Transgenic nonhuman mammals capable of secreting a lysosomal protein containing mannose 6-phosphate into milk

摘要

Transgenic nonhuman mammals (mice, rabbits, goat, sheep, pigs and cows) producing phosphorylated lysosomal proteins in their milk, and methods of generating the same are discussed. Phosphorylation occurs at the 6' position of a mannose side chain residue. Methods of purifying lysosomal proteins from milk and incorporating the proteins into pharmaceutical compositions for use in enzyme replacement therapy are also discussed. The purified protein can then be mixed with a pharmaceutical carrier for intravenous, intradermal, intramuscular or oral administration. The transgene in the transgenic mammal comprises a mammary gland specific promoter, a mammary-gland specific enhancer, a secretory DNA segment encoding a signal peptide functional in mammary secretory cells, and a recombinant DNA segment encoding a lysosomal protein, usually an enzyme. The recombinant DNA segment is operably linked to the secretory DNA segment to form a secretory recombinant DNA segment. The secretory-recombinant DNA segment is in turn operably linked to the promoter and to the enhancer. The transgene, in an adult, is capable of expressing the secretory-recombinant DNA segment in the mammary secretory cells to produce a form of the lysosomal protein that is processed and secreted by the mammary secretory cells into milk as a mannose 6-phosphate containing lysosomal protein. The concentration of the mannose 6-phosphate containing lysosomal protein in the milk is usually at least 100 pg/ml.