Lentiviral vectors for therapeutic expression of btk in hematopoietic cells

Abstract

The present application, in some embodiments, is directed to a polynucleotide including: (a) a first nucleic acid molecule including a sequence of a human endogenous Bruton's tyrosine kinase (BTK) promoter; and (b) a second nucleic acid molecule including a codon optimized sequence encoding a BTK or a functional analog thereof. Further provided are an expression vector, a cell, and a composition, all of which include the polynucleotide of the invention, and a method of using same, such as for treating X-linked Agammaglobulinemia (XLA) in a subject in need thereof.

1 . A polynucleotide comprising:
 a. a first nucleic acid molecule comprising a sequence of between 799 to 1,533 nucleotides of a human endogenous Bruton's tyrosine kinase (BTK) promoter; and   b. a second nucleic acid molecule comprising a codon optimized sequence encoding a BTK or a functional analog thereof,   
       wherein said first nucleic acid molecule and said second nucleic acid molecule are operably linked, and wherein said codon optimized is for BTK expression in a subject, cell derived therefrom, or both. 
     
     
         2 . The polynucleotide of  claim 1 , further comprising a third nucleic acid molecule comprising a sequence of at least one expression regulatory element, wherein said third nucleic acid molecule is contiguous to said first nucleic acid molecule, optionally wherein said third nucleic acid molecule is located between said first nucleic acid molecule and said second nucleic acid molecule, and optionally wherein said expression comprises transcription, translation, or both. 
     
     
         3 .- 4 . (canceled) 
     
     
         5 . The polynucleotide of  claim 1 , wherein said regulatory element comprises a sequence derived from an untranslated region (UTR) of a human BTK transcript. 
     
     
         6 . The polynucleotide of  claim 1 , wherein said first nucleic acid molecule comprises a nucleic acid sequence set forth in any one of SEQ ID Nos: 1-7. 
     
     
         7 . The polynucleotide of  claim 1 , wherein said second nucleic acid molecule comprises a nucleic acid sequence set forth in any one of SEQ ID Nos: 8-13. 
     
     
         8 . The polynucleotide of  claim 5 , wherein said sequence derived from a UTR of a human BTK transcript comprises a nucleic acid sequence set forth in SEQ ID NO: 14. 
     
     
         9 . The polynucleotide of  claim 1 , comprising a nucleic acid sequence set forth in SEQ ID Nos: 19-30, and 32-33. 
     
     
         10 . An expression vector comprising the polynucleotide of  claim 1 , and optionally wherein said expression vector is a lentivirus-based expression vector. 
     
     
         11 . (canceled) 
     
     
         12 . A cell comprising any one of:
 a. the polynucleotide of  claim 1 ; and   b. an expression vector comprising the polynucleotide and optionally wherein said expression vector is a lentivirus-based expression vector, and optionally wherein said cell is a B cell, a myeloid cell, or a hematopoietic stem cell.   
     
     
         13 .- 15 . (canceled) 
     
     
         16 . A composition comprising any one of:
 a. the polynucleotide of  claim 1 ;   b. an expression vector comprising the polynucleotide and optionally wherein said expression vector is a lentivirus-based expression vector; and   c. a cell comprising the polynucleotide or the expression vector,   
       and an acceptable carrier. 
     
     
         17 .- 19 . (canceled) 
     
     
         20 . A method for enhancing B cell development, viability or activity in a subject in need thereof, the method comprising transducing a cell derived or obtained from said subject with the expression vector of  claim 10 , thereby enhancing B cell development, viability or activity in the subject. 
     
     
         21 . The method of  claim 20 , wherein said transducing comprises contacting said cell with said expression vector ex vivo. 
     
     
         22 . The method of  claim 20 , further comprising a step of selecting a subject in need of enhancement of B cell development, viability or activity, said selecting comprises determining any one of: B cell survival in said subject, B cell proliferation in said subject, B cell differentiation in said subject, and any combination thereof. 
     
     
         23 . The method of  claim 20 , wherein said viability or activity comprises: survival, proliferation, differentiation, or any combination thereof. 
     
     
         24 . The method of  claim 20 , further comprising a step of transplanting said cell transduced with said expression vector to said subject. 
     
     
         25 . The method of  claim 20 , wherein said cell is a B cell. 
     
     
         26 . The method of  claim 20 , wherein said cell is a myeloid cell. 
     
     
         27 . The method of  claim 20 , wherein said cell is a hematopoietic stem cell, and optionally herein said hematopoietic stem cell comprises a CD34+ hematopoietic stem cell. 
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 20 , wherein said subject is afflicted with X-linked agammaglobulinemia (XLA). 
     
     
         30 . The method of  claim 20 , wherein said subject is characterized by: reduced B cell survival rate, reduced B cell proliferation and/or differentiation rate, or any combination thereof.