Recombinant human naglu protein and uses thereof

Abstract

The present invention provides compositions comprising an isolated mixture of recombinant human NaGlu proteins in which a substantial amount of the NaGlu proteins in the mixture has increased levels of phosphorylated mannose that confer the proteins to be efficiently internalized into human cells. The present invention also provides methods of producing such mixture of NaGlu proteins, vectors used in transgenesis and expression, host cells harboring such vectors, and methods of isolating and purifying the mixture of NaGlu proteins. The invention further provides methods of treating NaGlu associated diseases.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is related and claims priority to U.S. Provisional Application Ser. No. 61 / 546,248, filed Oct. 12, 2011, the entire contents of which are expressly incorporated herein by this reference.BACKGROUND OF THE INVENTION[0002]Sanfilippo Syndrome B is an autosomal recessive lysosomal storage disease (LSD) caused by a deficiency in a lysosomal enzyme known as N-acetyl-alpha-D-glucosaminidase (NaGlu). NaGlu is required for the degradation of heparan sulfate as part of the stepwise breakdown of glycosaminoglycans (GAG) in the lysosome. The deficiency or absence of NaGlu leads to accumulation and urinary excretion of heparan sulfate. With over 70 different mutations identified to date, Sanfilippo Syndrome B exhibits extensive molecular and genetic heterogeneity.[0003]Approximately 1 out of 200,000 births is affected by Sanfilippo Syndrome B and the deficiency mainly manifests in young children. After initial symptom-free interval, pa...

AI Technical Summary

Benefits of technology

The present invention is about using a recombinant human NaGlu protein (rhNaGlu) for therapy, specifically in treating Sanfilippo Syndrome B. The inventors have discovered that this protein has specific patterns of glycosylation that allow it to efficiently cross the blood brain barrier and be taken up into cells in the central nervous system of animals deficient in the enzyme. This results in a dramatic increase in α-N-acetylglucosaminidase activity in the brain, as well as a reduction of substrate levels. The rhNaGlu is efficiently taken up into mammalian cells and produces increased enzymatic activity compared to unmodified mammalian cells that are not designed to produce specific glycosylation. The use of the rhNaGlu also minimizes the need for an increased amount and frequency of dose, thereby reducing the potential risk of immunogenicity. In one embodiment, the rhNaGlu contains sufficient amounts of M6P to allow for internalization into a mammalian cell via M6P receptor-mediated endocytosis and restoration of normal levels of NaGlu activity. The therapeutically effective amount of the rhNaGlu is an amount effective to reduce heparan sulfate levels, increase NaGlu activity in the brain or liver, or treat Sanfilippo Syndrome B.

Problems solved by technology

The deficiency or absence of NaGlu leads to accumulation and urinary excretion of heparan sulfate.

After initial symptom-free interval, patients suffering from Sanfilippo Syndrome B usually present with a slowing of mental development and behavioral problems, followed by progressive intellectual decline resulting in severe mental retardation, dementia and motor disease.

Acquisition of speech is slow and incomplete.

The disease usually progresses to increasing behavioral disturbance and sleep disturbance.

Although the clinical features are mainly neurological, patients often develop diarrhea, carious teeth, an enlarged liver and spleen, stiff joints, hirsteness and / or coarse hair and may exhibit blood-clotting problems.

In the final stage of the illness, patients become immobile and unresponsive and develop swallowing difficulties and seizure.

No or weak phosphorylation of N-glycans in the NaGlu proteins secreted from the mammalian cells has posed a major obstacle for the development of a recombinant human NaGlu protein suitable for enzyme replacement therapy as all the aforementioned attempts has failed to produce an enzyme which is efficiently taken up by target cells as the concentration of the internalized proteins, if detectable at all, was nearly a thousand times less than wild-type levels (see, Zhao et al., Protein Expression and Purification, 19:202-211 (2000)).

To date, no approved product is available for the treatment of Sanfilippo Syndrome B.

Direct administration of mammalian cell-produced recombinant human NaGlu protein (rhNaGlu) having the native amino acid sequence into the central nervous system (CNS) (e.g., intrathecal administration into the cerebrospinal fluid (CSF)) of NaGlu deficient mice has been attempted, but failed to demonstrate successful biodistribution of the enzyme to the brain due to excessive accumulation of the protein on the ependymal ling of the ventricles as well as lack of requisite M6P residues for efficient cellular uptake.

Similarly, systemic administration (i.e., intravenous (IV) injection) of mammalian cell-produced rhNaGlu having the native amino acid sequence also failed to demonstrate successful localization of the protein to the brain.

In addition to known risks associated with highly invasive intrathecal administration, these obstacles in targeting rhNaGlu to the brain have been too great a challenge to achieve effective therapy for the treatment of Sanfilippo Syndrome B.

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