecting a suspension containing recombinant AAV9 viral particles and AAV9 capsid intermediates to high-performance liquid chromatography, in which 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 monitoring the eluent for UV absorbance at about 260 and about 280. The pH can range from about 10.0 to 10.4, although this is less optimal for rAAV9. In this method, elution occurs when the A260 / A280 ratio reaches an inflection point. Intact AAV9 capsids are recovered from the fractions containing the diafiltration product. In one example, for an affinity chromatography step, the diafiltration product may be applied to Capture Select™ Poros-AAV2 / 9 affinity resin (Life Technologies), which efficiently captures the AAV2 / 9 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured.

[0073] The rAAV.hIDS vector may be produced as shown in the flow diagram shown in Figure 11, which is described in further detail in Section 5.4 and Example 4 below.

[0074] 5.1.3 Pharmaceutical Formulations of rAAV hIDS The rAAV9.hIDS formulation is a suspension containing an effective amount of the AAV.hIDS vector suspended in an aqueous solution containing saline, surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, e.g., pH 6-9, or pH 6.5-7.5, pH 7.0-7.7, or pH 7.2-7.8. Because the pH of cerebrospinal fluid is about 7.28 to about 7.32, a pH within this range may be desirable for intrathecal delivery; for intravenous delivery, a pH of 6.8 to about 7.2 may be desirable. However, other pH values ​​within the broadest range and subranges thereof may be selected for other delivery routes.

[0075] A suitable surfactant or surfactant combination may be selected from non-toxic non-ionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated with primary hydroxyl groups, such as Pluronic® F68 (BASF), also known as Poloxamer 188, having a neutral pH and an average molecular weight of 8400, may be selected. Other surfactants and other Poloxamers (poloxamers), i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), such as SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), non-ionic triblock copolymers composed of ethanol and polyethylene glycol, may also be selected. In one embodiment, the formulation includes a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 indicate the approximate molecular weight of the polyoxypropylene core, and the last digit x 10 indicates the percentage polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount from about 0.0005% to about 0.001% of the suspension.

[0076] In one embodiment, the formulation is prepared as described, for example, in M. Lock et al., Hu Gene at least about 1 x 10 as measured by oqPCR or digital droplet PCR (ddPCR), as described in 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 ~ 3 × 1013 It may also include the concentration of GC / al.

[0077] In one embodiment, a frozen composition is provided containing rAAV in frozen form in a buffer solution as described herein. Optionally, the composition contains one or more surfactants (e.g., Pluronic F68), stabilizers, or preservatives. Suitably, for use, the composition is thawed and diluted with a suitable diluent, such as sterile saline or buffered saline, as desired. Titrate to a dose of

[0078] In one example, the formulation may comprise a buffered saline solution containing, for example, one or more of sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate·7H₂O), potassium chloride, calcium chloride (e.g., calcium chloride·2H₂O), dibasic sodium phosphate, and mixtures thereof, in water. Suitably, for intrathecal delivery, the osmolality is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, e.g., http: / / emedicine.medscape.com / article / 2093316-overview. If desired, commercially available diluents may be used as suspending agents, and in combination with other suspending agents and other excipients, for intrathecal delivery. See, e.g., Elliotts B® Solution [Lukare Medical]. In other embodiments, the formulation may include one or more permeation enhancers. Examples of suitable permeation enhancers include, for example, mannitol, sodium glycolate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether or EDTA.

[0079] In certain embodiments, kits are provided that include a concentrated vector suspended in a formulation (optionally frozen), an optional dilution buffer, and a device and other components necessary for intrathecal administration. In other embodiments, the kit may additionally or alternatively include components for intravenous delivery. In one embodiment, the kit provides sufficient buffer to allow injection. Such a buffer may allow for approximately a 1:1 to 1:5 dilution of the concentrated vector or greater. Other embodiments include larger or smaller volumes of buffer or sterile water to allow for dose titration and other adjustments by the treating clinician. In still other embodiments, one or more components of the device are included in the kit.

[0080] 5.2. Gene Therapy Protocol 5.2.1 Target patient population Provided herein are methods for treating mucopolysaccharidosis type II, comprising delivering a therapeutically effective amount of the rAAV.hIDS described herein to a patient in need thereof. Specifically, provided herein are methods for preventing, treating, and / or ameliorating neurocognitive decline in patients diagnosed with MPS II, comprising delivering a therapeutically effective amount of the rAAV.hIDS described herein to a patient in need thereof. A "therapeutically effective amount" of the rAAV.hIDS vector described herein may correct one or more of the symptoms identified in any one of the following paragraphs.

[0081] Candidates for treatment include children and adults with MPS II (Hunter syndrome) and / or symptoms associated with MPS II. MPS II is characterized as a mild / attenuated phenotype or a severe phenotype. Death occurs at a mean age of 11.7 years in patients with the severe phenotype (characterized by neurocognitive decline) and 21.7 years in patients with the mild or attenuated phenotype. The majority of patients (two-thirds) have the severe form of the disease. Patients with MPS II appear normal at birth, but signs and symptoms of the disease typically present between 18 months and 4 years of age in the severe form and between 4 and 8 years of age in the attenuated form. Signs and symptoms common to all affected patients include short stature, coarse facies, macrocephaly, macroglossia, hearing loss, hepatomegaly and splenomegaly, dysostosis multiplex, arthrogryposis, spinal stenosis, and carpal tunnel syndrome. Frequent upper respiratory tract and ear infections occur in most patients, and progressive airway obstruction is common, leading to sleep apnea and often death. Heart disease is the leading cause of death in this population and is characterized by valvular dysfunction leading to left and right ventricular hypertrophy and heart failure. Death is generally due to obstructive airway disease or heart failure.

[0082] In severe MPS II, developmental delays are readily apparent by 18 to 24 months, although early developmental milestones may be achieved. Some patients fail hearing screening tests in the first year, and other milestones, including the ability to sit unsupported, walk, and speak, are delayed. Developmental progress begins to plateau between the ages of 3 and 5 years, with regression reported beginning at approximately 6.5 years. Of the approximately 50% of children with MPS II who are toilet trained, most, if not all, lose this ability as the disease progresses.

[0083] Patients with significant neurological involvement exhibit severe behavioral disturbances, including hyperactivity, stubbornness, and aggression, beginning in the second year of life and continuing until age 8–9 years, when neurodegeneration diminishes this behavior.

[0084] Epileptic seizures are reported in more than half of severely affected patients who reach the age of 10 years, and by the time of death, most patients with CNS involvement have severe mental disabilities and require constant care. Patients with weakened disease exhibit normal intellectual function, but MRI imaging reveals gross abnormalities in all patients with MPS II, including white matter lesions, enlarged ventricles, and brain atrophy.

[0085] The compositions of the present 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 lifelong complications of peripheral administration, such as local and systemic infections. In contrast to ERT, the compositions of the present invention do not require repeated weekly injections for life. Without being bound by theory, the treatment methods described herein are believed to be useful for correcting at least the central nervous system phenotype associated with MPS II disorder by providing efficient, long-term gene transfer delivered by highly transduction-efficient vectors that result in continuously elevated circulating IDS levels, providing therapeutic leverage outside the CNS compartment. Furthermore, methods are provided herein for providing active tolerance and preventing antibody formation against the enzyme by various routes, including direct systemic delivery of the enzyme in protein form or in the form of AAV-hIDS, prior to AAV-mediated delivery to the CNS.

[0086] In some embodiments, patients diagnosed with severe MPS II are treated according to the methods described herein. In some embodiments, patients diagnosed with attenuated MPS II are treated according to the methods described herein. In some embodiments, pediatric subjects with MPS II who have early neurocognitive impairment are treated according to the methods described herein. In certain embodiments, patients aged 2 years or older who have been diagnosed with Hunter syndrome and who have or are at risk of developing neurocognitive impairment are treated according to the methods described herein. In certain embodiments, patients aged 2 years or older with the presence of a major rearrangement or deletion mutation known to be correlated with severe Hunter syndrome are treated according to the methods described herein.

[0087] In certain embodiments, newborns (3 months of age or younger) are treated according to the methods described herein. In certain embodiments, newborns 3 to 9 months of age are treated according to the methods described herein. In certain embodiments, children 9 to 36 months of age are treated according to the methods described herein. In certain embodiments, children 3 to 12 years of age are treated according to the methods described herein. In certain embodiments, children 12 to 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.

[0088] Suitably, patients selected for treatment include those with one or more of the following characteristics: lack of IDS enzyme activity measured in serum, plasma, fibroblasts or leukocytes. A confirmed diagnosis of MPS II confirmed by a decrease or a decrease in DQ (BSID-III) that is at least one standard deviation below the mean or confirmed historical evidence of a decrease of more than one standard deviation on sequential testing, if not explained by other neurological or psychiatric factors. Alternatively, elevated GAGs in urine, serum, CSF, or genetic testing may be used.

[0089] Prior to treatment, subjects, e.g., infants, preferably undergo genotyping to identify MPS II patients, i.e., patients with mutations in the gene encoding hIDS. Prior to treatment, MPS II patients may be assessed for neutralizing antibodies (NABs) against the AAV serotype used to deliver the hIDS gene. Such NABs may interfere with transduction efficiency and reduce treatment titers. MPS II patients with baseline serum NAB titers ≤ 1:5 are good candidates for treatment with rAAV.hIDS gene therapy protocols. Treatment of MPS II patients with serum NAB titers > 1:5 may require concomitant therapy, such as transient cotreatment with immunosuppressants, before and / or during treatment with rAAV.hIDS vector delivery. If necessary, immunosuppressant cotreatment 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. Prior immunosuppressive therapy may be desirable to prevent a potentially adverse immune response to the hIDS transgene product, particularly in patients with substantially no levels of IDUA activity that would cause the transgene product to be considered "foreign." The results of the nonclinical studies in mice, dogs, and NHPs described below are consistent with the development of an immune response to hIDS and neuroinflammation. Although a similar response may not occur in human subjects, as a precaution, immunosuppressive therapy is recommended for all recipients of rAAV-hIDS.

[0090] Immunosuppressants for such combination therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalogs), and cytostatics, such as alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents acting on immunophilins. Immunosuppressants include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25-) or CD3-specific antibodies, anti-IL-2 antibodies, cyclosporine, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids, or TNF-α (tumor necrosis factor-α) binding agents. In certain embodiments, immunosuppressive therapy may be initiated 0, 1, 2, 7, or more days prior to gene therapy administration. Such treatment may include the co-administration of two or more drugs (e.g., prednisone, minophenolate mofetil (MMF), and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs may be continued at the same or adjusted doses after gene therapy administration. Such treatment may be for about 1 week (7 days), about 60 days, or longer, as needed. In certain embodiments, a tacrolimus-free regimen is selected.

[0091] In certain embodiments, patients with one or more of the following characteristics may be excluded from treatment at the discretion of their caring physician: Have a neurocognitive disorder not attributable to MPS II that, in the opinion of either the investigator or the medical monitor, could confound the interpretation of the study results · Have a condition (e.g., medical history, evidence of current illness, findings on physical examination, or abnormal laboratory values) that, in the opinion of the investigator, poses undue risk to the subject or interferes with the assessment of the trial results or the subject's safety or interpretation of the trial results. Diagnosis of neuropsychiatric symptoms Contraindications for intrathecal / intracranial administration, including contraindications for fluoroscopy -Contraindications for MRI -Having acute hydrocephalus at the time of enrollment Currently enrolled in another clinical trial using an investigational drug within 4 weeks prior to screening or within 5 half-lives of the investigational drug used in that clinical trial, whichever is longer. -Having undergone hematopoietic stem cell transplantation (HSCT) -Received intrathecal idursulfase within 6 months prior to screening ·Receiving intrathecal idursulfase at any time and having a serious adverse event (AE) associated with intrathecal administration that, in the opinion of the investigator and / or medical monitor, poses undue risk to the subject.

[0092] In other embodiments, 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.

[0093] 5.2.2. Dosage and Mode of Administration Pharmaceutical compositions suitable for administration to patients include suspensions of rAAV.hIDS vectors in a formulation buffer containing a physiologically compatible aqueous buffer, surfactant, and optional excipients. In certain embodiments, the pharmaceutical compositions described herein are administered intrathecally. In other embodiments, the pharmaceutical compositions described herein are administered intracisternally. In other embodiments, the pharmaceutical compositions described herein are administered intravenously. In certain embodiments, the pharmaceutical compositions are delivered by infusion over 20 minutes (±5 minutes) via a peripheral vein. However, this time period may be adjusted as needed or desired. However, additional routes of administration may also be selected. Alternatively or additionally, routes of administration may be combined, if desired.

[0094] Although a single dose of rAAV is expected to be effective, administration may be repeated (e.g., quarterly, twice-yearly, annually, or otherwise as needed, particularly in the treatment of neonates). If necessary, the initial therapeutically effective dose may be delivered over a period of split infusions / injections, taking into account the age and ability of the subject to tolerate infusions / injections. However, repeated weekly injections of the full therapeutic dose are not required, providing benefits to the patient in terms of both comfort and treatment outcome.

[0095] In some embodiments, the rAAV suspension comprises at least 1.0 x 10 13 The rAAV genome copy (GC) titer is GC / mL. In certain embodiments, the rAAV empty / full particle ratio in the rAAV suspension is 0.01-0.05 (95%-99% free of empty capsids). In some embodiments, an MPS II patient in need thereof has a rAAV genome copy (GC) titer of at least about 4 x 10 8 GC / g brain mass ~ approx. 4×10 11 A dose of rAAV suspension of GC / g brain mass is administered.

[0096] MPS II patients in the indicated age groups may be administered the following therapeutically effective flat doses of rAAV.hIDS: Newborn: Approx. 3.8 x 10 12 ~Approx. 1.9×10 14 GC; 3 to 9 months: Approx. 6 x 10 12 ~Approx. 3×10 14 GC; 9-36 months: Approx. 10 13 ~Approx. 5×10 14 GC; ·3~12 years: Approx. 1.2×10 13 ~about 6×10 14 GC; Ages 12+: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 GC; 18+ (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0097] In some embodiments, the dose administered to MPS II patients aged 12+ years (including 18+ years) is 1.4 x 10 13 genome copies (GC) (1.1 × 10 10 In some embodiments, the dose administered to MPS II patients aged 12+ (including those aged 18+) is 7×10 13 GC(5.6×10 10 In yet a further embodiment, the dose administered to an MPS II patient is at least about 4 x 10 8 GC / g brain mass ~ approx. 4×10 11 GC / g brain mass. In certain embodiments, the dose administered to an MPS II newborn is about 1.4 x 10 11 ~Approx. 1.4×10 14 The dose administered to infants 3 to 9 months of age is approximately 2.4 x 10 11 ~Approx. 2.4×10 14 The dose administered to children 9 to 36 months of age with MPS II is approximately 4 × 10 11 ~Approx. 4×10 14 GC ranges from 3 to 12 years old; MPS The dose given to children is approximately 4.8 x 10 11 ~Approx. 4.8×10 14 The dose administered to children and adults aged 12+ is approximately 5.6 x 10 11 ~Approx. 5.6×10 14 This is the scope of GC.

[0098] Appropriate volumes for delivery of these doses and concentrations can be determined by one of skill in the art. For example, volumes between about 1 μL and 150 mL may be selected, with larger volumes being selected for adults. Typically, for newborns, an appropriate volume is between about 0.5 mL and about 10 mL, and for older infants, between about 0.5 mL and about 15 mL may be selected. For infants, volumes between about 0.5 mL and about 20 mL may be selected. For children, volumes up to about 30 mL may be selected. For preteens and teenagers, volumes up to about 50 mL may be selected. In yet other embodiments, patients may be administered intrathecally in volumes between about 5 mL and about 15 mL, or between about 7.5 mL and about 10 mL. Other appropriate volumes and doses may be determined. The dosage is adjusted to balance therapeutic benefit against any side effects, and such dosage may vary depending on the therapeutic application for which the recombinant vector is used.

[0099] 5.2.3. Monitoring Efficacy The efficacy of the treatments described herein can be measured by assessing (a) prevention of neurocognitive decline in MPS II (Hunter Syndrome) patients; and (b) reduction of disease biomarkers, such as GAG levels and / or enzyme activity in CSF, serum, and / or urine, and / or liver and spleen volume. Neurocognition can be determined by measuring intelligence quotient (IQ), for example, as measured by the Bayley's Infantile Development Scale for Hurler or the Wechsler Abbreviated Scale of Intelligence (WASI) for Hurler-Scheie subjects. Other appropriate measures of neurocognitive development and function, such as assessment of developmental quotient (DQ) using the Bayley Scale of Infant Development (BSID-III), memory assessment using the Hopkins Verbal Learning Test, and / or assessment using the Tests of Variables of Attention (TOVA), can also be utilized. Other neuropsychological functions, such as Vineland Adaptive Behavior Scale, visual processing, fine motor skills, communication, socialization, daily living skills, and emotional and behavioral health, will be monitored. Magnetic resonance imaging (MRI) of the brain will also be performed to obtain volumetric, diffusion tensor imaging (DTI), and resting-state data, median nerve cross-sectional area by ultrasound, spinal cord compression improvement, safety, liver size, and spleen size.

[0100] Optionally, other measures of efficacy include assessment of biomarkers (e.g., polyamines as described herein) and clinical outcomes. Urine is assessed for total GAG content, GAG relative to creatinine concentration, and MPS II-specific pGAG. Serum and / or plasma are assessed for IDUA activity, anti-IDS antibodies, pGAG, and heparin cofactor II-thrombin complex concentrations and markers of inflammation. CSF is assessed for IDUA activity, anti-IDS antibodies, hexosaminidase (hex) activity, and pGAG (such as heparan sulfate and dermatan sulfate). The presence of neutralizing antibodies to the vector (e.g., AAV9) and binding antibodies to anti-IDS antibodies can be assessed in CSF and serum. T cell responses to the vector capsid (e.g., AAV9) or hIDS transgene product can be assessed by ELISPOT assay. Pharmacokinetics of IDS expression in CSF, serum, and urine, as well as vector concentration (PCR vs. AAV9 DNA), may also be monitored.

[0101] The combination of gene therapy delivery of rAAV.hIDS to the CNS with systemic delivery of hIDS is encompassed by the methods of the invention. Systemic delivery may be achieved using ERT (e.g., using Elaprase® (idursulfase)) or additional gene therapy using a rAAV.hIDS with liver tropism (e.g., rAAV.hIDS with an AAV8 capsid).

[0102] Additional clinical efficacy measures related to systemic delivery include orthopedic measurements such as bone mineral density, bone mineral content, bone geometry and strength, and bone density measured by dual-energy X-ray absorptiometry (DXA); height (standing height / lying length Z-score); markers of bone turnover: serum osteocalcin (OCN) and bone-specific alkaline phosphatase (BSAP) measurements, carboxy-terminal telopeptide of type I collagen (ICTP) and carboxy-terminal telopeptide α1 chain of type I collagen (CTX); flexibility and strength: Biodex and Physical Therapy assessments, including a 6-minute walking study (a Biodex III isokinetic strength testing system is used to assess each participant's knee and elbow strength); Active Joint Range of Motion (ROM); Child Health Assessment Questionnaire / Health Assessment Questionnaire (CHAQ / HAQ) Disability Index Score; Electromyographic (EMG) and / or Oxygen Utilization to Monitor an individual's cardiorespiratory Fitness: Maximum oxygen uptake (VO2 peak) during exercise testing; Apnea / Hypopnea Index (AHI); Forced Vital Capacity (FVC); Left Ventricular Mass (LVM).

[0103] In certain embodiments, a method for diagnosing and / or treating or monitoring treatment of MPS II in a patient is provided, comprising obtaining a cerebrospinal fluid or plasma sample from a human patient suspected of having MPS II; detecting a spermine concentration level in the sample; diagnosing the patient with a mucopolysaccharidosis selected from MPS II in patients with spermine concentrations greater than 1 ng / mL; and delivering an effective amount of a human IDS to the diagnosed patient as provided herein.

[0104] In another embodiment, the method includes monitoring and adjusting MPS II therapy.

[0105] Such methods include obtaining a cerebrospinal fluid or plasma sample from a human patient undergoing treatment for MPS II; detecting spermine concentration levels in the sample by performing mass spectrometry analysis; and adjusting the dosage of an MPS II therapeutic agent. For example, a "normal" human spermine concentration is about 1 ng / mL to 2 ng / mL or less in cerebrospinal fluid. However, patients with untreated MPS II may have spermine concentration levels greater than 2 ng / mL and up to about 100 ng / mL. As the patient's levels approach normal levels, Conversely, if a patient has higher than desired MPS II levels, the patient may be provided with a higher dose or additional treatment, such as ERT.

[0106] Spermine concentration can be determined using a suitable assay, such as the assay described in J. Sanchez-Lopez et al., "Underivatives polyamine analysis is performed on plant samples by ion pair liquid chromatography coupled" (J. Sanchez-Lopez et al., 2001). with electrospray tandem mass spectrometry,” Plant Physiology and Biochemistry, 47(2009):592-598, available online 28 Feb 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, quantitative isotope dilution liquid chromatography (LC) / mass spectrometry (MS) assay. Other suitable assays may also be used.

[0107] In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing neurocognition 52 weeks after administration in pediatric subjects with MPS II who have early neurocognitive impairment. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the relationship between CSF glycosaminoglycans (GAGs) and neurocognition in MPS II 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 II patients as measured by magnetic resonance imaging (MRI), e.g., by volumetric analysis of gray matter and CSF ventricles. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the pharmacodynamic effect of the therapeutic agent on biomarkers (e.g., GAGs, HS) in the cerebrospinal fluid (CSF), serum, and urine of MPS II patients. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the impact of the therapeutic agent on quality of life (QOL) in MPS II patients. In some embodiments, the efficacy of a therapeutic agent described herein is determined by assessing the effect of the therapeutic agent on MPS II 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 milestones in MPS II patients.

[0108] When expressed from the rAAV vectors described herein, an expression level of at least about 2% of hIDS detected in CSF, serum, or other tissues may provide a therapeutic effect. However, higher expression levels may be achieved. Such expression levels may be 2% to about 100% of normal functional human IDS levels. In certain embodiments, higher-than-normal expression levels may be detected in CSF, serum, or other tissues.

[0109] In certain embodiments, the methods of treating, preventing, and / or ameliorating MPS II and / or symptoms thereof described herein result in a significant increase in the neurocognitive development quotient (DQ) in treated patients as assessed using the Bayley Scales of Infant Development. In certain embodiments, the methods of treating, preventing, and / or ameliorating MPS II and / or symptoms thereof described herein result in a decrease in DQ of 15 points or less in treated patients relative to untreated / natural historical control data in patients with Hunter syndrome.

[0110] In certain embodiments, the methods of treating, preventing, or preventing MPS II and / or its symptoms described herein are provided. The methods of preventing and / or ameliorating MPS II and / or its symptoms described herein result in a significant increase in functional human IDS levels. In certain embodiments, the methods of treating, preventing, and / or ameliorating MPS II and / or its symptoms described herein result in a significant decrease in GAG levels as measured in a patient's serum, urine, and / or cerebrospinal fluid (CSF) sample.

[0111] Combination therapy The combination of gene therapy delivery of rAAV.hIDS to the CNS with systemic delivery of the hIDS is encompassed by certain embodiments of the invention. Systemic delivery may be achieved using ERT (e.g., with Elaprase®) or additional gene therapy with a liver-tropic rAAV.hIDS (e.g., rAAV.hIDS carrying an AAV8 capsid).

[0112] In certain embodiments, intrathecal administration of rAAV9.hIDS is coadministered with a second AAV.hIDS injection, e.g., to the liver. In such cases, the vectors may be the same. 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), such as a liver-specific promoter and a CNS-specific promoter. Additionally or alternatively, each of the vector stocks may have a different capsid. For example, a vector stock directed to the liver may have a capsid selected from, among others, AAV8, AAVrh64R1, AAVrh64R2, rh8, rh10, AAV3B, or AAVdj. In such a regimen, the dose of each vector stock is adjusted so that the total vector delivered intrathecally is approximately 1 x 10 8 GC~1×10 14 In other embodiments, the combined vector delivered by both routes may be adjusted to be within the GC range; 11 GC~1×10 16 Alternatively, each vector contains approximately 10 8 GC~about 10 12 Such doses may be delivered at substantially the same time or at different times, for example, from about 1 day to about 12 weeks, or from about 3 days to about 30 days, or any other suitable time.

[0113] In some embodiments, the treatment method includes: (a) administering a hIDS enzyme or liver-specific rAAV-hIDUA in an amount sufficient to induce transgene-specific tolerance to a patient with MPS II and / or Hunter syndrome symptoms; and (b) administering rAAV.hIDS to the patient's CNS, wherein the rAAV.hIDS directs the expression of therapeutic levels of hIDS in the patient.

[0114] In further embodiments, methods are provided for treating a human patient with symptoms associated with MPS II and / or Hunter syndrome, comprising tolerizing a patient with symptoms associated with MPS II and / or Hunter syndrome with a sufficient amount of hIDS enzyme or hepatotropic rAAV-hIDS to induce transgene-specific tolerance, followed by rAAV-mediated delivery of the hIDS to the patient. In certain embodiments, the patient is administered the rAAV.hIDS by hepatotropic injection, e.g., if the patient is under 4 weeks of age (neonatal) or an infant, to tolerize the patient to the hIDS, followed by intrathecal injection, if the patient is an infant, child, and / or adult, to express therapeutic levels of the hIDS in the CNS.

[0115] In one example, an MPS II patient is tolerized by delivering a hIDS to the patient within two weeks of birth, e.g., within about 0 to about 14 days, or about 1 to about 12 days, or about 3 to about 10 days, or about 5 to about 8 days, i.e., the patient is a newborn. In other embodiments, older infants may be selected. The tolerizing dose of hIDS is administered by administering rAAV However, in another embodiment, the dose is delivered by direct delivery of the enzyme (enzyme replacement therapy). Methods for producing recombinant hIDS have been described in the literature.

[0116] Additionally, recombinant hIDS, commercially produced as Elaprase® (idursulfase), may be useful for systemic delivery. Although less preferred at present, the enzyme may be delivered via "naked" DNA, RNA, or another suitable vector. In one embodiment, the enzyme is delivered to the patient intravenously and / or intrathecally. In another embodiment, another route of administration (e.g., intramuscular, subcutaneous, etc.) is used. In one embodiment, MPS II patients selected for tolerization may not express detectable amounts of hIDS prior to the initiation of the tolerization dose. When recombinant human IDS enzyme is delivered, an intravenous rhIDS injection may consist of approximately 0.5 mg / kg body weight. Alternatively, higher or lower doses may be selected. Similarly, when expressed from a vector, lower levels of expressed protein may be delivered. In one embodiment, the amount of hIDS delivered for tolerization is lower than the therapeutically effective amount. However, other doses may be selected.

[0117] Typically, after 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, although other time points within these ranges may also be selected, as may longer or shorter waiting periods.

[0118] Alternatively, in addition to the vector, immunosuppressive therapy may be administered before, during, and / or after vector administration. Immunosuppressive therapy may include prednisolone, mycophenolate mofetil (MMF), and tacrolimus or sirolimus, as described above. Tacrolimus-free regimens, as described below, may be preferred.

[0119] 5.4.Manufacturing One embodiment provides for the production of the rAAV.hIDS pharmaceutical composition described herein (Example 4 below). An exemplary production process is shown in Figures 10A and 10B. The rAAV.hIDS vector may be produced as shown in the flow diagrams shown in Figures 10A and 10B. Briefly, cells are produced in suitable cell culture (e.g., HEK 293) cells. Methods for producing the gene therapy vectors described herein include methods well known in the art, such as generating the plasmid DNA used to produce the gene therapy vector, producing the vector, and purifying the vector. In some embodiments, the gene therapy vector is an AAV vector, and the generated plasmids are an AAV cis plasmid encoding the AAV genome and the gene of interest, an AAV trans plasmid containing the AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process may include method steps such as initiating cell culture, passaging the cells, seeding the cells, transfecting the cells with plasmid DNA, changing the medium after transfection into serum-free medium, and harvesting the vector-containing cells and culture medium. The harvested vector-containing cells and culture medium are referred to herein as the crude cell harvest.

[0120] This crude cell harvest may then be subjected to process steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare bulk vector.

[0121] Two-step affinity chromatography purification at high salt concentrations, followed by anion exchange resin chromatography, is used to purify the vector drug and remove empty capsids. These methods are described in International Patent Application No. PCT / US2016, filed December 9, 2016. This is described in further detail in U.S. Patent Application No. 62 / 322,071, filed April 13, 2016, and U.S. Patent Application No. 62 / 226,357, filed December 11, 2015, entitled "Scalable Purification Method for AAV9," which are incorporated by reference herein. International Patent Application No. PCT / US2016 / 065976, filed December 9, 2016, and its priority document, filed April 13, 2016, for a method for purifying AAV8; U.S. Patent Application No. 62 / 322,098, filed December 11, 2015, and its priority document, 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 document, U.S. Patent Application No. 62 / 322,055, filed April 13, 2016, for a method for purifying AAV8; No. 62 / 266,347, also filed December 11, 2015, entitled "AAVrh10," 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 No. 62 / 26,351, filed December 11, 2015, entitled "Scalable Purification Method for AAV1," are all incorporated herein by reference.

[0122] 5.5. DEVICES AND METHODS FOR DELIVERY OF PHARMACEUTICAL COMPOSITIONS TO THE CEREBROSPINAL FLUID In one aspect, 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. 11. Alternatively, other devices and methods may be selected. This method includes the steps of advancing a spinal needle into a patient's cisterna magna, connecting a length of flexible tubing to the proximal hub of the spinal needle, and connecting an output port of a valve to the proximal end of the flexible tubing. After the advancing and connecting steps and after allowing the tubing to self-prime 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 then connecting a second container containing a quantity of pharmaceutical composition to the vector inlet port of the valve. After connecting the first and second containers to the valve, a passage 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 spinal needle. After the pharmaceutical composition is injected, a fluid flow path is opened through the flush inlet and outlet ports of the valve, and an isotonic solution is injected into the spinal needle to flush the pharmaceutical composition into the patient.

[0123] In another aspect, a device for intracisternal delivery of a pharmaceutical composition is provided. The device includes a first container containing a quantity of the pharmaceutical composition, a second container containing an isotonic solution, and a spinal needle capable of directly releasing the pharmaceutical composition from the device into cerebrospinal fluid in the cisterna magna of a patient. The device further includes a first inlet port interconnected to the first container, a second inlet port interconnected to the second container, an outlet port interconnected to the spinal needle, and a valve with a luer lock for controlling the flow of the pharmaceutical composition and the isotonic solution through the spinal needle.

[0124] As used herein, the term Computed Tomography (CT) refers to radiography 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.

[0125] 11 includes one or more containers 12 and 14 interconnected via a valve 16. The containers 12 and 14 provide a fresh source of a substance, such as a pharmaceutical composition, a drug, a vector, 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 injection of a fluid into a patient.

[0126] 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 pharmaceutical composition, referred to herein as a "vector syringe." By way of example only, container 12 may contain approximately 10 cc of pharmaceutical composition, etc.

[0127] Similarly, container 14 may be provided in the form of a separate syringe, cannula, etc. containing a quantity of saline, and may be referred to as a "flush syringe." By way of example only, container 14 may contain approximately 10 cc of saline.

[0128] Alternatively, containers 12 and 14 may be provided in forms other than syringes and may be integrated into a single device, such as an integrated medical injection device having a pair of separate chambers, one for the pharmaceutical composition and one for saline, and the size of the chambers or containers may be provided as needed to contain the desired amount of fluid.

[0129] In the illustrated embodiment, valve 16 is provided as a four-way stopcock having a swivel male luer lock 18. Valve 16 interconnects containers 12 and 14 (i.e., a vector syringe and a flush syringe in the illustrated embodiment) and the swivel male luer lock allows the path through valve 16 to be closed or opened to the respective containers 12 and 14. In this manner, the path through valve 16 may be closed to both the vector syringe and the flush syringe, or may be opened to selected ones of the vector syringe and the flush syringe. As an alternative to a four-way stopcock, the valve may be a three-way stopcock or a fluid control device.

[0130] In the illustrated embodiment, the valve 16 is connected to one end of a length of extension tubing 20 or similar conduit for 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 long.

[0131] In the illustrated embodiment, the opposite end 22 of the tubing 12 is connected to a T-connector extension set 24, which in turn is connected to a spinal needle 26. By way of example, the needle 26 may be a 5-inch 22- or 25-gauge spinal needle. Additionally, optionally, the spinal needle 26 may be connected to an introducer needle 28, such as a 3.5-inch 18-gauge introducer needle.

[0132] During use, the spinal needle 26 and / or optional introducer needle 28 may be advanced into the patient toward the cisterna magna. After needle advancement, a computed tomography (CT) image may be obtained, allowing visualization of the needle 26 and / or 28 and associated soft tissues (e.g., paraspinal muscles, bone, brainstem, and spinal cord). Correct needle placement is confirmed by observation of cerebrospinal fluid (CSF) within the needle hub and visualization of the needle tip within the cisterna magna. A relatively short extension tube 20 may then be attached to the inserted spinal needle 26, and a four-way stopcock 16 may then be attached to the opposite end of the tube 20.

[0133] The above assembly is capable of "self-priming" with the patient's CSF. A prefilled saline flush syringe 14 is then attached to the flush inlet port of the four-way stopcock 16, and then a vector syringe 12 containing a pharmaceutical composition is attached 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 vector syringe are slowly injected into the patient through the valve 16 and the assembled device over a period of time. By way of example only, this period may be approximately 1-2 minutes and / or any other desired period of time.

[0134] After the contents of the vector syringe 12 have been injected, the swivel lock 18 of the stopcock 16 is turned to a second position so that the stopcock 16 and needle assembly can be flushed with a desired amount of saline using the attached prefilled flush syringe 14. By way of example only, 1-2 cc of saline may be used; however, more or less may be used as needed. The saline forces all or most of the pharmaceutical composition through the assembled device and into the patient, leaving little or no pharmaceutical composition remaining within the assembled device.

[0135] After the assembled apparatus has been flushed with saline, slowly remove the entire assembled device, including the needle(s), extension tubing, stopcock, and syringe, from the subject and place it on a surgical tray for disposal in a biohazard waste receptacle or hard container (in the case of needle(s)).

[0136] The screening process may be conducted by the investigator, which may ultimately lead to an intracisternal (IC) procedure. The investigator may describe the process, procedures, administration procedures themselves, and any potential safety risks to provide sufficient information to the subject (or designated caregiver). Medical history, concomitant medications, physical examination, vital signs, electrocardiogram (ECG), and laboratory test results are obtained, performed, and provided to the neuroradiologist, neurosurgeon, and anesthesiologist for use in screening assessment of subject eligibility for the IC procedure.

[0137] To allow sufficient time to screen for eligibility, the following procedures may be performed any time between the initial screening visit and up to 1 week before the study visit. For example, on "Day 0," a head / neck magnetic resonance imaging (MRI) with or without gadolinium (i.e., eGFR > 30 mL / min / 1.73 m) is required. 2 In addition to a head / neck MRI, investigators may determine the need for further evaluation of the neck with flexion / extension testing. MRI protocols may include T1, T2, DTI, FLAIR, and CINE protocol images.

[0138] Additionally, head / neck MRA / MRV may be obtained according to institutional protocols (i.e., subjects with a history of intradural / transdural surgery may be excluded or may require further testing (e.g., radionucleotide cisternalography)) that allow for adequate assessment of the CSF and identification of possible blockages or lack of communication between CSF spaces.

[0139] Neuroradiologists, neurosurgeons, and anesthesiologists ultimately discuss and determine each subject's eligibility for the IC procedure based on all available information (scans, medical history, physical exam, laboratory values, etc.) Preoperative anesthesia evaluations may be obtained from "Day -28" to "Day 1," where detailed assessments of the airway, neck (shortening / thickening), and head range of motion (cervical flexion) are obtained, keeping in mind the special physiological needs of MPS subjects.

[0140] Prior to the IC procedure, the CT suite will verify that the following equipment and medications are present: Adult lumbar puncture (LP) kits (provided by each facility); BD (Becton Dickinson) 22 or 25 gauge x 3-7 inch spinal needle (Quincke bevel); a coaxial introducer needle (for introducing spinal needles) used at the discretion of the interventionalist; 4-way small diameter stopcock with swivel (spin) male luer lock; T-connector extension set (tubing) with female Luer lock adapter, approximately 6.7 inches long; Omnipaque 180 (iohexol) for intrathecal administration; iodinated contrast media for intravenous (IV) administration; 1% lidocaine injection (if not included in the adult LP kit); Prefilled 10cc saline (sterile) flush syringe; radiopaque marker(s); surgical preparation equipment / shaving razors; Pillows / supports to allow proper positioning of the intubated subject; endotracheal intubation equipment, general anesthesia machines and mechanical ventilators; Intraoperative neurophysiological monitoring (IONM) equipment (and personnel); and A vector-containing 10 cc syringe; prepare according to a separate pharmacy manual and transport to the CT / operating room (OR) room.

[0141] Informed consent for this procedure will be confirmed and documented in the medical record and / or study file. Separate consent for the procedure by the radiologist and anesthesiologist will be obtained according to institutional requirements. The subject will have intravenous access in an appropriate hospital care unit according to institutional guidelines (e.g., two IV access sites). Intravenous fluids will be administered at the discretion of the anesthesiologist. At the discretion of the anesthesiologist and institutional guidelines, the subject may be induced with the administration of general anesthesia and undergo endotracheal intubation in an appropriate patient care unit, holding area, or surgical / CT procedure room.

[0142] A lumbar puncture is performed, first removing 5 cc of cerebrospinal fluid (CSF), followed by intrathecal injection of contrast agent (Omnipaque 180) to aid in visualization of the cisterna magna. Appropriate subject positioning maneuvers may be performed to facilitate diffusion of the contrast agent into the cisterna magna.

[0143] An intraoperative neurophysiological monitoring (IONM) device is attached to the subject. The subject is placed on the CT scanner table in the prone or lateral position. Appropriate staff must be present to ensure the subject's safety during transport and positioning. If deemed appropriate, the subject may be positioned in a manner that results in neck flexion to the degree deemed safe during preoperative evaluation and has a demonstrated normal neurophysiological monitor signal after positioning.

[0144] The following staff will be confirmed to be present and may be verified on-site: interventionalist / neurosurgeon to perform procedures; anesthesiologist and respiratory technician(s); nurses and physician assistants; CT (or OR) technicians; neurophysiology technicians; and site coordinator. A "time-out" may be completed per Joint Commission / hospital protocol to confirm correct subject, procedure, location, positioning, and presence of all necessary equipment in the room. The site investigator may then confirm with staff that the subject can be prepared.

[0145] The subject's skin below the skull base is shaved appropriately. A CT scout image is performed, followed by a pre-procedure planning CT with IV contrast if deemed necessary by the interventionalist to localize the target location and image the vasculature. After the target site (cisterna magna) is identified and the needle trajectory is planned, the skin is prepared and draped using aseptic technique according to institutional guidelines. A radiopaque marker is placed at the target skin location indicated by the interventionalist. The skin below the marker is anesthetized by infiltration with 1% lidocaine. A 22G or 25G spinal needle is advanced toward the cisterna magna, with the option of using a coaxial introducer needle.

[0146] After needle advancement, CT images are obtained using the thinnest CT slice thickness feasible using the facility's equipment (ideally ≤2.5 mm). Serial CT images are obtained using the lowest possible radiation dose that allows adequate visualization of the needle and associated soft tissues (e.g., paraspinal muscles, bone, brainstem, and spinal cord). Accurate needle placement is confirmed by observation of CSF within the needle hub and visualization of the needle tip within the cisterna magna.

[0147] The interventionalist ensures that the vector syringe is located near, but outside, the sterile field. Gloves, masks, and eye protection are applied by staff assisting the procedure within the sterile field before handling or administering the pharmaceutical composition within the vector syringe.

[0148] The extension tube is attached to the inserted spinal needle and then attached to the 4-way stopcock. Once the device is "self-primed" with the subject's CSF, a 10 cc prefilled 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.

[0149] After opening the stopcock outlet port to the vector syringe by placing the stopcock swivel lock in the first position, slowly inject the contents of the vector syringe (over approximately 1-2 minutes), being careful not to apply excessive force to the syringe plunger during injection. After the vector syringe contents have been injected, the stopcock swivel lock can be turned to the second position so that the attached prefilled flush syringe can be used to flush the stopcock and needle assembly with 1-2 cc of saline.

[0150] When ready, the interventionalist alerts the staff to remove the device from the subject. In one movement, slowly remove the needle, dilator tube, stopcock, and syringe from the subject and place them on a surgical tray for disposal in a biohazard waste receptacle or rigid container (for needles).

[0151] The needle insertion site is inspected for signs of bleeding or CSF leakage and treated as directed by the investigator. The site is dressed using gauze, surgical tape, and / or Tegaderm bandages as indicated. The subject is then removed from the CT scanner and placed supine on a stretcher. Appropriate staff are present to ensure the subject's safety during transport and placement.

[0152] Anesthesia is discontinued and the subject is cared for according to institutional guidelines for post-anesthesia care. Neurophysiological monitors are removed from the subject. The head of the gurney on which the subject is lying should be elevated slightly (approximately 30 degrees) during recovery. The subject is transported to the appropriate post-anesthesia care unit according to institutional guidelines. After the subject has sufficiently regained consciousness and is stable, they are admitted to the appropriate floor / unit for protocol-mandated assessments. A neurological evaluation is performed according to protocol, and the Investigator will oversee the subject's care in collaboration with hospital and study staff.

[0153] In one embodiment, a method of delivering a composition provided herein comprises the steps of advancing a spinal needle into the patient's cisterna magna; connecting a length of flexible tubing to the proximal hub of the spinal needle and connecting an output port of a valve to the proximal end of the flexible tubing; after this systemic and connecting step, and after allowing the tubing to self-prime the patient's cerebrospinal fluid, connecting a first container containing a quantity of isotonic solution to the flush inlet port of the valve and then connecting a second container containing a quantity of pharmaceutical composition to the vector inlet port of the valve; after connecting the first and second containers to the valve, opening a fluid flow path between the vector inlet and outlet ports of the valve and injecting the pharmaceutical composition into the patient through the spinal needle; after injecting the pharmaceutical composition, opening a passage for fluid flow through the flush inlet and outlet ports of the valve and injecting isotonic solution into the spinal needle to flush the pharmaceutical composition into the patient. In certain embodiments, the method further comprises confirming proper placement of the distal tip of the spinal needle within the cisterna magna prior to connecting the tubing and valve to the hub of the spinal needle. In certain embodiments, the confirming step comprises visualizing the distal tip of the spinal needle within the cisterna magna using computed tomography (CT) imaging. In certain embodiments, the confirming step comprises observing the presence of the patient's cerebrospinal fluid within the hub of the spinal needle.

[0154] In the above method, the valve may be a stopcock with a swivel luer lock adapted to swivel to a first position, allowing flow from the vector inlet port to the outlet port while simultaneously blocking flow through the flush inlet port to a second position, and allowing flow from the flush inlet port to the outlet port while simultaneously blocking flow through the vector inlet port, wherein the swivel luer lock is disposed in the first position when the pharmaceutical composition is injected into the patient and in the second position when the pharmaceutical composition is being flushed into the patient with an isotonic solution. In certain embodiments, after injecting the isotonic solution into the spinal needle to flush the pharmaceutical composition into the patient, the spinal needle is withdrawn from the patient with the tubing, valve, and first and second containers connected thereto as an assembly. 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 located at the hub of the spinal needle and interconnects the tubing to the spinal needle. Optionally, the spinal needle comprises an introducer needle at the distal end of the spinal needle. The spinal needle may be a 5 inch, 22 or 24 gauge spinal needle. In a specific embodiment, the introducer needle is a 3.5 inch, 18 gauge introducer needle.

[0155] In certain aspects, the method utilizes a device comprising at least: a first container for containing a quantity of a pharmaceutical composition; a second container for containing an isotonic solution; a spinal needle through which the pharmaceutical composition can be directly expelled from the device into the cerebrospinal fluid in the patient's cisterna magna; and 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 spinal needle, and a Luer lock needle for controlling the flow of the pharmaceutical composition and the isotonic solution through the spinal cord. In certain embodiments, the valve is a stopcock with a swivel Luer lock adapted to swivel to a first position, allowing flow from the first inlet port to the outlet port while simultaneously blocking flow through the first inlet port to the second position, and allowing flow from the second inlet port to the outlet port while simultaneously blocking flow through the first inlet port. 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 spinal needle is interconnected to the valve via a length of flexible tubing. A T-shaped connector may interconnect the tubing to the spinal needle. In certain embodiments, the spinal needle is a 5-inch, 22- or 24-gauge spinal needle. In certain embodiments, the device further comprises an introducer needle connected to the distal end of the spinal needle. Optionally, the introducer needle is a 3.5-inch, 18-gauge introducer needle.

[0156] The following examples are illustrative only and are not intended to limit the invention described herein. [Example]

[0157] 6. Working Example Example 1: Protocol for Treatment of Human Subjects This example relates to a gene therapy treatment for patients with MPS II, or Hunter syndrome. In this example, the gene therapy vector AAV9.CB.hIDS, a replication-deficient adeno-associated viral vector 9 (AAV9) expressing a modified hIDS gene encoding the wild-type hIDS enzyme, is administered to the central nervous system (CNS) of MPS II patients. Under anesthesia, a dose of AAV vector is injected directly into the CNS. As described herein, the effectiveness of treatment is assessed using clinical measures of neurocognitive development and / or surrogate markers, e.g., biomarkers, such as reductions in pathogenic GAG and / or heparin sulfate (HS) levels in the subject's CSF or serum.

[0158] A. Gene Therapy Vectors The gene therapy vector is a non-replicating recombinant adeno-associated virus (AAV) vector of serotype 9 expressing human iduronate-2-sulfatase (IDS), designated in this example as AAV9.CB.IDS (see Figure 1). The AAV9 serotype allows for efficient expression of the hIDS product in the CNS after IC administration.

[0159] The IDS expression cassette is flanked by inverted terminal repeats (ITRs), and expression is driven by a hybrid of the cytomegalovirus (CMV) enhancer and chicken beta-actin promoter (CB7). The transgene contains a chicken beta-actin intron and a rabbit beta-globin polyadenylation (polyA) signal.

[0160] The vector is suspended in formulation buffer (Elliots B Solution, 0.001% Pluronic F68). The construct is packaged into AAV9 capsids and purified and titered as previously described in M. Lock et al., Human Gene Ther, 21:1259-1271 (2010). The manufacturing process is described in more detail in Example 4 below.

[0161] Vector Generation: A series of vectors were generated. One plasmid contains a codon-optimized IDS sequence (nt 1177 to nt 2829 of SEQ ID NO:11). Two others use a shortened version of the CB7 promoter (CB6) to express a second protein called SUMF1 [SEQ ID NO:7 and SEQ ID NO:10]. The resulting vector genome, AAV.CB7.CI.hIDSco.RBG, has the sequence from nt 2 to nt 3967 of SEQ ID NO:11, while AAV.CB6.hIDSco.IRES.hSUMF1co has the sequence from nt 11 to nt 4964 of SEQ ID NO:8. Plasmids were constructed by codon-optimizing and synthesizing the hIDS sequence, and the resulting constructs were then cloned into the AAV2 ITR-flanked expression cassette, CI, and RBG expression elements containing plasmids pENN.AAV.CB7.CI.RBG or pENN.AAV.CB6.CI.RBG, CB7, or CB6, to generate the vector genomes described above.

[0162] Additional plasmids containing native human IDS cDNA were generated; these plasmids are referred to herein as pAAV.CB7.CI.hIDS.RBG and pAAV.CB6.CI.hIDS.IRES.SUMF1.RBG. The vector genomes derived from these plasmids [nt2 to nt3967 of SEQ ID NO:3 and nt11 to nt4964 of SEQ ID NO:5] are single-stranded DNA genomes with AAV2-derived ITRs flanking the hIDS expression cassette. Expression from the transgene cassette is driven by the CB7 or CB6 promoter, a hybrid between the CMV immediate-early enhancer (C4) and the chicken beta-actin promoter; transcription from this promoter is enhanced by the presence of the chicken beta-actin intron (CI). The poly(A) signal of the expression cassette is RBG poly(A). A plasmid is constructed by synthesizing the hIDS sequence and then cloning it into the expression cassette flanked by AAV2 ITRs containing CB7, CI, and RBG expression elements in the plasmid pENN.AAV.CB7.CI.RBG or pENN.AAV.CB6.CI.RBG, resulting in the vector genome described above.

[0163] Array element description: Inverted terminal repeats (ITRs): AAV ITRs (GenBank #NC001401) are sequences that are identical at both ends but in opposite orientations. The AAV2 ITR sequence functions as both an origin of vector DNA replication and a packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. Thus, the ITR sequence represents the only cis sequence required for vector genome replication and packaging.

[0164] CMV immediate-early enhancer (382 bp, C4; GenBank #K03104.1). This element is present in the vector genome plasmid.

[0165] The chicken beta-actin promoter (282 bp; CB; GenBank #X00182.1) is used to drive high levels of hIDS expression.

[0166] Chicken beta-actin intron: A 973-bp intron from the chicken beta-actin gene (GenBank #X00182.1) is present in the vector expression cassette. The intron is transcribed but is removed from the mature messenger RNA (mRNA) by splicing, flanking it with sequences. The presence of an intron in an expression cassette has been shown to facilitate transport of mRNA from the nucleus to the cytoplasm, thereby facilitating the accumulation of stable levels of mRNA for translation. This is a common feature in genetic vectors intended to increase the level of gene expression. This element is present in both the vector genome and the plasmid.

[0167] The iduronate-2-sulfatase coding sequence:hIDS sequence was synthesized [SEQ ID NO:1]. The encoded protein is 550 amino acids, as described earlier in this specification [SEQ ID NO:2; Genbank NP_000193, UnitProtKB / Swiss-Prot (P22304.1)]. See SEQ ID NOs:1 and 2. The codon-optimized hIDS coding sequence is shown in SEQ ID NO:8 from nt 3423 to nt 4553 and in SEQ ID NO:11 from nt 1937 to nt 3589.

[0168] 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 the vector genome and the plasmid.

[0169] B. Medication and Route of Administration Patients were 2.5 × 10 10 ~3.6×10 11 GC / g brain mass equivalent to 1.0 × 10 13 ~5.0×10 14 Receive a single intrathecal / intracisternal administration of rAAV9.CB7.hIDS within the GC (flat dose) range. Alternatively, administer the following flat doses to patients in the indicated age groups: Newborn: Approx. 3.8 x 10 12 ~Approx. 1.9×10 14 GC; 3 to 9 months: Approx. 6 x 10 12 ~Approx. 3×10 14 GC; 9-36 months: Approx. 1 x 10 13 ~Approx. 5×10 14 GC; ·3~12 years: Approx. 1.2×10 13 ~about 6×10 14 GC; Ages 12+: Approx. 1.4 x 10 13 ~Approx. 7.0×10 14GC; 18+ (adult): Approx. 1.4 x 10 13 ~Approx. 7.0×10 14 G.C.

[0170] To ensure that empty capsids are removed from the dose of rAAV9.CB7.hIDS administered to patients, empty capsids are removed from vector particles by cesium chloride gradient ultracentrifugation or ion exchange chromatography during the vector purification process, as discussed herein. Isolate the capsids.

[0171] C. Patient Subpopulations Suitable patients include male or female subjects of the following ages: · Newborns; · 3-9 months old; 9-36 months of age; 3-12 years old; 12+ years old; 18+ years old (adult).

[0172] D. Clinical Objective Measurements A primary clinical objective is to prevent and / or optionally reverse neurocognitive decline associated with MPS II deficiency. Clinical objectives can be determined by measuring neurocognition, such as that measured by the Bayley Scales of Infant and Toddler Development, Third Edition, BSID-III. Other suitable measures are adaptive behavior assessments, such as those measured by the Vernacular and Adaptive Behavior Scales, Second Edition (VABS-II), and quality of life measures, such as those measured by the Infant Toddler Quality of Life Questionnaire™ (ITQOL).

[0173] Secondary endpoints include assessment of biomarkers and clinical outcomes. Optionally, hearing may be assessed as a secondary endpoint. Urine will be assessed for total GAG content and heparan sulfate. Serum will be assessed for IDS activity, anti-IDS antibodies, GAG, and heparin cofactor II-thrombin complex concentrations. CSF will be assessed for GAG, IDS activity, anti-IDS antibodies, and heparin sulfate. The presence of anti-IDS antibodies will be assessed, as will the pharmacokinetics of IDS expression in CSF and serum. Volumetric analysis of gray matter and CSF ventricles will also be performed by MRI.

[0174] Example 2: Studies in a murine model of mucopolysaccharidosis type II - Delivery of adeno-associated viral vectors to the CSF attenuates central nervous system disease in mucopolysaccharidosis type II mice A. AAV9 delivery to the cerebrospinal fluid corrects central nervous system disease Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder that typically manifests in early childhood with bone and joint deformities, cardiac and respiratory disease, and developmental delay. Systemic delivery of the defective enzyme, iduronate-2-sulfatase (IDS), ameliorates many symptoms of MPS II, but because the enzyme does not cross the blood-brain barrier, there is currently no effective method to prevent central nervous system (CNS) disease progression. Using a mouse model of MPS II, AAV serotype 9 vector-mediated delivery of the IDS gene was evaluated as a means to achieve continuous IDS expression in the CNS. IDS knockout mice were treated with three vector doses (low-3 × 10 8 , medium-3×10 9 or high-3×10 10Mice received a single injection of IDS into one lateral ventricle of the 1000-kDa genome copies of the 1000-kDa gene and were sacrificed either 3 weeks after vector administration (n = 7–8 mice / group) for evaluation of vector biodistribution and IDS expression, or 3 months after vector administration (n = 7–8 mice / group) to assess the impact of gene transfer on disease progression. IDS activity was detectable in cerebrospinal fluid, reaching 15% of wild-type levels in the low-dose cohort and 268% of normal at the highest dose. Brain enzyme activity ranged from 2.7% of normal in the low-dose cohort to 32% of normal in the high-dose cohort. Quantification of brain accumulation lesions by staining for ganglioside GM3 demonstrated a dose-dependent correction, with reductions of 35%, 46%, and 86% in the low-, mid-, and high-dose cohorts, respectively. Treated mice also showed a significant improvement in IDS activity. , and showed improved cognitive function in a novel object recognition test. These findings indicate that intrathecal AAV-mediated gene transfer can serve as a platform for sustained enzyme delivery to the CNS, addressing this important unmet need for patients with MPS II.

[0175] B. Pharmacological and neurobehavioral effects of AAV9.CB.hIDS A knockout mouse model of MPS II (IDS knockout; IDS) was developed by replacing exons 4 and 5 of the IDS gene with a neomycin resistance gene. y / -(See Garcia et al., 2007, J Inherit Metab Dis, 30:924-34.) This model shows no detectable enzyme activity and develops histological storage lesions similar to those seen in MPS II patients. IDS knockout mice exhibit many clinical features of MPS II, including skeletal abnormalities. The neurobehavioral phenotype of the mice has not been extensively evaluated, but some studies have shown abnormalities (Muenzer et al., 2001, Acta Paediatr Suppl, 91(439):99-9), and therefore represents a relevant model for studying the efficacy of AAV9.CB.hIDS as a treatment for MPS II. In fact, this is the same knockout mouse model used to evaluate the efficacy of enzyme replacement therapy in MPS II, supporting clinical trials in this population. The following studies conducted in this MPS II mouse model established therapeutic activity of AAV9.CB.hIDS.

[0176] C. Materials and Methods Vectors. Human IDUA and IDS cDNAs were cloned into expression constructs containing the chicken beta-actin promoter, CMV enhancer, intron, and rabbit beta-globin polyadenylation sequence. The expression constructs were flanked by AAV2 inverted terminal repeats. AAV9 vectors were generated from these constructs by triple transfection of HEK293 cells and iodixanol purification as previously described (Lock et al. (2010). Hum Gene Ther 21:1259-71).

[0177] Animal Procedures. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. IDS knockout mice were obtained from the Jackson Laboratory (Stock no: 024744) and bred in-house. Wild-type male C57BL / 6 mice from the colony served as controls. At 2–3 months of age, animals were anesthetized with isoflurane and injected ICV with 5 μL of vector diluted in sterile phosphate-buffered saline. CSF was collected at necropsy by suboccipital puncture using a 32-gauge needle connected to polyethylene tubing. Terminal serum samples were collected by cardiac puncture. Animals were euthanized by exsanguination under ketamine / xylazine anesthesia. Death was established by cervical dislocation. The brain, heart, lungs, liver, and spleen were collected on dry ice. For histological experiments, brains were divided into an anterior half, which was fixed for LIMP2 immunohistochemistry, and a posterior half, which was fixed for GM3 immunohistochemistry. Two cohorts of mice were used for behavioral experiments. Initial cohorts of wild-type and IDS KO mice were tested in a series of procedures to determine whether gene deletion affected learning and memory. Low, medium, or high vector doses of AAV9.CB.hIDS (3 × 10 each) were injected into the brain. 8 , 3×10 9 or 3×10 10 A second cohort of mice treated with 1000 genome copies (GC) was used to investigate the feasibility of IT delivery as a strategy to rescue behavioral deficits.

[0178] Behavioral Procedures: All behavioral procedures were performed by an operator blinded to genotype and treatment group. Open field activity: Spontaneous activity in the open field was measured using the Photobeam Activity System (PAS)-Open Field (San Diego Instruments). Mice were individually placed in the arena for a 10-minute trial. Horizontal and vertical beam breaks were collected to assess general locomotion and rearing activity.

[0179] Y-maze: Short-term memory was assessed in a standard Y-maze (San Diego Instruments). The sequence and number of arm entries were recorded during an 8-minute trial. Spontaneous alternation (SA) was defined as consecutive entries into all three arms of the maze without immediately returning to the previously entered arm. Total arm entries (AE) were collected as a measure of motor activity. Spontaneous alternation was calculated as %SA = (SA / (AE-2) * 100).

[0180] Contextual fear conditioning: Conditioning experiments were performed as described by Abel et al. Cell. 1997 M ar 7;88(5):615-26. On the training day, mice were allowed to explore the specific conditioning chamber (Med Associates) for 300 seconds. A continuous 1.5 mA foot shock was delivered between 248 and 250 seconds. After an additional 30 seconds in the chamber, mice were returned to their home cage. 24 hours later, recall of the spatial context was assessed for 5 consecutive minutes in the same chamber where training had taken place. Memory was assessed using software (Freezescan, CleverSystems) used to score freezing behavior. Freezing rates during the 2.5-minute pre-stimulus period of the training session were compared with freezing rates upon re-exposure to the chamber. Increased freezing indicated learning had occurred.

[0181] Novel object recognition: The experimental apparatus consisted of a gray rectangular arena (60 cm x 50 cm x 26 cm) on a white floor and two unique objects: a 3.8 x 3.8 x 15 cm metal bar and a 3.2 cm diameter x 15 cm PVC pipe. Prior to exposure to the apparatus, mice were handled for 1–2 min per day for 5 days. During the 5-day habituation phase, mice were allowed to explore the empty arena for 5 min per day. During the training phase, mice explored the same two objects for 15 min to establish habituation. In the recall phase 24 h later, mice were returned to the arena, now containing the familiar and novel objects. Mice preferentially explored the novel object. A decreased preference for novelty suggests a lack of recall of the familiar object and therefore poor learning. All sessions were recorded and the time spent exploring the object was scored using an open-source image analysis program (Patel et al., Front Behav Neurosci. 2014 Oct 8;8:349).

[0182] Enzyme and GAG Assays. GAG, Hex, and IDUA assays were performed as previously described (Hinderer et al., 2015, Mol Ther 23:1298-307). IDS activity was measured using 1.25 mM IDUA in 0.1 M sodium acetate. 4-Methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate (Santa The 4-MU concentration was measured by incubating 20 μL of 0.01 M lead acetate (pH 5.0) solution (Cruz Biotechnology) with 10 μL of sample. After 2 h of incubation at 37°C, 45 μL of McIlvain buffer (0.4 M sodium phosphate, 0.2 M sodium citrate, pH 4.5) and 5 μL of recombinant human iduronidase (Aldurazyme, 0.58 mg / mL, Genzyme) were added to the reaction mixture and incubated overnight at 37°C. The mixture was diluted with glycine buffer (pH 10.9), and the released 4-MU was quantified by fluorescence (excitation 365 nm, emission 450 nm) relative to a standard dilution of free 4-MU.

[0183] Histology. The anterior half of the brain was fixed for LIMP2 immunohistochemistry and the posterior half was fixed for GM3 immunohistochemistry. The posterior halves were fixed for histochemistry. LIMP2 and GM3 immunohistochemistry were performed as previously described ( Hinderer et al., 2015 ). Ther 23:1298-307). The number of cells staining positive for LIMP2 and GM3 was quantified in four brain sections from each animal by a blinded reviewer.

[0184] Vector biodistribution. Tissues for vector biodistribution analysis were rapidly dissected and frozen on dry ice. Samples were stored at -80°C until the time of analysis. QIAmp DNA DNA was isolated from tissues using a Mini Kit, and vector genomes were quantified by TaqMan PCR as described (Wang et al., 2011, Hum. Gene Ther. 22:1389-1401).

[0185] ELISA for anti-hIDS antibodies. Polystyrene ELISA plates were coated overnight with 5 μg / mL of recombinant human IDS (R&D Systems) in PBS titrated to pH 5.8. Plates were washed and blocked for 1 hour with 2% bovine serum albumin in neutral PBS. Plates were then incubated with serum samples diluted 1:1000 in PBS. Bound antibodies were detected with HRP-conjugated goat anti-mouse antibody (Abcam) diluted 1:10,000 in PBS containing 2% BSA. The assay was performed using tetramethylbenzidine substrate and stopped with 2N sulfuric acid, followed by measuring absorbance at 450 nm. Titers were determined from a standard curve generated by serial dilutions of appropriately assigned positive serum samples at a titer of 1:10,000.

[0186] Statistics. Tissue GAG ​​content, Hex activity, and brain accumulation lesions in treated and untreated mice were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Open field and Y-maze dates were analyzed with a Students' t-test. Two-way ANOVA and Dunnett's post hoc analysis were applied to fear conditioning data to assess the effects of trial and genotype. In the novel object recognition test, the time to explore the novel object versus the familiar object was compared using a t-test for each group followed by Bonferroni correction for multiple comparisons.

[0187] Four age-matched groups of male IDS knockout mice and one group of wild-type littermates (8 mice per group (n=8)) received the following treatments at 2-3 months of age: Group 1: Untreated Group 2: Intracerebroventricular AAV9.CB.hIDS low dose 3×10 8 GC(1.875×10 9 GC / g brain mass) Group 3: Intracerebroventricular AAV9.CB.hIDS medium dose 3×10 9 GC(1.875×10 10 GC / g brain mass) Group 4: Intracerebroventricular AAV9.CB.hIDS high dose 3×10 10 GC(1.875×10 11 GC / g brain mass) Group 5: Untreated wild-type littermates.

[0188] D. Results: 2-3 month old male IDS y / - Mice were treated by intracerebroventricular (ICV) injection of an AAV9 vector expressing human IDS, driven by the chicken beta-actin promoter with a cytomegalovirus enhancer (AAV9.CB.hIDS). In one initial cohort, three vector doses [3 × 10 8 , 3×10 9 or 3×10 10 Mice were treated with one of the 1000 genome copies (GC) of the untreated IDS and sacrificed 3 weeks after vector administration for evaluation of vector biodistribution and IDS expression.y / - Mice and wild-type male littermates served as controls. Tissues were harvested for biodistribution analysis. Analysis of vector biodistribution in the high-dose group demonstrated efficient brain targeting, with an average of one vector genome per host diploid genome (Figure 3). Previous studies of AAV delivery to the CSF have demonstrated that CSF delivery is efficient and efficient. Consistent with previous studies, there was also vector escape to the periphery and efficient liver targeting with two or more vector genomes per host diploid genome (Figure 3). Brain tissue, CSF, and serum all showed a dose-dependent increase in IDS activity, approaching or exceeding wild-type levels in the high-dose group (Figures 2A-2C). In the mid- and high-dose cohorts, antibodies to human IDS were detected in the serum of some animals, but this did not appear to significantly affect circulating enzyme activity (Figure 8).

[0189] To evaluate the therapeutic potential of IT AAV9-mediated delivery of the IDS gene, y / - Additional cohorts of mice were treated with equivalent vector doses and evaluated at subsequent time points to assess the impact of gene transfer on disease progression. Two months after vector administration, mice were subjected to behavioral and neurocognitive testing. Three months after treatment, animals were sacrificed and tissues were harvested for histological and biochemical assessment of disease activity.

[0190] Consistent with the high levels of serum enzyme activity, GAG accumulation was reduced in the liver, with no significant trend toward normalization of GAG accumulation in the heart (Figures 4A-4B). Furthermore, activity of the lysosomal enzyme hexosaminidase (Hex), which was upregulated in the setting of GAG accumulation, was normalized in both tissues (Figures 4C-4D). These data demonstrate the potential for systemic therapeutic activity following intrathecal administration of AAV9.CB.hIDS.

[0191] Brains of untreated MPS II mice showed clear histological evidence of lysosomal accumulation in neurons, including accumulation of the lysosomal membrane protein LIMP2, as well as secondary accumulation of gangliosides, including GM3 (Figure 5L). Treated mice showed a dose-dependent reduction in neuronal accumulation lesions evident by both LIMP2 and GM3 staining (Figures 5A-5L). Based on this data, a 1.875 x 10 9 The low dose of GC / g was estimated to be the minimal effective dose (MED) in mice, as it was the lowest dose at which mice showed a significant (approximately 50%) reduction in both GM3 and LIMP2 accumulation pathology.

[0192] Behavioral testing was performed on mice 2 months after vector administration, when they reached 4-5 months of age. A comprehensive battery of tests was performed to assess general behavior, as well as short-term and long-term memory. y / - Mice exhibited normal exploratory activity in the open field arena (Figures 9A-9C). Spontaneous alternation behavior in the Y-maze (Figure 9D) was used to assess short-term working memory. y / - Mice had a similar number of arm entries and comparable spontaneous alternation behavior to wild-type littermates, indicating intact short-term memory. Long-term memory was assessed using classical contextual fear conditioning (FC) and novel object recognition (NOR). In FC, association of an aversive stimulus with a specific context elicits freezing behavior upon re-exposure to that context. All mice showed an increase in the percentage of freezing time during the recall phase of testing, demonstrating that learning had occurred, but IDS y / - Mice showed reduced freezing compared to wild-type littermates (Figure 6A). Treated animals showed reduced freezing compared to normal and untreated IDS. y / -Although it was difficult to assess treatment effects due to the small differences between mice, there was no clear improvement in contextual fear conditioning (Figure 6B). In NOR, mice are allowed to explore a pair of similar objects. 24 hours after training, one now-familiar object is replaced with a novel object. Mice have an innate tendency to explore the novel object; failure to do so indicates a lack of recognition of the familiar object, revealing memory deficits. Wild-type mice showed a preference for the novel object, but IDS mice showed no preference for the novel object. y / - The mice showed no preference. Notably, intrathecal AAV9 gene therapy was effective in preventing IDS y / - The long-term NOR deficit observed in IDS mice was rescued (Figure 6C). All treated IDS groups showed a trend toward a greater proportion of time exploring the novel object compared to the familiar object. y / - Object discrimination appeared to be improved in mice. Although the study was not sufficiently powered to compare the relative degree of rescue of behavioral deficits between treatment groups (Figures 6A-6C), preference for the novel object was statistically significant only in the mid-dose cohort.

[0193] One drawback of evaluating IT AAV delivery in mouse disease models is that the extremely small mouse CNS, with a brain mass of only 0.4 g and a CSF volume of 40 μL, cannot accurately model the spread of vectors and secreted enzymes in the 3,500-fold larger human brain. Previous studies of the related lysosomal storage disease mucopolysaccharidosis type I (MPS I) utilized naturally occurring large animal models to address this issue [Hinderer et al., Mol Therapy: the Journal of the Am Soc Gen Therapy, 2014:22:2018-2027; Hinderer et al., Mol Therapy: Journal of the Am Soc Gen Therapy, 2015:23:1298-1307]. MPS I cats and dogs, with average brain masses of 30 g and 72 g, respectively, provided a much more realistic model for addressing the challenge of achieving widespread vector and enzyme delivery in the human brain. Studies conducted in these models provided significant evidence of the efficacy of IT AAV9 delivery for MPS I [Hinderer et al., Mol Therapy: the Journal of the Am Soc Gen Therapy, 2014:22:2018-2027; Hinderer et al., Mol Therapy: Journal of the Am Soc Gen Therapy, 2015:23:1298-1307]. To determine whether similar efficacy is possible using this same approach in MPS II, we performed a parallel dose-ranging study in MPS I mice to examine the relative efficiency of enzyme expression and cross-correction in MPS II. MPS I mice were treated with the same α-L-iduronidase (IDUA) transgene as for IDS, except for the α-L-iduronidase (IDUA) transgene instead of IDS. y / - MPS I mice were treated with 3 × 10 vectors at 2–3 months of age, similar to the MPS II study. 8 , 3×10 9 , or 3 × 10 10Mice were treated with GC (n = 8 per group) and sacrificed at 21 days post-treatment for measurement of brain enzyme activity or 3 months post-treatment for histological analysis. Although IDUA expression was somewhat more efficient compared to IDS, both in terms of absolute enzyme activity and wild-type expression levels (Fig. 7A), IDS y / - There was a dose-response of brain enzyme expression similar to that observed in mice. Correction of brain storage lesions was similar between the two disease models, although treatment appeared to be slightly more effective in MPS I mice at the lowest vector dose (Figures 7A-7B). These results suggest that IT AAV9-mediated gene transfer of MPS II results in correction of brain storage lesions with approximately the same efficiency as observed in MPS I and that this approach remains effective even when scaled up from mice. However, slightly higher vector doses may be required in MPS II compared to MPS I to overcome less efficient enzyme expression in the brain.

[0194] In this study, IT delivery of an AAV9 vector carrying the human IDS gene in MPS II mice resulted in therapeutic levels of expression in the CNS and resolution of brain accumulation lesions. Functional improvement was demonstrated by greater novel object discrimination in treated mice compared with untreated controls. Neurocognitive impairment is associated with IDS. y / - These findings suggest that IDS has not previously been well characterized in mice. y / - Mice are shown to have normal exploratory activity in the open field and similar arm entries and activity in the Y-maze compared to wild-type littermates. These locomotion assessments are important when examining other types of behaviors that require normal exploration. y / - Mice have been found to have intact short-term working memory, as indicated by similar spontaneous alternation behavior in the Y-maze compared to controls. In contrast, Higuchi et al. [Mol Genet and Metabolism, 2012:107:122-128] found that 32-week-old IDS mice y / -We reported a decrease in spontaneous alternation and an increase in arm saturation in mice. The different Y-maze results were due to different ages at the time of testing, highlighting the progressive nature of MPS II. We evaluated the effects of IDS deficiency on two forms of long-term memory. The defect is IDS y / - Identified in contextual fear conditioning in mice. DS y / - Mice were able to recall the aversive stimulus, but showed a significantly reduced freezing response compared to wild-type littermates. y / - The mice showed a freezing response similar to that of uninjected mice. Therefore, no recovery was detected. Long-term memory loss was also observed in novel object recognition. In NOR, IDS y / - Mice showed a dislike of the novel object compared to the familiar object. y / - The results showed a reversal of NOR deficits seen in mice. Object discrimination was statistically significant only in the mid-dose cohort, but with eight mice per group, the study was not powerful enough to assess dose-response for behavioral endpoints. The ability of AAV9 to deliver IT to differentially affect the recovery of the two types of long-term memory may be due to the different neural substrates required for each task. Contextual fear conditioning is a hippocampal-dependent task in contrast to NOR, which requires the perithalamic cortex [Oliveira, et al., Post-training reversible inactivation of the hippocampus enhances novel object recognition memory]. Learning & memory (Cold Spring Harbor, NY) 2010;17:155-160;Abel et al., Cell 1997;88:615-626].

[0195] Comparison of the efficiency of IT AAV9 delivery in MPS II and MPS I mouse models showed similar enzyme expression and tissue correction between the two diseases, demonstrating that IT AAV delivery of MPS II is efficient enough to produce widespread disease correction in the context of significantly larger brain size and CSF volume, as previously shown in large animal models of MPS I [Hinderer et al., Mol Therapy: the Journal of the Am Soc Gen Therapy, 2014:22:2018-2027; Hinderer et al., Mol Therapy: Journal of the Am Soc Gen Therapy, 2015:23:1298-1307]. Interestingly, expression of the defective enzyme was significantly reduced in MPS II mice. The efficiency was somewhat lower in MPS II compared with MPS I. This was simply a product of the expression efficiency of these particular vectors, although the regulatory elements in the expression constructs were identical. Some studies have suggested that expression of sulfatases such as IDS may be limited by the availability of the sulfatase modifier SUMF1, which is required for post-translational modification of IDS [Fraldi et al., Biochemical J, 2007:403:305-312]. However, pilot experiments evaluating coexpression of IDS and SUMF1 did not demonstrate more active IDS expression (data not shown). Nevertheless, correction of storage lesions was nearly as efficient in MPS II mice as in MPS I mice, indicating that gene transfer should be similarly effective for both MPS I and MPS II patients, although in the latter case, a moderately high vector dose may be essential for optimal outcomes.

[0196] In addition to CNS gene transfer, there was significant hepatic transduction and peripheral enzyme expression in treated MPS II mice, indicating a potential systemic benefit of IT AAV9 delivery in MPS II. This is consistent with studies of IT AAV delivery in various other species [Passini et al., Human Gene Therapy, 2014;25:619-630; Hinderer et al., Molecular Therapy-Methods & Clinical Development, 2014;1; Hinderer et al., Molecular therapy: the journal of the American Society of Gene Therapy, 2014;22:2018-2027; Hinderer et al., Molecular therapy: the journal of the American Society of Gene Therapy, 2014;22:2018-2027]. American Society of Gene Therapy 2015;23:1298-1307; Gurda et al., Molecular therapy: the journal of the American Society of Gene Therapy 2015; Higuchi et al., cited above; Haurigot et al., J Clin Invest, 2013:123:3254-3271; Gray et al., Gene Therapy, 2013:20:450-459]. Notably, liver transduction after IT AAV vector delivery varies substantially between species, so it is not yet clear whether humans will exhibit significant peripheral expression.

[0197] This study demonstrates that IT AAV9 delivery achieves effective IDS gene transfer to the brain and resolves the CNS manifestations of MPS II, supporting the advancement of this approach to the clinic.

[0198] These data provide preliminary evidence that AAV9.CB.hIDS can ameliorate neurocognitive impairment in MPS II mice.

[0199] Example 3: Non-human primate studies A study in non-human primates (NHPs) will be conducted to evaluate the safety and efficacy of AAV9.CB.hIDS in adult male rhesus monkeys. The objectives of this study are to evaluate the local, acute, and chronic toxicity of intrathecal (IT) AAV9.CB.hIDS and to define the biodistribution of the vector. AAV9.CB.hIDS (5.6 × 10) was administered intrathecally. 11 or 1.875 x 10 11 A total of 21 male macaques will be treated with IT injections of AAV9.CB.hIDS at one of two doses of 1.875 x 10 GC / g brain mass or vehicle control diluent. The lower dose will be used to treat MPS II mice (MED = 1.875 x 10 9 This dose is approximately 100-fold greater than the MED (mg / g) and corresponds to the lowest dose that produces statistically significant histological improvement and neurobehavioral changes believed to reflect cognitive function in MPSII mice. The higher dose (approximately 300-fold greater than the MED) represents the maximum concentration at which the vector can be formulated and is limited by the requirement to maintain an injection volume that can be safely administered into the CSF (<10% CSF volume). During the injection procedure, animals are maintained under general anesthesia. A spinal needle is placed in the suboccipital space; placement is confirmed via fluoroscopy, and the dose is administered in a total volume of 1 mL. Serum and CSF are collected for clinical pathology evaluation at days 0, 3, 7, 14, 21, and 30 post-injection and monthly thereafter. At days 14, 90, and 180 post-injection, three male macaques from each dose group (a total of six per time point) are sacrificed for pathology and biodistribution. Vehicle-treated animals serve as controls. Complete histopathology will be performed on tissues from all animals, and vector biodistribution analysis will be performed on the high-dose cohort. DNA extraction and detection of vector genomes by quantitative PCR will be performed as previously described (see Chen et al., 2013, Hum Gene Ther Clin Dev, 24(4):154-160). The assay has a sensitivity of greater than 20 vector genome copies per μg of DNA, and spike controls will be included to verify the reaction efficiency of individual samples. Biodistribution, biochemistry, and immunogenicity data will be collected at 14 days, 3 months, and 6 months.

[0200] Example 4: Generation of rAAV9.CB7.hIDS vector AAV9.CB7.hIDS is generated by transfection of human HEK293 MCB cells with the following triple plasmids: (i) the hIDS vector genome plasmid, (ii) an AAV helper plasmid called pAAV29 containing the AAV rep2 and cap9 wild-type genes, and (iii) a helper adenovirus plasmid called pAdΔF6(Kan).

[0201] Cloning of the plasmid pAAV.CB7.CI.hIDS.RGB was performed as described above. As described in Example 1, the vector genome derived from this plasmid is a single-stranded DNA genome with ITRs from AAV2 flanking the hIDS 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 beta-actin promoter; transcription from this promoter is enhanced by the presence of the chicken beta-actin intron (CI). The polyA signal of the expression cassette is rabbit beta-globin (RBG) polyA. Plasmids were constructed by codon-optimizing and synthesizing the hIDS sequence [nt 1177 to nt 2829 of SEQ ID NO:8 and nt 1937 to nt 3589 of SEQ ID NO:11], and the resulting constructs were designated plasmids pENN.CBV, CI.RBG (p1044), and AAV2 containing the CBV, CI, and RBG expression elements. The ITRs were flanked by an expression cassette to generate pAAV.CB7.CI.hIDS.RBG.

[0202] Cloning of the cis-plasmid pAAV.CB7CIhIDS.RGB.KanR: The vector genome was excised from this plasmid using PacI restriction enzyme and cloned into a pKSS-based plasmid backbone (p2017) containing a kanamycin resistance gene. The final vector genome plasmid is pAAV.CB7.CI.hIDS.RBG.KanR.

[0203] AAV2 / 9 helper plasmid pAAV29KanRGXRep2: The AAV2 / 9 helper plasmid pAAV29KanRGXRep2 encodes four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins from AAV9. To generate the chimeric packaging construct, the AAV2 cap gene from plasmid p5E18, which contains the wild-type AAV2 rep and cap genes, was first deleted 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). The AAV2 / 9 helper plasmid pAAV29KanRGXRep2 normally promotes rep expression. Note that the p5 promoter is moved from the 5' end of the cap to the 3' end of rep in this construct. This arrangement serves to introduce a spacer between the promoter and the rep gene (i.e., the plasmid backbone), down-regulating rep expression and increasing its ability to support vector production. The plasmid backbone in p5E18 is derived from pBluescript KS. The AAV2 / 9 helper plasmid pAAV29KanRGXRep2 encodes four wild-type AAV2 rep proteins, three wild-type AAV VP capsid proteins from AAV9, and kanamycin resistance.

[0204] The pAdDeltaF6(Kan) adenoviral helper plasmid is 15,770 bp in size. This plasmid contains regions of the adenoviral genome important for AAV replication, namely E2A, E4, and VA RNAs (the adenoviral E1 function is provided by 293 cells), but does not contain any other adenoviral replication or structural genes. This plasmid does not contain cis elements important for replication, such as the adenoviral inverted terminal repeats, and therefore is not expected to produce infectious adenovirus. It was derived from an E1, E3 deleted molecular clone of Ad5 (pBHG10, a pBR322-based plasmid). Deletions were introduced into Ad5 DNA to eliminate expression of unnecessary adenoviral genes and reduce the amount of adenoviral DNA from 32 kb to 12 kb. Finally, the ampicillin resistance gene was replaced with a kanamycin resistance gene to obtain pAdΔF6(Kan). Functional elements of the E2, E4, and VAI adenoviral genes required for AAV vector production remain in this plasmid. The adenoviral E1 essential gene functions are supplied by HEK293 cells. DNA plasmid sequencing was performed by Qiagen. Genomic Services showed 100% homology with the following key functional elements of the reference sequence pAdDeltaF6(Kan)p1707FH-Q: E4 ORF6 3692-2808bp; E2A DNA-binding protein 11 784-10194bp; VA RNA region 12426-13378bp.

[0205] A flow chart summarizing the manufacturing process is shown in Figures 10A-10B.

[0206] Cell seeding: A qualified human embryonic kidney 293 cell line is used for the manufacturing process. Cells are seeded at 5 x 10 in Corning T-flasks and CS-10. 9 ~5×10 10Growth to the cells generates a cell population sufficient to seed up to 50 HS-36 cells for vector production per lot of BDS. Cells are cultured in a medium consisting of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% gamma-irradiated fetal bovine serum (FBS) from the United States. Cells are anchorage-dependent, and cell dissociation is achieved using TrypLE Select, an animal-product-free cell dissociation reagent. Cell seeding is achieved using sterile, disposable bioprocess bags and tubing sets. Cells are maintained at 37°C (±2°C) in a 5% (±0.5%) CO2 atmosphere. The cell culture medium is replaced with fresh serum-free DMEM medium and transfected with the three production plasmids using an optimized PEI-based transfection method. All plasmids used in the manufacturing process are produced in the context of a CMO quality system and infrastructure that utilizes the most salient features of cGMP manufacturing: traceability, document control, and material segregation.

[0207] Prepare enough DNA plasmid transfection complexes in a BSC to transfect up to 50 HS-36 cells (per BDS batch). First, add 7.5 mg of pAAV.CB7.CI.hIDS.RBG.KanR vector genome plasmid, 150 mg of pAdDeltaF6(Kan), 75 mg of pAAV29KanRGXRep2 AAV helper plasmid, and GMP-grade PEI (PEIPro, PolyPlus A DNA / PEI mixture containing 100% HS-36 mAb (SA) was prepared. This plasmid ratio was determined to be optimal for AAV production in small-scale optimization studies. After mixing well, the solution was left at room temperature for 25 minutes. Serum-free medium was then added to quench the reaction, which was then added to the HS-36 cells. The transfection mixture was distributed equally among all 36 layers of the HS-36 cells, and the cells were incubated at 37°C (±2°C) in a 5% (±0.5%) CO2 atmosphere for 5 days.

[0208] Cell Media Harvesting: Transfected cells and media are harvested from each HS-36 using a disposable bioprocess bag by aseptically draining the media from the unit. After media harvest, the approximately 80 liter volume is supplemented with MgCl2 to a final concentration of 2 mM (a cofactor for Benzonase) and Benzonase Nuclease (Cat. No. 1.016797.0001, Merck Group) is added to a final concentration of 25 units / ml. The product (in the disposable bioprocess bag) is incubated in an incubator at 37°C for 2 hours to allow sufficient time for enzymatic digestion of cell and plasmid DNA remaining during 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 is added to a final concentration of 500 mM to aid in product recovery during filtration and downstream tangential flow filtration.

[0209] Clarification: Cells and cell debris are removed from the product using depth filter capsules (1.2 μm / 0.22 μm) connected in series as a sterile closed tubing and bag set driven by a peristaltic pump. Clarification protects downstream filters and chromatography columns from fouling and ensures bioburden-reducing filtration, removing bioburden potentially introduced during the upstream manufacturing process, prior to downstream purification at the end of the filter train. The harvest is filtered through Sartorius Sartoguard PES capsule filters (1.2 / 0.22 μm) (Sartorius St edim Biotech Inc.

[0210] Large-scale tangential flow filtration: Volume reduction (10-fold) of clarified product is achieved by tangential flow filtration (TFF) using custom sterile closed bioprocessing tubing, bags, and membrane sets. The principle of TFF is to flow a solution under parallel pressure through a membrane of appropriate porosity (100 kDa). The pressure difference drives smaller molecules through the membrane, effectively transferring them to the waste stream while retaining molecules larger than the membrane pores. By recirculating the solution, the parallel flow sweeps across the membrane surface, preventing fouling of the membrane pores. By selecting the appropriate membrane pore size and surface area, the liquid sample can be rapidly reduced in volume while retaining and concentrating the desired molecules. Diafiltration in TFF applications involves adding new buffer to the recirculated sample at the same rate as the liquid passes through the membrane to the waste stream. As the amount of diafiltration increases, increasing amounts of small molecules are removed from the recirculated sample. This results in moderate purification of the clarified product, but also achieves a buffer exchange that is compatible with a subsequent affinity column chromatography step. Therefore, we utilize a 100 kDa PES membrane for concentration, followed by diafiltration with 4 volumes of buffer composed of: 20 mM Tris pH 7.5 and 400 mM NaCl. The diafiltered product is stored overnight at 4° C. and then further clarified with a 1.2 μm / 0.22 μm depth filter capsule to remove precipitated material.

[0211] Affinity chromatography: The diafiltered product is applied to Capture Select™ Poros-AAV2 / 9 affinity resin (Life Technologies), which efficiently captures the AAV2 / 9 serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured. After application, the column is washed to remove additional feed impurities, followed by a low-pH step elution (400 mM NaCl, 20 mM sodium citrate; pH 2.5), which is immediately neutralized by collection in 1 / 10 volume of neutralization buffer (Bis Tris Propane, 200 mM, pH 10.2).

[0212] Anion Exchange Chromatography: To further reduce in-process impurities, including empty AAV particles, the Poros-AAV2 / 9 elution pool was 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). After a low-salt wash, the vector product was eluted using a 60 CV NaCl linear salt gradient (10-180 mM NaCl). This shallow salt gradient effectively separated vector genome-free capsid particles (empty particles) from vector genome-containing particles (intact particles), resulting in a preparation enriched for intact capsids. To minimize nonspecific binding to the tubes and the length of exposure to high pH, ​​fractions were collected in tubes containing 1 / 100th the volume of 0.1% Pluronic F68 and 1 / 27th the volume of Bis-Tris pH 6.3, respectively. Collect appropriate peak fractions, assess peak area and compare with previous data to determine approximate vector yield.

[0213] Final formulation and sterile filtration to obtain BDS: Using TFF, final formulation of the pooled AEX fractions with a 100 kDa membrane was achieved by diafiltration with 4 volumes of formulation buffer (Elliot's B solution, 0.001% Pluronic F68) and concentration to obtain BDS, whereby the peak area from anion exchange chromatography was increased by 5×10 compared to previous data. 13 The concentration factor to achieve a titer of GC / ml or greater is estimated. Samples are removed for BDS testing (described in the following section). The filtered purified bulk is stored in sterile polypropylene tubes and frozen at or below -60°C in a quarantined location until released for final filling. Preliminary stability studies have confirmed the efficacy of our proposed It is shown that DP does not lose activity after freezing and thawing in the proposed formulation buffer. Further studies are underway to assess stability after long-term storage at -80°C.

[0214] Final Fill: Frozen BDS is thawed, pooled, diluted to target titer with final formulation buffer, and finally filtered through a 0.22 μm filter (Millipore, Billerica, MA) and filled into West Pharmaceutical "Ready-to-Use" (prefilled) 2 ml glass vials with 13 mm stoppers and seals at a fill volume of not less than 0.6 ml and not more than 2.0 ml per vial. Individually labeled vials are labeled according to the following specifications: Labeled vials are stored at -60°C or below.

[0215] Vectors (drug products) are vialed at a single fixed concentration, with the only variable being the volume per vial. To achieve lower dose concentrations, the drug product is diluted with Elliots B solution, 0.001% Pluronic F68. High-dose vectors are used directly without dilution, while low-dose vectors require a 1:5 dilution in formulation buffer, which is performed by the pharmacy at the time of administration.

[0216] Example 5: Testing of vectors Characterization assays including serotype identity, empty particle content, and transgene product identity are performed. A description of the assays is provided below.

[0217] A. Vector genome identity: DNA sequencing Isolate viral vector genomic DNA and sequence it using primer walking at 2x sequencing coverage. Align the sequences and compare them with the expected sequence.

[0218] B. Vector Capsid Identity: AAV Capsid Mass Spectrometry of VP3 Confirmation of the AAV2 / 9 serotype of the vector is achieved by an assay based on the analysis of peptides from the VP3 capsid protein by mass spectrometry (MS). This method involves multienzyme 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 has been developed that allows subtraction of host protein products and derivation of capsid peptide sequences from the mass spectrum.

[0219] C. Genome copy (GC) titer oqPCR-based genome copy titers were determined across a range of serial dilutions and compared to a cognate plasmid standard (pAAV.CB7.CI.hIDS.RBG.KanR). The oqPCR assay utilizes sequential digestion with DNase I and proteinase K, followed by qPCR analysis to measure encapsidated vector genome copies. DNA detection is achieved using sequence-specific primers targeting the RBG polyA region in combination with a fluorescently labeled probe hybridizing to this same region. Comparison to a plasmid DNA standard curve allows titer determination without the need for post-PCR sample manipulation. Multiple standards, validation samples, and controls (for background and DNA contamination) were introduced into the assay. The assay is quantified by establishing and defining assay parameters, including sensitivity, detection limit, qualification range, and intracellular and intercellular assay precision. An internal AAV9 reference lot was established and used to perform qualification testing. Previous experience has shown that titers obtained with the optimized qPCR assay described herein are generally 2x the titers achieved with our standard qPCR technique used to generate the preclinical data. This suggests that it is 0.5 times higher.

[0220] D. Ratio of empty particles to total particles The total particle content of the drug product is determined by SDS-PAGE analysis. An iodixanol gradient-purified reference vector preparation is analyzed by various methods (analytical centrifugation, electron microscopy, and absorbance at 260 / 280 nm) to establish that the preparation contains >95% genome-containing (intact) particles. This reference material is serially diluted to known genome copy numbers (and, by extension, particle numbers), and each dilution is run on an SDS-PAGE gel along with a similar dilution series of the drug product. The peak area volumes of both the reference material and the drug product VP3 protein band are determined by densitometry, and the reference material volume is plotted against particle number. The total particle concentration of the drug product is determined by extrapolation from this curve, and the genome copy (GC) titer is then subtracted to obtain the empty particle titer. The empty particle to total particle ratio is the ratio of the empty particle titer to the GC titer.

[0221] E. Infectious titer An infectious unit (IU) assay is used to determine productive vector uptake and replication in RC32 cells (rep2-expressing HeLa cells). Similar to previously published studies, a 96-well endpoint format is employed. Briefly, RC32 cells are co-infected with serial dilutions of rAAV9.CB.hIDS and a uniform dilution of Ad5 with 12 replicates at each rAAV dilution. At 72 h postinfection, cells are lysed and qPCR is performed to detect rAAV vector amplification relative to input. End-point dilution TCID50 calculations (Spearman-Karber) are performed to determine replication titers, expressed as IU / ml. Since "infectivity" values ​​depend on particle contact with the cell, receptor binding, internalization, nuclear transport, and genome replication, they are influenced by the assay format and the presence of appropriate receptors and post-binding pathways in the cell line used. Because receptors and post-binding pathways are typically not maintained in immortalized cell lines, the infectivity assay titer 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) may be used as a measure of lot-to-lot product consistency.

[0222] The GC / IU ratio is a measure of product consistency. Divide the oqPCR titer (GC / ml) by the infectious units (IU / ml) to obtain the calculated GC / IU ratio.

[0223] F. Replication-competent AAV (rcAAV) assay A sample is analyzed for the presence of replication-competent AAV2 / 9 (rcAAV), which may potentially arise during the production process. A three-passage assay has been developed, consisting of cell-based amplification and passaging followed by detection of rcAAV DNA by real-time qPCR (Cap9 target). The cell-based component consists of inoculating a monolayer of HEK293 cells (P1) with dilutions of the test sample and wild-type human adenovirus type 5 (Ad5). 10 10The vector product of GC is the largest amount of product tested. Due to the presence of adenovirus, replication-competent AAV amplifies in cell culture. After two days, cell lysates are generated and Ad5 is heat-inactivated. The clarified lysates are then passaged to a second round of cells (P2) (again in the presence of Ad5) to increase sensitivity. After two days, cell lysates are generated and Ad5 is heat-inactivated. The clarified lysates are then passaged to a third round of cells (P3) (again in the presence of Ad5) to maximize sensitivity. After two days, cells are lysed to release DNA, which is then subjected to qPCR to detect the AAV9 cap sequence. 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 and AAV9 cap genes allowed the limit of detection (LOD) of the assay to be determined (0.1, 1, 10, and 100 IU) and serial dilutions of the rAAV9.CB.hIDS vector (1 × 10 10 , 1×10 9 , 1×10 8 , 1×10 7 GC) to determine the rc present in the test sample. The approximate level of AAV may be quantified.

[0224] G. In Vitro Titer To correlate qPCR GC titers with gene expression, an in vitro bioassay is performed by transducing HEK293 (human embryonic kidney) cells with a known multiplicity of GC per cell and assaying the supernatant for IDS activity 72 hours post-transduction. IDS activity is measured by incubating samples diluted in 0.1 ml of water with 0.1 ml of 100 mmol / L 4MU-iduronide-2-sulfate at 37°C for 1-3 hours. The reaction is stopped by adding 2 ml of 290 mmol / L glycine, 180 mmol / L sodium citrate, pH 10.9, and liberated 4MU is quantified by comparing fluorescence with a 4MU standard dilution. Comparison with highly active preclinical and toxin vector preparations allows interpretation of product activity.

[0225] H. Total protein, capsid protein, protein purity determination and capsid protein ratio Vector samples are first quantified for total protein using the bicinchoninic acid (BCA) assay against a bovine serum albumin (BSA) protein standard curve. Determination is performed by mixing an equal volume of sample with the Micro-BCA reagent provided in the kit. The same procedure is applied to dilutions of the BSA standard. The mixture is incubated at 60°C, and the absorbance is measured at 562 nm. A standard curve is generated from the absorbance of standards of known concentrations using a four-parameter fit. Unknown samples are quantified according to the four-parameter regression.

[0226] To obtain a semiquantitative measure of AAV purity, samples were normalized for genome titer and analyzed on SDS-polyacrylamide (SDS-PAGE) gels under reducing conditions at 5 × 10 9 The samples were separated by GC. The gel was then stained with SYPRO Ruby stain. Any impurity bands were quantified by densitometry by comparison with co-electrophoresed BSA standards of 25, 50, and 100 ng of protein per lane. These amounts correspond to 1%, 2%, and 4% of the total AAV protein sample. Stained bands that appear in addition to the three AAV-specific proteins VP1, VP2, and VP3 are considered protein impurities. All impurity bands were compared to reference proteins, and the impurity mass percentage and approximate molecular weight were reported. SDS-PAGE gels were also used to quantify VP1, VP2, and VP3 proteins and determine their ratios.

[0227] Example 6: MPS II biomarkers In the current study, metabolite profiling of CSF samples from dogs with MPS I revealed substantial disease-associated changes in the CSF metabolome. The most striking difference was a more than 30-fold increase in spermine levels compared to normal controls. This finding was confirmed in MPS I patient samples as well as in a feline model of MPS I and is expected to be found in MPS II. Spermine binds to HS, and cellular uptake of spermine depends on this interaction [M. Belting, S. Persson, L.-A. Fransson, Proteoglycan involvement in polyamine uptake]. Biochemical Journal 338, 317-323 (1999); J.E. Welch, P. Bengtson, K. Svensson, A. Wittrup, G.J. Jenniskens, G.B. Ten Dam, T.V. An 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); online publication EpubApr]. Cell surface proteoglycans such as glypican-1 can bind spermine through their HS moieties, and after endocytosis of the glypican protein, intracellular cleavage of The HS chain releases bound spermine into the cell (cell surface proteoglycans such as glypican-1 can bind spermine through their HS moiety, and after endocytosis of the glypican protein, intracellular cleavage of the HS chain releases bound spermine into the cell) (Belting et al., K. Ding, S. et al., The Journal of Biological Chemistry 276, 46779-46791 (2001); published online in Epub December 14). Thus, intact HS recycling is essential for spermine uptake. In MPS I, accumulation of extracellular spermine could occur through inhibition of this uptake mechanism due to inefficient HS recycling or through simple binding of spermine to extracellular GAGs that accumulate in MPS, shifting the spermine binding equilibrium and promoting extracellular distribution. Future studies should address the relative contributions of these mechanisms to spermine accumulation in MPS I CSF.

[0228] 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 patient CSF. Gene therapy in a canine model of MPS I reversed spermine accumulation and normalized GAP43 expression, suggesting that the same pathway is affected in vivo. We were unable to directly assess the effects of spermine synthesis inhibition in vivo because available inhibitors do not cross the blood-brain barrier and chronic direct CNS administration from birth is not feasible in animal models. While our in vitro findings support a role for spermine in abnormal neurite outgrowth in MPS I, it is important to note that inhibition of spermine synthesis did not completely reverse the phenotype, and addition of spermine to normal neurons did not increase neurite outgrowth relative to the level of MPS I neurons. The effects of spermine modulation may have been limited by the relatively short duration of treatment. Spermine accumulation is a key factor in the development of MPS I. It is possible that spermine accumulation is not the only mediator contributing to neurite outgrowth in MPS I. In particular, many neurotrophic factors bind through HS-modified receptors, and their interaction with HS in the extracellular matrix may affect neurite outgrowth [D. Van Vactor, D.P. Wall, K.G. Johnson, Heparan sulfate proteoglycans and the emergence of neuronal connectivity. Current opinion in neurobiology 16, 40–51 (2006); published online Feb (10.1016 / j.conb.2006.01.011)]. Therefore, spermine accumulation may be one of several factors promoting abnormal neurite outgrowth in MPS I.

[0229] Of 15 MPS I dog CSF samples screened, only one had spermine concentrations within the normal range. At 28 days of age, this was the youngest animal included in the study. This finding indicates that spermine accumulation may be age-dependent. Future studies should assess CSF spermine levels longitudinally in MPS patients. If spermine increases with age in MPS patients, this could explain the kinetics of cognitive decline, as most patients experience 1–2 years of normal development before the onset of developmental delay.

[0230] The possibility that impairment of HS metabolism induces the accumulation of metabolites that alter neuronal growth is raised. These findings may point to a novel link between enzyme deficiency and abnormal neurite outgrowth phenotypes in MPS I, potentially explaining the cognitive dysfunction associated with these disorders. These findings also indicate the utility of CSF spermine as a noninvasive biomarker for assessing the pharmacodynamics of novel CNS-directed therapies for MPS I.

[0231] Materials and Methods: Experimental Design: This study was initially designed to detect metabolites present at significantly different levels in CSF samples from MPS I patients compared with samples from healthy controls. Due to the limited availability of CSF samples from children with MPS IH and healthy controls, initial screening was subsequently performed using CSF samples from MPS I dogs, which were available in larger numbers, with the aim of evaluating candidate biomarkers in human samples. A total of 15 CSF samples from individual untreated MPS I dogs were available for analysis, and an additional 15 samples were obtained from healthy controls. Following the identification of elevated spermine in MPS I canine CSF in the prospective metabolite screen, spermine was retrospectively measured in CSF samples from a previous study of MPS I dogs and cats treated with gene therapy, as well as in patient samples. The number of subjects included in each group for these analyses was limited by sample availability and not based on statistical considerations; therefore, in some cases, numbers were insufficient for statistical comparison. For in vitro neurite outgrowth studies, the number of cells quantified for each condition was based on pilot experiments showing that >30 cells per condition were required to detect a 20% difference in branch length, neurite number, or neurite branching per cell. After plating cells and treating them with the indicated drugs, wells were coded, and cell image acquisition and manual quantification of neurite length and branching were performed by a blinded reviewer. Comparisons of wild-type and MPS mouse neurons were repeated using a different substrate (poly-L-lysine (Sigma)-coated tissue culture plates rather than chamber slides (Sigma S6815)) with similar results. Comparisons of wild-type neurons were performed four times using both substrates, with and without spermine addition, with similar results.

[0232] CSF metabolite profiling: CSF metabolite profiling was performed by Metabolon. Samples were stored at -80°C until processing. Samples were prepared using a MicroLab STAR® system (Hamilton Company). A recovery standard was added before the first stage of the extraction process for QC purposes. Proteins were precipitated with methanol with vigorous shaking for 2 minutes, followed by centrifugation. The resulting extract was divided into five fractions: one for reversed-phase (RP) UPLC-MS / MS analysis with positive-ion mode electrospray ionization, one for RP / UPLC-MS / MS analysis with negative-ion mode electrospray ionization, one for analysis by hydrophilic interaction chromatography (HILIC) / UPLC-MS / MS with negative-ion mode electrospray ionization, one for analysis by GC-MS, and one sample reserved for backup. Samples were briefly placed on a TurboVap® (Zymark) to remove organic solvents. For LC, samples were stored under nitrogen overnight before preparation for analysis. For GC, each sample was dried under vacuum overnight before preparation for analysis.

[0233] The LC / MS portion of the platform consisted of a Waters ACQUITY ultra-performance liquid chromatograph (UPLC) and Thermo Scientific Q-Exactive high-resolution / accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and an Orbitrap mass spectrometer, operating at 35,000 mass resolution. Sample extracts were dried and then reconstituted in solvents compatible with each LC / MS method. Each reconstitution solvent contained a set of standards at fixed concentrations to ensure injection and chromatographic consistency. For RP chromatography, acidic cations were used. The analysis was performed using the optimized conditions and other basic anion-optimized conditions. Each method utilized a separate dedicated column (Waters UPLC BEHC18-2.1 x 100 mm, 1.7 μm). Extracts reconstituted under acidic conditions were gradient-eluted with water and methanol containing 0.1% formic acid. The basic extract was similarly eluted with methanol and water, but with 6.5 mM ammonium bicarbonate. A third aliquot was analyzed by anionization after elution from a HILIC column (Waters UPLC BEH Amide 2.1 x 150 mm, 1.7 μm) using a gradient consisting of water and acetonitrile containing 10 mM ammonium formate. MS analysis was performed by alternating between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slightly between methods but covered 80-1000 m / z.

[0234] Samples intended for analysis by GC-MS were dried under vacuum for a minimum of 18 hours and then derivatized under dry nitrogen with bistrimethylsilyltrifluoroacetamide. The derivatized samples were separated on a 5% diphenyl / 95% dimethylpolysiloxane fused silica column (20 m x 0.18 mm ID; 0.18 μm film thickness) with helium as the carrier gas and a temperature ramp from 60 min to 340 °C over a 17.5 min period. Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scan single quadrupole mass spectrometer using electron impact ionization (EI) and operated at unit mass resolution. The scan range was 50–750 m / z.

[0235] Several controls were analyzed in concert with the experimental samples: pooled matrix samples generated by taking a small amount of each experimental sample served as technical replicates across the entire data set; extracted water samples served as process blanks; and a cocktail of QC standards, carefully selected so as not to interfere with the measurement of endogenous compounds, was spiked into every analyzed sample to enable monitoring of instrument performance and aid in chromatographic alignment. Instrument variability was determined by calculating the median relative standard deviation (RSD) of standards added to each sample prior to injection into the mass spectrometer. Overall process variability was determined by calculating the median RSD of all endogenous metabolites (i.e., non-instrument standards) present in 100% of the pooled matrix samples. Experimental samples were randomized across platform runs, with QC samples evenly spaced between injections.

[0236] Metabolites were identified by automated comparison of ion characteristics in experimental samples to a reference library of chemical standards, including retention time, molecular weight (m / z), preferred adducts, and in-source fragments, as well as associated MS spectra, and overseen by visual inspection for quality control using software developed at Metabolon. Identification of known chemicals was based on comparison with metabolomics library entries of purified standards. Peaks were quantified using area-under-the-curve measurements. The raw area counts for each metabolite in each sample were normalized by the median value for each run day to correct for variations resulting from differences in instrument calibration between days, thereby setting the median to 1.0 for each run. This conserved variation between samples allowed metabolites with widely different raw peak areas to be compared on a similar graphical scale. Missing values ​​were considered the minimum value observed after normalization.

[0237] Quantitative MS assay: CSF samples (50 μL) were mixed with spermine-d8 internal standard (IsoSciences). The samples were deproteinized by mixing with a four-fold excess of methanol and centrifuging at 12,000 × g at 4 °C. The supernatant was dried under a stream of nitrogen and then resuspended in 50 μL of water. A 5 μL aliquot was subjected to LC-MS analysis. LC separation was performed using a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA) equipped with an Xbridge® C18 column (3.5 μm, 150 × 2.1 mm). The flow rate was 0.15 mL / min. Solvent A was 0.1% formic acid, and solvent B was 98 / 2 acetonitrile / HO (v / v) containing 0.1% formic acid. The elution conditions were as follows: 2% B for 0 min, 2% B for 2 min, 60% B for 5 min, 80% B for 10 min, 98% B for 11 min, 98% B for 16 min, 2% B for 17 min, and 2% B for 22 min. The column temperature was 35°C. MS / MS analysis was performed in positive ion mode using a TSQ Quantum Ultra spectrometer (Thermo Fisher, San Jose, CA) with the following parameters: spray voltage of 4000 V, capillary temperature of 270 °C, sheath gas pressure of 35 arbitrary units, ion sweep gas pressure of 2 arbitrary units, auxiliary gas pressure of 10 arbitrary units, vaporizer temperature of 200 °C, tube lens offset of 50, capillary offset of 35, and skimmer offset of 0. The following transitions were monitored: 203.1 / 112.1 (spermine); 211.1 / 120.1 (spermine-d8), with a scan width of 0.002 m / z and a scan time of 0.15 s.

[0238] Animal Procedures: All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. For CSF metabolite screening, samples were collected by retrosplenial bone marrow aspiration from normal dogs aged 3 to 26 months and MPS I dogs aged 1 to 18 months. Gene transfer studies in MPS I dogs and cats were performed as previously described (20, 22). CSF samples were collected 6 to 8 months after vector administration. For mouse cortical neuron experiments, primary cortical neuron cultures were prepared from E18 IDUA- / - or IDUA+ / + embryos.

[0239] Patient samples: Informed consent was obtained from each subject's parent or legal guardian. The protocol was approved by the University of Minnesota Institutional Review Board. CSF samples were collected by lumbar puncture. All MPS I patients had a diagnosis of Hurler syndrome and had not received enzyme replacement therapy or hematopoietic stem cell transplantation prior to sample collection. MPS I patients were 6 to 26 months of age. Healthy controls were 36 and 48 months of age.

[0240] Statistical Analysis: Random forest analysis and heatmap generation were performed using MetaboAnalyst 3.0 [R.G. Kalb, Development 120, 3063-3071 (1994); J. Zhong et al., Journal of Neurochemistry 64, 531-539 (1995); D. Van Vactor, D.P.W. et al., Current Opinion in Neurobiology 16, 40-51 (2006); published online in Epub Feb (10.1016 / j.conb.2006.01.011)]. Raw peak data were log-transformed and normalized to the mean of normal sample values. All other statistical analyses were performed using GraphPad Prism 6. Cultured nerve branch length, neurite number, and branching were compared by ANOVA followed by Dunnett's test. CSF spermine and cortical GAP43 were compared by Kruskal-Wallis test followed by Dunnett's test.

[0241] GAP43 Western: Frontal cortex samples were homogenized in 0.2% Triton X-100 using a Qiagen Tissuelyser at 30 Hz for 5 minutes. Samples were clarified by centrifugation at 4°C. Protein concentration was measured in the supernatant by BCA assay. Samples were incubated in NuPAGE LDS buffer containing DTT (Thermo Fisher Scientific) at 70°C for 1 hour and separated on a Bis-Tris 4-12% polyacrylamide gel in MOPS buffer. Proteins were transferred to a PVDF membrane and blocked in 5% nonfat dry milk for 1 hour. The membrane was then probed with rabbit polyclonal anti-GAP43 antibody (Abcam) diluted to 1 μg / mL in 5% nonfat dry milk, followed by HRP-conjugated polyclonal anti-rabbit antibody (Thermo Fisher Scientific) diluted 1:10,000 in 5% nonfat dry milk. The bands were probed using SuperSignal West Pico substrate (Thermo Fisher Scientific). Bands were detected using SuperSignal West Pico substrate (Thermo Fisher Scientific). Densitometry was performed using Image Lab 5.1 (Bio-Rad).

[0242] Neurite outgrowth assay: Day 18 embryonic cortical neurons were harvested as described above and plated at a concentration of 100,000 cells / mL onto chamber slides (Sigma S6815) or poly-L-lysine (Sigma)-coated tissue culture plates in serum-free Neurobasal medium (Gibco) supplemented with B27 (Gibco). Treatments were administered in duplicate wells 24 hours after plating (day 1). Phase-contrast images for quantification were captured with a Nikon Eclipse Ti at 20x magnification using a 600 ms manual exposure and a 1.70x gain setting for high contrast. Individual blinded treatment conditions were coded by capturing 10–20 images per well. Images were converted to 8-bit format using ImageJ (NIH) and traced by a blinded reviewer using NeuronJ [E. Meijering, M. Jacob, J.C. Sarria, P. Steiner, H. Hirling, M. Unser, Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry. Part A: the journal of the International Society for Analytical Cytology 58, 167–176 (2004); published online April 10, 2004 (10.1002 / cyto.a.20022)]. Soma diameter, neurite number, branching points, and branch length were manually traced. Images traced in NeuronJ were converted to micrometers using a conversion factor based on the image size: a 2560 × 1920 pixel image was converted to micrometers using a conversion factor of 0.17 micrometers per pixel.

[0243] Histology: Brain tissue processing and LIMP2 immunofluorescence were performed as previously described [C. Hinderer et al., Molecular therapy: the journal of the American Society of Gene Therapy 22, 2018-2027 (2014); published online in EpubDec (10.1038 / mt.2014.135)].

[0244] RT-PCR: Frontal cortex samples from three normal dogs and five MPS dogs were immediately frozen on dry ice at necropsy. RNA was extracted with TRIzol reagent (Thermo Fisher Scientific), treated with DNAse I (Roche) for 20 minutes at room temperature, and purified using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Purified RNA (500 ng) was reverse transcribed using a High Capacity cDNA Synthesis Kit (Applied Biosystems) with random hexamer primers. Transcripts of arginase, ornithine decarboxylase, spermine synthase, spermidine synthase, spermine-spermidine acetyltransferase, and glyceraldehyde phosphate dehydrogenase were quantified by Sybr green PCR using an Applied Biosystems 7500.

[0245] Real-time PCR system. A standard curve was generated for each target gene using a 4-fold dilution of a pooled standard consisting of all individual samples. The highest standard was assigned an arbitrary transcript number, and the Ct values ​​of individual samples were converted to transcript numbers based on the standard curve. Values ​​are expressed relative to the GAPDH control.

[0246] Statistical analysis: Random forest analysis and heatmap generation were performed using MetaboAnalyst 3.0 [J. Xia et al., MetaboAnalyst 2.0 - a comprehensive server for metabolomic data analysis. Nucleic Acids Research, (2012); published online May 2, 2012 (10.1093 / nar / gks374); J. Xia, et al., MetaboAnalyst: a web server for metabolomic data Analysis and interpretation (Web server for metabolomic data analysis and interpretation). Nucleic Acids Research 37, W652-W660 (2009); published online Epub July 1, 2009 (10.1093 / nar / gkp356). J. Xia et al., MetaboAnalyst 3.0—Making Metabolomics More Meaningful. Nucleic Acids Research, (2015); published online Epub April 20, 2015 (10.1093 / nar / gkv380). Undetectable values ​​in metabolite screening were considered the minimum observed in the dataset. Raw peak data were normalized to the mean of normal sample values ​​and log-transformed. All other statistical analyses were performed using GraphPad Prism 6. Cultured nerve branch length, neurite number, and branching were compared by ANOVA followed by Dunnett's test. CSF spermine and cortical GAP43 were compared by Kruskal-Wallis test followed by Dunnett's test.

[0247] result 1. Identification of Elevated CSF Spermine by Metabolite Profiling An initial screening of CSF metabolites was performed using a canine model of MPS I. These animals harbor a splice-site mutation in the IDUA gene, resulting in the complete loss of enzyme expression and the development of clinical and histological features similar to those of MPS I patients [KM Menon et al., Genomics 14, 763-768 (1992); R. Shull et al., The American Journal of Pathology 114, 487 (1984)]. CSF samples were collected from 15 normal dogs and 15 MPS I dogs. CSF samples were evaluated for relative metabolite abundance by LC and GC-MS. A total of 281 metabolites were identified as positive in CSF samples by mass spectrometry. Of these, 47 (17%) were significantly elevated in MPS1 dogs compared with controls, and 88 (31%) were decreased compared with controls. A heat map of the 50 metabolites most different between groups is shown in Figure 19A. Metabolic profiling identified significant differences in polyamine, sphingolipid, acetylated amino acid, and nucleotide metabolism between MPS I and normal dogs. Random forest clustering analysis identified the polyamine spermine as the largest contributor to metabolite differences between MPS I and normal dogs (Figure 23). On average, spermine was elevated more than 30-fold in MPS1 dogs, except for one MPS1 dog that was less than 1 month old at the time of sample collection. A stable isotope dilution (SID)-LC-MS / MS assay was developed to quantitatively measure spermine in CSF. Samples were screened from six children with Hurler syndrome (6-26 months of age) and two healthy controls (36 and 48 months of age). Both healthy controls had CSF spermine levels below the assay's limit of quantitation (1 ng / mL), whereas CSF samples from MPS I patients were on average 10-fold higher than the limit of quantitation (Figure 19B). The elevated spermine in MPS IH patients appeared to be consistent with the known role of HS in spermine binding and uptake [M. Belting et al. al., Journal of Biological Chemistry 278, 47181-47189(2003);M. Belting et al., Proteoglycan involvement in polyamine uptake.Biochemical Journal 338, 317-323(1999);JEWellch, et al, International journal of oncology 32, 749-756 (2008)); increased synthesis did not appear to be the cause of elevated CSF spermine, as normal and MPS I canine brain samples had similar mRNA expression levels for transcriptionally regulated enzymes in the polyamine synthesis pathway (Figure 24). To determine whether elevated spermine is a general feature of heparan sulfate storage diseases or specific to MPS I, spermine was measured in CSF samples from a canine model of MPS VII, which showed similar levels (Figure 25).

[0248] 2. Role of spermine in abnormal neurite outgrowth associated with MPS After axonal injury, neurons upregulate polyamine synthesis, which promotes neurite outgrowth [D. Cai, et al., Neuron 35, 711-719 (2002). Published online Epub Aug 15; K. Deng, et al., The Journal of neuroscience: the official journal of the Society for Neuroscience 29, 9545-9552 (2009). Published online Epub Jul 29; Y. Gao, et al., Neuron 44, 609-621 (2004). Published online Epub Nov 18; R. C. Schreiber, et al., Neuroscience 128, 741-749 (2004)]. Therefore, we evaluated the role of spermine in the abnormal neurite hyperplasia phenotype described in MPS neurons [Hocquemiller, S. et al., Journal of Neuroscience Research 88, 202-213 (2010)]. Cultures of E18 cortical neurons from MPS I mice exhibited greater neurite number, branching, and total branch length after 4 days in culture than neurons from wild-type mice in the colony (Figures 20A-20F). Treatment of MPS neurons with APCHA, an inhibitor of spermine synthesis, significantly reduced neurite outgrowth and branching. This effect was reversible by replacing spermine (Figures 20A-20F). The same APCHA concentration did not affect normal neuronal growth (Figure 26). Addition of spermine to wild-type neuronal cultures at concentrations similar to those identified in vivo significantly increased neurite outgrowth and branching (Figures 20A-20F).

[0249] 3. Effect of gene therapy on CSF spermine and GAP43 expression GAP43, a central regulator of neurite outgrowth, is overexpressed by MPS III mouse neurons both in vitro and in vivo, suggesting that the same neurite outgrowth pathways abnormally activated in neuronal cultures are also active in vivo. To assess the effects of IDUA deficiency on GAP43 expression and spermine accumulation in vivo, we measured CSF spermine and brain GAP43 levels in untreated MPS I dogs and dogs treated with CNS-directed gene therapy. We previously described five MPS I dogs treated with intrathecal injection of an adeno-associated virus serotype 9 vector carrying a canine IDUA transgene [C. Hinderer et al., Molecular therapy: the journal of the American Society of Gene Therapy 23, 1298-1307 (2015); online publication Epub Aug]. Because MPS I dogs can produce antibodies against the normal IDUA enzyme, two dogs were pretreated as neonates with liver IDUA gene transfer to induce immune tolerance to this protein. Both tolerized dogs exhibited significantly higher than normal brain IDUA activity after AAV9 treatment. Three non-tolerized dogs exhibited varying levels of expression, with one animal reaching levels above normal and the other two exhibiting near-normal expression (Figure 21A). CSF spermine reduction was inversely proportional to brain IDUA activity, decreasing 3-fold compared to untreated animals in the two dogs with the lowest IDUA expression and over 20-fold in the animal with the highest expression (Figure 21A, Figures 21B-21H, and 21K). GAP43 was upregulated in the frontal cortex of MPS1 dogs, and expression was normalized in all vector-treated animals (Figure 2). 1I~21J).

[0250] We further evaluated the relationship between CSF spermine levels and IDUA reconstitution in MPS1 dogs treated with a range of vector doses. MPS1 dogs previously tolerized to human IDUA by neonatal liver gene transfer were treated with three doses (1010 , 10 11 , 10 12 GC / kg, n=2 per dose) via intrathecal injection [(C. Hinderer, et al., Neonatal tolerance induction enables accurate evaluation of gene therapy Induction of neonatal tolerance allows accurate evaluation of gene therapy for MPS I in a canine model. Molecular Genetics and Metabolism, dx.doi.org / 10.1016 / j.ymgme.2016.06.006]. CSF spermine was assessed 6 months after injection (Figure 22A). The decrease in CSF spermine was dose-dependent, with mid- and high-vector-dose animals reaching the normal range, whereas low-dose animals only partially reduced CSF spermine. To independently confirm the relationship between IDUA deficiency and CSF spermine accumulation, we assessed CSF spermine levels in a feline model of MPS I. Using CSF samples from a previously reported gene therapy study, we found that untreated MPS I cats exhibited elevated CSF spermine (Figure 22B) [C. Hinderer, P. Bell, B. L. Gurda, Q. Wang, J. P. Louboutin, Y. Zhu, J. Bagel, P. O'Donnell, T. Sikora, T. Ruane, P. Wang, M. E. Haskins, J. M. Wilson, Intrathecal gene therapy corrects CNS pathology in a feline model of Mucopolysaccharidosis I. Molecular therapy (Intrathecal gene therapy corrects CNS pathology in a feline model of mucopolysaccharidosis I: the journal of the American Society of Gene Therapy 22, 2018-2027 (2014); published online in Epub December (10.1038 / mt.2014.135)). Intrathecal administration of a high-dose AAV9 vector expressing feline IDUA normalized CSF spermine levels (Figure 22B).

[0251] C. Consideration In the current study, we performed metabolite profiling of CSF samples from dogs with MPS I, which revealed substantial disease-related changes in the CSF metabolome. The most striking difference was a more than 30-fold increase in spermine levels compared to normal controls. This finding was confirmed in samples from MPS I patients as well as in cat and canine models of MPS I and MPS VII. Spermine directly binds to HS with high affinity, and cellular uptake of spermine depends on this interaction [M. Belting, S. PERSSON, L.-A. Fransson, Proteoglycan involvement in polyamine uptake. Biochemical Journal 338, 317-323 (1999); J.E. Welch et al., International Journal of Oncology 32, 749-756 (2008)]. Cell surface proteoglycans, such as glypican-1, can bind spermine through their HS moieties, and after endocytosis of the glypican protein, intracellular cleavage of the HS chains releases the bound spermine into the cell (Belting et al., supra; K. Ding et al., The Journal of Biological Chemistry 276, 46779-46791 (2001); published online in Epub December 14). Therefore, intact HS recycling is essential for spermine uptake. Inefficient HS recycling due to DUA deficiency may inhibit this spermine uptake mechanism, leading to extracellular spermine accumulation. Alternatively, extracellular GAGs may sequester spermine, shifting the equilibrium and favoring its extracellular distribution. The methanol deproteinization step used for LC-MS sample preparation in this study also precipitates soluble HS, suggesting that the spermine detected in CSF is unbound, thus suggesting that uptake inhibition, rather than GAG binding, is responsible for extracellular spermine accumulation [N. Volpi, Journal of Chromatography. B, Biomedical Applications 685, 27-34 (1996); published online Epub Oct 11]. The formation and maintenance of functional neural networks requires precise control of neurite outgrowth and synaptogenesis. During development, the CNS environment becomes increasingly inhibitory to neurite formation, and myelin-associated proteins primarily block neurite outgrowth in the adult brain. This developmental shift toward reduced neurite outgrowth is paralleled by a decrease in GAP43 expression [SMDe [La Monte et al., Developmental Brain Research 46, 161-168 (1989); online publication Epub 4 / 1 / ]. The persistent GAP43 expression and exaggerated neurite outgrowth exhibited by MPS neurons disrupt this normal balance of inhibitory and growth-promoting signals, resulting in abnormal connectivity and cognitive impairment. It is unclear how HS accumulation leads to this increased neuronal growth. Many studies have suggested the involvement of polyamines in neurite outgrowth; after axonal injury, the rate-limiting enzymes for the synthesis of spermine and its precursors, putrescine and spermidine, are elevated, allowing enhanced neurite outgrowth even in the presence of inhibitory signals from myelin [Cia (2002), Deng (2009), Gao (2004), all cited above]. Furthermore, treatment of neurons with putrescine induces neurite outgrowth when injected directly into the CSF, an effect that is blocked by inhibitors of spermine synthesis (Deng (2009) cited above). The mechanism by which polyamines affect neurite outgrowth is unknown. One potential target is the NMDA receptor, whose activation is enhanced by spermine binding (J. Lerma, Neuron 8, 343-352 (1992); published online in Epub 2 / / (http: / / dx.doi.org / 10.1016 / 0896-6273(92)90300-3)). NMDA signaling induces neurite outgrowth, and the spermine-sensitive subunit of the receptor is highly expressed during development [D. Georgiev et al., Experimental cell research 314, 2603-2617 (2008); published online Epub Aug 15 (10.1016 / j.yexcr.2008.06.009); R.G. Kalb, Regulation of motor neuron dendrite growth by NMDA receptor activation. Development 120, 3063-3071 (1994); J. Zhong, et al., Journal of neurochemistry 64, 531-539 (1995)].In particular, many neurotrophic factors bind via HS-modified receptors, and their interaction with HS in the extracellular matrix can affect neurite outgrowth (D. Van Vactor et al., Current Opinion in Neurobiology 16, 40–51 (2006); online publication Epub Feb). Therefore, spermine accumulation may be one of several factors promoting abnormal neurite outgrowth in MPS I. Of 15 MPS I canine CSF samples screened, only one had spermine concentrations within the normal range. At 28 days of age, this was the youngest animal included in the study. This finding indicates that spermine accumulation may be age-dependent, but this study demonstrates that spermine is already elevated in infants with Hurler syndrome by 6 months of age. Future studies will assess CSF spermine levels longitudinally in MPS patients. If spermine increases with age in MPS patients, this could explain the kinetics of cognitive decline, as most patients experience 1–2 years of normal development before the onset of developmental delay. between enzyme deficiency and abnormal neurite outgrowth phenotype in MPS. Inducing the accumulation of metabolites that alter neuronal growth markers in novel associations may contribute to HS metabolic disorders and explain the cognitive dysfunction associated with these disorders. Future studies will confirm the presence of elevated spermine in other MPSs, such as MPS II. These findings also indicate that CSF spermine may be useful as a noninvasive biomarker for assessing the pharmacodynamics of novel CNS-directed therapies for MPS. Future trials of CNS-directed therapies will evaluate the correlation between cognitive endpoints and changes in CSF spermine.

[0252] Example 7: CT-guided ICV delivery device A. Pre-Procedure Screening Evaluation 1. Protocol Visit 1: Screening The investigator will explain the screening process leading up to the intracisternal (IC) procedure, the administration procedure itself, and all potential safety risks so that the subject (or designated caregiver) is fully informed when signing the informed consent.

[0253] During the screening evaluation of subject eligibility for IC procedures, the following will be performed and provided to the neuroradiologist / neurosurgeon / anesthesiologist: medical history; concomitant medications; physical examination; vital signs; electrocardiogram (ECG); and laboratory test results.

[0254] 2. Interval: Screening visit 2 of the study To allow sufficient time to screen for eligibility, the following procedures will be conducted any time between the initial screening visit and up to one week prior to Study Visit 2 (Day 0): Head / neck magnetic resonance imaging (MRI) with or without gadolinium [Note: Subjects must be suitable candidates to receive gadolinium (i.e., eGFR >30 mL / min / 1.73 m 2 )] In addition to head / neck MRI, investigators will determine the need for further evaluation of the neck with flexion / extension studies. MRI protocols include T1, T2, DTI, FLAIR, and CINE protocol images Head / neck MRA / MRV per institutional protocol (Note: Subjects with a history of intra / transcranial surgery may be excluded or may require further testing (e.g., radionucleotide ultrasound) to allow proper assessment of CSF flow and identification of potential blockages or lack of communication between CSF spaces). ·Neuroradiologist / Neurosurgeon Subject Procedure Assessment Meeting: Representatives from the three sites will have a teleconference (or web conference) to discuss each subject's eligibility for the IC procedure based on all available information (scans, medical history, physical examination, laboratory tests, etc.). Every attempt should be made to reach a consensus on proceeding with the IC procedure or failing to screen the subject (i.e., each member should be prepared to accept the decision). ·Preoperative assessment of anesthesia from Day 28 to Day 1, with a detailed assessment of the airway, neck (shortening / thickening), and head range of motion (cervical flexion), keeping in mind the special physiological requirements of MPS subjects. 3. Day 1: Computed tomography room and vector preparation for administration. Prior to the IC procedure, the CT room will ensure the following equipment and medications are present: Adult lumbar puncture (LP) kit (provided by each facility) Becton Dickinson (BD) 22 or 25 gauge x 3-7 inch spinal needle (Quincke bevel) Coaxial introducer needle (e.g., 18G x 3.5 inches) (for spinal needle introduction) used at the discretion of the interventionalist 4-way small diameter stopcock with swivel (spin) male luer lock T-connector extension set (tubing) with female Luer lock adapter, approx. length 6.7 inches Omnipaque 180 (iohexol) for intrathecal administration Iodinated contrast agents for intravenous (IV) administration 1% Lidocaine Injection (if not included in the adult LP kit) Prefilled 10cc saline (sterile) flush syringe Radiopaque marker(s) Surgical preparation equipment / shaving razors Pillow / support to allow proper positioning of intubated subject Endotracheal intubation devices, general anesthesia machines and ventilators Intraoperative neurophysiological monitoring (IONM) equipment (and personnel) A 10cc syringe containing the AAV9.hIDUA vector; prepare and move to the CT / operating room (OR) according to the individual pharmacy manual. 4. Day 1: Subject Preparation and Dosing Informed consent for research and procedures will be confirmed and documented in the medical record and / or study file. Separate consent for procedures by the radiologist and anesthesiologist will be obtained according to institutional requirements. Study subjects will have intravenous access within an appropriate hospital care unit according to institutional guidelines (e.g., two IV access sites). Intravenous fluids will be administered at the anesthesiologist's discretion. ·At the discretion of the anesthesiologist, administer general anesthesia and induce study subjects in the appropriate patient care unit, holding area, or surgical / CT procedure room in accordance with institutional guidelines, and perform endotracheal intubation. A lumbar puncture is performed to remove 5 cc of cerebrospinal fluid (CSF), followed by the injection of intrathecal contrast (Omnipaque 180) to aid in visualization of the cisterna magna. Appropriate subject positioning maneuvers are performed to facilitate diffusion of the contrast into the cisterna magna. If not already done, attach an intraoperative neurophysiological monitoring (IONM) device to the subject. The subject is placed on the CT scanner table in the prone or lateral position. If deemed appropriate, the subject will be positioned in a manner that results in neck flexion to the degree deemed safe during preoperative evaluation and has a demonstrated normal neurophysiological monitor signal after positioning. The following research staff and / or researchers are confirmed to be present and identified on-site: o The interventionalist / neurosurgeon performing the procedure o Anesthesiologist and Respiratory Technician(s) Nurses and physician assistants oCT (or OR) technician Neurophysiology Technician o Field Research Coordinator Properly shave the subject's skin below the skull. · Perform a CT scout image followed by a pre-procedure planning CT with IV contrast if deemed necessary by the interventionalist to identify target location and image the vasculature. Once the target site (cisterna magna) has been identified and the needle trajectory planned, prepare and drape the skin using sterile technique according to institutional guidelines. Radiopaque markers are placed at the target skin location as directed by the interventionalist. The skin under the marker is anesthetized by infiltration with 1% lidocaine. Advance a 22G or 25G spinal needle toward the cisterna magna, using a coaxial introducer needle if necessary. After needle advancement, CT images are acquired using the thinnest CT slice thickness feasible using institutional equipment (ideally 2.5 mm or less). Serial CT images should be taken to visualize the needle and associated soft tissue. The lowest possible dose that allows adequate visualization of tissues (e.g., paraspinal muscles, bone, brainstem, and spinal cord) should be used. Correct needle placement is confirmed by observation of CSF in the needle hub and visualization of the needle tip within the cisterna magna. The interventionalist ensures that the syringe containing the vector is located near, but outside, the sterile field. Before handling or administering the vector, ensure that gloves, masks, and eye protection are worn by staff assisting with the procedure within the sterile field (other staff outside the sterile field do not need to undergo these procedures). Attach a short (~6 inch) extension tube to the inserted spinal needle, which in turn is attached to the 4-way stopcock. Once the device is "self-primed" with the subject's CSF, attach a 10 cc prefilled saline flush syringe to the 4-way stopcock. · Give the syringe containing the vector to the interventionalist and attach it to the port on the 4-way stopcock. Once the stopcock port to the syringe containing the vector has been opened, slowly inject the contents of the syringe (over approximately 1-2 minutes), being careful not to apply excessive force to the plunger during injection. Once the contents of the syringe containing AAV9.hIDUA have been injected, you may turn the stopcock and flush the stopcock and needle assembly with 1-2 cc of saline using the attached prefilled syringe. When ready, the interventionalist alerts the staff to remove the device from the subject. In one movement, 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 receptacle or hard container (for needles). Inspect the needle insertion site for signs of bleeding or CSF leak and treat as directed by the investigator. The site will be dressed using gauze, surgical tape and / or Tegaderm bandages as indicated. The subject is removed from the CT scanner and placed in a supine position on a stretcher. Discontinue anesthesia and care for the subject according to institutional guidelines for post-anesthesia care. Neurophysiological monitors may be removed from the study subject. The head of the stretcher on which the subject lies should be elevated slightly (approximately 30 degrees) during recovery. Subjects will be transported to the appropriate post-anesthesia care unit according to institutional guidelines. Once the subject has regained sufficient consciousness and is stable, they will be admitted to the appropriate floor / unit for protocol-specific assessment. Neurological assessment will be performed according to protocol, and the Investigator will oversee the subject's care in collaboration with hospital and study staff.

[0255] Example 8: Evaluation of the intrathecal route of administration in large animals The purpose of this study was to evaluate more conventional administration methods into the CSF, including intracerebroventricular (ICV) injection and injection via lumbar puncture. Briefly, this study compared ICV and IC AAV administration in dogs. Vector administration was assessed by lumbar puncture in nonhuman primates, and some animals were placed in Trendelenburg position after injection, a maneuver suggested to improve intracranial distribution of vector. In the dog study, ICV and IC vector administration resulted in similarly efficient transduction throughout the brain and spinal cord. However, animals in the ICV cohort developed encephalitis, apparently due to a severe T cell response to the transgene product. Only in the ICV cohort was this transgene-specific immune response observed, which is suspected to be related to the presence of localized inflammation from the injection procedure at the site of transgene expression. In nonhuman primate (NHP) studies, very large injection volumes (approximately 40% of the total CSF volume) were used. This improved transduction efficiency after vector administration into the lumbar cistern compared to previous studies. However, this approach was still not as efficient as IC administration. Placing the animals in Trendelenburg after injection did not provide any additional benefit. However, it was found that a larger injection volume could improve intracranial distribution of the vector.

[0256] To maximize the efficacy of intrathecal AAV delivery, it will be important to determine the optimal route of vector administration to the CSF. We previously reported that vector injection into the cerebellomedullary cistern via suboccipital puncture achieved effective vector distribution in nonhuman primates, whereas injection via lumbar puncture substantially reduced spinal cord transduction and resulted in virtually no distribution to the brain, highlighting the importance of the administration route [Hinderer, Molecular Therapy-Methods & Clinical Development. 12 / 10 / Online 2014;1]. Other researchers have suggested that vector delivery into the lateral ventricle, a common clinical procedure, results in effective vector distribution [Haurigot et al., J Clin Invest., 123(8):3254-3271]. It has also been reported that delivery via lumbar puncture can be improved by placing the animal in Trendelenburg position after injection to promote intracranial vector distribution [Meyer et al., Molecular therapy: the journal of the American Society of Gene Therapy, October 31, 2014]. This study compared intraventricular and intracisternal administration of an AAV9 vector expressing a green fluorescent protein (GFP) reporter gene in dogs. Although intraventricular delivery may carry the additional risk of transgene-specific immune responses, the inventors found that both routes achieved effective distribution throughout the CNS. The inventors also evaluated vector delivery via lumbar puncture in NHPs and the effect of placing the animal in Trendelenburg position after injection. Although there was no clear effect of positioning after injection, the inventors found that a larger injection volume could improve intracranial vector distribution.

[0257] A. Materials and Methods: 1. Vector Generation: The GFP vector was constructed from an AAV serotype 9 capsid carrying an expression cassette containing the chicken beta-actin promoter, a cytomegalovirus immediate-early enhancer, an artificial intron, enhanced green fluorescent protein cDNA, woodchuck hepatitis virus posttranscriptional regulatory elements, and a rabbit beta-globin polyadenylation sequence. The GUSB vector was constructed from an AAV serotype 9 capsid carrying an expression cassette containing the chicken beta-actin promoter, a cytomegalovirus immediate-early enhancer, an artificial intron, canine GUSB cDNA, and a rabbit beta-globin polyadenylation sequence. The vectors were produced by triple transfection of HEK293 cells and purified on an iodixanol gradient as previously described [Lock et al., Human gene therapy. Oct 2010;21(10):1259-1271].

[0258] 2. Animal Experiments: All dogs were examined at the National Referral Center for Animal Models of Humans at the University of Pennsylvania. Animals were maintained at the National Referral Center for Animal Models of Human Genetic Disease at the School of Veterinary Medicine (NIH OD P40-010939) in accordance with the National Institutes of Health and USDA guidelines for the care and use of laboratory animals.

[0259] 3. NHP Study: This study included six cynomolgus monkeys aged 9–12 years. The animals weighed 4–8 kg at the time of injection. The vector (2 × 10 13 GC) before injection. The vector was diluted with Nipaque (Iohexol) 180 contrast agent. Injection of the vector via lumbar puncture was performed as previously described [Hinderer, Molecular Therapy - Methods & Clinical Development. 12 / 10 / online 2014;1]. Accurate injection into the intrathecal space was confirmed by fluoroscopy. For animals in the Trendelenburg group, the head of the bed was lowered 30 degrees for 10 minutes immediately after injection. Euthanasia and tissue collection were performed as previously described [Hinderer, Molecular Therapy - Methods & Clinical Development. 12 / 10 / online 2014;1].

[0260] 4. Dog Study: This study included six 1-year-old MPS I dogs and six 2-month-old MPS I dogs. VII dogs were included. Baseline MRI was performed in all ICV-treated dogs to plan injection coordinates. Intracisternal injections were performed as previously described [Hinderer et al., Molecular therapy: the journal of the American Society of Gene Therapy. Aug 2015;23(8):1298-1307]. For ICV injection, dogs were anesthetized with intravenous propofol, endotracheally intubated, maintained under anesthesia with isoflurane, and positioned in a stereotactic frame. The skin was aseptically prepared, and an incision was made at the injection site. A single burr hole was drilled at the injection site, through which a 26-gauge needle was advanced to the desired depth. Placement was confirmed by CSF return. Vector (1.8 × 10 in 1 mL) was administered. 13 GC) was slowly infused over 1-2 minutes. Euthanasia and tissue collection were performed as previously described [Hinderer et al., Molecular therapy: the journal of the American Society of Gene Therapy. Aug 2015;23(8):1298-1307].

[0261] 5. Histology: Brains were processed as described for evaluation of GFP expression [Hinderer, Molecular Therapy - Methods & Clinical Development. 12 / 10 / Online 2014;1]. GUSB enzyme staining and GM3 staining were performed as previously described [Gurda et al., Molecular therapy: the journal of the American Society of Gene Therapy. October 8, 2015].

[0262] 6. ELISPOT: At the time of necropsy, blood was collected from vector-treated dogs into heparinized tubes. Peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation. T cell responses to AAV9 capsid peptides and GFP peptides were assessed by interferon gamma ELISPOT. AAV9 and GFP peptide libraries were synthesized as 15-mers with 10 amino acid overlaps (mimotopes). The AAV9 peptide library was grouped into three pools: Pool A, peptides 1–50; Pool B, peptides 51–100; and Pool C, peptides 101–146. The GFP peptide library was also grouped into three pools. Phorbol 12-myristate 13-acetate plus ionomycin salt (PMA+ION) was used as a positive control. DMSO was used as a negative control. Cells were stimulated with peptides, and interferon gamma secretion was detected as described. Responses were considered positive if they exceeded 55 spot-forming units (SFU) per million lymphocytes and were at least three times the DMSO negative control value.

[0263] 7. Biodistribution: At the time of necropsy, tissues for biodistribution were immediately frozen on dry ice. DNA isolation and quantification of vector genomes by TaqMan PCR were performed as described [Wang et al., Human gene therapy. Nov. 2011;22(11):1389-1401].

[0264] 8. GUSB Enzyme Assay: GUSB activity was measured in CSF as described [Gurda et al., Molecular therapy: the journal of the American Society of Gene Therapy. Oct 8 2015].

[0265] B. Results 1. Comparison of intraventricular and intracisternal vector delivery in dogs Our previous study using a canine model of the lysosomal storage disease mucopolysaccharidosis type I (MPS I) demonstrated that intracisternal AAV9 injection can effectively target the entire brain and spinal cord [Hinderer et al., Molecular therapy: journal of the American Society of Gene Therapy. Aug 2015;23(8):1298-1307]. In this study, we compared the distribution of AAV9 vectors expressing a GFP reporter gene administered into the cisterna magna or lateral ventricle of adult MPS I dogs. Three dogs were injected with 1 mL of vector (1.8 × 10 13 Dogs were treated with a single injection of 1000 copies of the vector (1 genome copy) into the cisterna magna. Three additional dogs received a single injection of the same vector into the lateral ventricle. In dogs treated by ICV injection, the larger lateral ventricle was selected for injection, and baseline MRI was performed to define the target coordinates. Injections were performed using a stereotaxic frame to accurately target the designated ventricle.

[0266] The three dogs treated with IC vector injection appeared healthy throughout the study. They were euthanized 2 weeks after vector injection for evaluation of vector biodistribution and transgene expression. No gross or microscopic brain lesions were observed in any IC-treated dog (Figures 12A-12F). Measurement of vector genomes by quantitative PCR revealed vector deposition throughout all harvested regions of the brain and spinal cord (Figure 13). Consistent with the distribution of vector genomes, strong transgene expression was detectable throughout most regions of the cerebral cortex as well as the entire spinal cord (Figures 14A-14H). Spinal cord histology was remarkable for strong transduction of alpha motor neurons with a transduction gradient favoring thoracic and lumbar segments.

[0267] The three dogs treated with ICV vector injections initially appeared healthy after the procedure. However, one animal (I-567) was found dead 12 days after injection. The other two animals survived to the designated 14-day necropsy time point, but one animal (I-565) became comatose before euthanasia, and the other (I-568) began to show facial muscle weakness. These clinical findings correlated with significant gross brain lesions (Figures 12A–12F). The brains of all three animals showed discoloration surrounding the needle track, and associated hemorrhage in those animals led to their death. Histological evaluation revealed severe lymphocytic inflammation in the area surrounding the injection site. Perivascular lymphocytic infiltrates were also observed throughout the brain of each animal (Figures 12G and 12H). Given this evidence of immunotoxicity, we assessed T cell responses to both the AAV9 capsid protein and the GFP transgene in peripheral blood samples collected from one of the ICV-treated dogs (I-565) at necropsy. Interferon gamma ELISPOT showed a strong T cell response to GFP, with no evidence of a response to the capsid peptide (Figure 12I). This suggests that the observed encephalitis was caused by a cell-mediated immune response to the transgene product.

[0268] Vector distribution in ICV-treated animals was similar to that observed in the IC-treated group, although spinal cord transduction was somewhat greater in the IC cohort (Figure 13). GFP expression was observed throughout the CNS regions examined in ICV-treated animals (Figures 14A-14H).

[0269] 2. Effect of Trendelenburg position on CNS transduction after AAV9 administration by lumbar puncture in NHPs We previously compared AAV9 injection into the cisterna magna or lumbar cisterna of NHPs and found that the lumbar route was 10-fold less effective for targeting the spinal cord and 100-fold less effective for targeting the brain [C. Hinderer, et al., Molecular Therapy - Methods & Clinical Development. 12 / 10 / Online 2014;1]. Since then, other investigators have demonstrated better transduction using AAV9 administration by lumbar puncture, with improved vector brain distribution achieved by placing the animals in the Trendelenburg position after injection [Myer et al. [Also see: [Bernie et al., Molecular Therapy: the journal of the American Society of Gene Therapy. Oct 31, 2014]. In this approach, the vector was diluted into an excess volume of contrast medium to increase the density of the solution and promote gravity-driven distribution via Trendelenburg. Six adult cynomolgus monkeys were transfected with AAV9 (2 × 10) expressing GFP in the L3-4 interspace. 13Animals were treated with a single injection of 1000 genome copies. The vector was diluted to a final volume of 5 mL with Iohexol 180 contrast agent. Four animals were placed on the treatment table with their heads at a -30° angle for 10 minutes immediately after injection. Fluoroscopic images were captured 10 minutes later to confirm the distribution of contrast agent in the CSF. Remarkably, this large injected volume (approximately 40% of the animal's total CSF volume) [Reiselbach et al., New England Journal of Medicine. 1962;267(25):1273-1278] rapidly distributed throughout the spinal cord subarachnoid space and into the basal cisterns, even in animals not positioned in Trendelenburg (Figures 16A and 16B). Analysis of vector genome distribution by PCR (Figure 17) and GFP expression (Figures 18A-18H) demonstrated transduction throughout the brain and spinal cord. There was no apparent effect of postinjection position on the number or distribution of transduced cells. As previously reported, there was peripheral vector escape and liver transduction after intrathecal AAV administration [Hinderer et al., Molecular Therapy-Methods & Clinical Development. 12 / 10 / Online 2014;1; Haurigot et al., Journal of Clinical Investigation. Aug 2013;123(8):3254-3271]. The extent of liver transduction was dependent on the presence of pre-existing neutralizing antibodies (nAb) to AAV9. Four of six animals had no detectable baseline AAV9 nAb (titer <1:5), and two animals (4051 and 07-11) had detectable pre-existing antibodies to AAV9 at a titer of 1:40. Consistent with previous results, pre-existing antibodies blocked liver transduction and resulted in increased vector distribution to the spleen [Wang et al, Human gene therapy. Nov 2011;22(11):1389-1401, but had no effect on CNS transduction; Haurigot et al, Journal of Clinical Investigation. Aug 2013;123(8):3254-3271].

[0270] C. Consideration Because suboccipital puncture is not a tolerated procedure in clinical practice, we evaluated more common CSF access sites, including the lateral ventricle and lumbar cistern. Here, we evaluated the use of higher density vector lysis and Trendelenburg positioning after injection to improve vector distribution cranially from the lumbar region.

[0271] In a canine study, both IC and ICV vector injections resulted in similarly effective vector distribution, but encephalitis occurred only in the ICV group. A T cell response against the GFP transgene was detectable in one ICV-treated dog, suggesting that the lymphocytic encephalitis observed in these animals was due to a transgene-specific immune response. Induction of a T cell response against a novel antigen requires recognition of epitopes from proteins by naive T cells. Two elements are required: a cellular response to the transgene, and an inflammatory "danger signal" that promotes T cell activation. AAV may be able to express a foreign transgene without eliciting immunity to the transgene product because it does not activate the innate immune system, thereby evading inflammatory signals and promoting tolerance rather than immunity when naive lymphocytes encounter the newly expressed antigen. Local inflammation caused by trauma penetrating the brain parenchyma at the same location where the foreign transgene product is expressed may provide the danger signal necessary to induce an immune response to the transgene product. This is supported by previous studies in MPS I dogs, which developed cell-mediated immune responses to enzymes expressed from AAV vectors delivered by direct brain injection, but not by IC injection [Ciron et al., Annals of Neurology. Aug 2006;60(2):204-213; Hinderer et al., Molecular therapy: the journal of the American Society of Gene Therapy. Aug 2015;23(8):1298-1307]. The possibility of such an immune response depends on whether the transgene product is recognized as foreign, as with delivering vectors expressing proteins that are also produced endogenously, even if the inflammatory response caused by the injection may not break tolerance to self-proteins. Results from studies of ICV vector delivery in MPS VII dogs support this concept, as similarity between the transgene product and endogenous proteins is likely responsible for the absence of the type of T cell response observed against GFP. This may also be true for patients with recessive disorders who have missense mutations that allow the production of proteins similar to the transgene product. Therefore, immune risks may vary depending on the patient population and the transgene product, and in some cases, immunosuppression may be necessary to prevent destructive T cell responses to the transgene. These findings suggest that the risk of adverse immune responses is likely to be mitigated by using IC rather than ICV administration.

[0272] A study of AAV9 administration via lumbar puncture in NHPs demonstrated greater transduction throughout the CNS than we previously observed with this route of administration. This difference is likely due to the larger injection volume in this study, which was necessary to dilute the vector into an excess of contrast agent. Previous studies have shown that such large injections (approximately 40% of the CSF volume) can deliver injected material directly to the basal cistern and even the ventricular CSF of macaques [Reiselbach, cited above]. Given the extremely large injection volumes (>60 mL) required, which are not routinely administered to patients, the translatability of this approach to humans is unclear. Furthermore, even with this large-volume approach, injection via lumbar puncture was less efficient than previous results with IC delivery. In this previous study, animals were dosed by weight, and only one animal received an IC vector dose equivalent to that used here [Hinderer et al., Molecular Therapy - Methods & Clinical Development. 12 / 10 / Online 2014;1]. The animals had an average of three times greater vector distribution in the brain and spinal cord, indicating that even very large volumes of vector delivered to the lumbar cistern are less efficient than IC delivery. In contrast to literature reports, we found no added benefit from placing the animals in Trendelenburg position after lumbar vector injection [Meyer et al. al,Molecular therapy: the journal of the American Society of Gene Therapy.Oct 31 2014].

[0273] Taken together, these findings support vector administration at the level of the cisterna magna, as this approach achieves more efficient vector distribution than administration by lumbar puncture and appears to pose less risk of immunity to the transgene product than ICV administration. Vector delivery to the cisterna magna can be performed clinically using the suboccipital puncture approach used in preclinical studies. Furthermore, injection into the subarachnoid space between the first and second cervical vertebrae using a lateral approach (C1-2 puncture) may result in similar vector distribution, given the proximity of the injection site to the cisterna magna. Unlike suboccipital puncture, the C1-2 approach has the added advantage of being widely used clinically for CSF access, especially for intrathecal contrast administration.

[0274] Example 9: Nonclinical pharmacology / toxicology study of intrathecally injected AAV2 / 9.CB7.CI.hIDS.RBG in rhesus monkeys To provide a sufficient safety margin beyond the minimum effective dose (MED) for human administration, a study designed to evaluate the safety of intrathecal administration of two doses of AAV2 / 9.CB7.CI.hIDS.RBG, a vector encoding human IDS, in rhesus monkeys will be conducted.

[0275] The control product is administered via suboccipital puncture to a single macaque randomized to Group 1. The test product is administered via suboccipital puncture to six rhesus macaques randomized to Groups 2-3. Macaques in Group 2 receive 5 x 10 13 The macaques in the three groups received the test article at a high dose of 1.7 x 10 genome copies (GC) (N = 3). 13 Subjects will be given the test article administered at a low dose of GC (N=3). Blood and cerebrospinal fluid will be collected as part of a general safety panel.

[0276] After the in-life phase of these studies is completed, 90±3 days after vector administration, the macaques are necropsied and tissues are collected for comprehensive histopathological examination. Lymphocytes are collected from the liver, spleen, and bone marrow and examined for the presence of cytotoxic T lymphocytes (CTL) in these organs at necropsy.

[0277] II. Experimental Design A. Material Test article-AAV9.CB7.hIDS is also known as AAV2 / 9.CB7.CI.hIDS.RBG and designated AAV2 / 9.CB7.CI.hIDS.RBG.KanR. These names are synonymous and may be used interchangeably.

[0278] The test product was measured using droplet digital (dd) PCR to determine 5.72 x 10 13 The vector was produced as a single lot with a concentration of 1000 GC / ml. After manufacture, the vector is stored at 60°C or below until the day of injection. On the day of injection, the vector is diluted with the control product. Once diluted, the vector is stored in the refrigerator or on wet ice at 2-4°C until the time of injection. The vector is prepared on the day of injection.

[0279] Control animals receive the control article Elliot's Formulation Buffer (EFB) and no test article (EFB + 0.001% Pluronic F68). Control articles are stored at room temperature after manufacture and until the day of injection.

[0280] B. Test System Justification for the choice of test system: This study involves intrathecal (IT) delivery of a gene therapy vector for CNS disease. The CNS landscape in non-human primates (NHPs) serves as the most representative model of our clinical target population. This study will provide data on dose-related toxicity of the vector following IT injection.

[0281] Seven (7) male Macaca mulatta (rhesus monkeys) weighing 3-10 kg and aged 3-7 years, supplied by Covance Research Products, Inc. (Alice, TX), are used.

[0282] II. General Design Procedure Seven male rhesus monkeys are used in the study. The animals are divided into three study groups as described in Table 1. All seven animals receive IT injections via suboccipital puncture. [Table 1]

[0283] Animals are anesthetized and the test article is administered via suboccipital puncture into the cisterna magna.

[0284] Anesthetized macaques are prepped in the procedure room, transferred to the fluoroscopy suite, and placed on the imaging table in a lateral decubitus position with the head flexed forward for CSF collection and administration into the cisterna magna. The injection site is aseptically prepared. Using aseptic technique, a 21- to 27-gauge, 1- to 1.5-inch Quincke spinal needle (Becton Dickinson) is advanced into the suboccipital space until CSF flow is observed. A maximum of 1.0 mL of CSF is collected for baseline analysis and prior to administration. Anatomical structures traversed include the skin, subcutaneous fat, epidural space, dura mater, and atlanto-occipital fascia. The needle is aimed toward the wider superior gap of the cisterna magna to avoid blood contamination and potential brainstem injury. Correct needle placement is confirmed by myelography using a fluorescent light (OEC9800 C-Arm, GE). The fluoroscopy equipment is operated according to the manufacturer's recommendations. After CSF collection, a Luer access extension catheter is attached to the spinal needle to facilitate administration of Iohexal (trade name: Omnipaque 180 mg / mL, General Electric Healthcare) contrast medium and the test or control product. One (1) mL of Iohexol is administered through the catheter and spinal needle. After confirming needle placement, a syringe containing the test product (a volume equal to 1 mL plus the volume of the syringe and the dead space of the linker) is attached to the flexible linker and injected slowly over 20 to 60 seconds. The needle is removed, and direct pressure is applied to the puncture site. Any residual test product remaining in the injection device is collected and stored at -60°C or below.

[0285] If the animal cannot be successfully dosed into the cisterna magna, dosing into the C1-C2 intrathecal space (atlas joint) may be used as an alternative site for dosing. The dosing procedure is as follows: The animal is placed in a lateral decubitus position, the head is flexed forward for CSF collection, and the dose is administered into the C1-C2 intrathecal space. The injection site is aseptically prepared. Using aseptic technique, a 21- to 27-gauge, 1- to 1.5-inch Quincke spinal needle (Becton Dickinson) is advanced into the intrathecal space until CSF flow is observed. A maximum of 1.0 mL of CSF is collected for baseline analysis and prior to dosing. Anatomical structures traversed include the skin, subcutaneous fat, epidural space, dura mater, and fascia. The needle should be inserted to avoid blood contamination and potential risk of infection to the cervical spinal cord. The catheter is inserted into the intervertebral space above the C2 vertebra to avoid any serious injury. The remaining procedure is similar to that described above for intracisternal administration.

[0286] Animals receive intrathecal injections of either AAV9.CB7.hIDS or a control. The dosing frequency is outlined in Table 3. The high dose of AAV9.CB7.hIDS is 5 x 10 13 GC and low dose 1.7 x 10 13 The total volume of diluted AAV9.CB7.hIDS or control injected per macaque is 1 mL.

[0287] III. Results CSF pleocytosis, expressed as CSF leukocyte counts, was observed in 2 of 3 animals in the high-dose treatment group (Group 2) and 1 of 3 animals in the low-dose treatment group (Group 3) (Figure 28). This pleocytosis resolved in all but one high-dose treated animal (RA2203) by Day 90.

[0288] In addition, axonal degeneration of the dorsal columns of the spinal cord is present (data not shown).

[0289] The current low dose is 1.9 × 10 11 GC / g brain weight, which corresponds to the proposed high clinical dose of 9.4 × 10 10The current high dose is approximately 5.6 x 10 11 This corresponds to GC / g brain weight, which is approximately five times the clinical dose.

[0290] ELISA assays for anti-hIDS antibodies were performed on serum samples collected from the animals. The results obtained on day 60 are plotted in Figure 29 and shown in Table 2 below. CSF samples were diluted 20-fold for evaluation via ELISA assay for anti-hIDS antibodies. The results are shown in Table 3. Reduced immunogenicity was observed in both serum and CSF in the low-dose treatment group (Group 2) compared to the high-dose group (Group 3). Immunogenicity to hIDS was not observed in untreated controls. [Table 2] [Table 3]

[0291] Example 10: Nonclinical pharmacology / toxicology study of intrathecally injected AAV9.CB7.hIDS in immunosuppressed rhesus monkeys A study designed to evaluate the effect of chemically induced immunosuppression (IS) on the safety of intrathecal administration of two doses of AAV9.CB7.hIDS, a vector encoding a human IDS, in rhesus monkeys is conducted. This study is described in Example 2 and is described below. with modifications noted below.

[0292] Control animals are repeated as in Example 9. Test articles are administered by suboccipital puncture to six rhesus monkeys randomized into groups 1 and 2. Group 1 macaques receive 5 x 10 13 The second group of macaques received the test product at a high dose of 1.7 x 10 genome copies (GC) (N = 3). 13Monkeys in Groups 1 and 2 will receive test article administered at a low dose of GC (N=3). Blood and cerebrospinal fluid will be collected as part of a general safety panel. Monkeys in Groups 1 and 2 will receive mycophenolate mofetil (MMF) at least 2 weeks prior to and up to 60 days after AAV9.CB7.hIDS dosing, and rapamycin at least 2 weeks prior to and up to 90 days (+ / - 3 days) after dosing. Plasma trough levels of both immunosuppressants will be monitored, and doses will be adjusted to maintain a range of 2-3.5 mg / L for mycophenolate acid (MPA, the active metabolite of MMF) and 10-15 μg / L for rapamycin.

[0293] After completing the in-life phase of these studies, 90±3 days after vector administration, the macaques will be necropsied and tissues will be collected for comprehensive histopathological examination. Lymphocytes will be collected from the blood, spleen, and bone marrow and examined for the presence of cytotoxic T lymphocytes (CTL) in these tissues at necropsy.

[0294] A. Material The test and control products and their preparation are described in Example 9.

[0295] B. Test System Macaca mulatta (rhesus monkeys) are utilized and maintained as described in Example 9. Cohorts of six animals (six males) are used in this study. Animals are assigned numbers 1-6, from lowest ID to highest ID. A random list of numbers 1-6 is generated by random.org, and once randomized, animals are assigned sequentially as follows: Group 1 is assigned 3 animals; Group 2 is assigned 3 animals.

[0296] C. General Design Procedure Sample size and group specification: Six rhesus monkeys are used in the study. The animals are divided into two study groups as listed in Table 4. All six animals receive an IT injection via suboccipital puncture. [Table 4]

[0297] An immunosuppressive regimen is administered to all animals in Groups 1 and 2. The drug combinations, doses, and administration schedules for each group of animals are summarized in Table 5. Animals may be treated with systemic antibiotics and / or antifungals to treat opportunistic infections associated with immunosuppression, if and when it occurs.

[0298] Animals receive a combination of mycophenolate mofetil (MMF) and rapamycin. Both immunosuppressants are administered via oral or nasogastric feeding tubes to anesthetized or conscious, chair-restrained macaques. Anesthetized animals are not fasted, allowing for aspiration of gastric contents through the tube (to confirm correct placement). The immunosuppressive regimen begins at least 2 weeks prior to intrathecal administration of the test article. The starting dose of the IS drug is based on previously described efficacy in rhesus monkeys, at doses previously used in GTP for other studies, and is adjusted upon monitoring of plasma trough levels.

[0299] The animal's weight on the first day of immunosuppression is used to calculate the initial dose. Animals are weighed every morning, and the dose is recalculated if the weight changes by more than + / - 10%.

[0300] Trough levels are monitored twice weekly initially and once weekly after 60 days (if rapamycin alone is administered). [Table 5]

[0301] c.IS drugs Rapamycin will be administered at a dose of 0.5-4 mg / kg PO, SID for at least 14 days prior to intrathecal administration and until study day 90 (inclusive) (+ / - 3 days). The starting dose is 1 mg / kg. Rapamycin doses will be calculated to maintain target trough levels as close to 10-15 μg / L as possible for the duration of the study. If target trough drug levels are not achieved within 1 week, the dose will be titrated in 0.25-2 mg / kg dose intervals. After the stabilization period, adjustments will be made if trough levels are too low for two consecutive blood draws. If trough levels are too high, adjustments will be made immediately.

[0302] MMF will be administered at a starting dose of 50 mg / kg PO BID for at least 14 days prior to intrathecal administration and up to and including day 60 of the study. Trough level results will then be used to adjust the dose, titrated in dose intervals of 5 to 25 mg / kg. MPA trough levels will be maintained as close to 2 to 3.5 mg / L as possible. After the stabilization period, adjustments will be made if trough levels are too low for two consecutive blood draws. If trough levels are too high, adjustments will be made immediately.

[0303] d. Prescribing IS drugs MMF is administered orally to the test system using a commercially available oral solution at a concentration of 200 mg / mL.

[0304] Rapamycin is administered orally to the test system using one or more of the following commercially available formulations: 0.5 mg coated tablets; 1 mg coated tablets; 2 mg coated tablets.

[0305] If a pill formulation is to be administered, tablets are prepared as follows: Each rapamycin pill is placed in a diluent, either room temperature or warm water (use warm water to accelerate dissolution if necessary), to dissolve the outer coating of the tablet. Use approximately 10 ml of water. The pills are white underneath the yellow coating. Once the outer coating has dissolved, crush each pill to a fine powder using a mortar and pestle to create a uniform solution. Record the total amount of diluent used to dissolve the tablet(s) in the study record.

[0306] The entire mixture is drawn into a dosing syringe and administered through an OG or NG tube as previously described in this example.

[0307] Following administration of MMF and / or rapamycin, the orogastric or nasogastric tube is flushed with drinking water, and the volume of the flush is recorded in the study record.

[0308] The following study was conducted to evaluate the efficacy of intracerebroventricular (ICV) delivery of AAV9.CB7.CI.hIDS in MPS II mice and to determine the minimum effective dose. Efficacy was based on evidence of a pharmacodynamic response to the vector (production of enzymatically active hIDS protein) and effects on the behavioral and histological manifestations of MPS II disease (IDS deficiency).

[0309] Example 11: Efficacy of intracerebroventricular AAV9.CB7.CI.hIDS.rBG in a mouse model of MPS II I. Summary Hunter syndrome, also known as mucopolysaccharidosis type II (MPS II), is an X-linked genetic disorder caused by a deficiency of iduronate-2-sulfatase (IDS), an enzyme involved in the lysosomal catabolism of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. This deficiency results in the intracellular accumulation of undegraded GAGs, ultimately leading to a progressively severe clinical phenotype. Numerous attempts have been made over the past few decades to identify potential therapeutic strategies for the disorder, including gene therapy and somatic cell therapy. A study was conducted to evaluate the short-term (21-day) biodistribution, expression, and activity of a single intracerebroventricular (ICV) administration of AAV9.CB7.CI.hIDS.rBG, an AAV9 vector expressing human IDS, in an MPS II mouse model. A further study was designed and conducted to determine the minimum effective dose (MED) of AAV9.CB7.CI.hIDS.rBG administered via the ICV route as a single dose with a 3-month post-injection (pi) observation period in a mouse model of MPS II. This study also included several safety endpoints (evaluation of humoral immune responses to the transgene and brain histopathology).

[0310] AAV9.CB7.CI.hIDS.rBG was administered at 3 x 10 8 GC or 3 × 10 9 C or 3 x 10 10A dose of 100 mg / kg of AAV9.CB7.CI.hIDS.rBG was administered intracerebroventricularly to 2-3 month-old C57BL / 6 IDSγ / - (MPS II) mice (16 males / group). On day 21, mice from groups 3-5 (Table 6) were euthanized and necropsied for assessment of CNS IDS activity, biodistribution, and anti-hIDS immunogenicity. Between days 60 and 89, wild-type and untreated MPS II mice were evaluated in a series of neurobehavioral assays (open field, Y-maze, contextual fear conditioning, and novel object recognition) to characterize the effect of disease state on these endpoints. Based on the results of these initial assays, the remaining treated mice were evaluated in two assays that assessed long-term memory (contextual fear conditioning and novel object recognition). Approximately 3 months after ICV administration of AAV9.CB7.CI.hIDS.rBG, all remaining mice were euthanized and necropsied. Serum and CSF were evaluated for IDS activity and serum anti-IDS antibodies. The liver and heart were evaluated for GAG tissue content. Lysosomal accumulation (both primary GAG accumulation and secondary ganglioside accumulation) throughout the brain was assessed by immunohistochemical staining for LIMP2 and GM3. Tissue hexosaminidase levels are higher in MPS II mice and patients and have been shown to be a biomarker of impaired lysosomal homeostasis. Tissue hexosaminidase enzyme activity was measured as a biomarker of lysosomal function secondary to AAV9.CB7.CI.hIDS.rBG administration. Brain histopathology was assessed to assess both efficacy and safety. [Table 6]

[0311] max 3×10 10 GC (5.2 × 10 by ddPCR) 10 C57BL / 6IDS in GC γ / - ICV administration of AAV9.CB7.CI.hIDS.rBG to (MPS II) mice was well tolerated without clinical signs or mortality and distributed to the CNS as well as peripheral tissues, particularly the liver, demonstrating partial redistribution of injected viral particles from the CSF to the peripheral blood.

[0312] There was evidence of brain IDS gene expression, as evidenced by vector detection and a dose-dependent increase in IDS activity in the brain at 21 days and in the CSF at 3 months p.i., with enzyme activity near wild-type levels at the highest dose (brain tissue), and comparable to wild-type levels at the mid and high doses (CSF). At all doses at 3 months p.i., there was a dose-dependent normalization of the lysosomal compartment, as indicated by a decrease in LIMP2 and GM3 staining in the CNS. Dose-dependent decreases in the amount and frequency of glial vacuolation and neuronal accumulation of amphiphiles, indicators of the MPS II CNS phenotype, were also observed in H&E-stained brain sections. Corresponding to the changes in CNS lysosomal content and the improvement in disease-related morphology in H&E-stained sections, there was an improvement in one measure of long-term memory (novel object recognition, NOR) but not in another, contextual fear conditioning (CFC). No clear dose response was evident in the improvement in NOR.

[0313] A dose-dependent increase in serum IDS activity was also observed at 3 months pi, with enzyme activity comparable to or greater than that of wild-type mice at the mid- and high-dose levels. Reflecting normalization of serum IDS activity, treated MPS II mice had a dose-dependent decrease in hexosaminidase activity and GAG content in the liver and heart. Hepatic hexosaminidase and GAG levels normalized at all dose levels in the liver and at the mid- and high-dose levels in the heart. The highly transduced liver (1–10 GCs per diploid genome) likely served as a storage organ for IDS secretion into the serum and cross-correction in the heart at high doses.

[0314] Although changes related to the ICV administration procedure itself were observed in some mice, there was no evidence of test article-related toxicity in the brain. Humoral immune responses to the transgene were minor and were observed only in some mid- and high-dose animals without affecting the health or brain histopathology of these animals.

[0315] In conclusion, AAV9.CB7.CI.IDS.rBG inhibited MPS at all dose levels. The lowest dose administered was 3 × 10 , which was well tolerated in MPS II mice and resulted in a dose-dependent increase in IDS levels (expression and enzyme activity) associated with improvement in both CNS and peripheral parameters of MPS II. 8 GC(5.2×10 8 GC ddPCR method) was the lowest effective dose in this study.

[0316] II. Materials and Methods The test article, identified as A.AAV9.CB7.CI.hIDS.rBG, had a viral load of 1.18 x 10 13 2.057 × 10 measured by GC / mL and ddPCR 13 It is a clear, colorless liquid with a titer of 1.0 GV / mL. Endotoxin is less than 1.0 EU / mL. Purity is 100%. Test samples were stored at ≤60°C.

[0317] B. Dosage Formulation and Analysis 1. Preparation of test articles: Test articles were diluted in sterile phosphate-buffered saline (PBS) to the appropriate concentration for each dose group. The diluted vectors were kept on wet ice and injected into animals within 4 hours of dilution.

[0318] C. Test System Mus musculus C57BL / 6J IDSγ / - (MPS II phenotype, N = 56 males) and C57BL / 6J IDSγ / + (wild-type phenotype, N = 8 males) were bred at Translational Research Laboratories (TRL) Vivarium from stocks originally obtained from Jackson Laboratories (stock number 024744). Animals were aged 2–3 months on day 0 (dosing day). He was 19 years old.

[0319] D. Experimental Design 1. Research W2356 All animals in groups 3-5 (16 males / group, W2356 and W2301 studies combined) received AAV9.CB7.CI.hIDS.rBG ICV on day 0. On day 21, mice in groups 3-5 (8 males / group, W2356 study) were euthanized, necropsied, and the brain, heart, lungs, liver, and spleen were harvested and snap-frozen on dry ice for assessment of brain IDS activity and tissue vector biodistribution. [Table 7]

[0320] 2. Research W2301 On day 0, all animals in groups 3-5 (16 males / group) were administered AAV9.CB7.CI.hIDS.rBG intravenously. At 2-3 months p.i., animals from groups 1 and 2 (8 mice / group) were evaluated in a battery of neurobehavioral assays, including open-field activity, Y-maze activity, contextual fear conditioning, and novel object recognition, to determine whether there was an effect of disease status (MPS II genotype) on these endpoints. If a clear disease effect was observed, 8 mice / group from groups 3-5 were also evaluated in those assays to determine whether treatment with the vector affected the response.

[0321] Animals in groups 1-5 (the same animals evaluated in the neurobehavioral assay, 8 mice / group) were deeply anesthetized (ketamine-xylazine) and CSF (cisternal puncture) and blood (cardiac puncture) were collected for evaluation of serum and CSF IDS activity and serum anti-hIDS antibodies. were euthanized and necropsied, and samples were collected for brain histopathology; brain lysosomal accumulation (assessed by immunohistochemistry and image analysis); liver and heart hexosaminidase activity; and liver and heart GAG content (assessed by tissue content). [Table 8]

[0322] 3. Administration of test article The ICV route was chosen because it is minimally invasive and does not require surgery in mice (compared to the cisternal route, which requires an incision in the skin and muscle of the neck). Because this study included several neurobehavioral endpoints, including activity recording in mice, a non-invasive injection without survival surgery was preferred. In both mice and large animals, we and others have previously demonstrated that a single injection of AAV9 into the cerebrospinal fluid (ICV or cisterna magna) targets neurons throughout the CNS (Dirren et al., Hum. Gene. Therapy 25, 109-120 (2014); Snyder et al., Hum. Gene Ther 22, 1129-1135 (2011); Federici et al., Gene Therapy 19, 852-859 (2012); Haurigot et al., J Clin Invest 123, 3254-3271 (2013); Bucher et al., Gene Therapy 21, 522-528 (2014); Hinderer et al., Mol Ther: 22, 2018-2027 (2014) and Mol Ther-Methods & Clin Dev 1, 14051 (2014)]. Data published by Haurigot et al. (2013) compare ICV injection with intracisternal injection in the setting of different lysosomal storage diseases. have specifically addressed the comparison between the two routes of administration and shown that the two routes are equivalent with respect to transgene expression and biodistribution.

[0323] The maximum achievable dose due to volume limitations (5 µL for ICV injection in adult mice) is approximately 6 x 10 per mouse. 10 Due to previous results obtained with GTP for treatment of other MPS, and due to concerns regarding scalability to larger animals, the vector was administered at 3 × 10 per mouse. 10 , 3×10 9 , or 3 × 10 8 The GC (qPCR titration method) was diluted to inject. Based on the ddPCR method, the actual dose was 5.2 × 10 per mouse. 10 , 5.2 × 109 , and 5.2 × 10 8 Considering the average brain mass of a young adult C57B16 mouse (0.4 g) (Biology of the laboratory mouse by the staff of the Jackson laboratory, 2nd ed., Earl L. Green Editor), this corresponds to 1.31 × 10 β-glucan per gram of brain mass at the highest dose. 11 GC equivalent, at the lowest dose, 1.31 × 10 per gram of brain mass 9 Equivalent to GC.

[0324] The vector was administered intravenously into the right lateral ventricle. Mice were anesthetized with isoflurane. Each anesthetized mouse was firmly grasped by the loose skin at the back of the head and injected freehand anterior and lateral to the bregma with a Hamilton syringe fitted with a 26-gauge needle adjusted to a depth of 3 mm. The injection method was previously validated in mice by injecting dye or substance P into the right lateral ventricle. Success was defined as visualization of the blue dye or itch behavior in the mouse after injection into the cerebrospinal fluid (after substance P administration).

[0325] F. Procedures, Observations and Measurements Animals were monitored daily for morbidity / mortality. Animals were monitored by daily cage-side visual observation for general appearance, signs of toxicity, distress, and behavioral changes. These data were not recorded in this non-GLP study.

[0326] Between days 60 and 90 (2–3 months after vector administration), behavioral and neurocognitive testing was performed on eight animals from groups 1 and 2 of Study W2356 to determine the effect of genotype on these endpoints. If a genotype effect was confirmed, additional testing was performed with eight animals from groups 3–5 of Study W2356 to determine whether there was a treatment effect. All behavioral procedures were performed by an operator blinded to genotype and group.

[0327] a) General locomotion (open field activity): Spontaneous activity in the open field was measured using the Photobeam Activity System (PAS)-Open Field (San Diego Instruments). For this assessment, mice were individually placed in the arena for a single 10-min trial. Horizontal and vertical beam breaks were collected to assess general locomotion and rearing activity.

[0328] b) Short-term memory (Y-maze activity): The Y Maze Spontaneous Alternation is a behavioral test to measure a rodent's willingness to explore a novel environment. The test is conducted in a Y-shaped maze with three white, opaque plastic arms at 120° angles to one another. After being introduced to the center of the maze, the animal is allowed to freely explore the three arms. Rodents typically prefer to explore new arms of the maze rather than returning to previously visited arms. Over the course of multiple arm entries, subjects should show a tendency to enter arms that have been less recently visited. The percentage of alternations is The number of arm entries and the number of triads were recorded to calculate the score. An entry is defined as all four limbs present in an arm. This test is used to quantify cognitive impairment in transgenic mouse strains and to evaluate novel chemicals for their effects on cognition. Many parts of the brain, including the hippocampus, septum, basal forebrain, and frontal cortex, are involved in this task. In this study, a standard Y-maze (San Diego Instruments) was used, and the order and number of arm entries were recorded during an 8-minute trial. Spontaneous alternation (SA) was defined as consecutive entry into all three arms of the maze without immediately returning to the previously entered arm. Total arm entries (AE) were collected as a measure of motor activity. The percentage of spontaneous alternation was calculated as %SA = (SA / (AE-2) × 100).

[0329] c) Long-term memory (contextual fear conditioning): In these tasks, animals learn to fear a novel environment, such as a tone, or an emotionally neutral conditioned stimulus (CS), due to its temporal association with an aversive unconditioned stimulus (US), usually a foot shock. When exposed to the same context or the same CS, conditioned animals exhibit freezing behavior (Abel et al., Cell 88:615–626, 1997). On the training day, mice were allowed to explore a unique conditioning chamber (Med Associates Inc.) for 300 s. A continuous 1.5 mA foot shock without a signal was delivered between 248 and 250 s of the 300 s period. After an additional 30 s in the chamber, mice were returned to their home cage. 24 h later, recall of the spatial context was assessed for 5 consecutive minutes in the same chamber where training took place. Memory was assessed using the software used to score freezing behavior (Freezescan, CleverSys Inc.). Freezing rates during the 2.5-minute pre-stimulus period of the training session (before the foot shock was administered) were compared to freezing rates upon re-exposure to the chamber. An increase in freezing indicates recall of the foot shock (i.e., learning has occurred and the animal now associates the chamber with the foot shock).

[0330] d) Long-term memory (novel object recognition): The novel object recognition (NOR) task is used to assess cognition, particularly recognition memory, in rodent models of CNS disorders. This test is based on the spontaneous tendency of rodents to spend more time exploring novel objects than familiar objects. The choice to explore novel objects reflects learning and the use of recognition memory. The novel object recognition task is generally performed in an open-field arena containing two different types of objects that are consistent in height and volume but differ in shape and appearance. During habituation, animals are allowed to explore the empty arena. 24 hours after habituation, animals are exposed to the familiar arena, which contains two identical objects placed at equal distances. The next day, mice are allowed to explore the open field in the presence of both familiar and novel objects to test long-term recognition memory. The time spent exploring each object and the discrimination index percentage are recorded. In this study, the experimental apparatus consisted of a gray rectangular arena (60 cm x 50 cm x 26 cm) with a white floor. The two unique objects were a 3.8 x 3.8 x 15 cm metal bar and a 3.2 cm diameter x 15 cm long PVC pipe. During the 5-day habituation phase, mice were handled for 1–2 min per day and allowed to explore the empty arena for 5 min per day. During the training phase, two identical objects were placed in the arena and mice were allowed to explore the objects for 15 min. During the recall phase, mice were returned to the arena containing one familiar object and one novel object. Normal mice preferentially explore the novel object. All sessions were recorded, and the time spent exploring the objects was scored using an open-source image analysis program [Patel et al. FrontBehav Neurosci 8:349(2014)].

[0331] I. Laboratory Evaluation 1. IDS activity in serum and CSF Blood samples for serum and CSF IDS activity were collected at autopsy approximately 3 months after injection. Serum was separated from the blood, and the serum and CSF were frozen on dry ice and stored at -80°C until analysis. IDS activity was measured by incubating 10 μL of the sample with 20 μL of 1.25 mM 4-methylumbelliferyl (MU)αL-idopyranosiiduronic acid 2-sulfate (Santa Cruz Biotechnology) dissolved in 0.1 M sodium acetate containing 0.01 M lead acetate (pH 5.0). After 2 h of incubation at 37°C, 45 μL of McIlvain buffer (0.4 M sodium phosphate, 0.2 M sodium citrate, pH 4.5) and 5 μL of recombinant human iduronidase (Aldurazyme, 0.58 mg / mL, Genzyme) were added to the reaction mixture, which was then incubated overnight at 37°C. The mixture was diluted in glycine buffer (pH 10.9) and the released 4-MU was quantified by fluorescence (excitation 365 nm, emission 450 nm) in comparison with a standard dilution of free 4-MU.

[0332] 2. Serum anti-IDS antibody Blood for serum anti-hIDS antibody measurement was collected by cardiac puncture at the terminal specimen endpoints of Study W2356 (day 21) and Study W2301 (approximately 3 months). Serum was separated, frozen on dry ice, and stored at -80°C until analysis. Polystyrene plates were coated overnight with 5 μg / mL recombinant human IDS (R&D Systems) in PBS, titrated to pH 5.8. Plates were washed and blocked for 1 hour in 2% bovine serum albumin (BSA) in neutral PBS. Plates were then incubated with serum samples diluted 1:1000 in PBS. Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Abcam) diluted 1:10,000 in PBS containing 2% BSA. The assay was developed using tetramethylbenzidine substrate and stopped with 2N sulfuric acid, after which absorbance was measured at 450 nm. Titers were determined from a standard curve generated by serial dilution of appropriately assigned positive serum samples at a titer of 1:10,000.

[0333] DNA was isolated from tissues from the high-dose group using a QIAamp DNA Mini Kit, and vector genomes were quantified by TaqMan PCR as previously described (Bell et al., 2006). Total cellular DNA was extracted from tissues using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). Detection and quantification of vector genomes in extracted DNA was performed by real-time PCR (TaqMan Universal Master Mix, Applied Biosystems, Foster City, CA, USA) using primer and probe sets targeting the rBG poly(A) sequence. Forward primer: 5V-TTCCCTCTGCCAAAAATTATGG-3V, SEQ ID NO: 16; Reverse primer: 5V-CCTTTATTAGCCAGAAGTCAGATGCT-3V, SEQ ID NO: 17; Probe: 6FAM-ACATCATGAAGCCCC-MGBNFQ, SEQ ID NO: 18.

[0334] PCR conditions were set as follows: 100 ng of total cellular DNA as template, 300 nM of primers, and 200 nM of probe. The cycle was 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. 1 × 10 per 100 ng of DNA. 4 The value of genome copies was calculated and corresponded to one genome copy per cell.

[0335] H&E staining was performed on formalin-fixed tissue from autopsies 3 months pi according to standard protocols. This study was performed on raffin-embedded rostral brain sections. Brain H&E sections were evaluated by a board-certified veterinary pathologist for evidence of toxicity and characterized the range of findings associated with the MPS II phenotype. The pathologist then re-examined the slides without knowledge of the treatment and scored the histological manifestations of the MPS II phenotype, including glial cytoplasmic vacuolization, neuronal cytoplasmic swelling, and amphiphile accumulation. The number of cells staining positive for LIMP2 and GM3 was quantified in two to four brain sections from each animal in the W2356 study by trained GTP morphology core personnel.

[0336] GM3 (frozen sections): GM3 immunostaining was performed on 30 μm-thick free-floating frozen sections as described using monoclonal antibody DH2 (Glycotech, Gaithersburg, MD) as the primary antibody, followed by a biotinylated secondary anti-mouse antibody (Jackson Immunoresearch, West Grove, PA) and detection with the Vectastain Elite ABC kit (Vector Labs, Burlingame, CA). Stained sections were transferred to glass slides and mounted with Fluoromount G (Electron Microscopy Sciences, Hatfield, PA).

[0337] LIMP2 (formalin-fixed sections): LIMP2 immunostaining was performed on 6 μm sections from formalin-fixed, paraffin-embedded brain tissue. Sections were deparaffinized in an ethanol and xylene series, microwaved for 6 minutes in 10 mmol / L citrate buffer (pH 6.0) for antigen retrieval, blocked with 1% donkey serum in PBS + 0.2% Triton for 15 minutes, and then sequentially incubated with primary antibody (1 hour) and labeled secondary antibody (45 minutes) diluted in blocking buffer. The primary antibody was rabbit anti-LIMP2 (Novus Biologicals, Littleton, CO, 1:200), and the secondary antibody was FITC- or TRITC-labeled donkey anti-rabbit (Jackson Immunoresearch).

[0338] The tissue sample (section b) obtained above was homogenized in lysis buffer (0.2% Triton-X100, 0.9% NaCl, pH 4.0) using a TissueLyser (Qiagen). The sample was freeze-thawed and clarified by centrifugation. Protein was quantified by BCA assay. IDS activity was measured using the fluorogenic substrate 4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate (Santa Cruz Biotechnology). Hexosaminidase activity and GAG concentration were measured using standard procedures as described (Hinderer et al. 2015).

[0339] IV. Computerized Systems For the open field, data were entered into Excel and analyzed using Graphpad Prism. For the Y-maze, data were entered into Excel and analyzed using Graphpad Prism. For CFC, Freezescan, CleverSys Inc. was used. For NOR, a MATLAB implementation and user guide was used: www.seas.upenn.edu / ~molneuro / autotyping.html.

[0340] V. Statistical analysis Tissue GAG ​​content, Hex activity, and brain accumulation lesions in treated and untreated mice were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Open field and Y-maze data were analyzed with Student's t-test. Two-way ANOVA and Dunnett's post hoc analysis were applied to fear conditioning data to evaluate the effects of trial and genotype. In the novel object recognition test, the time to explore the novel object versus the familiar object was compared using a t-test for each group, followed by Bonferroni correction for multiple comparisons. Used and compared.

[0341] VI.Results No deaths were observed. No clinical observations were considered related to treatment with the vector.

[0342] To assess general locomotor activity, an open field test was performed. IDSγ / - mice showed normal exploratory activity in the open field arena compared to wild-type littermates (Figures 9A-9C).

[0343] We compared the performance of wild-type and IDSγ / - (MPS II) mice in an open-field arena (horizontal movement) (Figure 9A). Spontaneous activity during a 10-min trial was automatically recorded based on the XY-axis beam breaks that capture horizontal movement. No differences were observed between WT and MPS II mice.

[0344] We compared the performance of wild-type and IDSγ / - (MPS II) mice in an open-field arena (vertical movement or rearing) (Figure 9B). Spontaneous activity during a 10-minute trial was automatically recorded based on the Z-axis beam break, capturing the rearing of the mouse's hind limbs. No differences were observed between WT and MPS II mice.

[0345] We compared the performance of wild-type and IDSγ / - (MPS II) mice in the open field arena (center activity) (Figure 9C). Spontaneous activity during a 10-min trial was automatically recorded based on the center beam break, capturing time spent in the open area as a marker of anxiety. No significant differences were observed between WT and MPS II mice.

[0346] To assess short-term memory, Y-maze activity was analyzed. Comparison of wild-type and IDS γ / - (MPS II) mice during an 8-min Y-maze test session. No differences were observed in the total number of arm entries between WT and MPS II mice. IDS γ / - mice also had a similar number of arm entries and comparable spontaneous alternation behavior in the Y-maze compared to wild-type littermates, indicating that the disease process does not affect short-term memory (Figure 9D).

[0347] To assess long-term memory, contextual fear conditioning (FC) was performed. The effects of treatment on cognition using contextual fear conditioning were assessed in wild-type, untreated, and treated MPS II mice. Percent freezing is plotted on the y-axis (Figure 6B). Freezing behavior before learning (Pre) is compared with freezing behavior after conditioning (Probe) for each group (Figure 6B). In the FC assay, all mice (IDSγ / - and wild-type) showed an increased percentage of freezing time during the recall phase of the test, indicating learning had occurred, but IDSγ / - mice showed reduced freezing compared to wild-type littermates (data not shown). Mice treated with AAV9.CB7.CI.hIDS.rBG showed no clear improvement in response to contextual fear conditioning, although the small differences between normal and untreated IDSγ / - mice made it difficult to assess treatment-related effects (Figure 6B).

[0348] To further evaluate long-term memory, novel object recognition was performed. The effect of treatment on cognition using novel object recognition was evaluated in wild-type, untreated, and treated MPS II mice. A comparison of the time spent exploring the novel object versus the familiar object was performed for wild-type, untreated, and treated IDSγ / - (MPS II) mice. An increase in the time spent exploring the novel object (indicating memory for the familiar object) was observed in all treated mouse groups, but was statistically significant only in the medium-dose group (Figure 6C). Wild-type mice As expected, IDSγ / - mice showed a preference for the novel object, but did not show a tendency to exhibit deficits in memory for the familiar object (long-term memory impairment). Intrathecal AAV9 gene therapy resulted in an improvement in the NOR deficit observed in IDSγ / - (MPS II) mice, but there was no clear dose-response. The preference for the novel object was statistically significant only in the mid-dose cohort, although the study was not sufficiently powered to compare the relative degree of rescue of behavioral deficits between treatment groups (Figure 6C).

[0349] A dose-dependent increase in brain IDS activity (day 21) was observed in wild-type, untreated, and treated MPS II mice (Figure 2B). At this early time point, only the high-dose group had levels similar to wild-type, although it was expected that expression had not yet reached its maximum.

[0350] A dose-dependent increase (at day 90) in IDS activity in wild-type, untreated, and treated MPS II mice was observed in CSF collected at necropsy 3 months pi (Fig. 2A). Essentially no IDS activity was detected in untreated IDSγ / - (MPS II) mice.

[0351] Furthermore, a dose-dependent increase in serum IDS activity (day 90) in wild-type, untreated, and treated MPS II mice was detected at necropsy 3 months p.i. (Fig. 2C). Essentially no activity was detected in the serum of untreated IDSγ / - (MPS II) mice.

[0352] At 3 months p.i., hexosaminidase activity (day 90) was normalized in a dose-dependent manner in both the liver (Figure 4C) and heart (Figure 4D) of wild-type, untreated, and treated MPS II mice. This normalization of the secondary increased enzyme activity indicated restoration of lysosomal homeostasis. GAG accumulation in the liver and heart was examined in wild-type, untreated, and treated MPS II mice. At 3 months p.i., GAG tissue content was dose-dependently reduced in both the heart (Figure 4B) and liver (Figure 4A) and was comparable to wild-type tissue content at the mid- and high-dose levels. Liver was corrected at all doses, with the heart showing the most partial improvement at the mid- and high-dose levels.

[0353] Additionally, humoral immunogenicity, as indicated by antibody responses to human IDS, was assessed in MPS II mice treated with ICV AAV9.CB7.CI.hIDS.rBG. Antibodies to human IDS were detected in the serum of only a few animals in the medium- and high-dose cohorts at both days 21 and 90 (Figure 8). At both time points, the majority of animals failed to mount a detectable humoral immune response (similar to controls). Approximately one-third of the medium-dose animals and approximately 20% of the animal group had antibody levels above background. Responses were less pronounced in the high-dose group. Figure 8 summarizes both necropsy endpoint data.

[0354] There were no treatment-related histopathological findings in the brain. All microscopic findings present in this study were considered to be related to the normal background, age and sex of this animal species, the disease model, or related to the vector administration procedure.

[0355] A scoring system based on cytoplasmic vacuolization was established and used for subsequent re-evaluation of all test animals without knowledge of treatment or phenotype. Cytoplasmic vacuolization in glia, characterized by large, clear vacuoles with eccentric or peripheral displacement of the nucleus, was a characteristic feature of the IDSγ / - genotype. There was regional variation in the amount of vacuolization in the brain regions evaluated. Neuronal accumulation of amphiphiles was less pronounced and was observed only in untreated IDSγ / - mice. Treatment with AAV9.CB7.CI.hIDS.RBG reduced the amount and frequency of glial vacuolization and neuronal accumulation of amphiphiles in all brain regions examined. Cumulative pathology scores demonstrate treatment-related improvements in glial and neuronal pathology (Figure 27).

[0356] Immunohistochemical staining for GM3 and LIMP2 was performed in wild-type, untreated, and treated MPS II mice. Brains from untreated IDSγ / - mice showed clear histological evidence of lysosomal accumulation in neurons, including accumulation of the lysosomal membrane protein LIMP2 and secondary accumulation of gangliosides, including GM3 (Figures 5A-5J). Images showed reduced lysosomal accumulation at all doses, with high-dose animals essentially similar to WT controls (Figures 5A-5J). Quantification of the dose-dependent reduction in GM3- and LIMP2-positive cells was performed in wild-type, untreated, and treated MPS II mice. There was a significant reduction in lysosomal accumulation at all doses, with high-dose animals essentially similar to WT controls. Treated mice showed a dose-dependent reduction in neuronal accumulation pathology, evidenced by reduced LIMP2 and GM3 staining (Figures 5K and 5L).

[0357] We analyzed the biodistribution of AAV9.CB7.CI.hIDS.rBG in MPS II mice from the high-dose group (day 21). Biodistribution data showed that brain tissue was transduced (1–10 GCs per diploid genome), as were the peripheral tissues evaluated, particularly the liver ...

Claims

1. 1. A pharmaceutical composition suitable for intrathecal administration in a human subject, comprising a suspension of a replication-deficient recombinant adeno-associated virus (rAAV) in a formulation buffer, (a) the rAAV comprises a heterologous nucleic acid encoding human iduronate-2-sulfatase (hIDS) packaged into an AAV9 capsid; (b) the formulation buffer comprises a physiologically compatible aqueous buffer, and optional surfactants and excipients; and (c) (i) the genome copy (GC) titer of the rAAV is at least 1.0 x 10 １３ GC / ml (+ / - 20%); and / or (ii) the rAAV has an empty particle / total particle ratio of at least about 80% free of empty capsids; and / or (iii) at least about 2.5 x 10 １０ GC / g brain mass ~ approx. 3.6×10 １１ The pharmaceutical composition, wherein the dose of the suspension of the rAAV is potent in GC / g brain mass.

2. 10. The pharmaceutical composition of claim 1, wherein the potency is measured by an in vitro assay.

3. 3. The pharmaceutical composition of claim 2, wherein the in vitro assay comprises transducing HEK293 cells with a known multiplicity of rAAVGC titer per cell and assaying the supernatant for hIDS activity 72 hours post-transduction using a 4MU-iduronide enzymatic assay.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the rAAV has an empty particle / total particle ratio of 0.01 to 0.05 (95 to 99% free of empty capsids).

5. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the encoded hIDS has a sequence selected from the following: (a) amino acids 1 to about 550 of SEQ ID NO:2 (Genbank NP_000193); and (b) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids from about 21 to about 550 of SEQ ID NO:2; (c) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids from about 21 to about 455 of SEQ ID NO:2; (d) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids from about 34 to about 550 of SEQ ID NO:2; or (d) A synthetic human enzyme comprising a heterologous leader sequence fused to amino acids from about 34 to about 455 of SEQ ID NO:

2.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the rAAV further comprises a 5' inverted terminal repeat (ITR) sequence, a CB7 promoter, a chicken beta-actin intron, a rabbit beta-globin polyadenylation (polyA) signal, and / or a 3' ITR sequence.

7. The pharmaceutical composition of claim 6 , wherein the AAV ITRs are heterologous to AAV9.

8. 8. The pharmaceutical suspension of claim 7, wherein the ITRs are derived from AAV2.

9. 9. The pharmaceutical suspension of any one of claims 1 to 8, wherein the suspension has a pH of 6 to 9.

10. 10. The pharmaceutical suspension of claim 9, wherein the suspension has a pH of 6.8 to 7.

8.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the suspension is formulated for delivery by intrathecal injection.

12. The suspension is formulated for delivery to neonatal patients and contains approximately 3.8 x 10 １２ genome copies (GC) of approximately 1.9 × 10 １４ The pharmaceutical composition according to any one of claims 1 to 11, comprising GC.

13. The suspension is formulated for delivery to patients between about 3 months and about 9 months of age and contains about 6 x 10 １２ GC ~ approx. 3×10 １４ The pharmaceutical composition according to any one of claims 1 to 11, comprising GC.

14. The suspension is formulated for delivery to patients between about 9 months and about 36 months of age and contains about 1 x 10 １３ GC ~ approx. 5×10 １４ The pharmaceutical composition according to any one of claims 1 to 11, comprising GC.

15. The suspension is formulated for delivery to a patient who is about 3 to about 12 years of age and contains about 1.2 x 10 １３ GC ~ approx. 6×10 １４ The pharmaceutical composition according to any one of claims 1 to 11, comprising GC.

16. The suspension is formulated for delivery to patients about 12 years of age or older and contains about 1.4 x 10 １３ GC ~ approx. 7×10 １４ The pharmaceutical composition according to any one of claims 1 to 11, comprising GC.

17. The pharmaceutical composition of any one of claims 1 to 11, which is administrable to a human subject in need thereof by intrathecal injection.

18. Use of a pharmaceutical composition according to any one of claims 1 to 11 for use in preparing a medicament that can be administered by intrathecal injection to a human subject in need thereof.

19. 19. The use of claim 18, wherein the human subject has been diagnosed with Mucopolysaccharidosis II (MPS II) or severe Hunter syndrome.

20. A kit comprising the components of a suspension according to any one of claims 1 to 16 and components necessary for intrathecal administration.

21. 21. The kit of claim 20, further comprising a dilution buffer useful for diluting the suspension.

22. An aqueous liquid suspension having a pH in the range of 6 to 9, comprising an aqueous suspension base and an rAAV9 vector having a genome including a 5' ITR, a chicken beta actin promoter, an intron, a human IDS coding sequence, a rabbit beta globin polyA, and a 3' ITR.

23. 1. A method of treating a human subject diagnosed with Mucopolysaccharidosis II (MPS II), comprising administering to a human subject in need thereof 2.5×10 １０ GC / g brain mass ~ approx. 3.6×10 １１ The present invention includes administering by intrathecal injection a suspension of replication-deficient recombinant adeno-associated virus (rAAV) in a formulation buffer at a dose of between GC / g brain mass, wherein: (a) the rAAV comprises a heterologous nucleic acid encoding human iduronate-2-sulfatase (hIDS) packaged in an AAV9 capsid; and (b) the formulation buffer comprises a physiologically compatible aqueous buffer, a surfactant, and optional excipients; and (c) (i) the genome copy (GC) titer of the rAAV is at least 1.0 x 10 １３ GC / ml (+ / - 20%); (ii) the rAAV has an empty particle / total particle ratio of 0.01 to 0.05 (95-99% free of empty capsids); and / or (iii) at least about 2.5 x 10 １０ GC / g brain mass ~ approx. 3.6×10 １１ The method, wherein the dose of the rAAV suspension in GC / g brain mass has a titer.

24. 21. The method of claim 20, wherein the human subject is 2 years of age or older and has been diagnosed with severe Hunter syndrome.

25. 24. The method of claim 23, wherein the human subject has or is at risk of developing a neurocognitive disorder.

26. 26. The method of any one of claims 23-25, wherein the human subject is 2 years of age or older and is diagnosed with the presence of a major rearrangement or deletion mutation known to be correlated with severe Hunter disease.

27. 26. The method of any one of claims 23 to 25, wherein the method results in an increase in the neurocognitive development quotient (DQ) in the subject as assessed using the Bayley Scales of Infant Development.

28. 26. The method of any one of claims 23 to 25, wherein the method results in a decline in DQ of 15 points or less in the subject compared to untreated / natural historical control data in patients with Hunter syndrome.

29. 26. The method of any one of claims 23 to 25, wherein said method results in an increase in the level of functional hIDS as measured in a serum sample from said patient.

30. 30. The method of any one of claims 23 to 29, wherein the method results in a reduction in GAG levels as measured in a sample of serum, urine and / or cerebrospinal fluid (CSF) from the patient.

31. 31. The method of any one of claims 23-30, further comprising administering AAV.hIDS to the patient by liver-directed injection.

32. 32. The method of claim 31, wherein the hepatotropic AAV.hIDS has a capsid selected from AAV8, AAVrh64R1, AAVrh64R2, rh8, rhlO, AAV3B, or AAVdj.

33. The method of any one of claims 23 to 32, wherein the titer is measured by an in vitro assay.

34. 34. The method of claim 33, wherein the in vitro assay comprises transducing HEK293 cells with a known multiplicity of the rAAVGC titer per cell and assaying the supernatant for hIDS activity 72 hours post-transduction using a 4MU-iduronide-2-sulfate enzymatic assay.

35. 36. The method of any one of claims 33 to 35, wherein the human hIDS coding sequence has the nucleotide sequence of SEQ ID NO: 1 or a sequence that is at least about 80% identical to SEQ ID NO: 1 that encodes a functional hIDS.

36. The method according to any one of claims 33 to 35, wherein the encoded (a) amino acids 1 to about 550 of SEQ ID NO:2 (Genbank NP_000193); and (b) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids from about 21 to about 550 of SEQ ID NO:2; (c) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids from about 21 to about 455 of SEQ ID NO:2; (d) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids from about 34 to about 550 of SEQ ID NO:2; or (d) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids from about 34 to about 455 of SEQ ID NO:

2.

37. The method of any one of claims 33 to 36, wherein the rAAV further comprises a 5' inverted terminal repeat (ITR) sequence, a CB7 promoter, a chicken beta-actin intron, a rabbit beta-globin polyadenylation (poly A) signal, and / or a 3' ITR sequence.

38. 38. The method of claim 37, wherein the AAV ITRs are heterologous to AAV9.

39. 38. The method of claim 37, wherein the ITRs are from AAV2.

40. 37. The method of any one of claims 33 to 36, wherein the suspension has a pH of from 6 to 9.

41. 41. The method of claim 40, wherein the suspension has a pH of 6.8 to 7.

8.

42. 42. The method of any one of claims 33 to 41, wherein the suspension is formulated for delivery by intrathecal injection.

43. The suspension is formulated for delivery to neonatal patients and contains approximately 3.8 x 10 １２ Genome copies (GC) ~ approximately 1.9 x 10 １４ The method of any one of claims 23 to 41, comprising GC.

44. The suspension is formulated for delivery to patients between about 3 months and about 9 months of age and contains about 6 x 10 １２３ GC ~ approx. 3×10 １４ The method of any one of claims 23 to 41, comprising GC.

45. The suspension is formulated for delivery to patients between about 9 months and about 36 months of age and contains about 1 x 10 １３ GC ~ approx. 5×10 １４ The method of any one of claims 23 to 41, comprising GC.

46. The suspension is about 3 to about 12 years old and contains about 1.2 x 10 １３ GC ~ approx. 6×10 １４ 42. The method of any one of claims 23 to 41, formulated for delivery to a patient comprising a GC.

47. The suspension is formulated for delivery to patients about 12 years of age or older and contains about 1.4 x 10 １３ GC ~ approx. 7×10 １４ The method of any one of claims 23 to 41, comprising GC.

48. 1. A method of treating a human patient having symptoms associated with MPS II and / or Hunter syndrome, comprising: (a) administering a hIDS enzyme to a patient with symptoms associated with MPS II and / or Hunter syndrome in an amount sufficient to induce transgene-specific tolerance; and (b) administering to said patient rAAV.hIDS, wherein the rAAV.hIDS directs the expression of therapeutic levels of hIDS in said patient.

49. 49. The method of claim 48, wherein the hIDS of (a) is administered as a recombinant protein.

50. 50. The method of claim 48 or 49, wherein the patient is an infant.

51. 49. The method of any one of claims 48, wherein the administration of (b) is performed about 3 days to about 14 days after the administration of (a).

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