Exogenous control of mammalian gene expression through aptamer-mediated modulation of polyadenylation

Abstract

Embodiments of the disclosure concern the use of expression constructs in which at least one polyA signal is embedded upstream of an expressible transcript, such as within a 5′ UTR for the transcript, for example. In certain embodiments, the polyA signal is comprised within a ligand-binding aptamer, and the binding of the ligand to the aptamer, or lack thereof, dictates the outcome for the expressible transcript. In specific embodiments, absence of the ligand causes the expressed transcript having a polyA in its 5′ UTR to be expressed but then degraded, whereas presence of the ligand causes inhibition of degradation upon expression of the expressible transcript. More than one ligand-binding aptamer may be present on the same expression construct.

What is claimed is: 
     
         1 . A system for modulating gene expression, comprising a polyA aptamer polynucleotide that comprises in a 5′ to 3′ direction:
 a) at least one ligand-binding aptamer comprising at least one polyA cleavage signal therein; and 
 b) an expressible polynucleotide. 
 
     
     
         2 . The system of  claim 1 , wherein the ligand binding aptamer comprising the polyA cleavage signal resides within the 5′ untranslated region of the expressible polynucleotide. 
     
     
         3 . The system of  claim 1  or  2 , wherein the system comprises a polynucleotide that expresses the ligand. 
     
     
         4 . The system of  claim 3 , wherein the polynucleotide that expresses the ligand is the same polyA aptamer polynucleotide that comprises the aptamer and expressible polynucleotide. 
     
     
         5 . The system of  claim 3 , wherein the polynucleotide that expresses the ligand is a different polynucleotide than the polyA aptamer polynucleotide that comprises the aptamer and expressible polynucleotide. 
     
     
         6 . The system of any one of  claims 3 - 6 , wherein the polyA aptamer polynucleotide comprises two, three, or more polyA signals in the 5′ UTR of the expressible polynucleotide. 
     
     
         7 . The system of any one of  claims 1 - 6 , wherein the polyA aptamer polynucleotide comprises:
 a) at least one polyA signal;   b) at least one ligand-binding aptamer comprising the at least one polyA signal; and   c) at least one U/UG rich region, at least one G rich region, or both of at least one U/UG rich region and at least one G rich region.   
     
     
         8 . The system of  claim 7 , wherein the ligand-binding aptamer comprises one, two, three, or more U/UG rich regions. 
     
     
         9 . The system of  claim 7 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide at least one polyA signal resides upstream of at least one U/UG rich region. 
     
     
         10 . The system of  claim 7 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide the ligand-binding aptamer resides upstream of one, two, or more U/UG rich regions. 
     
     
         11 . The system of  claim 7 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide at least one polyA signal resides upstream of at least one G rich region. 
     
     
         12 . The system of  claim 3 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide the ligand-binding aptamer resides upstream of one, two, or more G rich regions. 
     
     
         13 . The system of any one of  claims 1 - 12 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide the aptamer comprises two polyA signals and two U/UG rich regions. 
     
     
         14 . The system of any one of  claims 1 - 13 , wherein the polyA aptamer polynucleotide comprises 2, 3, 4, 5, or more aptamers. 
     
     
         15 . The system of  claim 14 , wherein a first aptamer and a second aptamer are in the same orientation in a 5′ to 3′ linear direction. 
     
     
         16 . The system of  claim 14 , wherein a first aptamer and a second aptamer are in a different orientation in a 5′ to 3′ linear direction. 
     
     
         17 . The system of any one of  claims 14 - 16 , wherein the polyA aptamer polynucleotide comprises one G-rich region. 
     
     
         18 . The system of  claim 17 , wherein the G-rich region is in the 3′-most aptamer in a 5′ to 3′ direction of the polyA aptamer polynucleotide. 
     
     
         19 . The system of  claim 17 , wherein the G-rich region is in the second aptamer in a 5′ to 3′ direction of the polyA aptamer polynucleotide. 
     
     
         20 . The system of any one of  claims 14 - 19 , wherein the polyA aptamer polynucleotide does not comprise base pairing between two or more aptamers. 
     
     
         21 . The system of any one of  claims 14 - 19 , wherein the polyA aptamer polynucleotide comprises base pairing between two or more aptamers. 
     
     
         22 . The system of any one of  claims 14 - 21 , wherein there is base pairing between at least part of a loop from two different aptamers in the polyA aptamer polynucleotide. 
     
     
         23 . The system of any one of  claims 1 - 22 , wherein the number of nucleotides between two loops within an aptamer is 10-25 nucleotides. 
     
     
         24 . The system of any one of  claims 1 - 22 , wherein the number of nucleotides between two loops within an aptamer is 18-20 nucleotides. 
     
     
         25 . The system of any one of  claims 1 - 24 , wherein the ligand is a polypeptide, peptide, nucleic acid, small molecule, drug, metabolite, or a combination thereof. 
     
     
         26 . The system of any one of  claims 1 - 25 , wherein an aptamer is between 14 and 250 nucleotides in length. 
     
     
         27 . The system of any one of  claims 1 - 26 , wherein the expressible polynucleotide is a reporter gene, a therapeutic gene, or a gene whose product alters the metabolic state of the cells. 
     
     
         28 . The system of any one of  claims 1 - 27 , wherein the polyA aptamer polynucleotide is at least part of a vector. 
     
     
         29 . The system of  claim 3 , wherein the polynucleotide that expresses the ligand is at least part of a vector. 
     
     
         30 . The system of  claim 28  or  29 , wherein the vector is a plasmid, a viral vector, or linear DNA. 
     
     
         31 . The system of any one of  claims 1 - 30 , wherein the expressible polynucleotide encodes the ligand. 
     
     
         32 . The system of any one of  claims 1 - 31 , wherein expression of the expressible polynucleotide is regulated by a tissue-specific promoter. 
     
     
         33 . A method of modulating gene expression, comprising the steps of:
 a) providing a system, said system comprising a polyA aptamer polynucleotide that comprises in a 5′ to 3′ direction:
 1) at least one ligand-binding aptamer comprising at least one polyA cleavage signal therein; 
 2) an expressible polynucleotide; and 
 3) optionally a ligand-expressing construct; and 
   b) subjecting the system to suitable conditions, wherein when mRNA from the expressible polynucleotide is not desired, the ligand does not bind the ligand-binding aptamer or is not present in the system or its environment, and mRNA from the expressible polynucleotide is degraded; or   c) subjecting the system that comprises the ligand-expressing construct to suitable conditions, wherein when expression of the expressible polynucleotide is desired, the ligand binds the aptamer and/or is present in the system or its environment, and mRNA from the expressible polynucleotide is not degraded.   
     
     
         34 . A method of modulating gene expression, comprising the steps of:
 a) providing a system, said system comprising a polyA aptamer polynucleotide that comprises in a 5′ to 3′ direction:
 1) at least one ligand-binding aptamer comprising at least one polyA cleavage signal therein; 
 2) an expressible polynucleotide; and 
 3) optionally a ligand-expressing construct; and 
   b) subjecting the system to suitable conditions, wherein when the ligand is not present in the system or its environment or does not bind the ligand-binding aptamer, mRNA from the expressible polynucleotide is degraded; or   c) subjecting the system that comprises the ligand-expressing construct to suitable conditions, wherein when the ligand binds the aptamer, mRNA from the expressible polynucleotide is not degraded, and a gene product is expressible from the expressible polynucleotide.   
     
     
         35 . The method of  claim 33  or  34 , wherein the method occurs in a cell. 
     
     
         36 . The method of  claim 35 , wherein the ligand is endogenous to the cell. 
     
     
         37 . The method of  claim 35  or  36 , wherein the cell is a stem cell, a cancer cell, or a diseased or defective cell in need of gene therapy of a gene. 
     
     
         38 . The method of  claim 37 , wherein the gene is dystrophin, albumin, or factor IX. 
     
     
         39 . The method of any one of  claims 33 - 38 , wherein the method occurs in vivo. 
     
     
         40 . The method of any one of  claims 33 - 39 , wherein the method occurs in a mammal. 
     
     
         41 . The method of  claim 40 , wherein the mammal is a human. 
     
     
         42 . The method of  claim 33  or  34 , wherein the method occurs in vitro. 
     
     
         43 . The method of any one of  claims 33 - 41 , wherein the method occurs in one or more cells of an individual, the ligand is glucose, the individual has diabetes, pre-diabetes, or complications from diabetes, and/or the expressible polynucleotide is insulin. 
     
     
         44 . The method of any one of  claims 33 - 41 , wherein the method occurs in one or more cells of an individual, the ligand is the gene product of a cancer biomarker, and the expressible polynucleotide is a suicide gene. 
     
     
         45 . The method of any one of  claims 33 - 41 , wherein the method occurs in an individual, the expressible polynucleotide is a reporter gene, and the location and/or intensity of the expression of the reporter gene provides information about spatial distribution, temporal fluctuation, or both, of a ligand in one or more cells of the individual. 
     
     
         46 . The method of any one of  claims 33 - 45 , further comprising the step of designing the aptamer to suitably bind the ligand. 
     
     
         47 . The method of any one of  claims 33 - 46 , wherein the method occurs in an individual, tissue, or cell, wherein the expressible polynucleotide encodes a detectable gene product, and wherein the respective individual, tissue, or cell is imaged. 
     
     
         48 . A method of monitoring the therapy for an individual, comprising the step of providing to the individual:
 a) a vector comprising a polyA aptamer polynucleotide that comprises in a 5′ to 3′ direction:
 1) at least one ligand-binding aptamer comprising at least one polyA cleavage signal therein; and 
 2) an expressible polynucleotide; and/or 
   b) one or more cells harboring the vector of a),   wherein the ligand is a specific gene product of a protein that is indicative of the efficacy of the therapy.   
     
     
         49 . The method of  claim 48 , wherein the vector of a) and/or the cells of b) are provided to the individual before the therapy, during the therapy, and/or after the therapy. 
     
     
         50 . A method of assaying for the presence, risk, or susceptibility for a medical condition in an individual, comprising the steps of providing to the individual:
 a) a vector comprising a polyA aptamer polynucleotide that comprises in a 5′ to 3′ direction:
 1) at least one ligand-binding aptamer comprising at least one polyA cleavage signal therein; and 
 2) an expressible polynucleotide; and/or 
   b) one or more cells harboring the vector of a),   wherein the expression of the expressible polynucleotide, or absence of expression of the expressible polynucleotide, identifies whether or not the ligand is present to bind the aptamer, wherein the respective presence or absence of the ligand in the individual or cells thereof is indicative of the presence, susceptibility or risk for the medical condition.   
     
     
         51 . A polyA aptamer polynucleotide, wherein said polynucleotide comprises in a 5′ to 3′ direction:
 a) at least one ligand-binding aptamer that comprises at least one polyA cleavage signal therein; and 
 b) an expressible polynucleotide. 
 
     
     
         52 . The polynucleotide of  claim 51 , wherein the ligand-binding aptamer is located in the 5′ UTR of the expressible polynucleotide. 
     
     
         53 . The polynucleotide of  claim 50  or  51 , wherein the polyA aptamer polynucleotide further comprises at least one U/UG rich region, at least one G rich region, or both of at least one U/UG rich region and at least one G rich region. 
     
     
         54 . The polynucleotide of  claim 53 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide at least one polyA signal resides upstream of at least one U/UG rich region. 
     
     
         55 . The polynucleotide of  claim 53 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide the ligand-binding aptamer resides upstream of one, two, or more U/UG rich regions. 
     
     
         56 . The polynucleotide of  claim 3 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide at least one polyA signal resides upstream of at least one G rich region. 
     
     
         57 . The polynucleotide of  claim 53 , wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide the ligand-binding aptamer resides upstream of one, two, or more G rich regions. 
     
     
         58 . A cell comprising the polynucleotide of any one of  claims 51 - 57 . 
     
     
         59 . The cell of  claim 58 , wherein the cell is a mammalian cell. 
     
     
         60 . The cell of  claim 59 , wherein the mammalian cell is a human cell. 
     
     
         61 . The cell of any one of  claims 58 - 60 , wherein the cell resides in a human. 
     
     
         62 . A cell comprising the system of any one of  claims 1 - 32 . 
     
     
         63 . The cell of  claim 62 , wherein the cell is a mammalian cell. 
     
     
         64 . The cell of  claim 63 , wherein the mammalian cell is a human cell. 
     
     
         65 . A vector comprising the polynucleotide of any one of  claims 51 - 457 . 
     
     
         66 . The vector of  claim 65 , wherein the vector is a viral vector. 
     
     
         67 . The vector of  claim 66 , wherein the viral vector is an adenoviral vector, an adeno-associated viral vector, a retroviral vector, or a lentiviral vector. 
     
     
         68 . The vector of  claim 65 , wherein the vector is a plasmid.