Methods and compositions for genomic integration

Abstract

Methods and composition for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed.

1 - 30 . (canceled) 
     
     
         31 . A method of expressing an exogenous therapeutic polypeptide from a genomically integrated DNA sequence of a target human cell population, the method comprising:
 (a) contacting a composition to the target human cell population, the composition comprising an isolated in vitro transcribed RNA molecule, wherein the RNA molecule comprises:
 (i) an RNA sequence that is a reverse complement of a sequence encoding the exogenous human therapeutic polypeptide, and 
 (ii) a human mobile genetic element comprising a sequence encoding a polypeptide, wherein the polypeptide encoded by the sequence of the human mobile genetic element promotes integration of the sequence encoding the exogenous therapeutic polypeptide into genomic DNA of a target human cell of the target human cell population via target-primed reverse transcription (TPRT); and 
   (b) integrating the sequence encoding the exogenous therapeutic polypeptide into the genomic DNA of the target human cell at a target site in the genomic DNA via the TPRT of the polypeptide encoded by the sequence of the human mobile genetic element, wherein   (c) after step (b) expressing only the exogenous human therapeutic polypeptide from the sequence integrated into the genomic DNA of step (b) in the target human cell, and wherein the exogenous human therapeutic polypeptide is expressed in at least 2% of the cells in the target human cell population.   
     
     
         32 . The method of  claim 31 , wherein the polypeptide encoded by the sequence of the human mobile genetic element comprises a human ORF2p or a functional fragment thereof. 
     
     
         33 . The method of  claim 31 , wherein the exogenous human therapeutic polypeptide is expressed in at least 5% of the cells in the target human cell population. 
     
     
         34 . The method of  claim 31 , wherein the exogenous human therapeutic polypeptide is expressed in at least 10% of the cells in the target human cell population. 
     
     
         35 . The method of  claim 32 , wherein the composition further comprises a sequence encoding a human ORF1p or a functional fragment thereof, wherein a first RNA molecule encodes the human ORF1p or functional fragment thereof and a second RNA molecule encodes the human ORF2p or functional fragment thereof. 
     
     
         36 . The method of  claim 35 , wherein the ratio of the first RNA molecule to the second RNA molecule in the composition is at least 2:1. 
     
     
         37 . The method of  claim 36 , wherein the ratio of the first RNA molecule to the second RNA molecule in the composition is at most 5:1. 
     
     
         38 . The method of  claim 35 , wherein the ratio of the first RNA molecule to the second RNA molecule in the composition is about 3:1. 
     
     
         39 . The method of  claim 35 , wherein the exogenous human therapeutic polypeptide is expressed in at least 10% of the cells in the target human cell population. 
     
     
         40 . The method of  claim 36 , wherein the exogenous human therapeutic polypeptide is expressed in at least 10% of the cells in the target human cell population. 
     
     
         41 . The method of  claim 35 , wherein the first RNA molecule comprises a first number of copies of a sequence encoding the human ORF1p or functional fragment thereof and the second RNA molecule comprises a second number of copies of a sequence encoding the human ORF2p or functional fragment thereof, wherein the ratio of the first number of copies to the second number of copies is at least 2:1. 
     
     
         42 . The method of  claim 32 , wherein the polypeptide encoded by the sequence of the human mobile genetic element comprises a sequence with at least 90% sequence identity to SEQ ID NO: 55. 
     
     
         43 . The method of  claim 31 , wherein the polypeptide encoded by the sequence of the human mobile genetic element comprises a C-terminal nuclear localization signal (NLS) or an N-terminal NLS. 
     
     
         44 . The method of  claim 32 , wherein the composition further comprises a sequence encoding a human ORF1p or a functional fragment thereof, wherein the human ORF1p or functional fragment thereof and the human ORF2p or functional fragment thereof are translated from different open reading frames (ORFs) of the same RNA molecule. 
     
     
         45 . The method of  claim 31 , wherein the RNA molecule is an mRNA molecule. 
     
     
         46 . The method of  claim 31 , wherein the target human cell is an immune cell selected from the group consisting of a T cell, a B cell, a myeloid cell, a monocyte, a macrophage and a dendritic cell. 
     
     
         47 . The method of  claim 31 , wherein the RNA molecule comprises homology arms complementary to the target site in the genomic DNA. 
     
     
         48 . The method of  claim 31 , wherein integrating comprises integrating the sequence encoding the exogenous therapeutic polypeptide into non-ribosomal genomic DNA of the target human cell or integrating the sequence encoding the exogenous therapeutic polypeptide into the genomic DNA at a locus that is not an rDNA locus. 
     
     
         49 . The method of  claim 31 , wherein the composition further comprises (i) one or more siRNAs and/or (ii) an RNA guide sequence or a polynucleic acid encoding the RNA guide sequence, and wherein the RNA guide sequence targets a DNA target site of the genomic DNA and the sequence encoding the exogenous therapeutic polypeptide is integrated into the genomic DNA at the DNA target site of the genomic DNA. 
     
     
         50 . The method of  claim 31 , wherein the RNA molecule has a total length of from 3 kb to 20 kb. 
     
     
         51 . The method of  claim 31 , wherein the exogenous human therapeutic polypeptide is selected from the group consisting of a ligand, an antibody, a receptor, an enzyme, a transport protein, a structural protein, a hormone, a contractile protein, a storage protein and a transcription factor. 
     
     
         52 . The method of  claim 31 , wherein the exogenous human therapeutic polypeptide is a receptor selected from the group consisting of a chimeric antigen receptor (CAR) and a T cell receptor (TCR). 
     
     
         53 . The method of  claim 31 , wherein the sequence encoding the exogenous human therapeutic polypeptide does not comprise introns. 
     
     
         54 . The method of  claim 32 , wherein the RNA molecule comprises a 5′ UTR sequence, a 3′ UTR sequence and a poly A sequence; wherein:
 (i) the 5′ UTR sequence is upstream of the sequence of the human mobile genetic element encoding a polypeptide, 
 (ii) the 3′ UTR sequence is downstream of the sequence encoding the exogenous therapeutic polypeptide; and 
 (iii) the 3′ UTR is upstream of the poly A sequence; and 
 wherein the 5′ UTR sequence, the 3′ UTR sequence or the poly A sequence comprises a binding site for the human ORF2p or a functional fragment thereof. 
 
     
     
         55 . The method of  claim 31 , wherein the RNA sequence that is a reverse complement of a sequence encoding the exogenous therapeutic polypeptide comprises an expression cassette comprising an RNA sequence that is a reverse complement of a promoter sequence, an RNA sequence that is a reverse complement of a 5′ UTR sequence, an RNA sequence that is a reverse complement of a 3′ UTR sequence and an RNA sequence that is a reverse complement of a poly A sequence; wherein:
 (i) the RNA sequence that is a reverse complement of a promoter sequence is downstream of the RNA sequence that is a reverse complement of a 5′ UTR sequence, 
 (ii) the RNA sequence that is a reverse complement of a 5′ UTR sequence is downstream of the RNA sequence that is a reverse complement of the sequence encoding an exogenous human therapeutic polypeptide, 
 (iii) the RNA sequence that is a reverse complement of a 3′ UTR sequence is upstream of the RNA sequence that is a reverse complement of the sequence encoding an exogenous human therapeutic polypeptide, and 
 (iv) the RNA sequence that is a reverse complement of a poly A sequence is upstream of the RNA sequence that is a reverse complement of a 3′ UTR sequence and downstream of the sequence of the human mobile genetic encoding a polypeptide. 
 
     
     
         56 . The method of  claim 32 , wherein the RNA molecule comprises a 5′ UTR sequence and a 3′ UTR sequence, wherein
 (a) the 5′ UTR comprises a 5′ UTR from LINE-1; and/or 
 (b) the 3′ UTR comprises a 3′ UTR from LINE-1. 
 
     
     
         57 . The method of  claim 31 , wherein the RNA molecule comprises a sequence encoding a nuclease domain, wherein the nuclease domain is a nuclease domain from megaTAL, TALEN, Cas9, Cas6, Cas7, or Cas8; wherein the nuclease domain is not from ORF2p. 
     
     
         58 . The method of  claim 32 , wherein the human ORF2p or functional fragment thereof is a modified human ORF2p that lacks endonuclease activity or has reduced endonuclease activity compared to wild type human ORF2p. 
     
     
         59 . The method of  claim 31 , wherein contacting comprises administering the composition to a human subject. 
     
     
         60 . The method of  claim 31 , wherein the RNA molecule
 (i) is formulated in a nanoparticle selected from the group consisting of a lipid nanoparticle and a polymeric nanoparticle; and/or   (ii) comprises a glycosylated RNA molecule, a circular RNA molecule or a self-replicating RNA molecule.