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 method for producing a genetically-modified human T cell, said method comprising:
 (a) introducing into a human T cell a nucleic acid molecule comprising:
 (i) a first expression cassette comprising a nucleic acid sequence encoding a chimeric antigen receptor; 
 (ii) a second expression cassette comprising a nucleic acid sequence encoding an RNA interference molecule that is inhibitory against beta-2 microglobulin; 
 (iii) a 5′ homology arm; and 
 (iv) 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 T cell receptor (TCR) alpha constant region gene; and   (b) introducing into said human T cell an mRNA encoding an engineered nuclease having specificity for said nuclease recognition sequence, wherein said engineered nuclease is expressed in said cell;   wherein said nucleic acid molecule is introduced into said human T cell using an adeno-associated virus (AAV) vector having a serotype of AAV6,   wherein said engineered nuclease recognizes and cleaves said nuclease recognition sequence in the genome of said human T cell to generate a cleavage site,   wherein said nucleic acid molecule is inserted into the genome of said human T cell at said cleavage site by homologous recombination,   and wherein cell surface expression of beta-microglobulin on said genetically-modified human T cell is reduced by about 90% to about 95% compared to cell surface beta-2 microglobulin expression on a control human T cell that is not genetically-modified to reduce cell surface beta-2 microglobulin expression.   
     
     
         2 . The method of  claim 1 , wherein said engineered nuclease is an engineered meganuclease, a TALEN, a zinc finger nuclease (ZFN), a CRISPR/Cas, a compact TALEN, or a megaTAL. 
     
     
         3 . The method of  claim 1 , wherein said engineered nuclease is an engineered meganuclease. 
     
     
         4 . The method of  claim 1 , wherein said nuclease recognition sequence consists of SEQ ID NO: 1.