Nucleic acid molecules encoding an engineered antigen receptor and an inhibitory nucleic acid molecule and methods of use thereof

Abstract

The present disclosure provides nucleic acid molecules encoding an engineered antigen receptor, such as a chimeric antigen receptor or exogenous T cell receptor, and an inhibitory nucleic acid molecule, such as an RNA interference molecule. The present disclosure further relates to nucleic acids, DNA constructs, vectors, pharmaceutical compositions, genetically-modified cells, and methods of treatment that utilize the nucleic acid molecules of the invention.

1 . A nucleic acid molecule comprising:
 (a) a first expression cassette comprising a nucleic acid sequence encoding an engineered antigen receptor;   (b) a second expression cassette comprising a nucleic acid sequence encoding an inhibitory nucleic acid molecule;   (c) a 5′ homology arm; and   (d) a 3′ homology arm;   
       wherein said 5′ homology arm and said 3′ homology arm have homology to chromosomal regions flanking a nuclease recognition sequence in a gene of interest. 
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein said inhibitory nucleic acid molecule is an RNA interference molecule. 
     
     
         3 . The nucleic acid molecule of  claim 1 , wherein said nuclease recognition sequence comprises SEQ ID NO: 1. 
     
     
         4 . The nucleic acid molecule of  claim 1 , wherein said inhibitory nucleic acid molecule is an shRNA inhibitory against beta-2 microglobulin, wherein said shRNA has a sequence comprising any one of SEQ ID NOs: 2-4. 
     
     
         5 . The nucleic acid molecule of  claim 4 , wherein said shRNA has a sequence comprising SEQ ID NO: 2. 
     
     
         6 . The nucleic acid molecule of  claim 4 , wherein said first expression cassette and said second expression cassette are in a 3′ to 5′ orientation relative to said 5′ and 3′ homology arms, and wherein said first expression cassette is 5′ upstream of said second expression cassette and wherein said first expression cassette comprises:
 a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous T cell receptor; 
 (ii) a JeT promoter which drives expression of said chimeric antigen receptor or said exogenous T cell receptor; and 
 (iii) a polyA sequence; 
 
       and wherein said second expression cassette comprises:
 (iv) a nucleic acid sequence encoding said shRNA; 
 (v) a U6 promoter which drives expression of said shRNA; and 
 (vi) a central polypurine tract and central terminator sequence (cPPT/CTS) sequence. 
 
     
     
         7 . A genetically-modified eukaryotic cell comprising said nucleic acid molecule of  claim 1 , wherein said engineered antigen receptor and said inhibitory nucleic acid molecule are expressed in said genetically-modified eukaryotic cell. 
     
     
         8 . The genetically-modified eukaryotic cell of any one of  claims 7 , wherein said inhibitory nucleic acid molecule is inhibitory against human beta-2 microglobulin. 
     
     
         9 . The genetically-modified eukaryotic cell of  claim 8 , wherein said genetically-modified eukaryotic cell is a genetically-modified human T cell, and wherein said engineered antigen receptor is a chimeric antigen receptor or an exogenous T cell receptor. 
     
     
         10 . The genetically-modified eukaryotic cell of  claim 7 , wherein said inhibitory nucleic acid molecule is inhibitory against human CD52. 
     
     
         11 . A genetically-modified eukaryotic cell comprising in its genome a nucleic acid sequence encoding an engineered antigen receptor which is expressed by said genetically-modified eukaryotic cell, wherein cell surface expression of beta-2 microglobulin on said genetically-modified eukaryotic cell is reduced by 10% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. 
     
     
         12 . A genetically-modified eukaryotic cell comprising in its genome a nucleic acid sequence encoding an engineered antigen receptor which is expressed by said genetically-modified eukaryotic cell, wherein cell surface expression of MHC class I molecules on said genetically-modified eukaryotic cell is reduced by 10% to 95% compared to cell surface expression of MHC class I molecules on a control cell. 
     
     
         13 . The genetically-modified eukaryotic cell of  claim 11 , wherein said genetically-modified eukaryotic cell is a genetically-modified human T cell. 
     
     
         14 . The genetically-modified eukaryotic cell of  claim 13 , wherein said genetically-modified human T cell expresses a chimeric antigen receptor or an exogenous T cell receptor. 
     
     
         15 . A method for producing a genetically-modified eukaryotic cell, said method comprising introducing into a cell said nucleic acid molecule of  claim 1  and:
 (a) a nucleic acid encoding an engineered nuclease having specificity for said nuclease recognition sequence, wherein said engineered nuclease is expressed in said cell; or 
 (b) an engineered nuclease protein having specificity for said nuclease recognition sequence; 
 
       wherein said engineered nuclease recognizes and cleaves said nuclease recognition sequence in the genome of said cell to generate a cleavage site, 
       and wherein said nucleic acid molecule is inserted into the genome of said cell at said cleavage site. 
     
     
         16 . The method of  claim 15 , wherein said inhibitory nucleic acid molecule is inhibitory against human beta-2 microglobulin. 
     
     
         17 . A method of using immunotherapy to treat a disease in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of said genetically-modified eukaryotic cell of  claim 9 ;
 wherein said genetically-modified eukaryotic cell is a genetically-modified human T cell expressing a chimeric antigen receptor or an exogenous T cell receptor;   and wherein cell surface expression of beta-2 microglobulin on said genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface beta-2 microglobulin expression on a control cell.   
     
     
         18 . A method of using immunotherapy to treat a disease in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of said genetically-modified eukaryotic cell of  claim 14 ;
 wherein said genetically-modified eukaryotic cell is a genetically-modified human T cell expressing a chimeric antigen receptor or an exogenous T cell receptor;   and wherein cell surface expression of WIC class I molecules on said genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to expression of WIC class I molecules on a control cell.   
     
     
         19 . A method of using immunotherapy to treat a disease in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of said genetically-modified eukaryotic cell of  claim 10 ;
 wherein said genetically-modified eukaryotic cell is a genetically-modified human T cell expressing a chimeric antigen receptor and an inhibitory nucleic acid against CD52;   and wherein cell surface expression of CD52 on said genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface CD52 expression on a control cell.   
     
     
         20 . A method for preparing an enriched population of genetically-modified eukaryotic cells comprising an engineered antigen receptor, said method comprising preparing a population of cells comprising said genetically-modified eukaryotic cell of claim  70 , and cells expressing a wild-type level of cell surface CD52, wherein cell surface expression of CD52 on said genetically-modified cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface CD52 expression on a control cell, said method comprising:
 (a) contacting said population of cells with beads conjugated to an anti-CD52-binding molecule, wherein cells expressing a wild-type level of cell surface CD52 are bound to said beads and said genetically-modified eukaryotic cell is not bound to said beads; and   (b) removing said beads from said population of cells to produce said enriched population of cells;   
       wherein said enriched population of cells is enriched for said genetically-modified eukaryotic cell.