# US 2024/0200104 A1 · Ltr transposon compositions and methods

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- Number: US 2024/0200104 A1
- Document ID: US20240200104A1
- Status: Published application
- Kind: A1
- Application number: 18/282,644
- Filed: 2022-03-18
- Published: 2024-06-20
- Earliest claimed: 2021-03-19
- Assignees: Flagship Pioneering Innovations VI, LLC
- Applicants: Flagship Pioneering Innovations VI, LLC
- Inventors: Robert James Citorik; William Edward Salomon; Zi Jun Wang; Jacob Rosenblum Rubens; Benjamin Harris Weinberg
- Web page: https://patentgrep.com/patent/US20240200104A1

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/907: {in mammalian cells}
- C12N 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase}
- C12N 15/86: Viral vectors
- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- C12Y 207/07049: RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
- C12N 2740/10022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2740/10023: Virus like particles [VLP]
- C12N 2740/10043: viral genome or elements thereof as genetic vector
- C12N 2800/90: Vectors containing a transposable element
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Methods and compositions for altering a genome at one or more locations in a host cell, tissue, or subject are disclosed.

## Claims

### Claim 1

A system for modifying DNA comprising:
a) a template RNA comprising a first long terminal repeat (LTR), a second 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); or a DNA molecule encoding the template RNA;
b) an LTR retrotransposon structural polypeptide domain (e.g., gag, e.g., a viral capsid (CA) protein), or a nucleic acid molecule encoding the structural polypeptide domain; and
c) 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.

### Claim 2

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.

### Claim 3

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.

### Claim 4

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).

### Claim 5

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.

### Claim 6

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.

### Claim 7

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.

### Claim 8

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.