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Methods and compositions for targeting proteins across the blood brain barrier

a technology of protein targeting and blood brain barrier, which is applied in the direction of drug compositions, peptide/protein ingredients, metabolic disorders, etc., can solve the problems of mild to serious neurological symptoms, blood brain barrier impedes the delivery of drugs to the cns, and methods are not available in the art to efficiently deliver drugs

Inactive Publication Date: 2006-06-08
BIOMARIN PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods and compositions for targeting therapeutic molecules from the blood stream to the brain or central nervous system (CNS) using an insulin-like growth factor (IGF) tagging system. The IGF tagging system uses an IGF moiety that is attached to a therapeutic peptide or protein to facilitate transport across the blood brain barrier. The IGF tag can be fused to the N-terminus, C-terminus, or within the body of the therapeutic protein. The invention is useful for delivering therapeutic molecules to the brain for treating brain or CNS disorders.

Problems solved by technology

However, the blood brain barrier impedes the delivery of drugs to the CNS.
In particular, many lysosomal storage diseases affect cells of the CNS and result in mild to serious neurological symptoms.
However, methods are not available in the art to efficiently deliver drugs, and particularly protein-based drugs, from the blood stream to the CNS through the blood brain barrier.

Method used

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  • Methods and compositions for targeting proteins across the blood brain barrier
  • Methods and compositions for targeting proteins across the blood brain barrier
  • Methods and compositions for targeting proteins across the blood brain barrier

Examples

Experimental program
Comparison scheme
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example 1

Fusion Protein Expressing Constructs

[0059] Nucleic acid constructs for expressing therapeutic protein fusions of the invention can be made recombinantly according to methods known in the art. For example, oligonucleotides complementary to genes encoding the different components described herein can be used to make synthetic genes or to amplify the natural genes and construct gene fusions. In preferred embodiments, proteins of the invention are expressed from a recombinant gene comprising a signal sequence. Examples of useful nucleic acids include nucleic acids that encode IGF targeting moieties of the invention. Such nucleic acids can be based on the sequences of IGF-1 shown in FIG. 4.

example 2

Expression and Purification Methods

[0060] Expression product can also be isolated from serum free media using other protozoa, including other Leishmania species. In general, the expression strain is grown in medium with serum, diluted into serum free medium, and allowed to grow for several generations, preferably 2-5 generations, before the expression product is isolated. For example, production of secreted recombinant LSD proteins can be isolated from Leishmania mexicana promastigotes that are cultured initially in 50 mL 1×M199 medium in a 75 cm2 flask at 27° C. When the cell density reaches 1-3×107 / mL, the culture is used to inoculate 1.2 L of M199 media. When the density of this culture reaches about 5×106 / mL, the cells were harvested by centrifugation, resuspended in 180 mL of the supernatant and used to inoculate 12 L of “Zima” medium in a 16 L spinner flask. The initial cell density of this culture is typically about 5×105 / mL. This culture is expanded to a cell density of abo...

example 3

Assays for Crossing the Blood Brain Barrier

[0063] According to the invention, a useful model system to determine whether a protein, particularly an LSD protein, tagged with an IGF tag crosses the blood-brain barrier, is the MPSVII mouse model (Wolfe and Sands (1996) Protocols for Gene Transfer in Neuroscience: Towards Gene Therapy of Neurological Disorders Chapter 20: 263-273). For example, recombinant human β-glucuronidase fused to an IGF tag can be produced in any convenient expression system such as Leishmania, yeast, mammalian, bacculovirus and other expression systems. L. mexicana expressing and secreting β-GUS is grown at 26° C. in 100 ml Standard Promastigote medium (M199 with 40 mM HEPES, pH 7.5, 0.1 mM adenine, 0.0005% hemin, 0.0001% biotin, 5% fetal bovine serum, 5% embryonic fluid, 50 units / ml penicillin, 50 μg / ml streptomycin and 50μg / ml nourseothricin). After reaching a density of approximately 5×106 promastigotes / ml, the promastigotes is collected by centrifugation fo...

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Abstract

Disclosed are methods and compositions for targeting therapeutic proteins to the brain. Methods and compositions of the invention involve associating an IGF moiety with a therapeutic protein in order to target the therapeutic protein to the brain. Soluble fusion proteins that include an IGF targeting moiety are transported to neural tissue in the brain from blood. Methods and compositions of the invention include therapeutic applications for treating lysosomal storage diseases. The invention also provides nucleic acids and cells for expressing IGF fusion proteins.

Description

RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Ser. No. 60 / 329,650, filed Oct. 16, 2001, the entire disclosure of which is incorporated herein by reference.FIELD OF THE INVENTION [0002] This invention provides a means for specifically delivering proteins to the brain. The ability to target proteins to the brain is of great utility in the treatment of neurological diseases. Methods and compositions of the invention are useful to target proteins to cells across the blood brain barrier, and in particular, to target proteins to the lysosomes of cells in the CNS, including neuronal cells, macrophage cells, and other cell types. Accordingly, the invention provides methods and compositions to deliver therapeutically useful proteins to treat lysosomal storage diseases that affect the CNS. BACKGROUND [0003] The blood-brain barrier maintains a homeostatic environment in the central nervous system (CNS). The capillaries that supply the blood to the brain have tight jun...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/47A61K38/30
CPCA61K38/30C07K2319/00C07K2319/02C07K2319/50C07K2319/74A61K2300/00
Inventor LEBOWITZ, JONATHAN
Owner BIOMARIN PHARMA INC
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