Methods and compositions for genomic integration

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 a plurality of the target human cell with a composition comprising:
• (I) a nanoparticle delivery vehicle comprising:
• one or more exogenous RNA molecules, wherein the one or more exogenous RNA molecules comprises a first RNA molecule, comprising:
• (i) a first RNA sequence that is a reverse complement of a sequence encoding the exogenous human therapeutic polypeptide, and
• (ii) a second RNA sequence comprising a mobile genetic element that is not controlled by a promoter;
• comprising a sequence that encodes a human LINE-1 open reading frame 2 (ORF2p) polypeptide or fragment thereof, wherein the human LINE-1 ORF2p polypeptide or the fragment thereof has a human LINE-1 ORF2p reverse transcriptase or a functional fragment thereof and a human LINE-1 ORF2p endonuclease domain that lacks a endonuclease activity,
• and a sequence encoding a heterologous RNA guided endonuclease,
• wherein the heterologous RNA guided endonuclease comprises a CRISPR-Cas (Cas) endonuclease;
• wherein the human LINE-1 ORF2p polypeptide or the fragment thereof that has the human LINE-1 ORF2p reverse transcriptase or a functional fragment thereof and the human LINE-1 ORF2p endonuclease domain that lacks endonuclease activity is fused to the Cas endonuclease;
• and
• (II) a guide RNA;
• wherein a target human cell uptakes the one or more exogenous RNA molecules and the guide RNA,
• (b) translating the sequence of the second RNA sequence of (a)(I)(ii), thereby producing the heterologous RNA guided endonuclease and the human LINE-1 ORF2p reverse transcriptase;
• (c) (i) cutting genomic DNA of the target human cell by the heterologous RNA guided endonuclease, thereby producing a DNA cut site in the genomic DNA of the target human cell;
• and
• (ii) reverse transcribing the sequence of (a)(i) by the human LINE-1 ORF2p reverse transcriptase translated in step (b), thereby producing a DNA sequence encoding the exogenous human therapeutic polypeptide;
• (d) integrating via the human LINE-1 ORF2p mediated target primed reverse transcription (TPRT), the DNA sequence encoding the exogenous human therapeutic polypeptide into the genomic DNA of the target human cell at the DNA cut site;
• and
• (e) after step (d) expressing the exogenous human therapeutic polypeptide from the DNA sequence integrated into the genomic DNA of step (d) in the target human cell,
• wherein the exogenous human therapeutic polypeptide expresses from the DNA sequence integrated into the genomic DNA in at least 2% target human cells of the plurality of the target human cell.