Production of butyrylcholinesterases in transgenic animals
AssigneeNexia Biotechnologies, Inc.
InventorsCostas Karatzas, Yue-Jin Huang, Anthoula Lazaris
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Abstract
Abstract of the Disclosure The present invention provides methods for the large-scale production of recombinant butyrylcholinesterase in cell culture, and in the milk and/or urine of transgenic mammals. The recombinant butyrylcholinesterases of this invention can be used to treat and/or prevent organophosphate pesticide poisoning, nerve gas poisoning, cocaine intoxication, and succinylcholine-induced apnea.
Claims62
A non-human transgenic mammal that upon lactation, expresses a BChE enzyme in its milk, wherein the genome of the mammal comprises a DNA sequence encoding a BChE enzyme, operably linked to a mammary gland-specific promoter, and a signal sequence that provides secretion of the BChE enzyme into the milk of the mammal.
A non-human transgenic mammal that expresses a BChE enzyme in its urine, wherein the genome of the mammal comprises a DNA sequence encoding a BChE enzyme, operably linked to a urinary endothelium-specific promoter, and a signal sequence that provides secretion of the BChE enzyme into the urine of the mammal.
A genetically-engineered DNA sequence, which comprises:
- (i) a sequence encoding a BChE enzyme;
- (ii) a mammary gland-specific promoter that directs expression of the BChE enzyme;
- and (iii) at least one signal sequence that provides secretion of the expressed BChE enzyme.
A method for making a genetically-engineered DNA sequence, which method comprises joining a sequence encoding a BChE enzyme with a mammary gland-specific promoter the directs expression of the BChE enzyme and at least one signal sequence that provides secretion of the expressed BChE enzyme.
A genetically-engineered DNA sequence, which comprises:
- (i) a sequence encoding a BChE enzyme;
- (ii) a urinary endothelium-specific promoter that directs expression of the BChE enzyme;
- and (iii) at least one signal sequence that provides secretion of the expressed BChE enzyme.
A method for making a genetically-engineered DNA sequence, which method comprises joining a sequence encoding a BChE enzyme with a urinary endothelium-specific promoter that directs expression of the BChE enzyme and at least one signal sequence that provides secretion of the expressed BChE enzyme.
A method for producing a transgenic mammal that upon lactation secretes a BChE enzyme in its milk, which method comprises allowing an embryo, into which at least one genetically-engineered DNA sequence, comprising (i) a sequence encoding a BChE enzyme;
- (ii) a mammary gland-specific promoter;
- and (iii) a signal sequence that provides secretion of the BChE enzyme into the milk of the mammal, has been introduced, to grow when transferred into a recipient female mammal, resulting in the recipient female mammal giving birth to the transgenic mammal.
A method for producing a transgenic mammal that upon lactation secretes a BChE enzyme in its milk, which method comprises cloning or breeding of a transgenic mammal, the genome of which comprises a DNA sequence encoding a BChE enzyme, operably linked to a mammary gland-specific promoter, wherein the sequence further comprises a signal sequence that provides secretion of the BChE enzyme into the milk of the mammal.
A method for producing a transgenic mammal that secretes a BChE enzyme in its urine, which method comprises allowing an embryo, into which at least one genetically-engineered DNA sequence, comprising (i) a sequence encoding a BChE enzyme;
- (ii) a urinary endothelium-specific promoter;
- and (iii) a signal sequence that provides secretion of the BChE enzyme into the urine of the mammal, has been introduced, to grow when transferred into a recipient female mammal, resulting in the recipient female mammal giving birth to the transgenic mammal.
A method for producing a transgenic mammal that secretes a BChE enzyme in its urine, which method comprises cloning or breeding of a transgenic mammal, the genome of which comprises a DNA sequence encoding a BChE enzyme, operably linked to a urinary endothelium-specific promoter, wherein the sequence further comprises a signal sequence that provides secretion of the BChE enzyme into the urine of the mammal.
A method for producing a BChE enzyme, which method comprises:
- (a) inducing or maintaining lactation of a transgenic mammal, the genome of which comprises a DNA sequence encoding a BChE enzyme, operably linked to a mammary gland-specific promoter, wherein the sequence further comprises a signal sequence that provides secretion of the BChE enzyme into the milk of the mammal;
- and
- (b) extracting milk from the lactating mammal.
The milk of a non-human mammal comprising a human BChE enzyme.
A method for producing a BChE enzyme, which method comprises extracting urine from a transgenic mammal, the genome of which comprises a DNA sequence encoding a BChE enzyme, operably linked to a urinary endothelium-specific promoter, where the sequence further comprises a signal sequence that provides secretion of the BChE enzyme into the urine of the mammal.
Urine of a non-human mammal comprising a human BChE enzyme.
A method for producing a BChE enzyme in a culture of MAC-T or BHK cells, which method comprises:
- (a) culturing said cells, into which a DNA sequence comprising (i) a DNA sequence encoding a BChE enzyme, (ii) a promoter that provides expression of the encoded BChE enzyme within said cells, and (iii) a signal sequence that provides secretion of the BChE enzyme into the cell culture medium, has been introduced;
- (b) culturing the cells;
- and
- (c) collecting the cell culture medium of the cell culture.
Cell culture medium from a culture of mammalian cells, which medium comprises a BChE enzyme, wherein at least 50% of the BChE enzyme is in tetramer form.
Dependencies are inferred from the claim text.