Ltr transposon compositions and methods

A system for modifying DNA comprising:

• a) a template RNA comprising a first LTR, a second LTR, and a heterologous object sequence encoding a therapeutic effector, positioned between the first LTR and the second LTR and optionally a primer binding site (PBS);
• or a DNA molecule encoding the template RNA;
• b) a retroviral structural polypeptide domain (e.g., gag), or a nucleic acid molecule encoding the structural polypeptide domain;
• c) a retroviral reverse transcriptase polypeptide domain (e.g., pol) capable of reverse transcribing the template RNA, thereby producing a template DNA, or a nucleic acid molecule encoding the reverse transcriptase polypeptide domain;
• and
• the system comprises neither an envelope polypeptide domain (e.g., a retroviral envelope polypeptide domain, e.g., a lentiviral envelope polypeptide domain) nor a nucleic acid molecule encoding the envelope polypeptide domain.

A cell-free system for modifying DNA comprising:

• a) a template RNA comprising a first LTR, a second LTR, and a heterologous object sequence encoding a therapeutic effector, positioned between the first LTR and the second LTR and optionally a primer binding site (PBS);
• or a DNA molecule encoding the template RNA;
• b) a first RNA encoding a retroviral structural polypeptide domain (e.g., gag);
• c) a second RNA encoding a retroviral reverse transcriptase polypeptide domain (e.g., pol) capable of reverse transcribing the template RNA, thereby producing a template DNA, or a nucleic acid molecule encoding the reverse transcriptase polypeptide domain;
• and
• wherein the first RNA sequence and the second RNA sequence are optionally part of the same nucleic acid molecule.

A template RNA comprising:

• a first retrotransposon LTR,
• a second retrotransposon LTR,
• a heterologous object sequence encoding a therapeutic effector, positioned between the first LTR and the second LTR, and
• optionally, a primer binding site (PBS).

A method of delivering a heterologous object sequence to a target cell, comprising:

• a) introducing into the target cell (e.g., contacting the target cell with) a template RNA comprising a first LTR, a second LTR, and a heterologous object sequence encoding a therapeutic effector, positioned between the first LTR and the second LTR, and optionally a primer binding site (PBS);
• and
• b) introducing into the target cell (e.g., contacting the target cell with) an LTR retrotransposon structural polypeptide domain (e.g., gag), or a nucleic acid molecule encoding the structural polypeptide domain, and an LTR retrotransposon reverse transcriptase polypeptide domain (e.g., pol) capable of reverse transcribing the template RNA, thereby producing a template DNA, or a nucleic acid molecule encoding the reverse transcriptase polypeptide domain;
• and
• c) incubating the target cell under conditions suitable for production of the template DNA.

A method of delivering a heterologous object sequence to a target cell, comprising:

• a) introducing into the target cell (e.g., contacting the target cell with) a template RNA comprising a first LTR, a second LTR, and a heterologous object sequence encoding a therapeutic effector, positioned between the first LTR and the second LTR, and optionally a primer binding site (PBS);
• and
• b) contacting the target cell with a first RNA encoding a retroviral structural polypeptide domain (e.g., gag) and a second RNA encoding a retroviral reverse transcriptase polypeptide domain (e.g., pol) capable of reverse transcribing the template RNA, thereby producing a template DNA, wherein the first RNA and the second RNA are optionally part of the same RNA molecule, and
• c) incubating the target cell under conditions suitable for production of the template DNA.

A method of delivering a heterologous object sequence to a target cell, comprising:

• a) introducing into the target cell (e.g., contacting the target cell with) a template RNA comprising a first LTR, a second LTR, and a heterologous object sequence encoding a therapeutic effector, positioned between the first LTR and the second LTR, and optionally a primer binding site (PBS);
• and
• b) introducing into the target cell (e.g., contacting the target cell with) a retroviral structural polypeptide domain (e.g., gag), or a nucleic acid molecule encoding the structural polypeptide domain and a retroviral reverse transcriptase polypeptide domain (e.g., pol) capable of reverse transcribing the template RNA, thereby producing a template DNA, or a nucleic acid molecule encoding the reverse transcriptase polypeptide domain;
• and
• c) incubating the target cell under conditions suitable for production of the template DNA;
• wherein the method does not comprise introducing into the target cell either of an envelope polypeptide domain or a nucleic acid molecule encoding the envelope polypeptide domain.

A method of delivering a heterologous object sequence to a target cell of a patient in need thereof (e.g., in vivo or ex vivo delivery), comprising:

• a) introducing into the target cell (e.g., contacting the target cell with) a template RNA comprising a first LTR, a second LTR, and a heterologous object sequence encoding a therapeutic effector, positioned between the first LTR and the second LTR, and optionally a primer binding site (PBS);
• and
• b) contacting the target cell with a first polynucleotide encoding a retroviral structural polypeptide domain (e.g., gag), and a second polynucleotide encoding retroviral reverse transcriptase polypeptide domain (e.g., pol) capable of reverse transcribing the template RNA, thereby producing a template DNA, wherein the first polynucleotide and the second polynucleotide are optionally part of the same polynucleotide molecule;
• and
• c) incubating the target cell under conditions suitable for production of the template DNA.