Transgenically produced non-secreted proteins

Abstract

The invention provides a method of making and secreting a non-secreted protein. The method includes expressing the protein from a nucleic acid construct which includes: (a) a mammary epithelial specific promoter; (b) a milk protein specific signal sequence which can direct the secretion of a protein; (c) optionally, a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal, of the non-secreted protein; and (d) a sequence which encodes a non-secreted protein, wherein elements (a), (b), optionally (c), and (d) are preferably operatively linked in the order recited. Both glutamic acid decarboxylase (GAD) and myelin basic protein (MBP), which are cytoplasmic proteins, have been produced by the methods of the present invention. The invention also provides methods for treating diabetes and multiple sclerosis using proteins produced by the methods of the present invention.

Description

[0001] This application claims the benefit of a previously filed Provisional Application No. 60 / 038,998, filed Feb. 25, 1997, which is hereby incorporated by reference.FIELD OF THE INVENTION [0002] This invention relates to the production and secretion of proteins which are not ordinarily secreted. BACKGROUND OF THE INVENTION [0003] A growing number of recombinant proteins are being developed for therapeutic and diagnostic applications. However, many of these proteins may be difficult or expensive to produce in a functional form and / or in the required quantities using conventional methods. Conventional methods involve inserting the gene responsible for the production of a particular protein into host cells such as bacteria, yeast, or mammalian cells, e.g., COS cells, and then growing the cells in culture media. The cultured cells then synthesize the desired protein. Traditional bacteria or yeast systems may be unable to produce many complex proteins in a functional form. While mamma...

AI Technical Summary

Benefits of technology

[0079] The application of transgenic technology to the commercial production of recombinant proteins in the milk of transgenic animals using milk protein specific signal and promoter sequences offers significant advantages over traditional methods of non-secreted protein production. These advantages include a reduction in the total amount of required capital expenditures, elimination of the need for capital commitment to build facilities early in the product development life cycle, and lower direct production cost per unit for complex proteins. Of key importance is the likelihood that, for certain non-secreted proteins, transgenic production may represent the only technologically and economically feasible method of commercial production.

[0080] Myelin basic protein (MBP) is membrane associated protein synthesized by oligodendrocytes and Schwann cells. It is not secreted in its natural environment. MBP is also an autoantigen of the disease multiple sclerosis. Animal model studies and clinical trial data have suggested that administrating MBP orally could establish peripheral immune tolerance and thereby suppress the symptoms of the disease.

[0081] Glutamic acid decarboxylase (GAD), another cytoplasmic protein, is an enzyme that catalyzes the biosynthesis of the neurotransmitter, γ-aminobutyric acid. The human genome has at least two homologous genes located on chromosomes 2 and 10. The GAD65 and the GAD67 cDNA derived primary amino acid sequences are 65% identical, with the difference between the two isomers in the first 250 amino acids being approximately 75%. GAD65 has been recently identified as a critical β-cell autoantigen in the disease insulin-dependent diabetes mellitus. Experiments in the NOD mouse model of insulin-dependent diabetes mellitus have shown an early appearance of GAD65-reactive antibodies and T cells, and have demonstrated protection of diabetes by early GAD treatment, indicating that GAD65 is a key antigen in the disease process (Kaufman et al. Nature 366:69-72, 1993; Tisch et al. Nature 366:72-5, 1993). The presence of anti-GAD antibodies in the sera of prediabetic individuals can serve as reliable makers for progression to overt diabetes. GAD is, therefore, thought to be a candidate for tolerance therapy.

[0083] The methods described herein allow the production of high levels of secreted proteins which are not normally secreted, e.g., glutamic acid decarboxylase (GAD), a protein that is a potential therapy for insulin-dependent diabetes mellitus (Type 1 diabetes), in the milk of a transgenic animal.

[0088] Oral tolerance is a mechanism that allows the human body to regulate the immune system so that it can absorb foreign materials as nourishment. Preclinical and clinical observations of oral tolerance show that digesting certain proteins can suppress autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Problems solved by technology

However, many of these proteins may be difficult or expensive to produce in a functional form and / or in the required quantities using conventional methods.

Traditional bacteria or yeast systems may be unable to produce many complex proteins in a functional form.

While mammalian cells can reproduce complex proteins, they are generally difficult and expensive to grow, and often produce only mg / L quantities of protein.

In addition, non-secreted proteins are relatively difficult to purify from procaryotic or mammalian cells as they are not secreted into the culture medium.

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