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 human therapeutic polypeptide from a genomically integrated DNA sequence of a target human cell, the method comprising:
 (a) contacting an isolated RNA molecule to the target human cell, wherein the target human cell uptakes the isolated RNA molecule and wherein the isolated RNA molecule comprises:
 (i) an RNA sequence that is a reverse complement of the DNA sequence encoding the exogenous human therapeutic polypeptide, and 
 (ii) a human mobile genetic element comprising an RNA sequence encoding a polypeptide with target-primed reverse transcription (TPRT) activity; 
   (b) translating the human mobile genetic element to produce the polypeptide with TPRT activity;   (c) reverse transcribing the RNA sequence that is the reverse complement of the DNA sequence encoding the exogenous human therapeutic polypeptide via the TPRT activity of the polypeptide translated in step (b), thereby producing the DNA sequence encoding the exogenous human therapeutic polypeptide;   (d) integrating the DNA sequence encoding the exogenous human therapeutic polypeptide into the genomic DNA of the target human cell at a target site; and   (e) after step (d), expressing the exogenous human therapeutic polypeptide from the genomically integrated DNA sequence encoding the exogenous human therapeutic polypeptide in the target human cell.   
     
     
         32 . The method of  claim 31 , wherein the isolated RNA molecule is a synthetic or in vitro transcribed RNA molecule. 
     
     
         33 . The method of  claim 31 , wherein the isolated RNA molecule is purified. 
     
     
         34 . The method of  claim 31 , wherein the exogenous human therapeutic polypeptide is expressed in at least 2% of the cells in the target human cell population. 
     
     
         35 . 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. 
     
     
         36 . The method of  claim 31 , wherein the polypeptide encoded by the RNA sequence of the human mobile genetic element comprises a human ORF2p or a functional fragment thereof. 
     
     
         37 . The method of  claim 31 , wherein the polypeptide encoded by the RNA sequence of the human mobile genetic element comprises a nuclear localization signal (NLS). 
     
     
         38 . The method of  claim 36 , wherein the polypeptide encoded by the RNA sequence of the human mobile genetic element comprises human ORF1p or a functional fragment thereof. 
     
     
         39 . The method of  claim 38 , wherein the human ORF1p or the functional fragment thereof and the human ORF2p or the functional fragment thereof are translated from different open reading frames of the same isolated RNA molecule. 
     
     
         40 . The method of  claim 38 , wherein different RNA molecules encode the human ORF1p or functional fragment thereof and the human ORF2p or functional fragment thereof. 
     
     
         41 . The method of  claim 36 , wherein integrating comprises integrating the DNA sequence encoding the exogenous human therapeutic polypeptide into the genomic DNA at a poly T site by endonuclease activity of an endonuclease domain of the human ORF2p or a functional fragment thereof. 
     
     
         42 . The method of  claim 41 , wherein the poly T site comprises the sequence TTTTTA. 
     
     
         43 . The method of  claim 31 , wherein the isolated RNA molecule comprises homology arms complementary to the target site in the genomic DNA. 
     
     
         44 . The method of  claim 31 , wherein integrating comprises integrating the DNA sequence encoding the exogenous human therapeutic polypeptide into non-ribosomal genomic DNA of the target human cell or integrating the DNA sequence encoding the exogenous human therapeutic polypeptide into the genomic DNA at a locus that is not an rDNA locus. 
     
     
         45 . The method of  claim 31 , comprising contacting the target human cell with (i) one or more siRNAs and/or (ii) an RNA guide sequence or a polynucleic acid encoding the RNA guide sequence, and wherein the siRNA or the RNA guide sequence targets a target site of the genomic DNA and the DNA sequence encoding the exogenous human therapeutic polypeptide is integrated into the genomic DNA at the target site of the genomic DNA. 
     
     
         46 . The method of  claim 31 , wherein the isolated RNA molecule has a total length of from 3 kb to 20 kb. 
     
     
         47 . The method of  claim 31 , wherein contacting comprises administering the isolated RNA molecule to a human subject. 
     
     
         48 . 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. 
     
     
         49 . The method of  claim 48 , 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). 
     
     
         50 . The method of  claim 49 , 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), and wherein the isolated RNA molecule is formulated in a pharmaceutical composition for systemic administration to a human subject. 
     
     
         51 . The method of  claim 31 , wherein the isolated 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 human mobile genetic element, 
 (ii) the 3′ UTR sequence is downstream of the human insert RNA sequence; 
 (iii) the 3′ UTR is upstream of the poly A sequence; 
 
       wherein the 5′ UTR sequence, the 3′ UTR sequence or the poly A sequence comprises a binding site for the human ORF2p or the functional fragment thereof. 
     
     
         52 . The method of  claim 31 , wherein the RNA sequence that is a reverse complement of the DNA sequence encoding the exogenous human 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 DNA sequence encoding the 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 DNA sequence encoding the 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 human mobile genetic element. 
 
     
     
         53 . The method of  claim 31 , wherein the isolated 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. 
 
     
     
         54 . The method of  claim 31 , wherein the DNA sequence encoding the exogenous human therapeutic polypeptide does not comprise introns. 
     
     
         55 . The method of  claim 31 , wherein the isolated RNA molecule comprises a sequence encoding a nuclease domain, wherein the nuclease domain is a nuclease domain from megaTAL, TALEN, Cas9, Cash, Cas7, or Cas8; wherein the nuclease domain is not from ORF2p. 
     
     
         56 . The method of  claim 55 , wherein the isolated RNA molecule comprises a sequence encoding the nuclease domain, wherein the nuclease domain does not have nuclease activity or comprises a mutation that reduces activity of the nuclease domain compared to the nuclease domain without the mutation. 
     
     
         57 . 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. 
     
     
         58 . The method of  claim 36 , wherein the human ORF2p or the functional fragment thereof is a modified human ORF2p that lacks endonuclease activity or has reduced endonuclease activity compared to a wild type human ORF2p. 
     
     
         59 . The method of  claim 31 , wherein the isolated 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.   
     
     
         60 . The method of  claim 31 , wherein the polypeptide encoded by the RNA sequence of the human mobile genetic element comprises a sequence with at least 90% sequence identity to SEQ ID NO: 55.