Patent Application
20230383268
The disclosure relates to the field of transposon vectors, in particular to a novel PiggyBac transposon system and its application.
It is a necessary condition for the realization of many cell therapy technologies, including immune cell therapy, to introduce exogenous genes into the target cells for stable expression to realize the transgenic transformation of the target cells. Compared with transient expression, stably expressed exogenous genes can be integrated into the genome of target cells, and can maintain stable expression for a long time even in the case of multiple cell passages or changes in culture conditions. Commonly used transgenic systems include viral-based vectors, eukaryotic expression plasmid vectors, and transposon vectors. Genetic modification of primary human T cells using non-viral vector-based approaches has proven to be extremely difficult. Therefore, worldwide, most laboratories are still using viral vector systems for transgenic modification of cells, including retroviral vectors, such as lentiviral vector systems. Although the virus vector system has been widely used, there are insurmountable problems such as complex virus preparation operations, relatively high safety risks, and high production costs.
In recent years, transposon vector systems have been increasingly used to modify immune cells for tumor immunotherapy. The earliest transposon used in mammals was the “Sleeping Beauty” transposon from fish, but it has defects such as the overproduction inhibition effect and small insert fragments (about 5 kb), etc. The application in transgenic manipulation is severely limited. PiggyBac transposon is another type of transposon system derived from Lepidoptera insects, which can carry larger fragments and can integrate into a variety of eukaryotic host cells. The PiggyBac (PB) transposon system mainly transposes through the “cut-paste” mechanism, and does not leave a footprint at the original site after transposition, and is increasingly used after modification in genome research, gene therapy, cell therapy, stem cell induction and induced differentiation, etc.
WO2019046815A1 discloses a traditional PiggyBac transposon-based binary system, which includes a vector containing PiggyBac transposase and a helper vector containing 5′ITR and 3′ITR. The PiggyBac transposon system combined with electroporation can introduce exogenous genes into T cells, NK cells and HSPCs. However, the binary system requires the PiggyBac transposase vector and the helper vector to be transfected into the cell simultaneously before transposition can occur, which is both demanding and difficult for transfection. Meanwhile, the mechanism of the PiggyBac transposition system, PiggyBac transposase inserting transposed fragments into the genome through the “cut-paste” mechanism, is reversible, and long as the expression of PiggyBac enzyme continues, the transposed fragments that have been integrated into the genome may also be re-cut, resulting in genome instability and substantially reducing the transposition efficiency. The transposition efficiency of the common PiggyBac binary transposition system in T cells is usually around 10%, which is relatively low. In one embodiment, WO2019046815A1 also describes that plasmid DNA is highly toxic to T cells, and its toxicity to T cells is related to the amount of DNA used for electroporation. The binary system undoubtedly requires a larger amount of plasmid DNA for electroporation, increases the toxicity to cells, especially T cells, and reduces the viability of T cells transfected with plasmid DNA.
CN105154473B discloses a unary PiggyBac transposon vector, which combines the PiggyBac transposase vector and the helper vector in the traditional binary PiggyBac transposition system into one vector, and through a mechanism in which the expression cassette of the PiggyBac transposase and the exogenous gene expression cassette share the same bidirectional polyA sequence in a single expression vector and the polyA in the PiggyBac transposase expression cassette is cut off and self-inactivated after integration, the continuous expression of the constitutive PiggyBac transposase was effectively reduced, and the transposition efficiency of PiggyBac transposase was improved. In the meantime, the binary system is reduced to a single vector, which greatly lowers the total amount of DNA and decreases the toxicity of exogenous DNA to T cells. However, the expression cassette of the PiggyBac transposase and that of the exogenous gene sharing the same bidirectional polyA sequence will cause mutual influence between the two expression cassettes opposite in direction, and in some types of cells, the integration efficiency of this unary transposon vector still needs further improvement.
Therefore, there is still a lack of a highly efficient unary transposon system that can efficiently integrate exogenous genes while shutting down the integration function in time.
The inventors constructed an integration system based on the PiggyBac transposon, which can mediate the efficient integration and stable expression of exogenous genes in host cells.
One embodiment of the present disclosure relates to a nucleic acid construct, which includes or consists of the following elements: a transposon 3′ terminal repeat sequence, first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence . In one or more embodiments, the nucleic acid construct further includes one or more elements selected from the group consisting of: a transposase coding sequence, a promoter controlling the expression of the transposase, a multiple cloning insertion site, an enhancer, 5′UTR, a second polyA sequence and an exogenous gene of interest.
The present disclosure further provides a nucleic acid construct, which includes the following elements: a transposon 3′ terminal repeat sequence, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a transposase encoding sequence and a promoter controlling the expression of the transposase.
In one or more embodiments, the nucleic acid construct further includes one or more elements selected from the group consisting of a multiple cloning insertion site, an enhancer, a 5′UTR, a second polyA sequence and an exogenous gene of interest.
In one or more embodiments, any one or more of the transposase coding sequence, the promoter controlling the expression of the transposase, the 5′UTR and the second polyA sequence are outside the region between the transposon 3′ terminal repeat and the transposon 5′ terminal repeat.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a promoter for controlling transposase expression, a transposase coding sequence and a second polyA sequence.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a transposase coding sequence and a promoter for controlling transposase expression.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a promoter for controlling transposase expression, a transposase coding sequence and a second polyA sequence.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a transposase coding sequence and a promoter for controlling transposase expression.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a promoter for controlling transposase expression, a transposase coding sequence and a second polyA sequence.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a transposase coding sequence, a 5′UTR and a promoter for controlling transposase expression.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a promoter for controlling transposase expression, a 5′UTR, a transposase coding sequence and a second polyA sequence.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a transposase coding sequence, a 5′UTR and a promoter for controlling transposase expression.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a promoter for controlling transposase expression, a transposase coding sequence and a second polyA sequence.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, an insulator sequence with a transcription termination function, a multiple cloning insertion site, a first polyA sequence, a transposon terminal repeat sequence, a promoter for controlling transposase expression, a 5′UTR, a transposase coding sequence and a second polyA sequence.
In one or more embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, an insulator sequence with a transcription termination function, a multiple cloning insertion site, a first polyA sequence, an enhancer, a transposon 5′ terminal repeat sequence, a promoter for controlling transposase expression, a 5′UTR, a transposase coding sequence and a second polyA sequence.
In one or more embodiments, the nucleic acid construct includes in turn: a transposon 3′ terminal repeat sequence, an enhancer, an insulator sequence with a transcription termination function, a multiple cloning insertion site, a first polyA sequence, a transposon 5′ terminal repeat sequence, a promoter for controlling transposase expression, a 5′UTR, a transposase coding sequence and a second polyA sequence.
In one or more embodiments, the multiple cloning insertion site is used to be inserted operably with the coding sequence of the exogenous gene and the promoter controlling the expression of the exogenous gene.
In one or more embodiments, the orientation of the tailing signal function of the first polyA sequence and the second polyA sequence is the same or opposite.
In one or more embodiments, the orientation of the expression cassette of the transposase is the same as or opposite to that of the exogenous gene.
In one or more embodiments, the orientation of the expression cassette of the transposase is the same as or opposite to the orientation of the sequence between the transposon 3′ terminal repeat and the transposon 5′ terminal repeat.
In one or more embodiments, each of the aforementioned elements is independently in single copy or multiple copies.
In one or more embodiments, each of the above-mentioned elements is connected directly or through a linker or restriction site.
The nucleic acid construct according to any embodiment of the present disclosure, wherein the positions of the transposon 5′ terminal repeat sequence and the transposon 3′ terminal repeat sequence can be exchanged.
In one or more embodiments, the transposon 3′ terminal repeat is a PiggyBac transposon 3′ terminal repeat. In one embodiment, the nucleotide sequence of the transposon 3′ terminal repeat sequence is as shown in SEQ ID NO:1.
In one or more embodiments, the sequence of the multiple cloning insertion site is as shown in SEQ ID NO:2.
In one or more embodiments, the first polyA sequence is as shown in SEQ ID NO:3, 13 or 16.
In one or more embodiments, the second polyA sequence is as shown in SEQ ID NO:3, 13 or 16.
In one or more embodiments, the enhancer is selected from the group consisting of: CMV enhancer sequence, SV40 enhancer, human epsilon globin 5′ HS2 enhancer, chicken beta globin gene 5′ HS4 enhancer. In one embodiment, the enhancer sequence is as shown in any one of SEQ ID NO:4, 26-28.
In one or more embodiments, an insulator sequence with a transcription termination function is as shown in SEQ ID NO:5 or 15.
In one or more embodiments, the transposon 5′ terminal repeat is a PiggyBac transposon 5′ terminal repeat. In one embodiment, the nucleotide sequence of the transposon 5′ terminal repeat sequence is as shown in SEQ ID NO:6.
In one or more embodiments, the transposase is PiggyBac transposase. In one or more embodiments, the amino acid sequence of the PiggyBac transposase is as shown in SEQ ID NO:36; and the coding sequence of the PiggyBac transposase is as shown in SEQ ID NO:7.
In one or more embodiments, the 5′UTR sequence is selected from the 5′UTR of C3 gene, ORM1 gene, HPX gene, FGA gene, AGXT gene, ASL gene, APOA2 gene, ALB gene. In one embodiment, the 5′UTR sequence is as shown in any one of SEQ ID NO:8, 17-24.
In one or more embodiments, the promoter is selected from: CMV promoter, miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, YB_TATA promoter, EF1α promoter, SV40 promoter, UbiquitinB promoter, CAG promoter, HSP70 promoter, PGK-1 promoter, β-actin promoter, TK promoter and GRP78 promoter. In one embodiment, the promoter is selected from the group consisting of miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter and YB_TATA. In one or more embodiments, the promoter sequence is as shown in any one of SEQ ID NO:9, 37-42. In one embodiment, the promoter is a miniCMV promoter, the sequence of which is as shown in SEQ ID NO:9.
In one or more embodiments, the transposase coding sequence contains or is operably linked to a single copy or multiple copies of a nuclear localization signal coding sequence. In one or more embodiments, the nuclear localization signal is a c-myc nuclear localization signal, preferably having the sequence shown in SEQ ID NO:35.
In one or more embodiments, the nucleic acid construct includes the sequence shown in SEQ ID NO:10 or 14.
In one or more embodiments, the nucleic acid construct is a recombinant vector.
In one or more embodiments, the nucleic acid construct is a recombinant cloning vector or a recombinant expression vector.
The present disclosure further provides a host cell including: (1) the nucleic acid construct described in any embodiment herein, and/or (2) the sequence between the transposon 3′ terminal repeat and the transposon 5′ terminal repeat of the nucleic acid construct described in any embodiment herein.
In one or more embodiments, the host cell is a mammalian cell.
In one or more embodiments, the host cells are selected from immune cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells.
In one or more embodiments, the immune cells are any one or more selected from the group consisting of T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T lymphocytes (CTL), dendritic cells (DC), tumor infiltrating lymphocytes (TIL), macrophages, NK T cells, and γδT cells.
The present disclosure further provides a pharmaceutical composition, including the nucleic acid construct or host cell described in any embodiment herein and a pharmaceutically acceptable excipient.
The present disclosure also provides the use of the nucleic acid construct or host cell described in any embodiment herein in the manufacture of or as a medicament, reagent or tool for integrating an exogenous gene expression cassette into a target cell genome, or for gene therapy, cell therapy, stem cell induction or differentiation.
In one or more embodiments, the target cells are mammalian cells.
In one or more embodiments, the target cells are selected from the group consisting of T cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells.
The present disclosure further provides a method for integrating an exogenous gene or its expression cassette into the genome of a cell, including introducing the nucleic acid construct described in any embodiment herein containing the exogenous gene and its promoter into the cells, and incubating the said cells under conditions in which the transposase integrates the exogenous gene or its expression cassette into the genome of the cell.
In one or more embodiments, the transposase is PiggyBac transposase.
In one or more embodiments, the introduction includes virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, electroporation, and the like. In one embodiment of the disclosure, the introduction is electroporation.
In one or more embodiments, the cells are incubated for at least three passages.
The present disclosure further provides a cell obtained by the method described herein in which exogenous genes or their expression cassettes are integrated in the genome.
It should be understood that within the scope of the present disclosure, the above-mentioned features of the present disclosure and the features described in the following (such as the examples) can be combined with each other to form other solutions.
As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Also, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. The term “comprises” and variations thereof, where these terms appear in the specification and claims, do not have a limiting meaning. Accordingly, the terms “comprises,” “comprising,” and “containing” are used interchangeably.
The term “nucleic acid construct”, defined herein as a single- or double-stranded nucleic acid molecule, preferably refers to an artificially constructed nucleic acid molecule. In one embodiment, the nucleic acid construct further includes one or more operably linked regulatory sequences, which can direct the expression of the coding sequence in a suitable host cell under compatible conditions. Expression is to be construed as including any step involved in the production of a protein or polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. The transposition system described herein is preferably a unary nucleic acid construct, that is, one nucleic acid construct can achieve high-efficiency transposition.
In the present disclosure, unless otherwise specified, the orientation of the transposase expression cassette is defined as the reverse orientation. The orientation and/or order referred to as “sequentially” in the above “sequentially including the following elements” refers to an upstream to downstream orientation. In the present disclosure, unless otherwise specified, the orientation along the above-mentioned “forward” is from upstream to downstream, and the orientation along the above-mentioned “reverse” is from downstream to upstream.
In the present disclosure, the term “expression cassette” refers to the complete set of elements required to express a gene, including promoter, gene coding sequence, and PolyA tailing signal sequence.
The term “operably inserted/linked” is defined herein as a conformation in which a regulatory sequence is located in an appropriate position relative to the coding sequence of a DNA sequence such that the regulatory sequence directs the expression of a protein or polypeptide. In the nucleic acid construct of the present disclosure, the multiple cloning site is operably inserted into one or more same or different exogenous genes and a promoter controlling the expression of the exogenous gene through DNA recombination technology, or its multiple cloning sites are replaced with one or more same or different coding sequences of the exogenous gene and a promoter controlling the expression of the exogenous gene. The “operably linked” can be achieved by means of DNA recombination, specifically, the nucleic acid construct is a recombinant nucleic acid construct.
The “exogenous gene” mentioned herein can be a nucleic acid molecule from any source, which is expressed or functions after being transposed into the host cell genome. Non-limiting examples of exogenous genes include luciferin reporter genes (e.g., green fluorescent protein, yellow fluorescent protein, etc.), luciferase genes (e.g., firefly luciferase, renilla luciferase, etc.), native functional protein genes, RNAi genes and artificial chimeric genes (such as chimeric antigen receptor genes, Fc fusion protein genes, full-length antibody genes).
The term “coding sequence” is defined herein as that portion of a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by the ribosome binding site (for prokaryotic cells) immediately upstream of the 5′ open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3′ open reading frame of the mRNA. A coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
The term “regulatory sequence” is defined herein to include all components necessary or advantageous for the expression of the peptides of the disclosure. Each regulatory sequence may be native or foreign to the nucleic acid sequence encoding the protein or polypeptide. These regulatory sequences include, but are not limited to, a leader sequence, polyA sequence, propeptide sequence, promoter, signal sequence, and transcription terminator. At a minimum, regulatory sequences will include a promoter and termination signals for transcription and translation. The control sequences with linkers may be provided for the purpose of introducing specific restriction sites for ligation of the control sequences with the coding region of the nucleic acid sequence encoding a protein or polypeptide.
The control sequence may be a suitable promoter sequence, which is a nucleic acid sequence recognized by the host cell in which the nucleic acid sequence is expressed. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein or polypeptide. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.
The regulatory sequence may also be a suitable transcription termination sequence, a sequence recognized by a host cell to terminate transcription. A termination sequence is operably linked to the 3′ end of a nucleic acid sequence encoding a protein or polypeptide. Any terminator that is functional in the host cell of choice may be used in the present disclosure.
The regulatory sequence may also be a suitable leader sequence, an untranslated region of an mRNA important for translation by the host cell. A leader sequence is operably linked to the 5′ end of a nucleic acid sequence encoding a polypeptide. Any terminator that is functional in the host cell of choice may be used in the present disclosure.
The control sequence can also be a signal peptide coding region, which codes an amino acid sequence connected to the amino terminal of the protein or polypeptide, and can guide the coded polypeptide into the secretory pathway of the cell. The 5′ end of the coding region of the nucleic acid sequence may naturally contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region of the secreted polypeptide. In one embodiment, the 5′ end of the coding region may contain a signal peptide coding region foreign to the coding sequence. It may be necessary to add a foreign signal peptide coding region when the coding sequence does not normally contain a signal peptide coding region. In one embodiment, the native signal peptide coding region can be simply replaced with a foreign signal peptide coding region to enhance polypeptide secretion. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the host cell used may be used in the present disclosure.
The nucleic acid construct of the present disclosure includes the following elements: a transposon 3′ terminal repeat sequence, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a transposase encoding sequence and a promoter controlling the expression of the transposase. The nucleic acid construct may also include one or more elements selected from the group consisting of a multiple cloning insertion site, an enhancer, a 5′UTR, and a second polyA sequence. In a particularly embodiments, the nucleic acid construct includes sequentially: a transposon 3′ terminal repeat sequence, a multiple cloning insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a transposase coding sequence, a 5′UTR and a promoter for controlling transposase expression of the transposase, as shown in
Herein, the position of the repeat sequence of the 5′ end of the transposon and the repeat sequence of the 3′ end of the transposon can be interchanged. In one embodiment, the 5′ end repeat sequence of the transpo son is the 5′ end repeat sequence of the PiggyBac transposon; the 3′ end repeat sequence of the transpo son is the 3′ end repeat sequence of the PiggyBac transposon.
Herein, the transposase is preferably PiggyBac transposase, the coding sequence of which contains or is operably connected with single copy or multiple copies of the nuclear localization signal coding sequence, to improve transposition efficiency. An exemplary PiggyBac transposase coding sequence is as shown in SEQ ID NO:7. An exemplary nuclear localization signal coding sequence is as shown in SEQ ID NO:35.
The nucleic acid constructs of the present disclosure can use polyA sequences for transposases and exogenous genes. This design can shorten the full length of the nucleic acid construct to an extent, and is helpful for the incorporation of longer exogenous genes for transposition. In one embodiment, the nucleic acid construct of the present disclosure can also use separate polyA sequences for the transposase and the exogenous gene, and the orientation of the tailing signal function of the two can be the same or opposite. This design avoids the mutual influence between two expression cassette in opposite orientations by sharing the same bidirectional polyA sequence. The polyA sequences described herein may or may not have a bidirectional transcriptional termination function. In one embodiment, the polyA sequence is independently selected from SEQ ID NO:3, 13 or 16.
The nucleic acid constructs of the present disclosure can also or further use insulator sequences for transcription termination for transposases and exogenous genes. Therefore, an insulator sequence may be incorporated at either end of any polyA sequence. In one embodiment, the insulator sequence of the disclosure is located between the 5′ terminal repeat of the transposon and the 3′ terminal repeat of the transposon. The insulator sequence described herein has the function of transcription termination, and the sequence may be any sequence known in the art that has the function of transcription termination. In one embodiment, the insulator sequence with transcription termination function is as shown in SEQ ID NO:5 or 15.
Therefore, transcription termination of the transposase can be achieved by using a polyA sequence, an insulator sequence, or a polyA sequence and an insulator sequence; transcription termination of the exogenous gene can be achieved by using a polyA sequence, an insulator sequence, or a polyA sequence and an insulator sequence. In one embodiment, any of said insulator sequence are located between said the terminal repeat sequence of the transposon and the 3′ terminal repeat sequence of the transposon.
In the nucleic acid construct of the present disclosure, a suitable promoter sequence for the transposase is a promoter sequence capable of driving high-level expression of the transposase operably linked thereto, including but not limited to the early stage of simian virus 40 (SV40) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoters, as well as human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. In one or more embodiments, the promoter of the transposase is selected from: CMV promoter, miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, YB_TATA promoter, EF1α promoter, SV40 promoter, UbiquitinB promoter, CAG promoter, HSP70 promoter, PGK-1 promoter, β-actin promoter, TK promoter and GRP78 promoter. In one embodiment, the promoter is selected from the group consisting of miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter and YB_TATA promoter. In one embodiment, the promoter is a miniCMV promoter. The miniCMV is much shorter than the CMV promoter, making the vector smaller and more conducive to the integration of larger exogenous genes. In one or more embodiments, a 5′UTR sequence is added between the miniCMV and the transposase to enhance transcription and translation. The 5′UTR sequence is as shown in any one of SEQ ID NO:8, 17-24.
A nucleic acid construct of the disclosure may contain an enhancer, which may be located at either end of any element in a nucleic acid construct described herein other than an enhancer. In one embodiment, the enhancer is located between the 3′ terminal repeat of the transposon and the 5′ terminal repeat of the transposon. In one embodiment, the enhancer is located downstream of the first polyA sequence. The enhancer sequence is as shown in any one of SEQ ID NO:4, 25-28. Herein, the nucleic acid construct may contain neither the 5′UTR sequence nor the enhancer sequence, or contain either or both, and the resulting nucleic acid construct can efficiently integrate the exogenous gene into the cell genome.
It may also be desirable to add regulatory sequences that regulate expression of the polypeptide according to the growth of the host cell. Examples of regulatory systems are those that switch gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those that enable gene amplification. In these instances, the nucleic acid sequence encoding the protein or polypeptide should be operably linked to the regulatory sequences.
In a specific embodiment containing one polyA signal sequence, the nucleic acid construct of the present disclosure sequentially includes a PiggyBac transposon 3′ terminal repeat sequence (3′ITR) (SEQ ID NO:1), multiple cloning site (SEQ ID NO :2), a polyA signal sequence (SEQ ID NO: 3, 13 or 16), an enhancer motif sequence (any in SEQ ID NO: 4, 25-28), an insulator sequence (SEQ ID NO: 5 or 15), the reverse complement sequence of a PiggyBac transposon 5′ terminal repeat (5′ITR) (SEQ ID NO:6), the reverse complement sequence of a PiggyBac transposase coding sequence (SEQ ID NO:7), the reverse complement of a 5′UTR sequence (any one of SEQ ID NO: 8, 17-24) and the reverse complement sequence of a miniCMV promoter sequence (SEQ ID NO: 9). In one or more embodiments, the nucleic acid construct of the present disclosure has the sequence shown in SEQ ID NO:10.
In a specific embodiment containing two polyA signal sequences, the nucleic acid construct of the present disclosure sequentially includes a PiggyBac transposon 3′ terminal repeat sequence (3′ITR) (SEQ ID NO:1), multiple cloning site (SEQ ID NO :2), a first polyA signal sequence (SEQ ID NO: 3, 13 or 16), an enhancer motif sequence (any in SEQ ID NO: 4, 25-28), an insulator sequence (SEQ ID NO: 5 or 15), a PiggyBac transposon 5′ terminal repeat sequence (5′ITR) (SEQ ID NO:6), a miniCMV promoter sequence (SEQ ID NO:9), a 5′UTR sequence (any one of SEQ ID NO: 8, 17-24), a PiggyBac transposase coding sequence (SEQ ID NO:7) and a second polyA signal sequence (SEQ ID NO:3, 13 or 16). In one or more embodiments, the nucleic acid construct of the present disclosure has the sequence shown in SEQ ID NO:14.
In one embodiment, the nucleic acid construct is a recombinant vector. The recombinant vector can be a recombinant cloning vector or a recombinant expression vector. The elements of the nucleic acid constructs of the present disclosure can be incorporated into many types of vectors, e.g., plasmids, phagemids, phage derivatives, animal viruses, and cosmids. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction sites and one or more selectable markers.
The vector introduced into the cells may also contain either or both of a selectable marker gene and a reporter gene for the purpose of assessing the expression of the carried gene to facilitate the identification and selection of cells from a cell population. Selectable markers can be carried on a single DNA fragment and used in co-transfection procedures. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include Flag, HA or V5. Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Expression of the reporter gene is measured at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene.
Recombinant cloning vectors can be used to provide coding sequences containing the various elements of the nucleic acid constructs of the disclosure and exogenous genes. The recombinant cloning vector can be a recombinant vector obtained by recombining each element of the nucleic acid construct of the present disclosure with pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315 series vectors.
The recombinant expression vector can be used to integrate the exogenous gene expression cassette into the genome and express it through the elements of the nucleic acid construct of the present disclosure in a suitable host cell. This vector may be suitable for replicating and integrating eukaryotic cells. A typical cloning vector contains transcriptional and translational terminators, initiation sequences and a promoter useful for regulating the expression of the desired nucleic acid sequence. The recombinant expression vector is a recombinant vector obtained by recombining the elements of the nucleic acid construct of the present disclosure with pCDNA3 series vectors, pCDNA4 series vectors, pCDNA5 series vectors, pCDNA6 series vectors, pRL series vectors, pUC57 vectors, pMAX vectors or pDC315 series vectors;
The recombinant vector (recombinant cloning vector or recombinant expression vector) can be a recombinant viral vector, including but not limited to a recombinant adenoviral vector, a recombinant adeno-associated viral vector, a recombinant retroviral vector, a recombinant herpes simplex virus vector or a recombinant vaccinia virus vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
The nucleic acid constructs described herein can generally be obtained by PCR amplification. In one embodiment, primers can be designed according to the nucleotide sequence disclosed herein, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method can be used as a template, and related sequences were amplified. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then assemble the amplified fragments in correct order. In one embodiment, the nucleic acid constructs described herein can also be directly synthesized.
The disclosure also provides a host cell including the nucleic acid construct described in any of the embodiments herein. Host cells include both mammalian cells and various cells used in the production of mammalian cells, such as E. coli cells, for providing nucleic acid constructs or vectors as described herein. In some embodiments, provided herein is a mammalian cell containing a nucleic acid construct or vector described herein, including but not limited to: T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T lymphocytes (CTL), dendritic cells (DC), tumor infiltrating lymphocytes (TIL), macrophages, NK T cells, γδT cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells.
The pharmaceutical composition of the present disclosure includes the nucleic acid construct or cell described herein and pharmaceutically acceptable excipients. In the present disclosure, “pharmaceutically acceptable excipients” are pharmaceutically or food-acceptable carriers, solvents, suspending agents or excipients used to deliver the nucleic acid constructs or cells of the present disclosure to animals or humans. Herein, pharmaceutically acceptable excipients are nontoxic to recipients of the composition at the dosages and concentrations employed. Various types of carriers or excipients commonly used in the delivery of therapeutics in therapy known in the art may be included. Exemplary excipients can be liquid or solid and include, but are not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, flavor enhancers, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers. In some embodiments, pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, Or other suitable inactive ingredients used in combination with medicinal compounds. In one embodiment, suitable adjuvants may be adjuvants commonly used in the art for administration of transposition systems or cells containing them. Examples of excipients include various lactoses, mannitols, oils such as corn oil, buffers such as PiggyBacS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethylsulfoxide, amides such as dimethylacetamide, proteins such as albumin, and detergents such as Tween 80, monosaccharides and oligopolysaccharides such as glucose, lactose, cyclodextrin and starch.
Other pharmaceutical compositions will be apparent, including formulations including the nucleic acid constructs or cells described herein in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery modes, such as liposomal vehicles, bioerodible microparticles or porous beads, and depot injections.
Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filter membranes. When the composition is lyophilized, it can be sterilized using this method before or after lyophilization and reconstitution. Compositions for parenteral administration may be in lyophilized form or stored in solution. Parenteral compositions are usually presented in containers with sterile access pores, such as intravenous solution strips or vials with a hypodermic needle pierceable stopper.
Typically, the composition contains a therapeutically effective amount of an agent described herein. A therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation and/or amelioration of a disease or condition in a subject. These effects can be realized by inserting exogenous genes with corresponding functions, and the exogenous genes with corresponding functions have functions corresponding to specific uses, such as therapeutic functions or induction functions. The therapeutically effective dose can be determined according to factors such as the patient's age, sex, disease and its severity, and other physical conditions of the patient. A therapeutically effective amount may be administered as a single dose, or may be administered in multiple doses in accordance with an effective treatment regimen. Herein, a subject or a patient generally refers to a mammal, especially a human. Exemplarily, the composition contains, for example, 0.001-50%, preferably 0.01-30%, more preferably 0.05-10% of the nucleic acid construct or cell described herein by weight.
The compositions described herein can be used in combination with other agents that have similar or corresponding functions to those performed by the exogenous genes. For example, it can be used in combination with an agent for treating the disease or condition treated by the exogenous gene. Dosages of other agents to be administered can be determined.
The dosage form of the pharmaceutical composition of the present disclosure can be varied, as long as the active ingredient can effectively reach the mammalian body, and can be made into the form of unit dosage. Dosage forms can be selected from, for example, gels, aerosols, tablets, capsules, pulvis, granules, syrups, solutions, suspensions, injections, powders, pills, controlled immediate releases, infusions, suspensions, etc. According to the types of diseases to be prevented and treated by the nucleic acid constructs or cells described herein, and convenient dosage forms can be chosen. From the standpoint of ease of preparation and storage, preferred compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. The nucleic acid constructs or cells described herein, or compositions thereof, may also be stored in sterile devices suitable for injection or infusion. The nucleic acid constructs or cells described herein, or compositions thereof, may also be stored in suitable containers and placed in kits or drug packages.
The inventors of the present disclosure found in research that the nucleic acid construct herein can efficiently realize genome integration of exogenous genes in a controllable manner. When the exogenous gene is integrated into the genome, it can effectively terminate the transcription and expression of PiggyBac transposase, and at the same time, it can function as an insulator of the exogenous gene expression cassette, reducing the impact of the integrated exogenous gene expression cassette on gene expression near the integration site. Therefore, the present disclosure relates to a method for integrating an exogenous gene or its expression cassette into the genome of a cell, including introducing the nucleic acid construct described in any embodiment herein containing the exogenous gene and its promoter into the cells, and incubating the cells under conditions in which the PiggyBac transposase integrates the exogenous gene or its expression cassette into the genome of the cell. The present disclosure also provides cells in which exogenous genes or its expression cassette is integrated in the genome obtained by the method described herein.
Methods for introducing nucleic acid constructs into cells are known in the art. Vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, vectors can be transfected into host cells by physical, chemical or biological means. Exemplary physical or chemical methods include: calcium phosphate precipitation, lipofection, microinjection, particle bombardment, microinjection, biolistic transformation, electroporation, colloidal dispersion systems, macromolecular complexes, nanocapsules, micropheres, beads, lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). Biological methods for introducing nucleic acid constructs into host cells include viral-mediated transformation, particularly retroviral vectors. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. Insertion of selected nucleic acid sequences into vectors and packaging into retroviral particles can be performed using techniques known in the art. The recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo. Reagents for virus packaging are well known in the art, for example, conventional lentiviral vector systems include pRsv-REV, pMD1g-pRRE, pMD2G and objective interfering plasmids.
The present disclosure also provides the use of the nucleic acid construct or host cell described in any embodiment herein in the preparation of or as a medicament, reagent or tool for integrating an exogenous gene expression cassette into a target cell genome, or for gene therapy, cell therapy, stem cell induction or differentiation. In one or more embodiments, the target cells are mammalian cells, including but not limited to T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T lymphocytes (CTL), dendritic cells (DC), tumor infiltrating lymphocytes (TIL), macrophages, NK T cells, γδT cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells.
Item 1, a nucleic acid construct, which includes or consists of the following elements: a transposon 3′ terminal repeat sequence, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence,
Item 2. The nucleic acid construct according to item 1, which includes the following elements: a transposon 3′ terminal repeat sequence, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5′ terminal repeat sequence, a transposase encoding sequence and a promoter controlling the expression of the transposase,
Item 3. The nucleic acid construct according to item 2, and
Item 4. The nucleic acid construct according to any one of items 1-3, and the nucleic acid construct has one or more characteristics selected from the group consisting of:
Item 5. The nucleic acid construct according to any one of items 1-3, and the nucleic acid construct has one or more characteristics selected from the group consisting of:
Item 6. The nucleic acid construct according to any one of items 1-3, characterized in that,
Item 7. A host cell including
Item 8. A pharmaceutical composition, including the nucleic acid construct described in any one of Items 1-6 or the host cell described in Item 8 and pharmaceutically acceptable excipients.
Item 9. Use of the nucleic acid construct described in any one of Items 1-6 or the host cell described in Item 7 in the preparation of or as a medicament, reagent or tool, and the medicament, reagent or tools are used for the integration of exogenous gene expression cassettes into the genome of target cells, or for gene therapy, cell therapy, stem cell induction or differentiation,
Item 10. A method for integrating an exogenous gene or its expression cassette into the genome of a cell, including introducing into said cells the nucleic acid construct described in any one of items 1-6 containing the exogenous gene and its promoter, and incubating said cells under conditions in which a transposase integrates an exogenous gene or its expression cassette into the genome of the cell, said exogenous gene and its promoter are located in the multiple cloning insertion site of said nucleic acid construct,
Embodiments of the present disclosure:
The embodiments involved in the present disclosure will be described in detail below with examples. The following examples are only used to illustrate the present disclosure, and should not be considered as limiting the scope of the present disclosure. Where techniques or conditions in the examples are not specified, experimental operation shall follow the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., “Molecular Cloning Experiment Guide” translated by Huang Peitang et al., third edition, Science Press) or follow the manufacturer's instructions. The used reagents or instruments, the manufacturers of which were not specifically indicated, are all commercially available conventional products. All cell lines used in the following examples were purchased from ATCC.
The PiggyBac transposon 3′ terminal repeat sequence (3′ITR) (SEQ ID NO: 1), multiple cloning site (SEQ ID NO: 2), bGH polyA signal sequence (SEQ ID NO: 3), enhancer motif sequence (SEQ ID NO:4), insulator sequence with transcription termination function (C2 transcription pause site, SEQ ID NO:5), the reverse complement sequence of PiggyBac transposon 5′ terminal repeat sequence (5′ITR) (SEQ ID NO:6), the reverse complement sequence of the PiggyBac transposase coding sequence (SEQ ID NO:7), the reverse complement sequence of the 5′UTR sequence (SEQ ID NO:8), and the reverse complement sequence of miniCMV promoter sequence (SEQ ID NO:9) were assembled sequentially into a long fragment (SEQ ID NO:10), and was synthesized by Shanghai Generay Biotechnology Co., Ltd. The long fragment was added with AgeI and AscI on both ends and was cloned into pUC57 (purchased from Shanghai Generay Biotech Co., Ltd.), The obtained vector was named pKB20. A schematic diagram of the plasmid is as shown in
pKB20-EGFP: the EF1A promoter sequence (SEQ ID NO:11) with NFAT motif was inserted between the XbaI and EcoRI sites in the multiple cloning site of pKB20, and the EGFP coding sequence was inserted between the EcoRI and SalI sites (SEQ ID NO:12). The obtained vector was named pKB20-EGFP. the EF1A promoter sequence with NFAT motif and the EGFP coding sequence were synthesized by Shanghai Generay Biotechnology Co., Ltd.
pKB205: the following elements were assembled into a long fragment (SEQ ID NO:14) sequentially: PiggyBac transposon 3′ terminal repeat sequence (3′ITR) (SEQ ID NO: 1), multiple cloning site (SEQ ID NO: 2), bGH polyA signal sequence (SEQ ID NO: 3), enhancer motif sequence (SEQ ID NO:4), insulator sequence with transcription termination function (C2 transcription pause site, SEQ ID NO:5), PiggyBac transposon 5′ terminal repeat sequence (5′ITR) (SEQ ID NO:6), miniCMV promoter sequence (SEQ ID NO:9), 5′UTR sequence (SEQ ID NO:8), PiggyBac transposase coding sequence (SEQ ID NO:7) and SV40 polyA signal sequence (SEQ ID NO:13). The long fragment was synthesized by Shanghai Generay Biotechnology Co., Ltd., with AgeI and AscI restriction sites added at both ends, and cloned into pUC57 (purchased From Shanghai Generay Biotechnology Co., Ltd). The obtained vector was named pKB205. A schematic diagram of the plasmid is as shown in
pKB205-EGFP: the EF1A promoter sequence (SEQ ID NO:11) with NFAT motif was inserted between the XbaI and EcoRI sites in the multiple cloning site of pKB205, and the EGFP coding sequence was inserted between the EcoRI and SalI sites (SEQ ID NO:12). The obtained vector was named pKB205-EGFP. The EF1A promoter sequence with NFAT motif and EGFP coding sequence was synthesized by Shanghai Generay Biotechnology Co., Ltd.
The following plasmids were obtained by the same method:
5×106 vigorously growing low-passage Jurkat cells were prepared, and 5 μg of pKB20-EGFP, pKB201-EGFP, pKB202-EGFP, and pKC20-EGFP were transfected into the nucleus through the Lonza2b-Nucleofector instrument (according to the manufacturer's instructions), and the cells were placed in 37° C., 5% CO2 incubator. After the cells were confluent, they were passaged at a ratio of 1:10 for further culture. On Day 7, 10 and 14 post-transfection, fluorescence imaging was performed; on day 7, and 14 (after 3 passages) post-transfection, flow cytometry assay was used to detect the change in the proportion of EGFP-positive cells, with non-transfected Jurkat cells acting as control. Cell fluorescence intensity was monitored by fluorescence microscope throughout the whole process of electrotransfection. Viable cell counts were performed on day 5, 7, 10 and 14 post-transfection to observe the effects of electrotransfection with pKB vector on the proliferation of Jurkat cells.
Cells were diluted and passaged at a ratio of 1:10, and the non-integrated plasmid were lost rapidly with cell division. After 3 passages (about 13 days), the green fluorescence-positive cells can be considered to have been stably integrated with the green fluorescent protein expression cassette. The efficiency of integration can be determined by measuring the proportion of green fluorescence-positive cells by flow cytometry.
The results are shown in
The above results show that pKB20-EGFP has a very high integration rate and positive expression rate of exogenous genes after electrotransfection into Jurkat cells. pKB201-EGFP and pKB202-EGFP also have high integration rates and positive expression rates of exogenous genes after electrotransfection into Jurkat cells, but lower than that of pKB20-EGFP. And the integration of the pKB series vectors basically has no effect on cell proliferation.
5×106 vigorously growing low-passage Jurkat cells were prepared, and 5 μg of pKB20-EGFP, pKB201-EGFP, and pKB202-EGFP were transfected into the nucleus with the Lonza2b-Nucleofector instrument (according to the manufacturers' instructions). Cells were incubated at 37° C., 5% CO2. On the 6th, 12th, 24th, 48th, 96th hour and 15th day after electrotransfection, cells were collected and RNA was extracted, and the expression level of PB transposase was quantitatively measured by RT-PCR with β-actin acting as the internal reference. The result is shown in
5×106 vigorously growing low-passage K562 cells were prepared, and 5 μg of pKB20-EGFP, pKB201-EGFP, pKB202-EGFP, and pKC20-EGFP each was tranfected into the nucleus through the Lonza2b-Nucleofector instrument (according to the manufacturer's instructions), and cells were cultured in a 37° C., 5% CO2 incubator. After the cells were confluent, they were subcultured at a ratio of 1:10. On Day 1, 7 and 10 post-transfection, fluorescence microscopic imaging was performed; on day 10 and 14 (after 3 passages) after the electrotransfection, flow cytometry assay was performed to detect the change in the proportion of EGFP-positive cells, with non-transfected K562 cells acting as control. Cell fluorescence intensity was monitored by fluorescence microscope throughout the whole process of electrotransfection. Viable cell counts were performed on day 5, 7, 10 and 14 pos-transfection to observe the effects of electrotransfection with pKB vector on the proliferation of K562 cells.
Diluting and passaging at a ratio of 1:10, the non-integrated plasmid is lost rapidly with cell division. After 3 passages (about 13 days), the green fluorescence-positive cells can be considered to have been stably integrated with the green fluorescent protein expression cassette. The efficiency of integration can be determined by measuring the proportion of green fluorescence-positive cells by flow cytometry.
The results are shown in
The above results indicated that pKB20-EGFP, pKB201-EGFP and pKB202-EGFP all had very high integration rate and positive expression rate of exogenous gene after electrotransfection into K562 cells.
Four groups of freshly isolated peripheral blood mononuclear cells (PBMC) were prepared, 5×106 cells in each group, and 5 μg of pKB20-EGFP, pKB201-EGFP, pKB202-EGFP and pKC20-EGFP each was electrotransfected into the cell nucleus (according to the manufacturer's instruction), and the cells were placed in AIM-V medium and cultured in a 37° C., 5% CO2 incubator post-tranfection. After 6 hours, cells were transferred to a 6-well plate containing 30 ng/mL anti-CD3 antibody and 3000 IU/mL IL-2 (purchased from Novoprotein), and cultured in a 37° C., 5% CO2 incubator. Upon reaching confluency, cells were diluted and passaged at a ratio of 1:10, and fluorescent microscopic imaging was performed on the cells electrotransfected with pKB20-EGFP, pKB201-EGFP, pKB202-EGFP, and pKC20-EGFP on the day 1, 7 and 10 post-transfection, respectively. Flow cytometric assay was conducted on the cells electrotransfected with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP on day 7, 10 and 14 post-transfection, and the results are shown in
1×107 vigorously growing Jurkat cells with low passage number were prepared and divided into two groups, A and B, with 5×106 cells in each group. 4 μg of pK201-PB+3 μg of pKC20-EGFP mixture and 3 μg of pK201-PB+4 μg of pKC20-EGFP mixture, were prepared, respectively, and were electrotransfected into the nucleus of cells in groups A and B via the Lonza2b-Nucleofector instrument (according to manufacturer's instruction), before they were cultured in a 37° C., 5% CO2 incubator. When the cells reached confluency, they were subcultured at a ratio of 1:10. Fluorescent microscopic imaging was conducted on the day 7, 10, 14 post-transfection, and the proportion of EGFP-positive cells was measured by flow cytometry on day 7, 10, and 14 (after 3 passages of culture) post-transfection, with non-transfected Jurkat serving as control.
The results are shown in
The above results indicated that the integration efficiency of the dual-vector transposition system expressing PB transposase and exogenous gene separately in Jurkat cells was much lower than that of the aforementioned single-vector pKB system.
5×106 freshly isolated peripheral blood mononuclear cells (PBMC) and 4 μg of pK201-PB+3 μg of pKC20-EGFP mixture were prepared, and Lonza Nucleofector-2b electroporator was used to electrotransfect the vector into the cell nucleus (according to the manufacturer's instruction). After electrotransfection, the cells were placed in AIM-V medium and cultured in a 37° C., 5% CO2 incubator. After 6 hours, the cells were transferred to a 6-well plate containing 30 ng/mL anti-CD3 antibody and 3000 IU/mL IL-2 (purchased from Novoprotein), and cultured in a 37° C., 5% CO2 incubator. Upon reaching confluency, the cells were diluted and passaged at a ratio of 1:10, and were submitted to flow cytometry assay on day 7 and 14 post-transfection.
The results are shown in
The above results indicated that the integration efficiency of the dual-vector transposition system expressing PB transposase and exogenous genes separately in primary T cells was significantly lower than that of the aforementioned single-vector pKB system.
5×106 vigorously growing low-passage Jurkat cells and 3 μg of pKB20-EGFP plasmid were prepared, and electrotransfected into the nucleus via Lonza2b-Nucleofector instrument (according to manufacturer's instruction), and cultured in a 37° C., 5% CO2 incubator. Upon reaching confluency, cells were subcultured at a ratio of 1:10. The proportion of EGFP-positive cells was measured by flow cytometry on day 7, 10 and 14 post-transfection (after 3 passages of culture), and non-transfected Jurkat cells were used as control.
The results are shown in
The above results show that when the amount of pKB20-EGFP plasmid used for electrotransfection is reduced by half on the basis of Example 2, the integration efficiency of the pKB vector system in cells remains basically unchanged, indicating that the pKB vector system of the present disclosure can achieve equivalent integration efficiency with reduced amount of DNA, which is greatly conducive to the reduction of amount of DNA used in electrotransfection, thus reducing the DNA-derived T cell toxicity as well as residual cellular plasmid DNA
According to the method of Examples 2 and 4, 5 μg of pKB20-EGFP was electrotransfected into Jurkat and K562 cells, respectively, and the cells were harvested on day 10, 14 and 20 post-transfection; according to the method of Example 8, 3 μg of pKB20-EGFP was electrotransfected into Jurkat cells per manufacturer's instructions, and the cells were harvested on day 10, 12 and 14 post-transfection, respectively. All the operations were repeated 3 times. The Taqman fluorescence probe quantitative PCR method was used to detect the amount of residual plasmid at different time points for all the above harvested cells containing the PB transposase expression cassette:
The results are shown in
The above results show that the vector of the present disclosure produces a very low residual level in host cells while fully exerting its genome integration function. In the meantime, combined with the results of Example 8, it is also shown that the pKB series vectors of the present disclosure enables the use of reduced amount of plasmid DNA while ensuring high integration efficiency after electrotransfection, thus further reducing the residual plasmid DNA in cells after electrotransfection.
According to the methods described in the above Examples 2 and 4, the pKB2003-EGFP plasmid was electrotransfected into Jurkat cells and K562 cells, respectively, and the positive proportion of the cells was detected by flow cytometry on day 7, 10, 14 post-transfection. The results are shown in
The above results show that the pKB2003 vector can efficiently integrate and express exogenous genes in cells.
According to the methods described in Example 2, 4 and 5, respectively, the amount of plasmid used in the electrotransfection was reduced to 3 μg, and the following vectors were electrotransfected into Jurkat, K562 and PBMC from healthy human blood, respectively: pKB20I1-EGFP, pKB20A1-EGFP, pKB20A1-EGFP, pKB20A2-EGFP, pKB20U1-EGFP, pKB20U2-EGFP, pKB20U3-EGFP, pKB20U4-EGFP, pKB20U5-EGFP, pKB20U6-EGFP, pKB20U7-EGFP, pKB20U8-EGFP, pKB20E1-EGFP, pKB20E2-EGFP, pKB20E3-EGFP, pKB20E4-EGFP, pKB20P1-EGFP, pKB20P2-EGFP, pKB20P3-EGFP, pKB20P4-EGFP, pKB20P5-EGFP and pKB20P6-EGFP. The positive proportion of cells were detected by flow cytometry on day 14 post-transfection. The results are shown in Table 2.
The results in Table 2 show that the above pKB series plasmid vectors with replaced regulatory element sequences can be efficiently integrated into the genomes of different cells, and the integration rates in the above types of cells are at the same level as that of pKB20-EGFP.
According to the method described in Example 5, the amount of plasmid used in electrotransfection was reduced to 3 μg, and pKB205-EGFP was electrotransfected into PBMCs from healthy human blood, and the positive proportions of cells were detected by flow cytometry on day 7 and 14 post-transfection.
The results are shown in
Two groups of Jurkat cells and K562 cells were prepared, respectively, and the pKB20-EGFP plasmid was electrotransfected to these two types of cells according to the methods described in Example 2 and 4. After the electrotransfection, cells were cultured for 14 days and collected for genomic DNA extraction. Whole-genome sequencing was conducted by Genergy Biotechnology (Shanghai) Co., Ltd., and the distribution of EGFP insertion sites in the genome was analyzed. The results are shown in
The results in
According to the methods described in the above-mentioned Example 2 and 4, the pKB20-EGFP plasmid was electrotransfected into Jurkat and K562 cells, respectively, and the amount of the plasmid was 4 μg. Cells were harvested 14 days after electrotransfection for mRNA sequencing and expression profile analysis, and the mRNA expression profiles were compared with that of non-transfected Jurkat cells and K562 cells. Two samples of K562 and Jurkat cells each were used for analysis.
The results of sequencing and analysis showed that compared with the non-transfected control cells, the mRNA expression of genes adjacent to the pKB20 integration sites after electrotransfection of pKB20-EGFP exhibited minor alteration, and the differential expression of related genes is as shown in Table 4-7. This shows that the integration of the pKB vector of the present disclosure into the genome has minor influence on cellular genome stability and gene expression profile.
The EF1A promoter sequence (SEQ ID NO: 11) with NFAT motif was inserted between the XbaI and EcoRI sites of the multiple cloning site of pKB20, and the coding sequence of HER2CAR (SEQ ID NO: 43) was inserted between the EcoRI and SalI sites. The obtained vector is named pKB20-HER2CAR. The EF1A promoter sequence with NFAT motif and the coding sequence of HER2CAR were synthesized by Shanghai Generay Biotechnology Co., Ltd.
PBMCs isolated from peripheral blood were electrotransfected with the pKB20-HER2CAR vector according to the following steps to prepare HER2-targeting CAR-T cells. The PBMCs used were purchased from AllCells and derived from the peripheral blood of healthy adults.
1) Collect the suspended cells in a 50 ml centrifuge tube, centrifuge at 1200 rmp for 3 min;
2) Discard the supernatant, resuspend cells in normal saline, centrifuge at 1200 rmp for 3 min, discard the normal saline, and repeat this step followed by cell counts;
3) Take two 1.5 ml centrifuge tubes, add 5×106 cells to each tube, and centrifuge at 1200 rmp for 3 min;
4) Discard the supernatant, use an electrotransfection kit (purchased from Lonza), add 18 μL of solution I, 82 μL of solution II, and 5 μg of pKB20 plasmid into the first tube as control, and add 5 μg of pKB20-HER2CAR plasmid into the second tube;
5) Transfer the cell suspension mixed with the plasmid in the centrifuge tube to the electroporation cuvette, put it into the electroporator, select program T020, and apply electroporation;
6) Use the micropipette in the kit to transfer the electrotroporated cell suspension into the 12-well plate (AIM-V culture medium containing 2% FBS), mix, and culture in a 37° C. 5% CO2 incubator; meanwhile, coat 2 wells of a 6-well plate with a mixture containing 5 μg/mL HER2 extracellular region antigen (SinoBiological 10004-H08H) and 5 μg/mL CD28 antibody (ThermoFisher 14-0281-82), add 1 mL to each well, and incubate the 6-well plate at 37° C.
7) After 6 hours, transfer the electroporated cells cultured in the 37° C., 5% CO2 incubator into the 6-well plate coated with HER2 extracellular domain antigen and CD28 antibody, add IL-2 to a final concentration of 100 IU/mL, and add culture medium to a final volume of 3 ml. After 4-5 days of culture, observe the growth of T cells, transfer the activated cells into AIM-V medium containing 2% FBS and continue to culture until the required cell amount is reached and collect HER2CAR-T cells; cells transfected with pKB20 empty vectors are Mock-T cells and serve as control.
1) 1×106 HER2CAR-T cells prepared in Example 15 were collected and centrifuged at 1000 rpm for 3 min;
2) The supernatant was discarded, normal saline was added to resuspend the cells, and cells were centrifuged at 1000 rpm for 3 min;
3) The supernatant was discarded, 100 μL of normal saline was added to resuspend the cells, and 1 μL of biotin-labeled HER2 antigen (purchased from KactusBiological with Product Number: HER-HM402) was added to each tube. The tubes were incubated at 4° C. for 30 minutes;
4) Appropriate amount of normal saline was added, cells were centrifuged at 1000 rpm for 3 min, washed twice, and the supernatant was discarded;
5) 100 μL of normal saline to was added to resuspend the cells. 1 μL of PE-labeled streptavidin (purchased from ThermoFisher with product number: S20982) was added, mixed well, and incubated at 4° C. for 30 min;
6) Appropriate amount of normal saline was added, cells were centrifuged at 1000 rpm for 3 min, washed twice, and the supernatant was discarded;
7) Cells were resuspended with 400 μL of normal saline, and submitted to flow cytometry.
The results are shown in
The in vitro killing activity of the HER2CAR-T cells obtained in Example 15 was detected using the real-time label-free cell function analyzer (RTCA) of ACEA Biosciences Inc., and the specific steps were as follows:
The results are shown in
Construction of pKB20-NY-ESO-1-TCR vector:
The gene DNA sequence encoding the α chain and β chain of the TCR that recognizes the NY-ESO-1 antigen peptide SLLMWITQC (HLA-*02:01) was synthesized, and the two chains were linked by the DNA sequence encoding the P2A peptide. the spliced sequence is as shown in SEQ ID NO:44. Then, the DNA sequence encoding EGFP was connected to the 3′ end of SEQ ID NO:44 by the DNA encoding the P2A peptide to obtain the NY-ESO-1-TCR gene covalently linked to the EGFP reading frame. The obtained sequence is as shown in SEQ ID NO :45. The EF1A promoter sequence (SEQ ID NO: 11) with NFAT motif was inserted between the XbaI and EcoRI sites of the multiple cloning site of pKB20, and the coding sequence of the NY-ESO-1-TCR gene (SEQ ID NO:45) covalently linked to EGFP reading frame was inserted between the EcoRI and SalI sites. The vector obtained was named pKB20-NY-ESO-1-TCR. The EF1A promoter sequence with the NFAT motif and the coding sequence of the NY-ESO-1-TCR gene covalently linked to the EGFP reading frame were synthesized by Shanghai Generay Biotechnology Co., Ltd. Preparation of NY-ESO-1 TCR-T cells:
The 6-well plate was coated with a coating solution containing 5 μg/ml anti-CD3 antibody (ThermoFisher 14-0037-82) and 5 μg/ml anti-CD28 antibody (ThermoFisher 14-0281-82) at room temperature for 2-4 hours. The coating solution was discarded, and the plate was washed with normal saline for 1-3 times, and AIM-V medium containing 2% FBS was added for use; human peripheral blood PBMC (HLA-*02:01, purchased from ALLCELLS) were recovered in a 37° C. water bath, and adherently cultured for 2-4 hours. The suspended cells that did not attach were the initial T cells. The suspended cells were collected into a 15 ml centrifuge tube, centrifuged at 1200 rmp for 3 min. The supernatant was discarded, normal saline was added, and cells were centrifuged at 1200 rmp for 3 min. The normal saline was discarded, and this step was repeated; the washed initial T cells were transferred to the antibody-coated wells filled with ready-to-use medium, cultured at 37° C., 5% CO2 for 3 to 4 days, and submitted to subsequent experiments.
Preparation of NY-ESO-1 TCR-expressing T cells by electrotransfection. The steps are as follows
1) 1×106 NY-ESO-1 TCR-T cells prepared in Example 18 was collected and centrifuged at 1000rpm for 3min;
2) The supernatant was discarded, and normal saline was added to resuspend the cells. Cells were then centrifuged at 1000 rpm for 3 min;
3) Cells were submitted to flow cytometry assay.
The results are shown in
The in vitro killing activity of the NY-ESO-1 TCR-T cells obtained in Example 18 was detected using the real-time label-free cell function analyzer (RTCA) of ACEA Biosciences Inc., and the specific steps are as follows:
The results are shown in
Comparative example 1, integration of pNB vector containing EGFP expression cassette in K562 cells The pNB vector and pNB328-EGFP were constructed according to the methods described in Example 1 on page 15 of the specification of Chinese patent CN105154473B and Example 2 on page 16 of the specification, respectively. According to the method described in Example 4 of the present application, pNB328-EGFP was used to prepare K562 stably integrated with and expressing EGFP by electrotransfection, and cells positive for EGFP expression were detected by flow cytometry on day 14 post-transfection (cultured for 3 passages).
The results are shown in
According to the method described in Example 5 of the present application, pNB328-EGFP was used to prepare primary T cells stably integrated with and expressing EGFP by electrotransfection, and cells positive for EGFP expression were detected by flow cytometry on day 14 post-transfection (cell culture 3 generations). The PBMCs used for electrotransfection are the same batch of PBMCs as that used in Example 5. 14 days after electrotransfection (cultured for 3 passages), flow cytometry was used to detecte cells positive for EGFP expression.
The results are shown in
Although specific embodiments of the present disclosure have been described in detail, based on all the teachings that have been disclosed, various modifications and substitutions can be made to details, and these changes are all within the scope of the disclosure. The full scope of the disclosure is given by the appended claims and any equivalents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011085981.6 | Oct 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/123191 | 10/12/2021 | WO |
