HLA Class II Deficient Cells, HLA Class I Deficient Cells Capable of Expressing HLA Class II Proteins, and Uses Thereof

Abstract

The invention provides isolated primate cells preferably human cells that comprise a genetically engineered disruption in a human leukocyte antigen (HLA) class II-related gene, which results in deficiency in MHC class II expression and function. This invention also provides isolated cells further comprising a genetically engineered disruption in a beta-2 microglobulin (B2M) gene, which results in HLA class I/class II deficiency. Also provided are the method of using the cells for transplantation and treating a disease condition.

1 . An isolated cell comprising a genetically engineered disruption in a human leukocyte antigen (HLA) class II-related gene, wherein the cell is a primate cell. 
     
     
         2 . The cell of  claim 1 , wherein the HLA class II-related gene is selected from the group consisting of regulatory factor X-associated ankyrin-containing protein (RFXANK) (SEQ ID NO: 24-27), regulatory factor 5 (RFX5) (SEQ ID NO: 28-31), regulatory factor X-associated protein (RFXAP) (SEQ ID NO: 32-33), class II transactivator (CIITA) (SEQ ID NO: 34-35), HLA-DPA (α chain) (SEQ ID NO: 36-37), HLA-DPB (β chain) (SEQ ID NO: 38-39), HLA-DQA (SEQ ID NO: 40-41), HLA-DQB (SEQ ID NO: 42-43), HLA-DRA (SEQ ID NO: 44-45), HLA-DRB (SEQ ID NO: 46-47), HLA-DMA (SEQ ID NO: 48-49), HLA-DMB (SEQ ID NO: 50-51), HLA-DOA (SEQ ID NO: 52-53) and HLA-DOB (SEQ ID NO: 54-55). 
     
     
         3 . The cell of  claim 2 , wherein the cell comprises genetically engineered disruptions in at least two, at least three, or in all four of the HLA class II-related genes. 
     
     
         4 . The cell of  claim 1 , wherein the HLA class II-related gene is regulatory factor X-associated ankyrin-containing protein (RFXANK) (SEQ ID NO: 24-27). 
     
     
         5 . The cell of  claim 1 , wherein the cell comprises genetically engineered disruptions in all copies of the HLA class II-related gene. 
     
     
         6 . The cell of  claim 1 , wherein the cell further comprises one or more recombinant immunomodulatory genes, each capable of expressing an immunomodulatory polypeptide in the human cell. 
     
     
         7 . The cell of  claim 6 , wherein the one or more immunomodulatory genes comprise a polynucleotide capable of encoding a single chain fusion HLA class II protein, or an HLA class II protein. 
     
     
         8 . The cell of  claim 1 , wherein the cell further comprises a genetically engineered disruption in the β2-microglobulin (B2M) gene (SEQ ID NO: 1). 
     
     
         9 . An isolated cell comprising
 (a) a genetically engineered disruption in a beta-2 microglobulin (B2M) gene (SEQ ID NO: 1); and   (b) one or more polynucleotides capable of encoding a single chain fusion HLA class II protein or an HLA class II protein;
 wherein the cell is a primate cell. 
   
     
     
         10 . (canceled) 
     
     
         11 . The cell of  claim 7 , wherein the one or more immunomodulatory genes comprise a polynucleotide capable of encoding one or more single chain fusion HLA class II proteins, and wherein the one or more single chain fusion HLA class II proteins comprises at least a portion of an HLA class II gene α chain covalently linked to at least a portion of an HLA class II gene β chain, wherein the HLA class II gene is selected from the group consisting of HLA-DP (SEQ ID NO: 36-39), HLA-DQ (SEQ ID NO: 40-43), HLA-DR (SEQ ID NO: 44-47), HLA-DM (SEQ ID NO: 48-51), and HLA-DO (SEQ ID NO: 52-55). 
     
     
         12 . The cell of  claim 11 , wherein the single chain fusion HLA class II protein comprises a plurality of different single chain fusion HLA class II proteins 
     
     
         13 . The cell of  claim 11 , wherein the single chain fusion HLA class II protein comprises one or more of:
 a. at least a portion of HLA-DQ α chain (SEQ ID NO: 41) and at least a portion of HLA-DQ (3 chain (SEQ ID NO: 43); and   b. at least a portion of HLA-DQ α chain allele HLA-DQA1*01 (SEQ ID NO: 41) and at least a portion of HLA-DQ β chain allele HLA-DQB1*02 (SEQ ID NO: 43).   
     
     
         14 . (canceled) 
     
     
         15 . The cell of  claim 11  wherein the single chain fusion HLA class II protein presents a first target peptide antigen on the cell surface. 
     
     
         16 . The cell of  claim 15  wherein the first target peptide antigen is covalently linked to the single chain fusion HLA class II protein. 
     
     
         17 . (canceled) 
     
     
         18 . The cell of  claim 8 , wherein the cell further comprises a polynucleotide capable of encoding a single chain fusion HLA class I protein. 
     
     
         19 . The cell of  claim 18 , wherein the single chain fusion HLA class I protein comprises one or more of:
 a. at least a portion of B2M (SEQ ID NO: 2) covalently linked to at least a portion of an HLA class I α chain selected from the group consisting of HLA-A (SEQ ID NO: 4), HLA-B (SEQ ID NO: 6), HLA-C (SEQ ID NO: 8), HLA-E (SEQ ID NO: 10), HLA-F (SEQ ID NO: 12) and HLA-G (SEQ ID NO: 14);   b. at least a portion of B2M (SEQ ID NO: 2) covalently linked to at least a portion of HLA-A (SEQ ID NO: 3); and   c. at least a portion of B2M covalently linked to at least a portion of HLA-A0201 (SEQ ID NO: 4).   
     
     
         20 .- 27 . (canceled) 
     
     
         28 . The cell of any  claim 1 , wherein the cell is a stem cell. 
     
     
         29 - 32 . (canceled) 
     
     
         33 . The cell of  claim 1 , wherein the cell is a human cell. 
     
     
         34 . A vaccine comprising the cell of  claim 16 , wherein the vaccine is capable of eliciting in a primate an immune response specific for the first and/or second target peptide antigen. 
     
     
         35 . (canceled) 
     
     
         36 . A method of transplantation in a patient in need thereof comprising the step of administering to the patient an effective amount of the cell of  claim 1 . 
     
     
         37 - 39 . (canceled)