Aptamers for the treatment of sickle cell disease

Abstract

The present invention provides polynucleotide aptamers that selectively bind to and inhibit polymerization of sickle hemoglobin (HbS), pharmaceutical compositions comprising the same, methods of use for diagnostics and treatment of sickle cell disease, methods of use as capture reagents, and methods of rational drug design.

1 . A polynucleotide aptamer that specifically binds sickle hemoglobin (HbS). 
     
     
         2 . The polynucleotide aptamer of  claim 1 , wherein the polynucleotide aptamer is an RNA aptamer. 
     
     
         3 . The polynucleotide aptamer of  claim 2 , wherein the polynucleotide aptamer inhibits polymerization of HbS. 
     
     
         4 . The polynucleotide aptamer of  claim 3 , wherein the polynucleotide aptamer specifically binds oxygenated HbS (oxy-HbS). 
     
     
         5 . The polynucleotide aptamer  claim 3 , wherein the polynucleotide aptamer specifically binds deoxygenated HbS (deoxy-HbS). 
     
     
         6 . The polynucleotide aptamer of  claim 3 , wherein the polynucleotide aptamer specifically binds both oxygenated HbS (oxy-HbS) and deoxygenated HbS (deoxy-HbS). 
     
     
         7 . The polynucleotide aptamer of  claim 2 , comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2-60;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2-60;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2-60;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2-60; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2-60.   
     
     
         8 . The polynucleotide aptamer of  claim 2 , comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2, 4, 31, and 37; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2, 4, 31, and 37.   
     
     
         9 . The polynucleotide aptamer of  claim 7  selected from the group consisting of:
 (a) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:2, 4, or 5, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:61; 
 (b) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:11 or 14, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:62; 
 (c) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 37, 38, 40, or 49, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:63; 
 (d) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:64; and 
 (e) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 2, 4, 5, 8, 34, or 57, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:65. 
 
     
     
         10 . The polynucleotide aptamer of  claim 2 , comprising a consensus sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS:61, 62, 63, 64, and 65. 
     
     
         11 . A polynucleotide aptamer of  claim 1 , wherein the polynucleotide aptamer is modified to increase its circulating half-life after administration to a subject. 
     
     
         12 . A polynucleotide encoding the polynucleotide aptamer of  claim 1 . 
     
     
         13 . A vector comprising the polynucleotide of  claim 12 . 
     
     
         14 . A cell comprising the polynucleotide aptamer of  claim 1 . 
     
     
         15 . A method of treating or preventing sickle cell disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polynucleotide aptamer that specifically binds sickle hemoglobin (HbS), wherein the polynucleotide aptamer inhibits polymerization of HbS. 
     
     
         16 . The method of  claim 15 , wherein the polynucleotide aptamer is an RNA aptamer. 
     
     
         17 . The method of  claim 16 , wherein the polynucleotide aptamer specifically binds oxygenated HbS (oxy-HbS). 
     
     
         18 . The method of  claim 16 , wherein the polynucleotide aptamer specifically binds deoxygenated HbS (deoxy-HbS). 
     
     
         19 . The method of  claim 16 , wherein the polynucleotide aptamer specifically binds both oxygenated HbS (oxy-HbS) and deoxygenated HbS (deoxy-HbS). 
     
     
         20 . The method of  claim 16 , wherein the polynucleotide aptamer comprises a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2-60;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2-60;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2-60;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2-60; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2-60.   
     
     
         21 . The method of  claim 16 , wherein the polynucleotide aptamer comprises a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2, 4, 31, and 37; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2, 4, 31, and 37.   
     
     
         22 . The method of either of  claim 20 , wherein the polynucleotide aptamer is selected from the group consisting of:
 (a) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:2, 4, or 5, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:61;   (b) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:11 or 14, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:62;   (c) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 37, 38, 40, or 49, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:63;   (d) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:64; and   (e) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 2, 4, 5, 8, 34, or 57, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:65.   
     
     
         23 . The method of  claim 16 , wherein the polynucleotide aptamer comprises a consensus sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS:61, 62, 63, 64, and 65. 
     
     
         24 . The method of  claim 16 , wherein the polynucleotide aptamer is modified to increase its circulating half-life after administration to the subject. 
     
     
         25 . The method of  claim 16 , wherein the polynucleotide aptamer is in a pharmaceutically acceptable carrier. 
     
     
         26 . The method of  claim 16 , wherein the sickle cell disease is sickle cell anemia. 
     
     
         27 . The method of  claim 16 , further comprising contacting the polynucleotide aptamer with an antidote. 
     
     
         28 . The method of  claim 27 , wherein the antidote is an oligonucleotide comprising a sequence complementary to at least a portion of the polynucleotide aptamer. 
     
     
         29 . A method for diagnosing or predicting a sickle cell disease in a subject having or at risk of developing a sickle cell disease or at risk of passing it on to offspring, the method comprising:
 (a) obtaining a biological sample from the subject;   (b) contacting the biological sample with a polynucleotide aptamer that specifically binds to HbS; and   (c) detecting binding of the polynucleotide aptamer with HbS in the biological sample;   
       wherein detection of binding of the polynucleotide aptamer with HbS in the biological sample is indicative of the subject having or at risk of developing a sickle cell disease or at risk of passing it on to offspring. 
     
     
         30 . The method of  claim 29 , wherein the polynucleotide aptamer is an RNA aptamer. 
     
     
         31 . The method of  claim 30 , wherein the polynucleotide aptamer specifically binds oxygenated HbS (oxy-HbS). 
     
     
         32 . The method of  claim 30 , wherein the polynucleotide aptamer specifically binds deoxygenated HbS (deoxy-HbS). 
     
     
         33 . The method of  claim 30 , wherein the polynucleotide aptamer specifically binds both oxygenated HbS (oxy-HbS) and deoxygenated HbS (deoxy-HbS). 
     
     
         34 . The method of  claim 30 , wherein the polynucleotide aptamer comprises a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2-60;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2-60;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2-60;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2-60; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2-60.   
     
     
         35 . The method of  claim 30 , wherein the polynucleotide aptamer comprises a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2, 4, 31, and 37; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2, 4, 31, and 37.   
     
     
         36 . The method of  claim 34 , wherein the polynucleotide aptamer is selected from the group consisting of:
 (a) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:2, 4, or 5, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:61;   (b) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:11 or 14, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:62;   (c) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 37, 38, 40, or 49, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:63;   (d) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:64; and   (e) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 2, 4, 5, 8, 34, or 57, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:65.   
     
     
         37 . The method of  claim 30 , wherein the polynucleotide aptamer comprises a consensus sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS:61, 62, 63, 64, and 65. 
     
     
         38 . The method of  claim 30 , wherein the sickle cell disease is sickle cell anemia. 
     
     
         39 . The method of  claim 30 , wherein the biological sample comprises whole blood, hemocytes, serum, or plasma. 
     
     
         40 . The methods of  claim 30 , wherein the polynucleotide aptamer is labeled for detection with a fluorescent, luminescent, phosphorescent, radioactive, or colorimetric compound. 
     
     
         41 . A method of purifying hemoglobin from a biological sample, the method comprising:
 (a) providing a biological sample containing hemoglobin;   (b) contacting the biological sample with a polynucleotide aptamer that specifically binds to HbS under conditions effective to bind hemoglobin to the aptamer; and   (c) recovering the hemoglobin bound to the aptamer.   
     
     
         42 . The method of  claim 41 , wherein the step of contacting the biological sample with the polynucleotide aptamer that specifically binds to HbS under conditions effective to bind hemoglobin to the polynucleotide aptamer comprises providing a solid support comprising an aptamer that specifically binds to HbS immobilized onto the solid support through a spacer. 
     
     
         43 . The method of  claim 41 , wherein the polynucleotide aptamer is an RNA aptamer. 
     
     
         44 . The method of  claim 43 , wherein the polynucleotide aptamer specifically binds oxygenated HbS (oxy-HbS). 
     
     
         45 . The method of  claim 43 , wherein the polynucleotide aptamer specifically binds deoxygenated HbS (deoxy-HbS). 
     
     
         46 . The method of  claim 43 , wherein the polynucleotide aptamer specifically binds both oxygenated HbS (oxy-HbS) and deoxygenated HbS (deoxy-HbS). 
     
     
         47 . The method of  claim 43 , wherein the polynucleotide aptamer comprises a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2-60;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2-60;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2-60;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2-60; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2-60.   
     
     
         48 . The method of  claim 43 , wherein the polynucleotide aptamer comprises a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2, 4, 31, and 37; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2, 4, 31, and 37.   
     
     
         49 . The method of  claim 47 , wherein the polynucleotide aptamer is selected from the group consisting of:
 (a) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:2, 4, or 5, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:61;   (b) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:11 or 14, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:62;   (c) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 37, 38, 40, or 49, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:63;   (d) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:64; and   (e) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 2, 4, 5, 8, 34, or 57, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:65.   
     
     
         50 . The method of  claim 43 , wherein the polynucleotide aptamer comprises a consensus sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS:61, 62, 63, 64, and 65. 
     
     
         51 . The method of  claim 43 , wherein the polynucleotide aptamer is modified to enable covalent immobilization or to prevent enzymatic degradation. 
     
     
         52 . The method of  claim 43 , wherein the biological sample comprises whole blood, hemocytes, serum, or plasma. 
     
     
         53 . A method of using a three-dimensional structure of a polynucleotide aptamer that specifically binds to HbS and inhibits polymerization of HbS in a drug screening assay comprising:
 (a) selecting a potential drug by performing rational drug design with the three-dimensional structure of the polynucleotide aptamer that specifically binds to HbS and inhibits polymerization of HbS determined from one or more sets of atomic coordinates; wherein said selecting is performed in conjunction with computer modeling;   (b) contacting the potential drug with HbS;   (c) detecting the binding of the potential drug with the HbS; and   (d) detecting the inhibition of polymerization of HbS by the potential drug;   
       wherein a potential drug is selected as a drug if the potential drug binds to HbS and inhibits polymerization of HbS. 
     
     
         54 . The method of  claim 53 , wherein the polynucleotide aptamer is an RNA aptamer. 
     
     
         55 . The method of  claim 54 , wherein the polynucleotide aptamer specifically binds oxygenated HbS (oxy-HbS). 
     
     
         56 . The method of  claim 54 , wherein the polynucleotide aptamer specifically binds deoxygenated HbS (deoxy-HbS). 
     
     
         57 . The method of  claim 54 , wherein the polynucleotide aptamer specifically binds both oxygenated HbS (oxy-HbS) and deoxygenated HbS (deoxy-HbS). 
     
     
         58 . The method of  claim 54 , wherein the polynucleotide aptamer comprises a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2-60;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2-60;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2-60;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2-60; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2-60.   
     
     
         59 . The method of  claim 54 , wherein the polynucleotide aptamer comprises a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence at least 80% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (b) a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (c) a nucleotide sequence at least 95% identical to any one of SEQ ID NOS:2, 4, 31, and 37;   (d) a nucleotide sequence at least 99% identical to any one of SEQ ID NOS:2, 4, 31, and 37; and   (e) the nucleotide sequence of any one of SEQ ID NOS:2, 4, 31, and 37.   
     
     
         60 . The method of  claim 58 , wherein the polynucleotide aptamer is selected from the group consisting of:
 (a) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:2, 4, or 5, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:61;   (b) wherein when the polynucleotide aptamer is any one of SEQ ID NOS:11 or 14, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:62;   (c) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 37, 38, 40, or 49, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:63;   (d) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 31, 37, 38, 40, 42, 45, 46, 47, 48, 49, 53, 56, 59 or 60, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:64; and   (e) wherein when the polynucleotide aptamer is any one of SEQ ID NOS: 2, 4, 5, 8, 34, or 57, the polynucleotide aptamer further comprises a consensus sequence consisting of SEQ ID NO:65.   
     
     
         61 . The method of  claim 54 , wherein the polynucleotide aptamer comprises a consensus sequence consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 61, 62, 63, 64, and 65.