Methods and Materials for GALGT2 Gene Therapy

Abstract

The present disclosure relates to recombinant adeno-associated virus (rAAV) delivery of a GALGT2 polynucleotide. The disclosure provides rAAV and methods of using the rAAV for GALGT2 gene therapy of neuromuscular disorders. Exemplary neuromuscular disorders include, but are not limited to, muscular dystrophies such as Duchenne muscular dystrophy, Congenital Muscular Dystrophy 1A and Limb Girdle Muscular Dystrophy 2D.

Claims

1-8. (canceled)

9. A method of treating a neuromuscular disorder in a human subject in need thereof comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises a genome comprising a nucleic acid comprising, in order from 5′ to 3′: (i) a first AAV2 inverted terminal repeat sequence (ITR); (ii) an MCK enhancer comprising nucleotides 236-442 of SEQ ID NO: 2; (iii) a muscle creatine kinase core promoter sequence comprising nucleotides 443-793 of SEQ ID NO: 2; (iv) a nucleotide sequence encoding a human β1-4-N-acetyl-D-galactosamine glycosyltransferase (GALGT2) polypeptide; and (v) a second AAV2 ITR sequence; wherein the human GALGT2 polypeptide has an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 or is 100% identical to SEQ ID NO: 3, or is encoded by a nucleotide sequence 90% identical to nucleotides 1002-2522 of SEQ ID NO: 2 or 100% identical to nucleotides 1002-2522 of SEQ ID NO: 2.

10. The method of claim 9 wherein the route of administration is an intramuscular route and the dose of the rAAV administered is about 3×10.sup.11 vector genomes/injection to about 5×10.sup.12 vg/injection, an intramuscular route and the dose of the rAAV administered is about 3×10.sup.11 vector genomes/injection, an intramuscular route and the dose of the rAAV administered is about 1×10.sup.12 v vector genomes/injection, an intramuscular route and the dose of the rAAV administered is about 5×10.sup.12 vector genomes/injection, inter-arterial limb perfusion and the dose of the rAAV administered is about 6×10.sup.12 vector genomes/kg/limb to about 4.8×10.sup.13 vector genomes/kg/limb, inter-arterial limb perfusion and the dose of the rAAV administered is about 6×10.sup.12 vector genomes/kg/limb, inter-arterial limb perfusion and the dose of the rAAV administered is about 1.2×10.sup.13 vector genomes/kg/limb, inter-arterial limb perfusion and the dose of the rAAV administered is about 2.4×10.sup.13 vector genomes/kg/limb, inter-arterial limb perfusion and the dose of the rAAV administered is about 4.8×10.sup.13 vector genomes/kg/limb, systemic intravenous administration and the dose of the rAAV administered is about 2×10.sup.14 vector genomes/kg to about 6×10.sup.15 vg/kg, systemic intravenous administration and the dose of the rAAV administered is about 4×10.sup.14 vector genomes/kg to about 6×10.sup.15 vector genomes vector genomes/kg, systemic intravenous administration and the dose of the rAAV administered is about 4×10.sup.14 vector genomes/kg, systemic intravenous administration and the dose of the rAAV administered is about 8×10.sup.14 vector genomes/kg, systemic intravenous administration and the dose of the rAAV administered is about 2×10.sup.15 vector genomes/kg, or systemic intravenous administration and the dose of the rAAV administered is about 6×10.sup.15 vector genomes/kg.

11. The method of claim 9, wherein the neuromuscular disorder is Duchenne Muscular Dystrophy (DMD); Becker Muscular Dystrophy; Congenital Muscular Dystrophy (MDC) 1A, 1B, 1C and 1D; Limb Girdle Muscular Dystrophy (LGMD) 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G 2H, 2I, 2J, 2K, 2L, 2M, 2N, 20 and 2Q; Bethlem Myopathy; Ullrich Congenital Muscular Dystrophy; Muscle Eye Brain Disease; Fukuyama Congenital Muscular Dystrophy; Walker Warburg Syndrome; Myotonic Dystrophy; Myasthenic syndromes; Congenital Myasthenias; Inclusion Body Myopathy; Inclusion Body Myositis; Emery Dreifuss Muscular Dystrophy; Distal Muscular Dystrophy; Dermatomyositis; Centronuclear Myopathy; Faciosacpulohumeral Muscular Dystrophy; Myoshi Myopathy; Mitochondrial Myopathy; Nemaline Myopathy; Nonaka Myopathy; Myasthenia Gravis; and Polymyositis.

12. The method of claim 9 whereby there is an improvement in the human subject in absolute muscle specific force; force decrement during eccentric muscle contractions; serum CK level; serum cardiac troponin level; serum MMP9 level; grip strength; limb torque; limb mobility or flexibility; ambulation; 6 minute walk test; knee flexor or extensor strength; maximal voluntary isometric muscle contraction; North Star Ambulatory Assessment; muscle mass, fat reduction, or edema by limb T2-weighted MRI measures; muscle contractures; limb joint angle; heart function (heart rate, cardiac output, percent fractional shortening, stroke volume); respiration (including respiratory rate, blood oxygenation, need for supplemental oxygen); muscle necrosis; muscle regeneration; muscle wasting; muscle inflammation; muscle calcification; muscle central nucleation; muscle size or myofiber size; lifespan; and dystrophin or laminin alpha 2 surrogate protein expression (utrophin, plectin 1, laminin alpha 5, agrin).

13. The method of claim 9, wherein the nucleic acid further comprises 3′ to said core promoter, a mouse MCK exon 1 sequence comprising nucleotides 794-846 of SEQ ID NO: 2.

14. The method of claim 13, wherein the nucleic acid further comprises 3′ to said core promoter, an SV40 intron sequence comprising set out in nucleotides 847-943 of SEQ ID NO: 2.

15. The method of claim 14, wherein the nucleic acid further comprise 3′ to said core promoter, a 5′ untranslated region comprising set out in nucleotides 944-1000 of SEQ ID NO: 2.

16. The method of claim 15, wherein the nucleic acid further comprises 3′ to said nucleotide sequence encoding a human GALGT2 polypeptide, a Syn pA synthetic polyadenylation signal sequence comprising set out in nucleotides 2531-2579 of SEQ ID NO: 2.

17. The method of claim 9, wherein in the nucleic acid said first ITR comprises nucleotides 53-230 of SEQ ID NO: 2, and/or said second ITR comprises nucleotides 2581-2762 of SEQ ID NO: 2.

18. The method of claim 16, wherein in the nucleic acid said first ITR comprises nucleotides 53-230 of SEQ ID NO: 2; and said second ITR comprises nucleotides 2581-2762 of SEQ ID NO: 2.

19. The method of claim 9, wherein the rAAV comprises an rAAV.rh74.MCK.GALGT2 genome that is at least 90% identical to the nucleotide sequence of set out in SEQ ID NO: 2.

20. A method of claim 9, wherein the AAV comprises the rAAV.rh74.MCK.GALGT2 genome set out in SEQ ID NO: 2.

21. The method of claim 9, wherein the rAAV is administered as is a recombinant adeno-associated virus particle.

22. The method of claim 9, wherein the rAAV is serotype AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, or AAVrh.74.

23. The method of claim 9, wherein the AAV DNA in the rAAV genome is from AAV rh.74.

24. The method of claim 24, wherein the polynucleotide sequence of the AAV rh.74 genome comprises the nucleotide sequence of SEQ ID NO: 1.

Description

DESCRIPTION OF DRAWING

[0063] FIG. 1 provides a schematic of The rAAVrh74.MCK.GALGT2 gene cassette described in Example 1.

EXAMPLES

[0064] Thus, aspects and embodiments of the invention are illustrated by the following examples.

Example 1

[0065] A non-replicating rAAV termed rAAVrh74.MCK.GALGT2 was generated. The rAAV vector contains the complete human GALGT2 cDNA (Genbank Accession #AJ517771) under the control of a muscle creatine kinase promoter (MCK; a muscle specific promoter). A MCK promoter/enhancer sequence was used to drive muscle-specific gene (expression and is composed of the mouse MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal). After the core promoter, the 53 bp endogenous mouse MCK Exon1 (untranslated) is present for efficient transcription initiation, followed by the SV40 late 16S/19S splice signals (97 bp) and a small 5′UTR (61 bp). The intron and 5′ UTR are derived from plasmid pCMVB (Clontech). The GALGT2 cassette has a consensus Kozak sequence immediately in front of the ATG start and a small 53 bp synthetic polyA signal for mRNA termination. The human GALGT2 cassette was previously described by Martin et al (2009), supra. The only viral sequences included are the inverted terminal repeats (ITR) of AAV2, which are required for both viral DNA replication and packaging. The pAAVrh74.MCK.GALGT2 plasmid contains the human GALGT2 cDNA expression cassette flanked by AAV2 inverted terminal repeat (ITR) sequences. The gene cassette includes an MCK promoter, a chimeric intron with a Kozak sequence for optimizing gene expression, human GALGT2 coding sequences and a polyA signal (see FIG. 1). The sequence of the gene cassette with flanking AAV ITRs is set out in SEQ ID NO: 2.

[0066] The AAV vectors including the GALGT2 polynucleotides were produced by a modified cross-packaging approach in an adenovirus-free, triple plasmid DNA transfection (CaPO4 precipitation) method in HEK293 cells [Rabinowitz et al., J. Virol., 76:791-801 (2002)]. Vector was produced by co-transfecting plasmid containing GALGT2 polynucleotide with an AAV helper plasmid rep2-cap rh.74 and an adenovirus helper plasmid in similar fashion as that previously described [Wang et al., Gene. Ther., 10:1528-1534 (2003)]. Plasmid rep2-cap rh.74 encodes the wild-type AAV2 rep gene and rh.74 cap gene, and the adenovirus helper plasmid (pAdhelper) expresses the adenovirus type 5 E2A, E4ORF6, and VA I/II RNA genes which are required for high-titer rAAV production.

[0067] Vectors were purified from clarified 293 cell lysates by sequential iodixanol gradient purification and anion-exchange column chromatography using a linear NaCl salt gradient as previously described [Clark et al., Hum. Gene Ther, 10:1031-1039 (1999)]. Vector genome (vg) titers were measured using QPCR based detection with a MCK specific primer/probe set and utilized the Prism 7500 Taqman detector system (PE Applied Biosystems) as previously described (Clark et al., supra). Vector stock titers ranged between 1-40×10.sup.12 vg/mL.

[0068] The vector is formulated in 20 mM Tris (pH 8.0), 1 mM MgCl.sub.2 and 200 mM NaCl containing 0.001% pluronic F68. The vector is supplied as a frozen liquid that is thawed before clinical administration.

Example 2

IM Delivery to Subjects with DMD

[0069] Human patients are subjects contemplated herein for treatment by IM delivery. In an exemplary clinical protocol, DMD patients receive bilateral injections with one extensor digitorum brevis (EDB) muscle of each patient receiving the vector rAAVrh74.MCK.GALGT2 and the other EDB muscle of each patient receiving saline alone. Subjects in a first cohort receive a low dose of vector of 3×10.sup.11 vg (total dose). Subjects in a second cohort receive a higher dose of vector of 1×10.sup.12 vg (total dose).

[0070] Immediately prior to transportation to the clinical setting, appropriate dilutions of the vector are prepared. The dilution for the injection is 1:1 with normal saline. The vector is kept on ice (not frozen) until administration and is administered to the subject within 8 hours of preparation. Handling of rAAVrh74.MCK.GALGT2 follows compliance standards for Biosafety Level 1 vectors. See, www4.od.nih.gov/oba/RAC/guidelines 02/APPENDIX_G.htm#_Toc7246561.

[0071] Subjects have muscle weakness by clinical exam. The genetic diagnosis of DMD is established on the basis of a DMD gene mutation consistent with DMD, in the setting of an appropriate clinical history. In this study, all subjects are non-ambulant, having lost ambulation in an age range diagnostic of DMD (i.e., less than 12 years old without steroid therapy, or less than 15 years old in the setting of longstanding steroid treatment). Subjects receive a stable dose of corticosteroid therapy (either prednisone or deflazacort, or their generic forms) for twelve weeks prior to treatment.

[0072] The vector or control is delivered via direct intramuscular injection into the extensor digitorum brevis (EDB) muscle of one foot of a subject, while the other foot receives saline alone. Conscious sedation is used on participants under 12 years of age. Patients over 12 years of age may receive conscious sedation or a sedative (like lorazepam) at least one hour prior to gene transfer. In addition, the skin over the gene transfer site is pre-treated with a lidocaine/prilocaine eutectic mixture incorporated in a cream base (EMLA cream) or a cellulose disk (EMLA patch). Comparable cream-based anesthesia such as xylocaine cream may be used. Procedures are performed under sterile conditions. The injection site is cleansed with three successive applications of non-iodine containing surgical prep swabs and draped with disposable sterile drapes. A standard clinical Doppler ultrasound is used with a sterile sheath around the transducer to maintain asepsis of the injection field. For vector injections of rAAVrh74.MCK.GALGT2 or placebo to the EDB, disposable MyoJect Needles that enable simultaneous EMG recording and fluid injection are used to increase the precision of muscle injection. The anatomical midline point of the muscle is identified on the skin and 2 to 6 separate vector injections are distributed into the muscle. The injections are 0.5 cm in depth from the muscle surface. The total dose of vector is 3×10.sup.11 vg in 1.5 ml in the low dose group and 1×10.sup.12 vg in 1.5 ml in the high-dose group. The proximal and distal extent of vector delivery as determined by ultrasound is marked with an indelible radiographic marker for reference at the time of post-gene transfer muscle biopsy.

[0073] Subjects are followed with close monitoring of vital signs. Concomitant medications are monitored and documented after injection. Subjects are discharged two days after gene transfer (if no side effects are observed).

[0074] Subjects return for follow up visits. Muscle biopsies are conducted at day 45 or day 90. Immune studies at 45 and 75 days post-gene transfer and at 9, 12, 18, and 24 months include testing for binding antibody to rAAVrh74 and antibody to GALGT2, as well as ELISpot to detect T cell response to capsid antigens. Subjects are seen at the end of first and second years for a physical exam, strength testing and immune studies.

Example 3

Efficacy Outcome Measurements

[0075] Muscle biopsies are taken from EDB muscles. Samples are coded to maintain a blind in all subsequent analysis as to which was injected with rAAVrh74.MCK.GALGT2.

[0076] Efficacy outcome measures include: expression of GALGT2 as demonstrated with anti-CT epitope antibodies; GALGT2 protein expression quantified by western blot and assessed by densitometry; transduction efficiency measured by qPCR of the GALGT transgene from muscle, and expressed as vector genomes normalized to a genomic single-copy control; the number of fibers containing central nuclei compared between muscles by paired t-tests; and analyses will also include: Dystrophin expression (with antibodies to N-terminal, C-terminal, and rod domains), utrophin expression, and leukocyte markers including CD45, CD3, CD4, CD8, and MAC 387. Muscle is examined for histological appearance. Antibodies to rAAVrh74 along with PBMC ELISpots to both rAAVrh74 capsid and GALGT protein are evaluated at different time points during the study up to two years. The muscle analysis of gene expression and inflammation is also done without breaking the blind.

[0077] Transgene expression is compared blindly between both EDB muscles from a single subject, and between those subject's biopsies at day 45 or day 90. A vector-specific primer probe set is used to amplify a unique 5′ untranslated leader sequence of the transgene that will distinguish transgene expressed GALGT2 protein from endogenous GALGT2. Quantification of protein is done using direct immunofluorescence (IF) and Western blot (WB) studies of muscle tissue. CD4+ and CD8+ mononuclear cells are quantified by immunostaining and reported as number of cells/mm2 area. MHCI and MHCII antigen expression are assessed on muscle sections. Muscle morphometrics are also be performed, including fiber size histograms and quantification of central nucleation. Analysis also includes PCR analysis for viral DNA.

[0078] Immune responses are assessed by IFN-γ ELISpots to GALGT2 and AAV capsid. A rise in IFN-γ of >2SD to either virus or transgene is considered significant. An additional measure of immune response is the binding antibody assay to AAV.

[0079] Measurements for improvements in one or more of absolute muscle specific force; force decrement during eccentric muscle contractions; serum CK level; serum cardiac troponin level; serum MMP9 level; grip strength; limb torque; limb mobility or flexibility; ambulation; 6 minute walk test; knee flexor or extensor strength; maximal voluntary isometric muscle contraction; North Star Ambulatory Assessment; muscle mass, fat reduction, or edema by limb T2-weighted MRI measures; muscle contractures; limb joint angle; heart function (heart rate, cardiac output, percent fractional shortening, stroke volume); respiration (including respiratory rate, blood oxygenation, need for supplemental oxygen); muscle necrosis; muscle regeneration; muscle wasting; muscle inflammation; muscle calcification; muscle central nucleation; muscle size or myofiber size; lifespan; and dystrophin or laminin alpha 2 surrogate protein expression (utrophin, plectin 1, laminin alpha 5, agrin) are among those contemplated. See, for example, Forbes et al., Radiology, 269(1): 198-207 (2013); Govoni et al., Cell Mol. Life Sci., 70(23): 4585-4602 (2013); and Chandrasekharan and Martin, Methods Enzymol., 479: 291-322 (2010).

[0080] For each of the measures, statistical analysis based on differences between pre- and post-gene transfer examinations (clinical, or on muscle biopsy) will be analyzed using a paired t test, with a p value of <0.05 indicating significance.

Example 4

Vascular Delivery by Isolated Limb Perfusion

[0081] A vascular delivery route termed isolated limb perfusion (ILP) is also contemplated for treatment of human patients. Multiple leg muscles can be targeted by ILP via delivery through the femoral artery. The method permits isolation of the limb from the general circulation, increasing transduction efficiency and preventing virus from escaping to the general circulation. [Rodino-Klapac et al., Mol. Ther., 18: 109-117 (2010)]. An exemplary clinical protocol is set out below.

[0082] The rAAVrh74.MCK.GALGT2 is prepared as described in Examples 1 and 2.

[0083] The subject receives a stable dose of corticosteroid therapy (either prednisone or deflazacort, or their generic forms) for twelve weeks prior to treatment. Prednisone treatment is also continued after gene transfer. The sedated and anesthetized subject is secured to a surgical bed. Proximal and distal tourniquets are loosely positioned above the knee and below the gastrocemius muscle of a macaque. A small incision is placed at the femoral triangle and the femoral artery is identified and dissected free and looped with proximal and distal ligatures to control bleeding and facilitate catheter introduction. The femoral artery is cannulated with a 3.0 Fr introducer sheath via a modified Seldinger method by passing the pre-flushed sheath over a wire previously placed in the artery. The sheath is advanced only a few centimeters and secured in place with a 3.0 braided silk suture.

[0084] Following sheath placement in the femoral arteries and veins, 100-200 u/kg of unfractionated heparin is administered and allowed to circulate for 3-5 minutes. A Choice PT coronary guide wire is then placed initially into the right femoral vein and artery, and then ultimately into the left femoral vein and artery. A 4-mm diameter Tyshak Mini Balloon catheter is passed through the 3.3-French sheath through the right femoral artery into position in the femoral iliac artery junction. An 8-mm diameter×2 cm long Tyshak Mini Balloon catheter is passed through the 4-French sheath into appropriate position in the right femoral-iliac vein junction. Small hand injections of diluted contrast are performed to confirm appropriate blockage of both the left femoral artery and the right femoral vein. If needed, sheaths and balloons can be exchanged for larger sizes. For example, the 4-French sheath in the right femoral vein can be exchanged for a 6-French sheath and a 12 mm×2 cm long Tyshak II Balloon catheter can be passed over the Choice PT guidewire into appropriate position. Small hand injections through the side arm of the sheath are performed to confirm location and complete occlusion of the femoral vein.

[0085] A pre-flush of 2 mL/kg of Ringer's lactate heparinized solution is infused after both right femoral artery and femoral venous balloons are inflated, with isolation of the right leg. After 1 minute, the Ringer's lactate flush at 2 mL/kg is completed. Next, the rAAVrh74.MCK.GALGT2 vector is infused at a dose of between 2×10.sup.12 vg/kg/limb and 4.8×10.sup.13 vg/kg/limb in a volume of 8 mL/kg LR over 1½ minutes (since bilateral limb perfusion is performed, leading to a total patient dose of between 4×10.sup.12 vg/kg and 9.6×10.sup.13 vg/kg). After the rAAVrh74.MCK.GALGT2 is delivered, there is 10 minutes of dwell time, and then the right femoral arterial sheath is then used to infuse 2 mL/kg of heparinized Ringer's lactate over 1 minute. The balloons are then deflated, and the catheters and guidewires are removed.

[0086] The left leg is then targeted for the same procedure. The left femoral artery is maintained with 3.3-French sheaths. Again, using a 4-mm diameter Tyshak Mini Balloon catheter in the left femoral artery over the Choice PT coronary guidewire, as well as the 12 mm×2 cm long Tyshak II Balloon catheter through the 5-French sheath in the left femoral vein with inflations up to 3 atmospheres of pressure, appropriate occlusion is demonstrated. The infusion protocol is repeated with 2 mL/kg of heparinized Ringer's lactate infused over 1 minute, and a dose of between 2×10.sup.12 vg/kg/limb and 4.8×10.sup.13 vg/kg/limb of rAAVrh74.MCK.GALGT2 is infused over 1 minute and 15 seconds, with the dwell time of 10 minutes. Finally, 2 mL/kg of heparinized Ringer's lactate is infused through left femoral arterial sheath, and then the sheaths are removed from all 4 access sites with pressure hemostasis and a HemCon patch.

[0087] Variations of this protocol can be used to deliver rAAVrh74.MCK.GALGT2 via other arteries to alternative groups of muscles as needed. For example, delivery via the phrenic, intercostal and/or subcostal arteries to supply the diaphragm muscle, or delivery via the coronary arteries to supply the heart are contemplated. Similar doses would be utilized for each procedure, and that multiple procedures might be done in a single patient or even in a single patient admission.

[0088] At the completion of dosing the tourniquets and catheter are removed and direct pressure is applied to the wound for 10 min to control bleeding. The wound is closed with a continuous subcuticular 4.0 Vicryl suture. A pressure dressing is applied to the site and kept in place until the subject awoke from anesthesia.

[0089] Following the ILP vector delivery protocol, subject follow up and efficacy outcome measurements/analyses similar to that described above for the IM-treated subjects are conducted.

Example 5

Systemic Vascular Delivery

[0090] Another contemplated route of delivery of the rAAVrh74.MCK.GALGT2 vector to muscle is systemic vascular delivery. An exemplary dose escalation study examining efficacy can be conducted as follows.

[0091] Determination of Dose Range

[0092] IV injection (via the tail vein) of 1.4×10.sup.15 vg/kg rAAVrh74.MCK.GALGT2 at day 1 of age causes transduction of over 90% of all limb skeletal muscles in a wild type mouse, including tibialis anterior, gastrocnemius, quadriceps and triceps, and the same does leads to over 50% transduction of all cardiomyocytes in the wild type mouse heart and over 70% of cardiomyocytes in the heart of mdx model mice heart. Notably, analysis of overall mdx mouse heart function at 3 months after treatment, relative to mock-treated mdx control animals, showed almost a doubling of cardiac output as the result of rAAVrh74.MCK.GALGT2 treatment with this dose of vector, either with or without stimulation with dobutamine, a beta agonist that stimulates heart rate. Thus, there is an 80% increase in blood flow from the dystrophin-deficient heart after rAAVrh74.MCK.GALGT2 treatment when vector is given prior to the onset of disease-related cardiac pathology. 5×10.sup.15 vg/kg is contemplated to be the maximal therapeutic dose to transduce all heart and skeletal muscle cells throughout the entire body using intravenous injection. Thus, it is contemplated that a dose range of about 5×10.sup.13 vg/kg to about 5×10.sup.15 vg/kg would cover the minimally effective dose and the optimally effective dose for rAAVrh74.MCK.GALGT2 treatment of the whole patient in a clinical IV study.

[0093] Protocol

[0094] The rAAVrh74.MCK.GALGT2 is prepared as described in Examples 1 and 2.

[0095] The subject is started on prophylactic enteral prednisolone (glucocorticoid) (approximately 1 mg/kg/day) one day prior to the rAAVrh74.MCK.GALGT2 administration. Prednisone treatment is also continued after gene transfer.

[0096] On the day of gene transfer (Day 0) prior to rAAVrh74.MCK.GALGT2 infusion, a physical exam is performed with vitals collected.

[0097] If a subject appears inadequately hydrated in the judgment of the PI, bolus(es) of 10-20 mL/kg normal saline may be given during the time between hospital admission and gene transfer. Subjects maintain their usual diet until eight hours prior to gene transfer, after which they have no solid food; clear liquids are allowed up until two hours prior to gene transfer, after which they will be fully NPO. They resume their usual PO intake after they return to pre-sedation baseline. Gene transfer will be performed under sterile conditions, under light to moderate sedation under the direction of a qualified anesthesiologist. Sedation may vary, but the subject can be sedated using inhaled nitrous prior to induction with propofol via an IV, and maintained with inhaled sevoflurane or a propofol drip. In those subjects who, in the opinion of the PI (and in consultation with the anesthesiologist), are determined to not need sedation in order to safely deliver the vector, sedation may be deferred.

[0098] All subjects in the trial receive an intravenous injection of rAAVrh74.MCK.GALGT2 via peripheral limb vein. The dose range contemplated is between 5×10.sup.13 vg/kg and 5×10.sup.15 vg/kg.

[0099] As one example, each vector dose is given undiluted, divided into 50 mL or less, to fill Becton Dickinson 60 mL capacity polypropylene syringes, prepared by the NCH Investigational Drug Pharmacy. The vector salt solution is approximately 400 mOsmol/L. Infusion is performed using a Smiths Medical Medfusion 4000 Syringe Infusion Pump with PharmGuard Infusion Management Software Suite, delivered via a Smiths Medical MX563 infusion tube. The infusion rate is not to exceed 2 ml/kg/min for any subject. The infusion is given over approximately 10 to 20 minutes. The vector is flushed from the infusion tubing using normal saline at the end of the infusion. It is contemplated that vector doses can be divided and administered differently as necessary.

[0100] Subjects are closely monitored for side effects during the infusion, including continuous heart rate, respiratory rate, and pulse oximetry; and intermittent blood pressure monitoring. Heart rate, respiratory rate, pulse oximetry, temperature, and blood pressure are measured before and immediately after the infusion, and every five minutes during the infusion, and repeated at 15 minutes post-infusion.

[0101] Subjects remain in an intensive care unit bed following gene transfer and remain admitted to the hospital for 48 hours after gene transfer. Vital signs are obtained hourly for 4 hours following the injection and then every 4 hours until discharge. Transfer out of intensive care may be undertaken after the initial 24 hours of post-infusion monitoring, if the PI has no concerns.

[0102] Following the vector delivery protocol, subject follow up and efficacy outcome analyses similar to that described above for the IM-treated subjects are conducted.

[0103] While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.

[0104] All documents referred to in this application are hereby incorporated by reference in their entirety with particular attention to the content for which they are referred. Also, this application claims the benefit of the filing date of U.S. Provisional Application Nos. 62/220,107 filed Sep. 17, 2015; 62/221,068 filed Sep. 20, 2015 and 62/301,260 filed February 2016; which are incorporated by reference in their entirety herein.