Intracellular genomic transplant and methods of therapy
Genetically modified compositions, such as non-viral vectors and T cells, for treating cancer are disclosed. Also disclosed are the methods of making and using the genetically modified compositions in treating cancer.
1. A method of making an engineered population of primary cells comprising:
a) introducing into a population of primary cells a ribonucleic acid encoding for at least a portion of an exogenous functional T cell receptor sequence or encoding for at least a portion of an exogenous functional chimeric antigen receptor sequence;
b) reverse transcribing said ribonucleic acid, thereby producing a deoxyribonucleic acid encoding for said at least a portion of an exogenous functional T cell receptor sequence or said at least a portion of an exogenous functional chimeric antigen receptor sequence; and
c) introducing said deoxyribonucleic acid into a genomic break comprising at least a portion of a cytokine inducible SH2-containing protein (CISH) gene in a genome of at least one cell in said population of primary cells, wherein said genomic break is performed by an endonuclease and partially reduces or completely suppresses expression of said CISH gene.
2. The method of claim 1 , wherein said endonuclease is from a system selected from the group consisting of CRISPR, TALEN, Zinc Finger, transposon-based, ZEN, meganuclease, and Mega-TAL.
3. The method of claim 2 , wherein said endonuclease is from a CRISPR system.
4. The method of claim 3 , wherein said CRISPR system performs said genomic break in said genome of said at least one cell.
5. The method of claim 1 , wherein a polypeptide encoded by said exogenous functional T cell receptor sequence or a polypeptide encoded by said exogenous functional chimeric antigen receptor sequence binds a cancer neo-antigen.
6. The method of claim 1 , wherein said population of primary cells comprises cellular viability from 50% to 100% following said introduction of said ribonucleic acid to said population of primary cells.
7. The method of claim 1 , wherein said ribonucleic acid comprises a nuclear localization sequence (NLS).
8. The method of claim 1 , wherein said population of primary cells is a population of human cells.
9. The method of claim 1 , wherein said population of primary cells is a population of T cells.
10. The method of claim 1 , further comprising introducing a reverse transcriptase.
11. The method of claim 10 , wherein said reverse transcriptase is selected from the group consisting of: Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT), Moloney murine leukemia virus reverse transcriptase (M-MLV RT), Human Immunodeficiency Virus (HIV) reverse transcriptase (RT), and derivatives or combinations thereof.
12. The method of claim 10 , wherein said reverse transcriptase is functional at 37 degrees Celsius.
13. The method of claim 1 , wherein said ribonucleic acid encoding for at least a portion of an exogenous functional T cell receptor sequence or encoding for at least a portion of an exogenous functional chimeric antigen receptor sequence is a mRNA.
14. The method of claim 1 , wherein said ribonucleic acid encoding for at least a portion of an exogenous functional T cell receptor sequence or encoding for at least a portion of an exogenous functional chimeric antigen receptor sequence incorporates a unique sequence.
15. The method of claim 14 , wherein said unique sequence is selected from SEQ ID NOS: 1-4.
16. The method of claim 7 , wherein said NLS is from a BMP2-OP1-responsive gene (BORG).
17. The method of claim 1 , wherein said ribonucleic acid encoding for at least a portion of an exogenous functional T cell receptor sequence or encoding for at least a portion of an exogenous functional chimeric antigen receptor sequence comprises a secondary structure.
18. The method of claim 17 , wherein said secondary structure comprises a hairpin.
19. The method of claim 1 , wherein said reverse transcribing occurs in a nucleus of said at least one cell.
20. The method of claim 1 , wherein at least a hundred copies of said deoxyribonucleic acid are detected in a nucleus of said at least one cell.
21. A method of making an engineered cell comprising:
a) introducing a genomic break in a cytokine inducible SH2-containing protein gene in a genome of said cell, wherein said genomic break is introduced by an endonuclease;
b) introducing into said cell a deoxyribonucleic acid encoding a construct comprising:
(I) at least a portion of an exogenous functional T cell receptor sequence, or
(II) at least a portion of an exogenous functional chimeric antigen receptor sequence,
and at least one homology arm, wherein said at least one homology arm binds to a nucleic acid sequence adjacent to a genomic break at:
(i) said cytokine inducible SH2-containing protein gene; or
(ii) a T cell receptor alpha gene or a T cell receptor beta gene in a genome of said cell;
such that said exogenous functional T cell receptor sequence or said exogenous functional chimeric antigen receptor sequence is introduced at said genomic break.
22. The method of claim 21 , wherein b) comprises introducing said exogenous functional T cell receptor sequence or said exogenous functional chimeric antigen receptor sequence into the genomic break of a).
23. The method of claim 21 , wherein said construct comprises a viral deoxyribonucleic acid.
24. The method of claim 23 , wherein said viral deoxyribonucleic acid is an adeno-associated virus (AAV) deoxyribonucleic acid.
25. The method of claim 1 , wherein said deoxyribonucleic acid is introduced into said genomic break in at least 15% of cells in said population of primary cells when quantified absent a selection step.
26. The method of claim 1 , wherein said population of primary cells comprises at least about 80% viable cells as measured by fluorescence-activated cell sorting (FACS) at about 3 days after said introducing said deoxyribonucleic acid.
27. The method of claim 1 , wherein said ribonucleic acid encodes for at least a portion of an exogenous functional T cell receptor sequence.
28. The method of claim 1 , wherein said ribonucleic acid encodes for at least a portion of an exogenous functional chimeric antigen receptor sequence.
29. The method of claim 21 , wherein said construct comprises at least a portion of an exogenous functional T cell receptor sequence.
30. The method of claim 21 , wherein said deoxyribonucleic acid encodes for at least a portion of an exogenous functional chimeric antigen receptor sequence.
31. The method of claim 29 , wherein said at least a portion of an exogenous functional T cell receptor sequence comprises at least a portion of an intracellular signaling domain sequence.