Lentivirus with altered integrase activity

A system for modifying DNA comprising:

• a) a template RNA comprising a DNA recognition sequence or a DNA molecule encoding the template RNA;
• b) a retroviral structural polypeptide domain;
• c) a retroviral reverse transcriptase polypeptide domain capable of reverse transcribing the template RNA, thereby producing a template DNA;
• wherein b) and c) are substantially unable to integrate the template DNA into a target DNA;
• d) a serine recombinase polypeptide domain, wherein the serine recombinase polypeptide domain binds the DNA recognition sequence and is capable of integrating the template DNA into the target DNA;
• or a nucleic acid molecule encoding the serine recombinase polypeptide domain;
• and
• e) a retroviral envelope polypeptide domain, or a nucleic acid molecule encoding the retroviral envelope polypeptide domain;
• wherein b), c), d), and e) are optionally part of the same polypeptide;
• wherein:
• (i) the template RNA further comprises a heterologous object sequence encoding a therapeutic effector;
• (ii) the DNA recognition sequence of the template DNA is capable of being recombined by the serine recombinase polypeptide domain with a cognate DNA recognition sequence in a naturally occurring human genome and/or in Genome Reference Consortium Human Build 38 (GRCh38), and wherein the target DNA comprises the cognate DNA recognition sequence;
• (iii) the serine recombinase polypeptide domain is capable of recombining the DNA recognition sequence of the template DNA with a cognate DNA recognition sequence in a naturally occurring human genome, and wherein the target DNA comprises the cognate DNA recognition sequence;
• and/or
• (iv) the retroviral reverse transcriptase polypeptide domain does not comprise a D64V mutation, or wherein the retroviral reverse transcriptase polypeptide domain comprises a D116 or E152 mutation.

A template RNA comprising:

• a) a region comprising a DNA recognition sequence that is recognized by a serine recombinase polypeptide domain;
• b) a retroviral attachment site;
• wherein:
• (i) the template RNA further comprises a heterologous object sequence encoding a therapeutic effector;
• or
• (ii) the serine recombinase polypeptide domain comprises an amino acid sequence of any of SEQ ID NOs:
• 1-12,677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

A system for modifying DNA comprising:

• a) a template RNA comprising a DNA recognition sequence, or a DNA molecule encoding the template RNA;
• b) a retroviral structural polypeptide domain, or a nucleic acid molecule encoding the retroviral structural polypeptide domain;
• c) a retroviral reverse transcriptase polypeptide domain capable of reverse transcribing the template RNA, thereby producing a template DNA, or a nucleic acid molecule encoding the retroviral reverse transcriptase polypeptide domain;
• d) a retroviral envelope polypeptide domain, or a nucleic acid molecule encoding the retroviral envelope polypeptide domain;
• wherein b) and c) together are integration-deficient or substantially unable to integrate the template DNA into a target DNA;
• wherein b), c), and d) are optionally part of the same polypeptide;
• wherein:
• (i) the DNA recognition sequence is recognized by a serine recombinase polypeptide domain that comprises an amino acid sequence of any of SEQ ID NOs:
• 1-12,677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto;
• or
• (ii) the system further comprises a serine recombinase polypeptide domain comprising an amino acid sequence of any of SEQ ID NOs:
• 1-12,677, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein the serine recombinase polypeptide domain binds the DNA recognition sequence and is capable of integrating the template DNA into the target DNA;
• or a nucleic acid molecule encoding the serine recombinase polypeptide domain.