Optimized promoter constructs
OwnerMichael W. Heartlein, Justin Chace Lamsa +4inventors
InventorsMichael W. Heartlein, Justin Chace Lamsa, Douglas A. Treco, Richard F. Selden, Michael F. Concino, Heidi Kempinski
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Abstract
The invention features constructs and related methods for expression of products in mammalian cells, e.g., human cells. Constructs include a human γ-actin, β-actin, fibronectin, YY1, or β-tubulin promoter region operably linked to a heterologous nucleic acid sequence.
Claims87
An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:
- 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
An isolated nucleic acid sequence which is at least 60% identical to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
A construct suitable for expression in a mammalian cell, comprising a human γ-actin promoter region operably linked to a heterologous nucleic acid sequence.
A construct suitable for expression in a human cell comprising a human β-tubulin promoter region operably linked to a heterologous nucleic acid sequence.
A construct suitable for expression in a human cell, comprising a human YY1 promoter region operably linked to a heterologous nucleic acid.
A construct suitable for expression in a human cell, comprising a human fibronectin promoter region operably linked to a heterologous nucleic acid sequence, provided that said promoter region includes at least two contiguous nucleotides from SEQ ID NO:2.
A construct suitable for expression in a human cell, comprising a human fibronectin promoter region operably linked to a heterologous nucleic acid sequence, provided that said promoter region includes at least one of the following elements:
- (a) an enhancer;
- (b) a U1 intron.
A construct suitable for expression in a human cell, comprising a human β-actin promoter region operably linked to a heterologous nucleic acid sequence, provided that said promoter region includes at least one of the following elements:
- (a) an MAR;
- (b) a β-actin 5′ flank addition;
- or
- (c) a β-actin 5′ UTS fusion.
A construct suitable for expression in a human cell, comprising a human β-actin promoter region operably linked to a heterologous nucleic acid sequence, wherein the construct includes at least one of the following elements:
- (a) an MAR;
- (b) an enhancer;
- (c) a heterologous 5′ UTS;
- (d) a U1 intron;
- or
- (e) a β-actin 5′ UTS fusion;
- provided that if the construct includes (b) it does not include (c) and if the construct includes (c) it does not include (b).
A cell transfected with a construct described herein.
A method of producing a substance, comprising:
- providing a human cell which includes a construct described herein;
- and
- allowing said cell to express a heterologous polypeptide, thereby producing a substance.
A method of supplying a substance to a subject, comprising:
- (a) providing a human cell which includes a construct described herein;
- and
- (b) allowing said cell to express a heterologous polypeptide;
- and
- (c) administering said heterologous polypeptide to the subject, thereby supplying a substance to a subject.
A method of treating a disorder in a subject, comprising:
- providing a human cell which includes a construct described herein;
- and
- allowing said cell to express a heterologous polypeptide in vivo in the subject, thereby treating a disorder in a subject.
A method of providing a heterologous protein to a subject, comprising:
- (a) providing a human cell which includes a construct described herein;
- and
- (b) allowing said cell to produce the heterologous protein in vivo in said subject, thereby providing a heterologous protein to a subject.
A method of treating a disorder in a subject, comprising:
- identifying a subject in need of a product;
- and
- introducing into the subject a construct described herein, wherein the construct causes the production of the product in an amount sufficient to ameliorate a symptom of said disorder;
- thereby treating the disorder in the subject.
A method of evaluating the level of Factor VIII in a blood sample of the subject, comprising:
- (a) contacting a blood sample of the subject with a first antibody to Factor VIII thereby forming a reaction mixture;
- (b) thereafter contacting the reaction mixture with a second labeled antibody to Factor VIII;
- (c) detecting the amount of label in the reaction mixture, the amount of label being correlated to the amount of Factor VIII in the sample,
- thereby evaluating the level of Factor VIII in a blood sample.
A kit for the detection of Factor VIII, comprising:
- (a) a first antibody to Factor VIII;
- (b) a second labeled antibody to Factor VIII;
- and
- (c) instructions for using the first and second antibodies to detect Factor VIII at a concentration of 3.5 mU/mL or more.
Dependencies are inferred from the claim text.