Provided herein is a dual vector transfection system for the production of recombinant adeno-associated virus (rAAV). The dual vector transfection system generally comprises: (1) a first nucleic acid vector comprising a first nucleotide sequence encoding an AAV Rep protein, a second nucleotide sequence comprising an rAAV genome comprising a transgene, and a third nucleotide sequence encoding an AAV capsid protein; and (2) a second nucleic acid vector comprising a helper virus gene.

The present invention provides, among other things, methods and compositions for production of recombinant I2S protein with improved potency and activity using cells co-express I2S and FGE protein. In some embodiments, cells according to the present invention are engineered to simultaneously over-express recombinant I2S and FGE proteins. Cells according to the invention are adaptable to various cell culture conditions. In some embodiments, cells of the present invention adaptable to a large-scale suspension serum-free culture.

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.

Disclosed is a method for production of a fusion protein in which an antibody and a lysosomal enzyme are fused. The method comprises; (a) a step of culturing mammalian cells producing the fusion protein in a serum-free medium to let the mammalian cells secrete the fusion protein in the culture medium, (b) a step of collecting culture supernatant by removing the mammalian cells from the culture medium, and (c) a step of purifying the fusion protein from the culture supernatant by using a column chromatography employing as a solid phase a material to which a substance having affinity for the fusion protein has been bound, a column chromatography employing as a solid phase a material having affinity for the phosphate group, and a size exclusion column chromatography.

Compositions and methods are described for the delivery of recombinant human iduronate-2-sulfatase (IDS) produced by human neuronal or glial cells to the cerebrospinal fluid of the central nervous system (CNS) of a human subject diagnosed with mucopolysaccharidosis II (MPS II).

Compositions and methods are described for the delivery of recombinant human iduronate-2-sulfatase (IDS) produced by human neuronal or glial cells to the cerebrospinal fluid of the central nervous system (CNS) of a human subject diagnosed with mucopolysaccharidosis II (MPS II).

Certain embodiments provide a method of treating Hunter syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective dose of a pharmaceutical composition comprising an ETV:IDS protein.

Described herein are engineered nucleases comprising mutations in the cleavage domain (e.g., FokI or homologue thereof) and/or DNA binding domain (zinc finger protein, TALE, single guide RNA) such that on-target specificity is increased.

Mutated Pseudomonas aeruginosa sulfatase (PaS) enzymes with enhanced enzymatic activity as compared to the parental wild type enzyme are provided. The variant sulfatases of the invention advantageously allow for accurate analysis of bodily samples for the presence of drugs. Methods of using the mutated enzymes for hydrolysis of sulfate ester linkages are also provided, including with substrates such as opioids or steroids containing a sulfate ester linkage.

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.