The present invention provides, among other things, compositions and methods for CNS delivery of lysosomal enzymes for effective treatment of lysosomal storage diseases. In some embodiments, the present invention includes a stable formulation for direct CNS intrathecal administration comprising an arylsulfatase A (ASA) protein, salt, and a polysorbate surfactant for the treatment of Metachromatic Leukodystrophy Disease.

The present invention provides, among other things, improved methods for purifying I2S protein produced recombinantly for enzyme replacement therapy. The present invention is, in part, based on the surprising discovery that recombinant I2S protein can be purified from unprocessed biological materials, such as, I2S-containing cell culture medium, using a process involving as few as four chromatography columns.

The present invention provides, among other things, methods of purifying a protein or polypeptide by affinity chromatography, for example, custom affinity chromatography, followed by downstream chromatographic steps, comprising eluting the recombinant protein from the affinity column using an elution buffer wherein the elution buffer comprises glycine and arginine at a concentration such that conductivity is no greater than the conductivity limit of the subsequent chromatographic resin.

Problem

To provide a method for producing a glycoprotein having N-linked sugar chains containing M6Ps in a wild type as a protein (M6P low-modified protein) in which the M6Ps originally contained in the glycoprotein are deleted or the number thereof is reduced, the M6P low-modified protein, and a method for utilizing the M6P low-modified protein.

Resolution Means

A protein in which at least one of one or more N-linked sugar chains that is linked to Asn is deleted by adding a mutation to at least one amino acid sequence represented by Asn-Xaa-Yaa (where Xaa represents an amino acid other than proline, and Yaa represents threonine or serine) contained in an amino acid sequence of a glycoprotein having, in a wild type, one or more N-linked sugar chains binding thereto, the one or more N-linked sugar chains each containing one or more mannose-6-phosphate (M6Ps).

The present invention provides an effective and less invasive approach for direct delivery of therapeutic agents to the central nervous system (CNS). In some embodiments, the present invention provides methods including a step of administering intrathecally to a subject suffering from or susceptible to a lysosomal storage disease associated with reduced level or activity of a lysosomal enzyme, a composition comprising a replacement enzyme for the lysosomal enzyme.

Certain embodiments provide a fusion protein comprising an iduronate 2-sulfatase (IDS) amino acid sequence, an IDS variant amino acid sequence, or a catalytically active fragment thereof; and an anti-transferrin receptor (TfR) antibody as described herein, as well as methods of use thereof.

Applicants sought to express human iduronate-2-sulfatase (hIDS) in the brain endothelium of a mouse model of Mucopolysaccharidosis type II (MPSII, Hunter's syndrome) to enable enzyme secretion into the brain parenchyma. In this disorder, IDS deficiency results in the pathophysiological accumulation of heparan and dermatan sulfate GAGs. To test the hypothesis, Applicants chose AAV-BI30, an AAV9-derived capsid that has an enhanced in vivo tropism specific to the endothelium in the rodent CNS and can transduce human brain vascular endothelial cells in vitro more efficiently than AAV9. Applicants show that systemic delivery of AAV-BI30: hIDS restored IDS enzyme activity in the brain, liver, and serum of IDS-KO mice (FIG. 1). Importantly, AAV-BI30-mediated gene transfer resulted in the correction of GAG accumulation in the brain (FIG. 2). This effect was not observed when using the AAV-BI30 vector packaging a non-secreting version of hIDS. These findings highlight that targeting endothelial cells throughout the CNS is a promising approach for delivering enzymes across the BBB and restoring lysosomal metabolism.

A co-therapeutic regimen comprising AAV9-mediated intrathecal/intracisternal and/or systemic delivery of an expression cassette containing a hIDS gene and two or more immunosuppressants is provided herein. Also provided are methods and kits containing these vectors and compositions useful for treating Hunter syndrome and the symptoms associated with Hunter syndrome.

The present invention provides an effective and less invasive approach for direct delivery of therapeutic agents to the central nervous system (CNS). In some embodiments, the present invention provides methods including a step of administering intrathecally to a subject suffering from or susceptible to a lysosomal storage disease associated with reduced level or activity of a lysosomal enzyme, a composition comprising a replacement enzyme for the lysosomal enzyme.

An object of the present invention is to provide a nucleic acid molecule, a vector, a recombinant cell, and a drug for treating a central nervous system disease, which easily migrate to the central nervous system. The nucleic acid molecule of the present invention contains a nucleotide sequence encoding a fusion protein, the fusion protein containing: an anti-transferrin receptor (TfR) antibody or an antigen-binding fragment thereof; and a protein to be brought into function in the central nervous system.