Transposon system, kit comprising the same, and uses thereof
Inventors
Assignees
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Publication Number
US-11345926-B2
Publication Date
2022-05-31
Expiration Date
Abstract
An in vitro method for integrating an exogenous DNA sequence into the genome of a cell using a transposon system. The transposon system includes a first vector carrying inverted repeat sequences and an exogenous DNA, and a second vector that expresses a transposases. Also provided is a kit including the transposon system for integrating an exogenous DNA into the genome of a cell. A method for treating a subject is described that includes engineering an immune cell to carry an exogenous DNA sequence using the method and/or the kit described above and administering an effective amount of the engineered immune cell to the subject.
Core Innovation
The invention relates to an in vitro transposon system for integrating an exogenous DNA sequence into the genome of a cell using a first vector and a second vector. The first vector contains a first inverted repeat having a nucleic acid sequence consisting of SEQ ID NO: 1, an exogenous DNA sequence downstream of the first inverted repeat, and a second inverted repeat downstream of the exogenous DNA sequence having a nucleic acid sequence consisting of SEQ ID NO: 2. The second vector contains a promoter operably linked to a nucleic acid encoding a transposase having the amino acid sequence of SEQ ID NO: 4.
In the cell, the transposase is expressed and catalyzes excision of the exogenous DNA sequence from the first vector and integration of the excised exogenous nucleic acid into the genome of the cell. The cell is a T cell, and the first vector is a minicircle DNA lacking prokaryotic sequences required for bacterial replication. The invention emphasizes higher transposition efficacy than that obtained from a corresponding first vector containing prokaryotic sequences required for bacterial replication.
The invention also provides a kit configured to include the transposon system and an instruction associated with a container. The kit uses a first vector with SEQ ID NO: 1 and SEQ ID NO: 2 inverted repeats flanking a cloning site for introducing the exogenous DNA sequence, and a second vector in which a promoter drives expression of a SEQ ID NO: 4 transposase.
Claims Coverage
The document identifies two independent claims: an in vitro method for integrating exogenous DNA into a T cell and a corresponding kit. Across these independent claims, the core inventive features include a SEQ ID NO: 1/SEQ ID NO: 2 inverted-repeat transposon layout, a SEQ ID NO: 4 transposase expression second vector, and the use of a minicircle DNA first vector lacking prokaryotic replication sequences to achieve higher transposition efficacy.
Inverted repeats flanking exogenous DNA sequence
A first vector containing a first inverted repeat having a nucleic acid sequence consisting of SEQ ID NO: 1, the exogenous DNA sequence downstream of the first inverted repeat, and a second inverted repeat downstream of the exogenous DNA sequence, the second inverted repeat having a nucleic acid sequence consisting of SEQ ID NO: 2.
Transposase expressed from a second vector
A second vector that contains a promoter operably linked to a nucleic acid encoding a transposase having the amino acid sequence of SEQ ID NO: 4; wherein the transposase is expressed in the T cell.
Minicircle DNA lacking prokaryotic replication sequences
The first vector is a minicircle DNA lacking prokaryotic sequences required for bacterial replication.
Excision and integration into the T cell genome with higher transposition efficacy
The transposase catalyzes excision of the exogenous DNA sequence from the first vector and integration of the excised exogenous nucleic acid into the genome of the T cell with higher transposition efficacy than that obtained from a corresponding first vector containing prokaryotic sequences required for bacterial replication.
Kit with transposon system and instruction
A kit comprising a container, a transposon system that includes the first vector and the second vector, and an instruction associated with the container and indicating how to use the transposon system, wherein the cell is a T cell and the first vector is a minicircle DNA lacking prokaryotic sequences required for bacterial replication.
The independent claims cover both the method and the kit for transposon-mediated genome integration in T cells. Both claims require the SEQ ID NO: 1/SEQ ID NO: 2 inverted-repeat first vector, a SEQ ID NO: 4 transposase second vector driven by a promoter, and the minicircle DNA design lacking prokaryotic replication sequences to achieve higher transposition efficacy.
Stated Advantages
Higher transposition efficacy than that obtained from a corresponding first vector containing prokaryotic sequences required for bacterial replication.
Documented Applications
Engineering immune cells, especially T cells, by integrating an exogenous DNA sequence into the genome for treatment of immune-related diseases.
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