Compositions and methods for treating beta-hemoglobinopathies

Abstract

The present disclosure provides expression vectors comprising at least two nucleic acid sequences, namely a nucleic acid sequence encoding an anti-HPRT RNAi, and a nucleic acid sequence encoding a gamma globin gene. In some embodiments, the viral vector is a self-inactivating lentiviral vector. In some embodiments, the gamma-globin gene is used to genetically correct sickle cell disease or β-thalassemia or to reduce symptoms thereof.

1 . A vector comprising a first expression control sequence operably linked to a first nucleic acid sequence, the first nucleic acid sequence encoding an RNA, wherein the first expression control sequence is a Pol III promoter; and a second expression control sequence operably linked to a second nucleic acid sequence, the second nucleic acid sequence encoding a gamma-globin gene, wherein the second expression control sequence is a pol II promoter. 
     
     
         2 . The vector of  claim 1 , wherein the RNA is an RNAi to knockdown HPRT. 
     
     
         3 . The vector of  claim 1 , wherein the RNA is an shRNA comprising a hairpin loop sequence of SEQ ID NO: 35. 
     
     
         4 . The vector of  claim 1 , wherein the RNA is an shRNA having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. 
     
     
         5 . The vector of  claim 1 , wherein the RNA is an shRNA having at least 95% sequence identity to a nucleic acid sequence selected from the group consist of SEQ ID NO: 67 and SEQ ID NO: 68. 
     
     
         6 . The vector of  claim 1 , wherein the RNA is an shRNA having at least 95% sequence identity to that of SEQ ID NO: 59. 
     
     
         7 . The vector of  claim 1 , wherein the Pol III promoter is 7sk. 
     
     
         8 . The vector of  claim 7 , wherein the 7sk promoter has at least 95% sequence identity to that of SEQ ID NO: 32. 
     
     
         9 . The vector of  claim 7 , wherein the 7sk promoter has SEQ ID NO: 33. 
     
     
         10 . The vector of  claim 1 , wherein the second nucleic acid encoding the gamma-globin gene has at least 90% sequence identity to that of SEQ ID NO: 55. 
     
     
         11 . The vector of  claim 1 , wherein the pol II promoter is a beta-globin promoter. 
     
     
         12 . The vector of  claim 11 , wherein the beta-globin promoter has at least 95% identity to that of SEQ ID NO: 66. 
     
     
         13 . The vector of  claim 1 , further comprising a cSH4 insulator. 
     
     
         14 . A pharmaceutical composition comprising the vector of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         15 . A host cell transduced with the vector of  claim 1 , wherein the host cell is substantially HPRT deficient and wherein the host cell expresses the gamma-globin gene. 
     
     
         16 . A vector comprising a first expression control sequence operably linked to a first nucleic acid sequence, the first nucleic acid sequence encoding an RNA, wherein the first expression control sequence is a 7sk promoter having at least 90% sequence identity to that of SEQ ID NO: 32; and a second expression control sequence operably linked to a second nucleic acid sequence, the second nucleic acid sequence encoding a globin gene, wherein the second expression control sequence is a pol II promoter. 
     
     
         17 . The vector of  claim 16 , wherein the RNA is an RNAi to knockdown HPRT. 
     
     
         18 . A vector comprising a first expression control sequence operably linked to a first nucleic acid sequence, the first nucleic acid sequence encoding a shRNA targeting hypoxanthine guanine phosphoribosyitransferase (HPRT); and a second expression control sequence operably linked to a second nucleic acid sequence, the second nucleic acid sequence encoding a therapeutic gene, wherein the therapeutic gene is selected from the group consisting of a gene encoding an enzyme adenosine deaminase, a gene encoding alpha-1-antitrypsin, a gene encoding a cystic fibrosis transmembrane conductance regulator, a gene encoding Galactose-1-phosphate uridylyltransferase, a gene encoding a clotting factor, a gene encoding a lipoprotein lipase gene, a gene encoding for glial cell line-derived neurotrophic factor (GDNF), a gene encoding interleukin-2 receptor subunit gamma (IL-2RG), a gene encoding Gp91phox, a gene encoding the Wiskott-Aldrich syndrome protein, and a gene encoding an anti-CD19 antibody. 
     
     
         19 . The vector of  claim 18 , wherein the shRNA has at least 95% identity to SEQ ID NO: 30. 
     
     
         20 . The vector of  claim 18 , wherein the shRNA has SEQ ID NO: 30. 
     
     
         21 . The vector of  claim 18 , wherein the first expression control sequence is a Pol III promoter. 
     
     
         22 . The vector of  claim 21 , wherein the Pol III promoter is a 7sk promoter.