Gene therapy for treating mucopolysaccharidosis type i

Abstract

A suspension useful for AAV9-mediated intrathecal/intracisternal and/or systemic delivery of an expression cassette containing a hIDUA gene is provided herein. Also provided are methods and kits containing these vectors and compositions useful for treating MPSI and the symptoms associated with Hurler, Hurler-Scheie and Scheie syndromes.

1 . A pharmaceutical composition suitable for intrathecal administration in human subjects, comprising a suspension of replication deficient recombinant adeno-associated virus (rAAV) in a formulation buffer, wherein:
 (a) the rAAV comprises a heterologous nucleic acid encoding human α-L-iduronidase (hIDUA), wherein said nucleic acid is operably linked to a CB7 promoter and packaged in an AAV9 capsid;   (b) the formulation buffer comprises a physiologically compatible aqueous buffer, a surfactant and optional excipients; and   (c)(i) the rAAV Genome Copy (GC) titer is at least 1×10 9  GC/mL (+/−20%);   (ii) the rAAV Empty/Full particle ratio is at least about 80% free of empty capsids; and/or   (iii) a dose of at least about 4×10 8  GC/g brain mass to about 4×10 11  GC/g brain mass of the rAAV suspension has potency.   
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein potency is measured by an in vitro assay. 
     
     
         3 . The pharmaceutical composition of  claim 2 , wherein the in vitro assay comprises transducing HEK293 or Huh7 cells with a known multiplicity of the rAAV GC titer per cell and assaying the supernatant for hIDUA activity 72 hours post-transduction using the 4 MU-iduronide enzymatic assay. 
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein the human hIDUA coding sequence has the nucleotide sequence of SEQ ID NO: 1 or a sequence at least about 80% identical to SEQ ID NO: 1 which encodes a functional hIDUA. 
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the encoded hIDUA has the sequence selected from:
 (a) about amino acid 1 to about 653 of SEQ ID NO: 2 (Genbank NP_000193); and   (b) a synthetic human enzyme comprising a heterologous leader sequence fused to about acids 27 to about 653 of SEQ ID NO: 2.   
     
     
         6 . The pharmaceutical composition of  claim 1 , wherein the rAAV further comprises a 5′ inverted terminal repeat (ITR) sequence, a chicken beta actin intron, a rabbit beta-globin polyadenylation (polyA) signal, and/or a 3′ ITR sequence. 
     
     
         7 . The pharmaceutical suspension of  claim 1 , wherein the empty/full ratio is between 0.01 and 0.05 (95%-99% free of empty capsids) 
     
     
         8 . The pharmaceutical suspension of  claim 1 , wherein the suspension has a pH of 6 to 8 or a pH of 6.8 to 7.8. 
     
     
         9 . A method of treating a human subject diagnosed with Mucopolysaccharidosis I (MPS I) and/or the symptoms associated Hunter syndrome, comprising administering to the human subject in need thereof by intrathecal injection, a suspension according  claim 1  at a dose of 4×10 8  GC/g brain mass to about 4×10 11  GC/g brain mass. 
     
     
         10 . The method of  claim 9 , wherein the human subject is diagnosed with Hurler syndrome. 
     
     
         11 . The method of  claim 9 , wherein the human subject is diagnosed with Hurler-Scheie syndrome. 
     
     
         12 . The method of  claim 9 , wherein the human subject is diagnosed with Scheie syndrome. 
     
     
         13 . The method of  claim 9 , further comprising co-administering the rAAV in an immunosuppressive regimen. 
     
     
         14 . The method of  claim 9 , wherein said method results in an increase in intelligence quotient (IQ) in said subject, as assessed using Bayley Scales of Infant Development or as assessed using WASI. 
     
     
         15 . The method of  claim 9 , wherein said method results in an increase in functional hIDUA levels, as measured in a serum sample from said patient. 
     
     
         16 . The method of  claim 9 , wherein said method results in a decrease in GAG levels, as measured in a sample of the patient's serum, urine and/or cerebrospinal fluid (CSF). 
     
     
         17 . The method of  claim 9 , further comprising administering to said patient by liver-directed injection a rAAV.hIDUA. 
     
     
         18 . The method of  claim 17 , wherein the liver-directed AAV.hIDUA has a capsid selected from AAV8, AAVrh64R1, AAVrh64R2, rh8, rh10, AAV3B, or AAVdj. 
     
     
         19 . The method of  claim 9 , wherein potency is measured by an in vitro assay. 
     
     
         20 . The method of  claim 19 , wherein the in vitro assay comprises transducing HEK293 or Huh7 cells with a known multiplicity of the rAAV GC titer per cell and assaying the supernatant for hIDUA activity 72 hours post-transduction using the 4 MU-iduronide enzymatic assay. 
     
     
         21 . The method of  claim 9 , wherein the suspension is formulated for delivery via intrathecal injection. 
     
     
         22 . The method of  claim 19 , wherein the suspension is formulated for:
 (a) delivery to newborn patients and comprises about 1.4×10 11  genome copies (GC) to about 1.4×10 14  GC;   (b) delivery to patients that are about 3 months to about 9 months of age and comprises about 2.4×10 11  to about 2.4×10 14  GC;   (c) delivery to patients that are about 9 months to about 36 months of age and comprises about 4×10 11  GC to about 4×10 14  GC;   (d) delivery to patients that are about 3 years to about 12 years of age and comprises about 4.8×10 11  GC to about 4.8×10 14  GC,   (e) delivery to patients that are about 12 years of age or older and comprises about 5.6×10 11  GC to about 5.6×10 14  GC; or   (f) delivery to patients that are about 18 years of age or older and comprises about 1.4×10 13  GC to about 7.0×10 13  GC.   
     
     
         23 . A method of treating a human patient having MPS I and/or the symptoms associated Hunter syndrome, the method comprising:
 (a) dosing a patient having MPS I and/or the symptoms associated with Hunter syndrome with a sufficient amount of hIDUA enzyme to induce transgene-specific tolerance; and   (b) administering an rAAV.hIDUA to the patient, which rAAV.hIDUA directs expression of therapeutic levels of hIDUA in the patient.   
     
     
         24 . The method of  claim 23 , wherein the hIDUA of (a) is dosed as a recombinant protein. 
     
     
         25 . The method of  claim 23 , wherein the patient is an infant. 
     
     
         26 . The method of  claim 23 , wherein the administering (b) is performed about three days to about 14 days post-dosing (a).