CN109312361A

CN109312361A - Transposon system and application method

Info

Publication number: CN109312361A
Application number: CN201780025816.1A
Authority: CN
Inventors: D·夏洛克; D·赫尔曼森; E·奥斯特泰格
Original assignee: Posey Ida Therapeutics Ltd By Share Ltd
Current assignee: Posey Ida Therapeutics Ltd By Share Ltd
Priority date: 2016-02-26
Filing date: 2017-02-24
Publication date: 2019-02-05
Also published as: JP2019506177A; AU2017224111A1; RU2018133691A; WO2017147538A1; SG11201807134RA; EP3420090A1; KR20180131545A; CA3015642A1; BR112018067536A2; MX2018010288A; IL261343A; RU2018133691A3

Abstract

Disclosed herein is the compositions and method for carrying out ex vivo genetic modification to immunocyte, it includes delivering to the immunocyte: (a) nucleic acid or amino acid sequence of the sequence comprising encoding transposase, and the DNA sequence dna (b) generated comprising the recombination and non-natural that encode the DNA sequence dna of transposons.

Description

Transposon System and application method

Related application

This application claims the priority for the U.S. Provisional Patent Application No. 62/300,387 submitted for 26th for 2 months in 2016, The full content of this article is included in herein for all purposes by reference.

Sequence table reference

It is created in 24 days, size 26KB 2 months 2017, with the submission of " POTH-007001WO_SeqList.txt " title Text document content be included in by way of quoting its full text herein.

Technical field

The present invention relates to the composition modified for target gene and methods.

Background technique

Using based on non-virus carrier gene transfer delivery system carry out non-transformed primary human T lymphocyte it is in vitro Genetic modification is extremely difficult.Therefore, most of team generally use the transduction based on viral vectors, such as retrovirus, packet Include slow virus.After tested, and the liposome including antibody target, nano particle, aptamer siRNA are chimeric for a variety of non-viral methods Body, electroporation, nuclear transfection, lipofection and peptide transduction.Generally, the transfection efficiency that these modes obtain is low, leads to cell toxicant Property, or lack experiment flux.

Application of the plasmid vector for genetic modification Human Lymphocytes is limited to the low of currently available plasmid transfection system Efficiency and most plasmid transfection reagents are for toxicity possessed by these cells.For carrying out non-viral gene in immunocyte The method of modification has long-term and unsatisfied needs.

Summary of the invention

Compared with the viral transduction of immunocyte (such as T lymphocyte), by DNA transposons (such as piggyBac and Sleeping beauty transposon stand) delivering transgenosis provide following aspect benefit: ease for use, delivering it is much bigger by the ability of loading, reality The speed and production cost of existing clinical application.Specifically, piggyBac DNA transposons provides additional benefit: transgenosis For a long time, high level and stable expression, and compare significant less mutagenesis, non-carcinogenic for retrovirus and completely may be used Inverse property.The previous trial of transgene delivery to T cell is being generated by commericially feasible product or manufacturing method side using DNA transposons Face is unsuccessful, because prior method is inefficient.For example, using DNA transposons by the previous of transgene delivery to T cell Method display is inefficient, and causes to need extended in vitro amplification.Other people carried out to solve this problem it is previous not at Function is attempted to be conceived to the amount for increasing the DNA transposons delivered to immunocyte, this has been a kind of for nonimmune cell Possible strategy.The invention shows the amount for increasing DNA transposons can make the efficiency in immunocyte even worse, because of this meeting Increase the toxicity that DNA is mediated.The problem is not solved, unusually, the method for the present invention reduces the DNA's for being delivered to immunocyte Amount.Using the method for the present invention, data provided herein not only show that the amount for reducing the DNA transposons for importing cell can increase work Power, and showing this method increase the percentage of the cell with transposition event obtains feasible business method and feasible Commercial product.Therefore, the method for the present invention demonstrates other people and fails the success of item.

The present invention is provided to the non-viral methods of ex vivo genetic modification are carried out to immunocyte comprising be immunized to described Cell delivering: (a) nucleic acid or amino acid sequence of the sequence comprising encoding transposase, and the DNA sequence (b) comprising coding transposons The recombination of column and the DNA sequence dna of non-natural generation.In some embodiments, the method also includes following steps: with one kind Or cytokine profiles stimulate immunocyte.

In the certain embodiments of methods described herein, the sequence of the encoding transposase is mRNA sequence.The coding The mRNA sequence of transposase can generate in vitro.

In the certain embodiments of methods described herein, the sequence of the encoding transposase is DNA sequence dna.The coding turns The DNA sequence dna of seat enzyme can generate in vitro.The DNA sequence dna can be cDNA sequence.

In the certain embodiments of methods described herein, the sequence of the encoding transposase is amino acid sequence.The volume The amino acid sequence of code transposase can generate in vitro.Protein sPBo can be delivered after with transposons DNA preincubate.

In the certain embodiments of methods described herein, delivery step includes the electroporation or nuclear transfection of immunocyte.

In the certain embodiments of methods described herein, with one or more cell factors stimulate immunocyte the step of It is carried out after delivery step.Or it or in addition, in some embodiments, is immunized with the stimulation of one or more cell factors thin The step of born of the same parents, carries out before delivery step.In some embodiments, one or more cell factors include IL-2, IL-21, IL-7 and/or IL-15.

In the certain embodiments of methods described herein, the immunocyte is the immunocyte of Autologous.It is described Immunocyte can be the immunocyte of human immune cells and/or Autologous.The immunocyte may originate from non-self same The source in source, including but not limited to: primary cell, the cell of culture or cell line, embryo or adult stem cell, induction type multipotency Stem cell or transdifferentiated cells.The immunocyte can be through previous genetic modification or from genetically modified cell or cell System.The cell or cell that the immunocyte can be modified or be may originate to have modified to inhibit one or more apoptosis pathway System.The immunocyte can be modified or be may originate to have modified (to control the situation for example, being related to adoptive cellular transfer Treat) in most of receptors' " generally " be allogeneic cell or cell line.

In the certain embodiments of methods described herein, the immunocyte is the immunocyte of activation.

In the certain embodiments of methods described herein, the immunocyte is tranquillization immunocyte.

In the certain embodiments of methods described herein, the immunocyte is T- lymphocyte.In certain embodiment party In formula, T- lymphocyte is the T- lymphocyte of activation.In some embodiments, T- lymphocyte is that tranquillization T- lymph is thin Born of the same parents.

In the certain embodiments of methods described herein, the immunocyte is natural kill (NK) cell.

In the certain embodiments of methods described herein, the immunocyte is cytokine-induced killer cell (CIK) Cell.

In the certain embodiments of methods described herein, the immunocyte is natural killer T (NKT) cell.

In the certain embodiments of methods described herein, the immunocyte is isolated from or is originated from the mankind.

In the certain embodiments of methods described herein, the immunocyte is isolated from or is originated from non-human mammal. In some embodiments, non-human mammal is rodent, rabbit, cat, dog, pig, horse, milk cow or camel.In certain embodiment party In formula, the immunocyte is isolated from or is originated from non-human primates.

In the certain embodiments of methods described herein, transposase is Super piggyBac^TM(sPBo) transposase. Super piggyBac (PB) transposase may include amino acid sequence identical with following sequence at least 75% or be made from it:

MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQN VIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEII SEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFL IRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKIL MMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVR SNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDT LDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF(SEQ ID NO:1)。

In the certain embodiments of methods described herein, transposase is sleeping beauty (Sleeping Beauty) transposase (see, for example, U.S. Patent number 9,228,180, content is included in herein by way of quoting its full text).In certain implementations In mode, sleeping beauty's transposase is high activity sleeping beauty's SB100X transposase.In some embodiments, sleeping beauty's transposase packet Containing amino acid sequence identical with following sequence at least 75%: MGKSKEISQDLRKKIVDLHKSGSSLGAISKRLKVPRSSV QTIVRKYKHHGTTQPSYRSGRRRYLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGR SARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGS IMLWGCFAAGGTGALHKIDGIMRKENYVDILKQHLKTSVRKLKLGRKWVFQMDNDPKHTSKVVAKWLKDNKVKVLE WPSQSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPTYCGKLVEGYPKRLTQVKQFKGNATKY(SEQ ID NO:2).Certain embodiments (including wherein sleeping beauty's transposase is those of high activity sleeping beauty's SB100X transposase) In, sleeping beauty's transposase includes amino acid sequence identical with following sequence at least 75%:

MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRSGRRRYLSPRD ERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRT FWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMDAVQYVD ILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTN LTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY(SEQ ID NO:3)。

In the certain embodiments of methods described herein, recombination and non-natural comprising encoding the DNA sequence dna of transposons The DNA sequence dna of generation can be ring-shaped.As non-limiting example, the DNA sequence dna for encoding transposons can be plasmid vector. As non-limiting example, the DNA sequence dna for encoding transposons can be small circular DNA carrier.

In the certain embodiments of methods described herein, the DNA sequence dna of the recombination of transposons and non-natural generation is encoded It can be linear.The DNA sequence dna of the linear recombination and non-natural generation that encode transposons can generate in vitro.Of the invention is linear The DNA sequence dna that recombination and non-natural generate can be the product of the restriction digest of annular DNA.In some embodiments, Annular DNA is plasmid vector or small circular DNA carrier.The DNA sequence dna that linear recombination and non-natural of the invention generates can be poly- Polymerase chain reacts the product of (PCR).The DNA sequence dna that linear recombination and non-natural of the invention generates can be double-strand doggybone^TMDNA sequence dna.Doggybone of the invention^TMDNA sequence dna can be generated by enzyme process, and only coding for antigens is expressed Box includes antigen, promoter, poly A tract and telomerase.

In the certain embodiments of methods described herein, the DNA sequence dna of the recombination of transposons and non-natural generation is encoded Sequence also comprising encoding chimeric antigen receptor or part thereof.Chimeric antigen receptor (CAR) of the invention may include (a) containing anti- The extracellular domain of former identification region, (b) transmembrane domain, and (c) comprising the intracellular domain of at least one costimulation structural domain (endodomain).In some embodiments, extracellular domain also may include signal peptide.Or or in addition, in certain embodiments In, extracellular domain also may include the hinge between antigen recognition region and transmembrane domain.In certain implementations of CAR as described herein In mode, signal peptide may include encoding human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, CD4, CD8 α, CD19, CD28,4-1BB or The sequence of GM-CSFR signal peptide.In the certain embodiments of CAR as described herein, signal peptide may include encoding human CD8 α letter The sequence of number peptide.In some embodiments, transmembrane domain may include encoding human CD2, CD3 δ, CD3 ε, CD3 γ, CD3 ζ, The sequence of CD4, CD8 α, CD19, CD28,4-1BB or GM-CSFR transmembrane domain.In certain embodiment party of CAR as described herein In formula, transmembrane domain may include the sequence of encoding human CD8 α transmembrane domain.In the certain embodiments of CAR as described herein In, intracellular domain may include people's CD3 ζ intracellular domain.In the certain embodiments of CAR as described herein, it is described at least one Costimulation structural domain may include people 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 section intracellular, or any combination thereof.? In the certain embodiments of CAR as described herein, at least one described costimulation structural domain may include CD28 and/or 4-1BB total Stimulus structure domain.In the certain embodiments of CAR as described herein, hinge may include being originated from people CD8 α, IgG4, and/or CD4 The sequence of sequence.In the certain embodiments of CAR as described herein, hinge may include the sequence from people CD8 α sequence.

In the certain embodiments of methods described herein, the DNA sequence dna of the recombination of transposons and non-natural generation is encoded Sequence also comprising encoding chimeric antigen receptor or part thereof.The part codified antigen of the sequence of encoding chimeric antigen receptor is known Other region.The antigen recognition region may include one or more complementary determining regions.The antigen recognition region may include antibody, Antibody analog, protein scaffolds or its segment.In some embodiments, the antibody is chimeric antibody, recombinant antibodies, people Source antibody or human antibody.In some embodiments, the antibody adjusts (affinity-tune) through compatibility.The present invention The non-limiting example of antibody includes single chain variable fragment (scFv), VHH, single domain antibody (sdAB), little module immune drug (SMIP) molecule or nano antibody.In some embodiments, VHH is Camelidae.Or or in addition, in certain embodiment party In formula, VHH is humanization.The non-limiting example of antibody fragment of the present invention includes complementary determining region, variable region, heavy chain, light Chain, or any combination thereof.The non-limiting example of antibody analog of the present invention includes: affine body (affibody), Afflilin Molecule, conglutnin (affimer), Affitin molecule, alpha body (alphabody), anti-transporter (anticalin), and Avimer molecule, DARPin, Fynomer, Kunitz domain peptides or monomer (monobody).Present protein bracket Non-limiting example includes Centyrin.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 10.0 μ g.In some embodiments, during electroporation or nuclear transfection are reacted, the amount of the DNA sequence dna of encoding transposase It is equal to or less than 100 μ g/mL with the concentration of the amount of the DNA sequence dna of coding transposons.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 7.5 μ g.In some embodiments, electroporation or nuclear transfection reaction in, the amount of the DNA sequence dna of encoding transposase and The concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 75 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 6.0 μ g.In some embodiments, electroporation or nuclear transfection reaction in, the amount of the DNA sequence dna of encoding transposase and The concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 60 μ g/mL.In some embodiments, transposase is to sleep beauty People's transposase.In some embodiments, sleeping beauty's transposase is sleeping beauty 100X (SB100X) transposase.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 5.0 μ g.In some embodiments, electroporation or nuclear transfection reaction in, the amount of the DNA sequence dna of encoding transposase and The concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 50 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 2.5 μ g.In some embodiments, electroporation or nuclear transfection reaction in, the amount of the DNA sequence dna of encoding transposase and The concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 25 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 1.67 μ g.In some embodiments, during electroporation or nuclear transfection are reacted, the amount of the DNA sequence dna of encoding transposase It is equal to or less than 16.7 μ g/mL with the concentration of the amount of the DNA sequence dna of coding transposons.In some embodiments, transposase is Super piggyBac (PB) transposase.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 0.55 μ g.In some embodiments, during electroporation or nuclear transfection are reacted, the amount of the DNA sequence dna of encoding transposase It is equal to or less than 5.5 μ g/mL with the concentration of the amount of the DNA sequence dna of coding transposons.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 0.19 μ g.In some embodiments, during electroporation or nuclear transfection are reacted, the amount of the DNA sequence dna of encoding transposase It is equal to or less than 1.9 μ g/mL with the concentration of the amount of the DNA sequence dna of coding transposons.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is DNA sequence dna, and every The amount etc. of the DNA sequence dna of the amount and coding transposons of the DNA sequence dna of the encoding transposase of electroporation or the nuclear transfection reaction of 100 μ L In or less than 0.10 μ g.In some embodiments, during electroporation or nuclear transfection are reacted, the amount of the DNA sequence dna of encoding transposase It is equal to or less than 1.0 μ g/mL with the concentration of the amount of the DNA sequence dna of coding transposons.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 10.0 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 100 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 7.5 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 75 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 6.0 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 60 μ g/mL.? In certain embodiments, transposase is sleeping beauty's transposase.In some embodiments, sleeping beauty's transposase is sleeping beauty 100X (SB100X) transposase.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 5.0 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 50 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 2.5 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 25 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 1.67 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 16.7 μ g/mL. In some embodiments, transposase is Super piggyBac (PB) transposase.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 0.55 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 5.5 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 0.19 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 1.9 μ g/mL.

In the certain embodiments of methods described herein, the nucleic acid sequence of encoding transposase is RNA sequence, and every The amount of the DNA sequence dna of the coding transposons of electroporation or the nuclear transfection reaction of 100 μ L is equal to or less than 1.0 μ g.In certain implementations In mode, in electroporation or nuclear transfection reaction, the concentration for encoding the amount of the DNA sequence dna of transposons is equal to or less than 1.0 μ g/mL.

The present invention provides the immunocyte modified through the method for the present invention.The immunocyte can be T- lymphocyte, from So killing (NK) cell, cytokine-induced killer cell (CIK) cell or natural killer T (NKT) cell.Immunocyte can also be by The modification of second gene editing tool, the including but not limited to endonuclease comprising being operatively connectable to Cas9 or TALE sequence Those of gene editing tool.In the certain embodiments of the second gene editing tool, endonuclease passes through covalent manner It is operatively connectable to Cas9 or TALE sequence.In the certain embodiments of the second gene editing tool, endonuclease is logical It crosses non-covalent fashion and is operatively connectable to Cas9 or TALE sequence.In some embodiments, Cas9 is the Cas9 of inactivation (dCas9).In some embodiments, the Cas9 of inactivation includes D10A and N580A in catalytic site.In certain embodiments In, Cas9 is small and inactivation Cas9 (dSaCas9).In some embodiments, dSaCas9 includes following amino acid sequence

The present invention provides the immunocyte modified through the method for the present invention.The immunocyte can be T- lymphocyte, from So killing (NK) cell, cytokine-induced killer cell (CIK) cell or natural killer T (NKT) cell.Immunocyte can also be by The modification of second gene editing tool, the including but not limited to endonuclease comprising being operatively connectable to Cas9 or TALE sequence Those of gene editing tool.Or or in addition, the second gene editing tool may include only excision type (excision-only) PiggyBac transposase to cut off the sequence or its any part of insertion again.For example, only excision type piggyBac transposase is available In " cutting off (re-excise) again " transposons.

The present invention provides the composition comprising immunocyte of the invention.

The present invention provides the composition comprising immunocyte of the invention and is used to treat the disease in the object for having this to need Or the purposes of illness.In some embodiments, the disease or illness are cancers.In some embodiments, the disease Or illness is infectious diseases.For example, infectious diseases can be caused by virus, bacterium, yeast, microorganism or any combination thereof. In some embodiments, the immunocyte of the composition is Autologous.In some embodiments, the composition Immunocyte be allogeneic.

The present invention is provided to enhance the culture medium of the vigor of modified immunocyte, it includes IL-2, IL-21, IL- 7, IL-15 or any combination thereof.The modified immunocyte can be T- lymphocyte, natural kill (NK) cell, thin Killing (CIK) cell or natural killer T (NKT) cell of intracellular cytokine induction.The modified immunocyte may include one Or multiple foreign DNA sequences.The modified immunocyte may include one or more exogenous rna sequences.The warp The immunocyte of modification can electroporated or nuclear transfection.

Detailed description of the invention

A series of figures of Fig. 1 illustrate to carry out transfection efficiency after Plasmid DNA nuclear transfection and thin in primary human T lymphocyte Born of the same parents' vigor.

DNA cytotoxicity of the system figure explanation of Fig. 2 for T cell.

The cytotoxicity that a series of figures of Fig. 3 show that the DNA in T cell is mediated is dose-dependent.

A series of figures of Fig. 4 show that extracellular Plasmid DNA does not have cytotoxicity.

A series of figures of Fig. 5 illustrate the high-efficiency transposon that sPBo mRNA is used in Jurkat cell.

A series of figures of Fig. 6 illustrate to use the high-efficiency transposon of sPBo mRNA in T lymphocyte.

A series of figures of Fig. 7 illustrate the high-efficiency delivery of the DNA transposons product of linearisation.

A series of figures of Fig. 8 show the addition of IL-7 and IL-15 and stimulate T cell to enhance cell at once after nuclear transfection Vigor.

A series of figures of Fig. 9 show the toxic effect that IL-7 and IL-15 rescue T cell is not mediated by DNA.

A series of figures of Figure 10 stimulate T cell to enhance cell viability at once after showing nuclear transfection.

A series of figures of Figure 11 A-C illustrate the T cell swivel base using not same amount DNA.The primary general T cell of people uses piggyBac^TMWith not same amount DNA nuclear transfection.The transposons of T cell indicatrix and 5 μ g sPBo mRNA nuclear transfections.Then, exist Cell was stimulated by CD3 and CD28 in the 2nd day after nuclear transfection.Such as expection, with the T cell of a large amount DNA nuclear transfection after nuclear transfection Show within 1st day the expression of high additive type, and in low DNA dosage almost without observing that additive type expresses.Unlike, in consideration convey After dye after amplification in the 21st day, the transgene positive cells of largest percentage are observed in the case where lower amount of DNA, for this Transposons, the 1.67 μ g of peak value of amount of DNA.(A) flow cytometer showed is carried out to transgene positive cells with the 21st day on day 1.(B) turn The percentage of gene masculine T cell.(C) percentage of the 1st day and the 21st day T cell living.For all figures shown in this Fig Table, Y-axis range are 0-100%, and increment 20%, X-axis range is 0-10⁵, exponential increment 10.

A series of figures explanation of Figure 12 A-B uses Sleeping Beauty^TM100X (SB100X) transposase low DNA amount The T cell swivel base of progress.Primary people's general T cell coding piggyBac^TM(PB) or Sleeping Beauty^TM(SB) ITR GFP plasmid nuclear transfection.(A) the SB transposons of cell indicatrix and 1 μ g SB transposase mRNA nuclear transfection.(B) cell indicates The SB transposase of amount and 0.75 μ g SB transposons nuclear transfection.For all samples, flow cytometer showed after nuclear transfection the 14th day into Row.For all charts shown in this Fig, Y-axis range is 0-250K, and increment 50K, X-axis range is 0-10⁵, index difference is 10。

Specific embodiment

Disclosed herein is the compositions and method for carrying out ex vivo genetic modification to immunocyte comprising exempts to described Epidemic disease cell delivering: (a) nucleic acid or amino acid sequence of the sequence comprising encoding transposase, and the DNA (b) comprising coding transposons The recombination of sequence and the DNA sequence dna of non-natural generation.In some embodiments, the method also includes following steps: with one Kind or cytokine profiles stimulate immunocyte.

Centyrin of the invention may include protein scaffolds, wherein the bracket being capable of molecule of the antigen binding.This hair Bright Centyrin may include protein scaffolds, contain the shared sequence of at least one fibronectin type III (FN3) structural domain Column, wherein the bracket being capable of molecule of the antigen binding.At least one fibronectin type III (FN3) structural domain may originate from Human protein.The human protein can be tenascin-C.The consensus sequence may include LPAPKNLVVSEVTEDS LRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT(SEQ ID NO:5) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERS YDLTGLKPGT EYTVSIYGVKGGHRSNPLSAEFTT(SEQ ID NO:6).The consensus sequence may include and following sequence at least 74% phase Same amino acid sequence: LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGL KPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO:5) or MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFL IQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT(SEQ ID NO:6).It is described total There is sequence can be by the nucleic acid sequence encoding comprising following sequence: atgctgcctgcaccaaagaacctggtggtgtctcatgtg acagaggatagtgccagactgtcatggactgctcccgacgcagccttcgatagttttatcatcgtgtaccgggaga acatcgaaaccggcgaggccattgtcctgacagtgccagggtccgaacgctcttatgacctgacagatctgaagcc cggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagcttccctctgtccgcaatcttcacc aca(SEQ ID NO:7).The consensus sequence can be at one or more positions in following part through modifying: (a) A-B ring, It comprising amino acid residue TEDS (SEQ ID NO:8) or is made from it at 13-16 of the consensus sequence；(b)B-C Ring comprising amino acid residue TAPDAAF (SEQ ID NO:9) or is made from it at 22-28 of the consensus sequence； (c) C-D ring, at 38-43 of the consensus sequence comprising amino acid residue SEKVGE (SEQ ID NO:10) or by it Composition；(d) D-E ring, 51-54 of the consensus sequence comprising amino acid residue GSER (SEQ ID NO:11) or by It is formed；(e) E-F ring, 60-64 in the consensus sequence include amino acid residue GLKPG (SEQ ID NO:12) Or it is made from it；(f) F-G ring, 75-81 in the consensus sequence include amino acid residue KGGHRSN (SEQ ID NO:13 it) or is made from it；Or (g) any combination of (a)-(f).Centyrin of the invention may include at least five fibronectin The consensus sequence of type III (FN3) structural domain, the consensus sequence or at least 15 of at least ten fibronectin type III (FN3) structural domain The consensus sequence of a fibronectin type III (FN3) structural domain.The bracket can be to come selected from following at least one compatibilities In conjunction with antigen: being less equal than 10^-9M is less equal than 10^-10M is less equal than 10^-11M is less equal than 10^-12M is less than Or it is equal to 10^-13M is less equal than 10^-14M, and it is less equal than 10^-15The K of M_D。K_DIt can be determined by surface plasma body resonant vibration.

Term " antibody analog " is intended to describe such organic compound, specifically binds target sequence and has and day The different structure of the antibody so generated.Antibody analog may include protein, nucleic acid or small molecule.Antibodies mimic of the invention The target sequence of object specific binding can be antigen.Antibody analog can provide the superior property better than antibody, including but unlimited In, superior dissolubility, tissue penetration, for the stability (such as resistance for enzymatic degradation) of heat and enzyme, and it is lower Production cost.Exemplary antibodies analogies include but is not limited to affine body (affibody), Afflilin molecule, conglutnin (affimer), Affitin molecule, alpha body (alphabody), anti-transporter (anticalin) and Avimer molecule (also referred to as affinity polymer), DARPin (ankyrin repeat protein through designing), Fynomer, Kunitz domain peptides, or Monomer (monobody).

Affine body molecule of the invention includes protein scaffolds, and it includes one or more α spirals without any disulphide bridges Or it is made from it.Preferably, affine body molecule of the invention includes three α spirals or is made from it.For example, of the invention is affine Body molecule may include immune globulin binding structural domain.Affine body molecule of the invention may include the Z structural domain of a-protein.

Affilin molecule of the invention includes protein scaffolds, by such as γ-B crystallin or ubiquitin Exposed amino acid is modified and is generated.Functionally analog antibody is to the compatibility of antigen for Affilin molecule, but in structure Not analog antibody.It, can quilt in the protein molecule correctly folded in any protein scaffolds for manufacturing Affilin It is considered as exposed amino acid that solvent or possible binding partners, which those of touch amino acid,.In the amino acid of these exposures Any one or more can be modified to be specifically bound to target sequence or antigen.

Conglutnin molecule of the invention includes protein scaffolds, and the protein scaffolds contain highly stable protein, Its is engineered to show peptide ring, and the peptide ring provides high-affinity binding site for specific target sequence.Of the invention shows Example property conglutnin molecule includes the protein scaffolds based on cystatin albumen or its tertiary structure.Exemplary adhesive element of the invention Molecule can share common tertiary structure, and it includes the alpha-helixes being located at the top of antiparallel β lamella.

Affitin molecule of the invention includes artificial protein scaffolds, and structure can be originated from, for example, DNA binding protein dna (such as DNA binding protein dna Sac7d).Affitin of the invention is selectively bound to target sequence, can be the complete of antigen Portion or part.Exemplary Affitin of the invention is generated in the following way: tying one or more amino acid sequences in DNA It is randomized on the mating surface of hop protein, and makes gained protein experience ribosomal display and selection.Affitin of the invention Target sequence may be present in, for example, in peptide, protein, virus or the genome of bacterium or on surface.In certain realities of the invention It applies in mode, Affitin molecule is used as the specific inhibitor of enzyme.Affitin molecule of the invention may include heat-resisting egg White matter or derivatives thereof.

Alpha body molecule of the invention is also referred to as cell-penetrating type alpha body (CPAB).Alpha body of the invention Molecule includes little albumen matter (typically less than 10kDa), is bound to different target sequences (including antigen).Alpha body molecule energy It gets at and reaches and be bound to target sequence intracellular.In structure, alpha body molecule of the invention includes to form single-stranded α spiral (to be similar to Naturally-produced coiled-coiled structure) artificial sequence.Alpha body molecule of the invention may include protein scaffolds, the albumen Matter bracket contains one or more amino acid that target protein is specifically bound through modifying.Combination independent of the molecule is special Property, alpha body molecule of the invention keeps correct folding and thermal stability.

Anti- transporter molecule of the invention includes the artificial of the target sequence that is bound in protein or small molecule or site Albumen.Anti- transporter molecule of the invention may include the artificial protein from human lipids transporter.Anti- fortune of the invention Carrying protein molecular can be used for substituting, for example, monoclonal antibody or its segment.Anti- transporter molecule can show anti-better than monoclonal The superior tissue penetration and thermal stability of body or its segment.Exemplary anti-transporter molecule of the invention can include about 180 A amino acid, the quality with about 20kDa.In structure, anti-transporter molecule of the invention includes barrel structure, the barrel structure packet α spiral containing the antiparallel β-strand and attachment connected in pairs by ring.In the preferred embodiment, anti-transporter of the invention point Attached bag contains barrel structure, which includes the α spiral by ring eight antiparallel β-strands connected in pairs and attachment.

Avimer molecule of the invention includes the artificial protein for being specifically bound to target sequence (it can also be antigen).This hair Bright Avimer can recognize multiple binding sites in identical target or in different targets.When Avimer identification of the invention is more than When one target, which simulates the function of bispecific antibody.Artificial protein Avimer may include respectively about 30-35 ammonia Two or more peptide sequences of base acid.These peptides can be connected by one or more joint peptides.The one or more of Avimer The amino acid sequence of peptide may originate from the A structural domain of membrane receptor.Avimer has rigid structure, optionally including disulfide bond And/or calcium.Compared to antibody, Avimer of the invention can show higher thermal stability.

DARPin (ankyrin repeat protein through designing) of the invention includes to have high specific and height for target sequence Compatibility through it is genetically engineered, recombination or chimeric protein.In some embodiments, DARPin of the invention is originated from anchor Albumen, and optionally, at least three comprising ankyrin repeat motif (also referred to as constitutional repeating unit).Ankyrin mediates high Compatibility protein-protein interaction.DARPin of the invention includes big target interactive surfaces.

Fynomer of the invention includes small conjugated protein (about 7kDa), is originated from people Fyn SH3 structural domain and through engineering Transformation is with the compatibility that is equal with antibody and equivalent is specifically bound to target sequence and molecule.

Kunitz domain peptides of the invention include protein scaffolds, which includes Kunitz structural domain. Kunitz structural domain includes to be used for the active active site of protease inhibition.In structure, Kunitz structural domain of the invention includes Alpha+beta rich in disulphide folds.The structure is illustrated by bovine pancreatic trypsin inhibitor.Kunitz domain peptides identify specific protein It matter structure and plays a role as competitive protein enzyme inhibitor.Kunitz structural domain of the invention may include Ai Kala peptide (Ecallantide) (it is originated from the relevant coagulation inhibitor of human lipoprotein (LACI)).

Monomer of the invention is little albumen matter (comprising about 94 amino acid and with the quality of about 10kDa), in ruler It is very little upper suitable with single-chain antibody.These are through genetically engineered protein specific combination target sequence (including antigen).This hair Bright monomer can selectively targeted one or more different protein or target sequence.In the preferred embodiment, of the invention Monomer includes protein scaffolds, the structure of protein scaffolds simulation people's fibronectin, and it is highly preferred that simulation fibronectin The tenth extracellular type III structural domain structure.The extracellular type III structural domain of the tenth of the fibronectin and monomer simulation Object includes seven β lamellas, they form bucket on each side of three complementary determining regions (CDR) for corresponding to antibody and three sudden and violent The ring of dew.It is different from the structure of the variable domains of antibody, monomer lack for metal ion any binding site and in Heart disulfide bond.Polyspecific monomer can be optimised by modifying BC and FG ring.Monomer of the invention may include A Di connection Plain (adnectin).

The singular as used in this paper full text "one", "an" and "the" include plural reference, unless literary It is expressly stated otherwise in this.Thus, for example, mentioning " method " includes a variety of such methods, mentioning " dosage " includes this field skill One or more dosage and its equivalent, etc. known to art personnel.

Term " about " or " about " indicate the acceptable error model of the special value determined in this field general technical staff In enclosing, this will depend on the measurement or method of determination of the numerical value, for example, the limit of measurement system.For example, " about " can indicate 1 In a or multiple Standard deviation-Ranges.Alternatively, " about " can indicate up to the 20% of given value, or up to 10%, or up to 5%, Or up to 1% range.Alternatively, the term can indicate the certain amount grade of certain value for particularly with biosystem or process It is interior, in preferably 5 times, in more preferable 2 times.When describing occurrence in the application and claims, unless otherwise indicated, it is assumed that art Language " about " indicates in the acceptable error range of the occurrence.

The present invention provides the polynucleotides or protein compositions of separation or basic purifying." separation " or " purifying " Polynucleotides or albumen or its biologically-active moiety are substantially or substantially without multicore glycosides usually adjoint in its naturally-produced environment Acid or albumen or the component interacted therewith.Therefore, separation or the polynucleotides or protein of purifying pass through recombinant technique Other cellular materials or culture medium are substantially free of when generation, or by being chemically synthesized when be substantially free of chemical precursors or Other chemical substances.Optionally, " separation " polynucleotides are without natural in the organism genomic DNA in the polynucleotides source Flank the sequence (optional albumen coded sequence) of the polynucleotides (sequence for being located at the end polynucleotides 5' and 3').Example Such as, in various embodiments, isolated polynucleotides in the cell genomic dna in the polynucleotides source comprising naturally flanking The nucleotide sequence of less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb of the polynucleotides.It is substantially free of cell The protein of substance includes the protein having less than about 30%, 20%, 10%, 5% or 1% (dry weight) protein pollutant Preparation.When protein of the invention or its biologically-active moiety are through recombinant production, optimal culture medium having less than about 30%, 20%, the chemical substance of the chemical precursors or non-proteins of interest of 10%, 5% or 1% (dry weight).

The present invention provides the segment and variant of disclosed DNA sequence dna and the protein by these DNA sequence encodings.As herein Term used in full text " segment " refers to the part of DNA sequence dna or the part of amino acid sequence and protein encoded by it.Packet Protein fragments as the segment codified of DNA sequence dna containing coded sequence retain the bioactivity of urporotein, because And retain and the DNA of target DNA sequence is identified or combined activity, as described herein.Alternatively, can be used as the DNA sequence of hybridization probe The segment of column does not encode the protein for retaining bioactivity or not retaining promoter activity generally.Therefore, the segment of DNA sequence dna Range can be overall length polynucleotides of the invention up at least about 20 nucleotide, about 50 nucleotide, about 100 nucleosides Acid, and it is up to the overall length polynucleotides.

Nucleic acid or protein of the invention can be constructed by modular approach, which is included in targeting vector preparatory group Dress monomeric unit and/or repetitive unit, the targeting vector then can be assembled into final purpose carrier.Polypeptide of the invention may include Repeated monomer (repeat monomer) of the invention, and can be constructed by modular approach, the modular approach in target by carrying Pre-assembly repetitive unit carries out in body, which subsequent can be assembled into final purpose carrier.The present invention is provided by this The polypeptide of method generation and the nucleic acid sequence for encoding these polypeptides.It includes what coding was generated by the modular approach that the present invention, which provides, The host organisms and cell of the nucleic acid sequence of polypeptide.

Term " antibody " especially covers single monoclonal antibody (including agonist and short of money with the use of most broad sense Anti-agent antibody) and antibody compositions with multi-epitope specificity.The scope of the invention further include the natural of this paper associated antibodies or The application of synthetic analogues, mutant, variant, allele, homologue and ortholog thing (collectively referred to herein as " analog "), As defined herein.Therefore, according to this paper embodiment, with it, most broad sense covers term " this paper associated antibodies " Such analog.In general, compared to this paper associated antibodies being defined herein, in such analog, one or more ammonia Base acid residue can be substituted, is deleted and/or added.

" antibody fragment " used herein and its whole grammatical variants, are defined as the antigen knot comprising complete antibody The part of coincidence point or the complete antibody of variable region, wherein the constant heavy structure in Fc area of the part without complete antibody Domain (i.e. CH2, CH3 and CH4, depending on antibody isotype).The example of antibody fragment includes Fab, Fab', Fab'-SH, F (ab')₂With Fv segment；Double antibody；The polypeptide for the primary structure being made of the uninterrupted sequence of continuous amino acid residue Any antibody fragment (referred to herein as " single chain antibody fragments " or " single chain polypeptide "), including but not limited to (1) scFv (scFv) Molecule, (2) single chain polypeptide, the single chain polypeptide only includes a light variable domains or it contains the light chain variable domain The segment of three CDR in domain, without the heavy chain moiety being connected, and (3) single chain polypeptide, the single chain polypeptide only include a heavy chain The segment of variable region or its three CDR for containing the heavy chain variable region, without the chain moiety being connected；With by antibody fragment shape At polyspecific or multivalent structure.In the antibody fragment comprising one or more heavy chains, one or more of heavy chains can Comprising any constant domain sequence (for example, CHI in IgG isotype) seen in the area complete antibody Fei Fc, and/or can It comprising any hinge sequence seen in complete antibody, and/or may include being fused to or positioned at hinge sequence or the heavy chain Leucine zipper sequences in constant domain sequence.The term further includes single domain antibody (" sdAB "), refers generally to have single One monomer can be changed the antibody fragment of antibody domain, (for example, coming from Camelidae).Those of ordinary skill in the art can be easily Understand such antibody fragment type.

" combination " refers between macromolecular the sequence-specific, noncovalent interaction of (such as between protein and nucleic acid). As long as interaction is as a whole sequence-specific, not requiring all components of binding interactions is all sequence specific (for example, being contacted with the phosphate moiety in DNA backbone) of property.

Term " includes " or "comprising" are intended to mean that the composition and method includes mentioned element, but are not precluded Other elements.When to define composition and method, "consisting essentially of" should indicate to exclude when for when being intended to purpose There are other elements of any basic meaning to the combination.Therefore, the basic combination being made of certain elements being defined herein Object will be not excluded for contaminant trace species or inert carrier." by ... form " it should indicate the microelement for excluding to be more than other ingredients With the part of basic method steps.The embodiment defined by these each transition terms is within the scope of the present invention.

Term " epitope " refers to the Antigenic Determinants of polypeptide.Epitope may include three amino in certain space conformation Acid is unique for the epitope.In general, epitope is by least 4,5,6 or 7, this amino acid is formed, and more Typically, by least 8,9 or 10, this amino acid is formed.Method for determining amino acid space conformation is known in the art , and including for example, x-ray crystallisation and two dimensional NMR method.

" expression " used herein refers to the process of that polynucleotides are transcribed into mRNA and/or the mRNA of transcription is then translated At the process of peptide, polypeptide or protein.If polynucleotides are originated from genomic DNA, expression may include mRNA in eukaryocyte Montage.

" gene expression ", which refers to, is converted to gene product for gene information contained.Gene product can be the direct transcription of gene Product is (for example, mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, Microrna, structure RNA or any other type RNA the protein) or by mRNA translation generated.Gene product further includes modified RNA, by processing modification, example as follows Such as capped, polyadenylation, methylation, and editor, and the protein by processing modification as follows, for example, methylation, acetyl Change, phosphorylation, ubiquitination, ADP- ribosylation, myristoylation and glycosylation.

" regulation " or " adjusting " of gene expression refers to the change of gene activity.The regulation of expression may include but be not limited to, base Because of activation and gene repression.

Term " being operatively connected " or its equivalent form (such as " being operably connected ") indicate two or more Molecule is positioned relative to each other, so that they can interact to influence to be caused by one or two molecule or combinations thereof Function.

Disclose the component that the component and the manufacture and use that connect by non-covalent fashion are connected by non-covalent fashion Method.Different components can have a variety of different forms as described herein.For example, by non-covalent fashion connection (that is, operation Property connection) protein can be used for allowing temporary interaction, the one or more problems being avoided that in this field.It is logical Cross non-covalent fashion connection component (such as protein) is associated and the ability that is separated make only or mainly in certain feelings Functional association under shape (such as needing such association to obtain required activity) is possibly realized.Connection can carry out time enough Length is to allow to obtain required effect.

It discloses the method for the particular locus in the genome of protein targeting organism.The method may include as Lower step: providing DNA positioning component and provides effector molecule, wherein the DNA positioning component and the effector molecule energy It is enough to be operatively connected by non-covalent linking mode.

Term " scFv " refers to single chain variable fragment.ScFv is the heavy chain variable region (VH) and light chain variable region of immunoglobulin (VL) fusion protein, is connected by joint peptide.The length of joint peptide can be about 5-40 amino acid or about 10-30 amino acid Or about 5,10,15,20,25,30,35 or 40 amino acid.Single chain variable fragment lacks constant present in complete antibody molecule The area Fc, to lack the common binding site (for example, protein G) for being used for antibody purification.The term further includes interior antibody (intrabody) scFv of form, to stablize in cell cytoplasm and in combination with the antibody to intracellular protein.

Term " single domain antibody " indicates that there is single monomer can be changed antibody structure for such antibody fragment, the antibody fragment Domain can selectively be bound to specific antigen.Single domain antibody is generally about the peptide chain of 110 amino acid longs, includes heavy chain One variable domains (VH) of antibody or common IgG, it is generally similar to complete antibody for the compatibility of antigen, but more resistant to It is hot and more stable to the urea of detergent and high concentration.Example is from those of Camelidae or fish antibody.Alternatively, single domain antibody It can be by having the common mouse of four chains or human IgG to be made.

Term " specifically combining " used herein and " specific binding " refer to antibody, antibody fragment or nano antibody Preferentially it is bound to the ability of specific antigen present in the not homogeneous mixture of synantigen.In some embodiments, specific Binding interactions will distinguish expectation antigen and undesirable antigen in sample, and in some embodiments, difference is more than about 10 - 100 times or more again (for example, being more than about 1000- or 10,000 times)." specificity " refers to immunoglobulin or immune globulin white tiles Section, such as nano antibody are preferentially bound to a kind of ability of antigenic target, not necessarily relative to different antigenic targets Indicate high-affinity.

" target site " or " target sequence " is the nucleic acid sequence for limiting the combining nucleic acid moiety of binding molecule, on condition that in the presence of In conjunction with adequate condition.

Term " nucleic acid " or " oligonucleotides " or " polynucleotides " refer at least two nucleotide being covalently joined together.It is right The sequence of complementary strand is also defined in single-stranded description.Therefore, nucleic acid can also cover the single-stranded complementary strand.Nucleic acid of the invention It also covers essentially identical nucleic acid and its retains identical structure or encode the complement of same protein.

Probe of the invention may include single-chain nucleic acid, be hybridized to target sequence under stringent hybridisation conditions.Therefore, of the invention Nucleic acid can refer to the probe hybridized under stringent hybridisation conditions.

Nucleic acid of the invention can be single-stranded or double-stranded.Nucleic acid of the invention may include double-stranded sequence, or even most It is in the case that number molecule is single-stranded and such.Nucleic acid of the invention may include single-stranded sequence, or even be in most of molecules It is also such in the case where double-strand.Nucleic acid of the invention may include genomic DNA, cDNA, RNA or its heterozygote.Of the invention Nucleic acid may include the combination of deoxyribonucleotide and ribonucleotide.Nucleic acid of the invention may include base (including uracil, Adenine, thymidine, cytimidine, guanine, inosine, xanthine hypoxanthine, iso-cytosine and isoguanine) combination. Nucleic acid of the invention can be modified through synthesizing comprising unnatural amino acid.Nucleic acid of the invention can be by chemical synthesis process or logical Recombination method is crossed to obtain.

Nucleic acid of the invention, their complete sequence or its any part can be non-natural generation.Core of the invention Acid may include one or more mutation, substitution, missing or the insertion that non-natural generates, and produce complete nucleic-acid sequences non-natural Raw.Nucleic acid of the invention may include one or more double, overturning or duplicate sequences, and gained sequence is non-natural It generates, so that complete nucleic-acid sequences are what non-natural generated.Nucleic acid of the invention may include non-natural generate it is modified, Artificial or synthesizing ribonucleotide, so that complete nucleic-acid sequences are what non-natural generated.

In view of the redundancy of genetic code, multiple any specific proteins of nucleotide sequence codified.Consider herein it is all this A little nucleotide sequences.

The term as used in this paper full text " being operatively connected " refers to the expression of gene in the promoter of its space connection Control under.Promoter can be the upstream 5'(of the gene controlled with it) or the downstream 3'().Promoter and intergenic distance Can in the gene of the derivative promoter promoter it is roughly the same at a distance from the gene that it is controlled.Promoter and gene it Between the variation of distance can be through adjusting without losing promoter function.

The term as used in this paper full text " promoter " refer to synthesis or it is natural derived from molecule, can assign, activate or Enhance expression of the nucleic acid in cell.Promoter may include one or more specific transcriptional regulatory sequences to further enhance core Acid expression and/or change its space expression and/or temporarily expression.Promoter also may include Distal enhancer or suppress matter-element Part can be located at away from the up to thousands of a base-pairs of transcription initiation site.Promoter may originate from a variety of sources, including viral, thin Bacterium, fungi, plant, insect and animal.Promoter is directed to cell, tissue or the organ wherein expressed or for wherein occurring The stage of development of expression or response external stimulation such as physiological stress, pathogen, metal ion or inducing substance, with composing type side Formula or otherness mode adjust the expression of gene component.The representative example of promoter include bacterial phage t7 promoter, Bacteriophage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 are early Phase promoter, RSV-LTR promoter, CMV IE promoter, EF-1 Α promoter, CAG promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.

The term as used in this paper full text " basic complementary " refer to the complement of First ray and second sequence 8,9,10, 11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、 80,85,90,95,100,180,270,360,450,540, or more at least 60% on the region of nucleotide or amino acid, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% are identical or the two sequences are rigorous miscellaneous Hybridize under the conditions of friendship.

The term as used in this paper full text " essentially identical " refer to the first and second sequences 8,9,10,11,12,13,14, 15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、 100, at least 60% on the region of 180,270,360,450,540 or more nucleotide or amino acid, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% are identical；Or for nucleic acid, First ray and the second sequence The basic complementary situation of complement.

The term as used in this paper full text " variant ", when for referring to the part of (i) reference nucleotide sequence when describing nucleic acid Or segment；(ii) complement of reference nucleotide sequence or part thereof；(iii) essentially identical with reference nucleic acid or its complement Nucleic acid；Or (iv) under high stringency conditions with reference nucleic acid, its complement or the nucleic acid hybridized with its essentially identical sequence.

The term as used in this paper full text " carrier " refers to the nucleic acid sequence containing replication orgin.Carrier can be viral vectors, Bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.Carrier can be DNA or RNA carrier.Carrier can be certainly The outer carrier of duplicated chromosome, and preferably, it is DNA plasmid.Carrier may include amino acid and DNA sequence dna, RNA sequence, or and DNA With the combination of RNA sequence.

The term as used in this paper full text " variant ", when for when describing peptide or polypeptide, referring to the insertion by amino acid, lacking Lose or conservative substitution and it is different in amino acid sequence, but retain the peptide or polypeptide of at least one bioactivity.Variant It can indicate such protein, the reference amino acid sequence of amino acid sequence and the protein for retaining at least one bioactivity It is essentially identical.

The conservative substitution of amino acid, that is, wherein amino acid, which is used, has similar quality (for example, hydrophily, the journey of charging zone Degree and distribution) different aminoacids substitution, be believed in the art that and usually cause small change.These small changes can partially lead to It crosses hydrophilic (hydropathic) index for considering amino acid and identifies, as is understood in the art.Kyte etc., J.Mol.Biol.157:105-132(1982).The hydrophilic index of amino acid is based on the considerations of to its hydrophobicity and charging property.Tool There is the amino acid of similar hydropathic index that can be substituted and still retaining protein function.In an aspect, replace hydrophilic with ± 2 The amino acid of index.The hydrophily of amino acid can also be used for disclosing the substitution that can obtain the protein for retaining biological function.Just For peptide, allows to calculate the maximum local average hydrophilicity of the peptide for the hydrophilic consideration of amino acid, be reported as To antigenicity and the well relevant useful metrics of immunogenicity.U.S. Patent number 4,554,101, mode by reference of text It is included in herein.

The substitution of amino acid with similar hydrophilicity score can lead to the peptide for retaining bioactivity (such as immunogenicity).It takes In generation, can be used amino acid of the mutual hydrophilicity value within ± 2 and carry out.The hydrophobicity index and hydrophilicity value of amino acid by The influence of the specific side chain of the amino acid.Consistent with the observation conclusion, the amino acid substitution compatible with biological function is interpreted as Depending on the side chain of the relative similarities of those amino acid, especially those amino acid, such as by hydrophobicity, hydrophily, charging property, Size and other properties are revealed like that.

" conservative " amino acid substitution used herein can define in following Table A, B or C.In some embodiments, it merges Polypeptide and/or the nucleic acid of the coding fused polypeptide include conservative substitution, and the conservative substitution has passed through the modification coding present invention The polynucleotides of polypeptide and be introduced into.Amino acid can classify according to physical property, and constitute second level and three-level protein structure. The case where conservative substitution is another amino acid substitution that an amino acid is had similar quality.Exemplary conservative, which replaces, to be shown in Table A.

Table A -- conservative substitution I

Alternatively, conserved amino acid can be by Lehninger (" biochemistry " (Biochemistry), the second edition；Butterworth is published Company (Worth Publishers, Inc.), New York New York, (1975), the 71-77 pages) classification, as shown in tableb.

Table B-- conservative substitution II

Alternatively, Exemplary conservative, which replaces, is shown in table C.

Table C-- conservative substitution III

It should be understood that polypeptide of the invention is intended to include one or more insertion, deletion or substitution containing amino acid residue, Or any combination thereof, and different from amino acid residue insertion, missing, or replace modification polypeptide.Polypeptide of the invention Or nucleic acid may include one or more conservative substitutions.

The term as used in this paper full text " more than one " aforementioned amino acid substitution refers to, 2,3,4,5,6,7,8,9,10, 11,12,13,14,15,16,17,18,19 or 20 or more the amino acid substitution.Term " more than one " can refer to 2,3,4 Or 5 amino acid substitutions.

Peptide and protein of the invention, their complete sequence or its any part can be non-natural generation.This The peptide and protein of invention may include one or more mutation, substitution, missing or the insertion that non-natural generates, so that complete ammonia Base acid sequence is non-naturally-produced.Peptide and protein of the invention may include one or more double, reverse or again Multiple sequence, gained sequence are what non-natural generated, so that complete amino acid sequence is what non-natural generated.Of the invention is more Peptide and protein may include modified, the artificial or synthesizing amino acid that non-natural generates, so that complete amino acid sequence is non- Naturally-produced.

As used in this paper full text " sequence identity " can be used for carrying out blast retrieval (bl2seq) to two sequences Independence BLAST engine program can be performed determine that the program is available from National Center for Biotechnology Information (National Center for Biotechnology Information, NCBI) ftp website, using default parameters (Tatusova and Madden,FEMS Microbiol Lett.,1999,174,247-250；It is included in herein by way of quoting its full text). Term " identical " or " the phase same sex " refer to when in the case where for two or more nucleic acid or polypeptide sequence in each sequence Specific region on identical residue specific percentage.The percentage can calculate in the following way: two sequences are optimal Ground alignment compares the two sequences in specific region, determines the positional number of the identical residue occurred simultaneously in the two sequences Amount, to generate the quantity of matching position, by the quantity of matching position divided by total number of positions amount in the specific region, and, by result Multiplied by 100, to generate the percentage of sequence identity.There is different length in two sequences, or compare and generate one or more In the case where stagger arrangement (staggered) is held, and the specific region compared only includes unique sequence, the residue of unique sequence is included in The denominator of the calculating rather than in molecule.When comparison dna and RNA, it is believed that thymidine (T) and uracil (U) are equivalent. Identification can be carried out manually or be carried out using computer sequence algorithm such as BLAST or BLAST 2.0.

The term as used in this paper full text " endogenic " refers to that the host cell with target gene or as importing object is natural Associated nucleic acid or protein sequence.

The term as used in this paper full text " exogenous " refers to that the host cell with target gene or as importing object is not natural Associated nucleic acid or protein sequence, the naturally-produced nucleic acid sequence including being located at the genomic locations that non-natural generates, Or multiple copies that the non-natural of naturally-produced nucleic acid (for example, DNA sequence dna) generates.

The present invention provides the method that the polynucleotide constructs comprising DNA sequence dna are imported host cell." importing " is intended to Polynucleotide constructs are presented to plant in this way by expression, and which is able to enter construct in host cell Portion.The method of the present invention is independent of the specific method for polynucleotide constructs to be imported to host cell, as long as polynucleotides Construct enters the cell interior of host.For polynucleotide constructs to be imported bacterium, plant, fungi and animal Method be it is known in the art, including but not limited to, stable conversion method, transient transformation methods and virus-mediated method.

" stable conversion " is intended to mean that, the polynucleotide constructs for importing plant are integrated into host genome, and energy Enough by its descendant inheritting." instantaneous conversion " is intended to mean that the polynucleotide constructs unconformity for importing host enters host gene Group.

The term as used in this paper full text " genetically modified plant (or genetically modified plants) " refers to and wraps in its genome Plant containing Exogenous polynucleotide.In general, and preferably, which enters genome, To which the polynucleotides are gone down by successive generation heredity.The Exogenous polynucleotide can be individually or as recombinant expression cassettes It is partially integrated into genome." transgenosis () " is herein for including any cell, cell line, callus, tissue, plant Object part or plant, genotype because Exogenous Nucleic Acid there are due to be changed, including those initial transgenosis through changing Those of and generated from initial transgenosis by sexual hybridization or vegetative propagation.The term as used herein " transgenosis " is not contained Lid is (such as random allogamy, non-recombinant virus infection, non-by conventional plant implantation methods or by naturally-produced event Recombinant bacteria conversion, non-recombinant swivel base or spontaneous mutation) change to caused by genome (outside chromosome or chromosome).

The term as used in this paper full text " modification " be intended to mean that the sequence be considered by polypeptide in conjunction with and simply modify.Its It is not intended to indicate that nucleotide sequence changes, although after these (and other) variation can cause polypeptide and nucleic acid interested It is continuous to combine.In some embodiments, the nucleic acid sequence is DNA.The modification of nucleic acid interested (is just modified comprising module weight For the combination of the polypeptide and nucleic acid of multiple unit) can by a variety of methods (such as gel mobility shift assay, using labeled Polypeptide -- marker may include radioactivity, fluorescence, enzyme or biotin/streptavidin marker) in any detect.Sense The modification (and its missing) of interest nucleic acid sequence can be form needed for whole (such as in medical diagnosis on disease).However, it is expected that into Row is further processed sample.For the sake of convenient, make the residue of polypeptide (and nucleic acid sequence specifically bound therewith) and sample It is partially separated.Advantageously, polypeptide-DNA compound is made to be bound to solid support, to promote the separation.For example, polypeptide can be deposited It is in acrylamide or agarose gel matrix, or it is highly preferred that is fixed in film surface or the hole in titer plate In.

Unless there are specific in addition explanation, all percentage and rate/ratio are by weight.

Unless otherwise indicated, all percentages and ratio are calculated based on total composition.

The each greatest measure limitation provided through present disclosure includes each lower numerical value limitation, as these compared with Low numerical value limitation is clearly write out the same herein.Each minimum value for being provided in disclosure full text limit include each compared with High numerical definiteness writes on here as these higher numerical definiteness understand.The each numerical value model provided in disclosure full text It encloses including falling into the relatively narrow numberical range of each of described broader numerical, as clearly write these relatively narrow numerical value models exactly herein It encloses.

Value as described herein should not be construed as being strictly defined as the accurate numerical value.On the contrary, unless otherwise specifically It is bright, the described value value and the on weekly duty function enclosed equivalent range described respectively for finger.For example, being disclosed as the value meaning of " 20 μm " At expression " about 20 μm ".

Unless expressly excluded or other modes limit, otherwise every document recited in this paper, including any intersection is drawn With with relevant patent or application, be incorporated into this article by reference.The reference of any document is not to it as disclosed herein Or claimed any invention the prior art or its individually or with any other bibliography Any combination or reference, proposition, suggestion or the approval for disclosing any such invention.In addition, when any of the terms contains When justice or definition and any meaning or definition contradiction of term in file incorporated by reference, tax herein should be obeyed Give the meaning or definition of the term.

Although the specific embodiment of the disclosure has been illustrated and described, spirit and scope of the present disclosure are not being departed from In the case where, various other changes and modification can be carried out.Scope of the appended claims include the institute within the scope of the disclosure There are these to change and modify.

Embodiment

In order to more effectively understand invention disclosed herein, embodiment is provided below.It should be appreciated that these embodiments It is for illustration purposes only, should not be construed as limiting the invention in any way.Unless otherwise stated, running through these embodiments Molecular cloning reaction and other standard recombinant dna technologies, according to Maniatis etc., " molecular cloning-laboratory manual " (Molecular Cloning-A Laboratory Manual), second edition, Cold Spring Harbor Publications (Cold Spring Harbor Press) method described in (1989) carries out, using commercially available reagent.

The ex vivo genetic of embodiment 1:T cell is modified

Using piggyBac^TM(PB) Transposon System carries out genetic modification to Human Lymphocytes, for Autologous CAR-T immunization therapy and other application production.Using Plasmid DNA transposons and transposase, to be purified from blood samples of patients or The T lymphocyte of serum method product carries out electroporation.The T cell that several difference electric perforating systems have been used for Transposon System is passed It send, including Neon (match Mo Feishier company (Thermo Fisher)), (Harvard Apparatus company (Harvard of BTX ECM 830 Apparatus)), Gene Pulser (radiation company (BioRad)), MaxCyte PulseAgile (mikey Saite company And Amaxa 2B and Amaxa 4D (Lonza Inc. (Lonza)) (MaxCyte)).Some electricity for providing or recommending using manufacturer Electroporation buffer and several independently developed buffer tests.As a result be especially difficult to resting T lymphocytes DNA transfection and seemingly Generally recognizing there are inversely related between electroporation efficiency (expressing measurement by the GFP of the plasmid from electroporation) and cell viability Know consistent.Fig. 1 shows the embodiment for testing the experiment of multiple electric perforating system and nuclear transfection program.

Whether there is toxicity to T cell in order to further test Plasmid DNA during nuclear transfection, primary people T lymph is thin Two kinds of different DNA plasmid electroporations of born of the same parents.First plasmid is pmaxGFP^TMPlasmid, in Lonza Amaxa nuclear transfection kit It is middle to be provided as control plasmid.It is highly purified by HPLC, and is free of the endotoxin of detectable level.Second plasmid for we from The PB transposons that row generates, the driving GFP of encoding human EF1 α promoter.Measurement is expressed by the GFP of the plasmid from electroporation Transfection efficiency and cell viability, assessed by FACS within the 2nd, 3 and 6 day after electroporation.Data are shown in Fig. 2.Although cut-off electricity The 6th day after perforation, simulate electroporation cell (plasmid-free DNA) show relative high levels cell viability, 54%, use is any The T cell vigor of plasmid electroporation is only 1.4-2.6%.These data show that Plasmid DNA has cell toxicant for T lymphocyte Property.In addition, these data show DNA mediate toxicity and be not attributed to transposable elements (such as the region ITR or core insulation Son), because of pmaxGFP^TMPlasmid lacks these elements and is equally cytotoxicity in identical DNA concentration.Two kinds of plasmids it is big It is small roughly the same, indicate that the DNA of analog quantity enters T cell by electroporation.

In order to test whether toxicity that DNA in T cell is mediated is dose-dependent, our PB-GFP plasmids to us It is titrated.Fig. 3 is shown, with dosage escalation (1.3,2.5,5.0,10.0 and of the Plasmid DNA to nuclear transfection reaction addition The Plasmid DNA of 20.0 μ g), cell viability decline, such as detection in the 1st day after nuclear transfection and the 5th day.End the 4th day, very Plasmid DNA to 1.3 μ g causes 2.4 times of T cell vigor to decline.

, it is clear that Plasmid DNA has toxicity for T cell during nuclear transfection, so we consider periplasms Whether grain DNA causes cell death.Fig. 4 shows extracellular Plasmid DNA for T cell no cytotoxicity.In this experiment, electroporation The Plasmid DNA of 5 μ g is added within 45 minutes to cell afterwards, and on day 1 or the 4th day almost without observing cell death.Similarly, When reacting the Plasmid DNA of 5 μ g of addition to nuclear transfection in the case where electroporation is not present, almost without cell death is observed. However, cell shows that 2.0 times of cell viability are reduced on day 1 when Plasmid DNA is added before electroporation reaction, and The 13.2 times of reductions of display in 4th day.

Because the toxicity that DNA is mediated be it is dose-dependent, next we are conceived to reduce is delivered to the swivel base of T cell The mode of required DNA total amount.Realize that a kind of relatively straightforward mode of the purpose is to be encoded to mRNA rather than is encoded to DNA Form deliver transposase.The delivering of from mRNA to primary human T-Cells are very efficient, obtain high transfection efficiency and high vigor.We By Super piggyBac^TM(sPBo) transposase subclone enters us and voluntarily grinds the mRNA generation carrier obtained and produce high-quality The sPBo mRNA of amount.PB-GFP transposons and the sPBo mRNA (30,10,3.3,3,1,0.33 μ g mRNA) of various dose exist The strong swivel base (Fig. 5) being shown under all dosage of test is delivered jointly in Jurkat cell.These data show that sPBo turns Seat enzyme can be delivered and have the effect of being equal with Plasmid DNA or mRNA.In addition, the amount of sPBo mRNA is in Jurkats Almost without generation difference (for the MFI that totality GFP+ cell percentages or GPF are expressed) in terms of overall transposition efficiency.In order to Whether research is also in this way, we are by PB-GFP and sBPo Plasmid DNA (3:1 ratio) or the sPBo of 5 μ g for T lymphocyte MRNA is delivered.Seven (7) days after nuclear transfection reaction and IL7 and IL15 addition, assess GFP swivel base.Fig. 6 shows sPBo MRNA has efficiently mediated swivel base of the GFP transposons into T lymphocyte.Importantly, working as in the form of mRNA (relative to pDNA) When common delivering sPBo, T cell vigor is improved；Respectively 32.4% comparison 25.4%.These are statistics indicate that mRNA form SPBo it is common delivering be dosage saving (dose-sparing) in the total amount for the Plasmid DNA for being delivered to T cell, thus With relatively low cell toxicant.

Because current plasmid transposons also includes that plasmid expands required main chain in bacterium, can be by excluding the sequence To substantially reduce DNA total amount.This can be realized by carrying out restriction digest to plasmid transposons before nuclear transfection is reacted. In addition, this can be by giving the transposons or doggybone of PCR product form^TM(it is by excluding the plasmid to DNA form The mechanism of initial main chain element needed for bacterium duplication and the double-stranded DNA that generates in vitro) transposons realize.

We conducted preliminary experiments, and to study, whether plasmid transposons needs to be ring-shaped or whether it can be in linear form It is delivered to cell.For this purpose, transposons and restriction enzyme (ApaLI) are incubated overnight so that the plasmid linearization.It has not been cut Or the plasmid of linearisation enters primary T lymphocyte by electroporation, and in assessment GFP expression two days later.Fig. 7 display linearisation Plasmid is also by high-efficiency delivery to nucleus.These data show that linear transposons product can also enter original by efficiently electroporation For human T-cell.

Plasmid DNA has been illustrated above in we has toxicity, but it is observed that the poison in primary T lymphocyte Property act in tumor cell line and other transformed cells there is no so acutely.Based on the observation result, it will be assumed that primary T Lymphocyte is likely difficult to carry out plasmid DNA transfection, this is attributed to the DNA induction approach of raising, will protect immunocyte From virus and bacterium infection.If these data be the DNA induction mechanism improved as a result, possibly through to core if so Reaction addition DNA incudes approach restrainer to enhance plasmid transfection efficiency and/or cell viability after transfection.Therefore, suppression is tested A variety of different reagents of approach those of involved in TLR-9 approach processed, Guang winter enzymatic pathway or the induction of cytoplasm double-stranded DNA.These examinations Agent includes bar bifilomycin A1, and the autophagy for interference interference endosomal acidification and the NFkB signal transduction of retardance TLR9 inhibits Agent, chloroquine is TLR9 antagonist, and quinacrine is TLR9 antagonist and cGAS antagonist, AC-YVAD-CMK, is target To 1 inhibitor of Guang winter enzyme of AIM2 approach, Z-VAD-FMK is general caspase inhibitor, Z-IETD-FMK, for by TLR9 8 inhibitor of Guang winter enzyme of approach triggering.In addition, we also pass through addition cell factor IL7 and IL15, and addition AntiCD3 McAb resists CD28Human T-Expander CD3/CD28 pearl tests the stimulation to the T cell of electroporation.As a result See Fig. 8.It was found that the 4th day after nuclear transfection, these compounds or caspase inhibitor are for cell under proof load Vigor is almost without any positive effect.However, it is appreciated that further dose study may be needed preferably to test this A little reagents.It can also be more effectively from genetically inhibiting these approach.Two nuclear transfection postconditions enhance T cell vigor really. When with only import plasmid transposons and the case where without extra process compared with, IL7 and IL15 is added, no matter they are 1 after electroporation Hour or 1 day add, and make vigor enhancing more than 3 times.In addition, using activator or augmentor (expander) pearl to nuclear transfection T cell afterwards carries out stimulation and has been significantly enhanced T cell vigor；Compared to for the case where being added for 2 days later, pearl is in core After transfection 1 hour or 1 day addition stimulation more preferably.Finally, we are also tested for ROCK inhibitor and dead cell from culture Removal (using the dead cell removal kit from U.S.A day Ni (Miltenyi)), but do not observe the improvement of cell viability.

In order to after these displays are to nuclear transfection T cell stimulate made in the discovery result that can improve vigor it is further Extension, we repeat the research using the addition of cell factor IL7 and IL15.Fig. 9 is shown, compared with non-treated situation, After nuclear transfection at once or 1 hour after, adding these cell factors with each 20ng/mL dosage keeps cell viability enhancing high Up to 2.9 times.It also invigorates adding within 1 day these cell factors after nuclear transfection, but is not so good as to tie at prior point Fruit is strong like that.

As we have found that cell viability can be increased by carrying out stimulation at once to T cell after nuclear transfection, it is assumed that in core The preceding stimulation cell of transfection can also invigorate and transfection efficiency.For this purpose, we stimulate for 2,3 or 4 days before transposons nuclear transfection Primary T lymphocyte.Figure 10 shows, delivers transposons and transposase after stimulation T cell jointly before nuclear transfection is reacted In situation, there is the swivel base of some levels.The effect of stimulation, may stimulated dynamic (dynamical) influence in advance, it is thus possible to according to The exact type of augmentor selected by Lai Yu (expander) technology.

Sequence table

<110>Bo Saiyida acology limited liability company (Poseida Therapeutics, Inc.)

<120>Transposon System and application method

<130> POTH-007001WO

<150> US 62/300,387

<151> 2016-02-26

<160> 13

<170> PatentIn version 3.5

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Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Ser Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Ser Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Asn Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 2

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Met Gly Lys Ser Lys Glu Ile Ser Gln Asp Leu Arg Lys Lys Ile Val

1 5 10 15

Asp Leu His Lys Ser Gly Ser Ser Leu Gly Ala Ile Ser Lys Arg Leu

20 25 30

Lys Val Pro Arg Ser Ser Val Gln Thr Ile Val Arg Lys Tyr Lys His

35 40 45

His Gly Thr Thr Gln Pro Ser Tyr Arg Ser Gly Arg Arg Arg Tyr Leu

50 55 60

Ser Pro Arg Asp Glu Arg Thr Leu Val Arg Lys Val Gln Ile Asn Pro

65 70 75 80

Arg Thr Thr Ala Lys Asp Leu Val Lys Met Leu Glu Glu Thr Gly Thr

85 90 95

Lys Val Ser Ile Ser Thr Val Lys Arg Val Leu Tyr Arg His Asn Leu

100 105 110

Lys Gly Arg Ser Ala Arg Lys Lys Pro Leu Leu Gln Asn Arg His Lys

115 120 125

Lys Ala Arg Leu Arg Phe Ala Thr Ala His Gly Asp Lys Asp Arg Thr

130 135 140

Phe Trp Arg Asn Val Leu Trp Ser Asp Glu Thr Lys Ile Glu Leu Phe

145 150 155 160

Gly His Asn Asp His Arg Tyr Val Trp Arg Lys Lys Gly Glu Ala Cys

165 170 175

Lys Pro Lys Asn Thr Ile Pro Thr Val Lys His Gly Gly Gly Ser Ile

180 185 190

Met Leu Trp Gly Cys Phe Ala Ala Gly Gly Thr Gly Ala Leu His Lys

195 200 205

Ile Asp Gly Ile Met Arg Lys Glu Asn Tyr Val Asp Ile Leu Lys Gln

210 215 220

His Leu Lys Thr Ser Val Arg Lys Leu Lys Leu Gly Arg Lys Trp Val

225 230 235 240

Phe Gln Met Asp Asn Asp Pro Lys His Thr Ser Lys Val Val Ala Lys

245 250 255

Trp Leu Lys Asp Asn Lys Val Lys Val Leu Glu Trp Pro Ser Gln Ser

260 265 270

Pro Asp Leu Asn Pro Ile Glu Asn Leu Trp Ala Glu Leu Lys Lys Arg

275 280 285

Val Arg Ala Arg Arg Pro Thr Asn Leu Thr Gln Leu His Gln Leu Cys

290 295 300

Gln Glu Glu Trp Ala Lys Ile His Pro Thr Tyr Cys Gly Lys Leu Val

305 310 315 320

Glu Gly Tyr Pro Lys Arg Leu Thr Gln Val Lys Gln Phe Lys Gly Asn

325 330 335

Ala Thr Lys Tyr

340

<210> 3

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Met Gly Lys Ser Lys Glu Ile Ser Gln Asp Leu Arg Lys Arg Ile Val

1 5 10 15

Asp Leu His Lys Ser Gly Ser Ser Leu Gly Ala Ile Ser Lys Arg Leu

20 25 30

Ala Val Pro Arg Ser Ser Val Gln Thr Ile Val Arg Lys Tyr Lys His

35 40 45

His Gly Thr Thr Gln Pro Ser Tyr Arg Ser Gly Arg Arg Arg Tyr Leu

50 55 60

Ser Pro Arg Asp Glu Arg Thr Leu Val Arg Lys Val Gln Ile Asn Pro

65 70 75 80

Arg Thr Thr Ala Lys Asp Leu Val Lys Met Leu Glu Glu Thr Gly Thr

85 90 95

Lys Val Ser Ile Ser Thr Val Lys Arg Val Leu Tyr Arg His Asn Leu

100 105 110

Lys Gly His Ser Ala Arg Lys Lys Pro Leu Leu Gln Asn Arg His Lys

115 120 125

Lys Ala Arg Leu Arg Phe Ala Thr Ala His Gly Asp Lys Asp Arg Thr

130 135 140

Phe Trp Arg Asn Val Leu Trp Ser Asp Glu Thr Lys Ile Glu Leu Phe

145 150 155 160

Gly His Asn Asp His Arg Tyr Val Trp Arg Lys Lys Gly Glu Ala Cys

165 170 175

Lys Pro Lys Asn Thr Ile Pro Thr Val Lys His Gly Gly Gly Ser Ile

180 185 190

Met Leu Trp Gly Cys Phe Ala Ala Gly Gly Thr Gly Ala Leu His Lys

195 200 205

Ile Asp Gly Ile Met Asp Ala Val Gln Tyr Val Asp Ile Leu Lys Gln

210 215 220

His Leu Lys Thr Ser Val Arg Lys Leu Lys Leu Gly Arg Lys Trp Val

225 230 235 240

Phe Gln His Asp Asn Asp Pro Lys His Thr Ser Lys Val Val Ala Lys

245 250 255

Trp Leu Lys Asp Asn Lys Val Lys Val Leu Glu Trp Pro Ser Gln Ser

260 265 270

Pro Asp Leu Asn Pro Ile Glu Asn Leu Trp Ala Glu Leu Lys Lys Arg

275 280 285

Val Arg Ala Arg Arg Pro Thr Asn Leu Thr Gln Leu His Gln Leu Cys

290 295 300

Gln Glu Glu Trp Ala Lys Ile His Pro Asn Tyr Cys Gly Lys Leu Val

305 310 315 320

Glu Gly Tyr Pro Lys Arg Leu Thr Gln Val Lys Gln Phe Lys Gly Asn

325 330 335

Ala Thr Lys Tyr

340

<210> 4

<211> 1053

<212> PRT

<213>artificial sequence

<220>

<223>construct is synthesized

<400> 4

Met Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val

1 5 10 15

Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly

20 25 30

Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg

35 40 45

Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile

50 55 60

Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His

65 70 75 80

Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu

85 90 95

Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu

100 105 110

Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr

115 120 125

Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala

130 135 140

Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys

145 150 155 160

Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr

165 170 175

Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln

180 185 190

Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg

195 200 205

Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys

210 215 220

Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe

225 230 235 240

Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr

245 250 255

Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn

260 265 270

Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe

275 280 285

Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu

290 295 300

Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys

305 310 315 320

Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr

325 330 335

Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala

340 345 350

Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu

355 360 365

Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser

370 375 380

Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile

385 390 395 400

Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala

405 410 415

Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln

420 425 430

Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro

435 440 445

Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile

450 455 460

Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg

465 470 475 480

Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys

485 490 495

Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr

500 505 510

Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp

515 520 525

Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu

530 535 540

Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro

545 550 555 560

Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys

565 570 575

Gln Glu Glu Ala Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu

580 585 590

Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile

595 600 605

Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu

610 615 620

Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp

625 630 635 640

Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu

645 650 655

Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys

660 665 670

Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp

675 680 685

Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp

690 695 700

Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys

705 710 715 720

Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys

725 730 735

Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu

740 745 750

Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp

755 760 765

Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile

770 775 780

Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu

785 790 795 800

Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu

805 810 815

Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His

820 825 830

Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly

835 840 845

Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr

850 855 860

Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile

865 870 875 880

Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp

885 890 895

Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr

900 905 910

Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val

915 920 925

Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser

930 935 940

Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala

945 950 955 960

Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly

965 970 975

Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile

980 985 990

Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met

995 1000 1005

Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys

1010 1015 1020

Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu

1025 1030 1035

Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly

1040 1045 1050

<210> 5

<211> 88

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 5

Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser Leu

1 5 10 15

Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu Ile

20 25 30

Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr Val

35 40 45

Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly Thr

50 55 60

Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg Ser Asn

65 70 75 80

Pro Leu Ser Ala Glu Phe Thr Thr

85

<210> 6

<211> 90

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 6

Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp

1 5 10 15

Ser Leu Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe

20 25 30

Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu

35 40 45

Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro

50 55 60

Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg

65 70 75 80

Ser Asn Pro Leu Ser Ala Glu Phe Thr Thr

85 90

<210> 7

<211> 270

<212> DNA

<213>homo sapiens (Homo sapiens)

<400> 7

atgctgcctg caccaaagaa cctggtggtg tctcatgtga cagaggatag tgccagactg 60

tcatggactg ctcccgacgc agccttcgat agttttatca tcgtgtaccg ggagaacatc 120

gaaaccggcg aggccattgt cctgacagtg ccagggtccg aacgctctta tgacctgaca 180

gatctgaagc ccggaactga gtactatgtg cagatcgccg gcgtcaaagg aggcaatatc 240

agcttccctc tgtccgcaat cttcaccaca 270

<210> 8

<211> 4

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 8

Thr Glu Asp Ser

1

<210> 9

<211> 7

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 9

Thr Ala Pro Asp Ala Ala Phe

1 5

<210> 10

<211> 6

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 10

Ser Glu Lys Val Gly Glu

1 5

<210> 11

<211> 4

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 11

Gly Ser Glu Arg

1

<210> 12

<211> 5

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 12

Gly Leu Lys Pro Gly

1 5

<210> 13

<211> 7

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 13

Lys Gly Gly His Arg Ser Asn

1 5

Claims

1. A method for ex vivo genetic modification of immune cells comprising delivering to said immune cells,

(a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase, and

(b) A recombinant and non-naturally occurring DNA sequence comprising a DNA sequence encoding a transposon.

2. The method of claim 1, wherein the sequence encoding the transposase is an mRNA sequence.

3. The method of claim 1, wherein the sequence encoding the transposase is a DNA sequence.

4. The method of claim 1, wherein the sequence encoding the transposase is an amino acid sequence.

5. The method of any one of claims 1-4, wherein the delivering step comprises electroporation or nucleofection of the immune cells.

6. The method of any one of claims 1-5, wherein the method further comprises the step of stimulating the immune cells with one or more cytokines.

7. The method of claim 6, wherein the step of stimulating the immune cells with one or more cytokines occurs after the delivering step.

8. The method of claim 6, wherein the step of stimulating the immune cells with one or more cytokines occurs prior to the delivering step.

9. The method of any one of claims 6-8, wherein the one or more cytokines comprise IL-2, IL-21, IL-7 and/or IL-15.

10. The method of any preceding claim, wherein the immune cells are autologous immune cells.

11. The method of any one of claims 1-10, wherein the immune cells are T lymphocytes.

12. The method of any one of claims 1-10, wherein the immune cells are natural killer (NK) cells.

13. The method of any one of claims 1-10, wherein the immune cells are cytokine-induced killer (CIK) cells.

14. The method of any one of claims 1-10, wherein the immune cells are natural killer T (NKT) cells.

15. The method of any one of claims 2 and 5-14, wherein the mRNA sequence encoding the transposase is generated in vitro.

16. The method of any preceding claim, wherein the transposase is a Super piggyBac ^™ (sPBo) transposase.

17. The method of claim 16, wherein the Super piggyBac (PB) transposase comprises an amino acid sequence that is at least 75% identical to:

MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF(SEQ ID NO:1)。

18. The method of any one of claims 1-15, wherein the transposase is a Sleeping Beauty transposase.

19. The method of claim 18, wherein the Sleeping Beauty transposase is a high activity Sleeping Beauty SB100X transposase.

20. The method of claim 18 or 19, wherein the Sleeping Beauty transposase comprises an amino acid sequence that is at least 75% identical to:

MGKSKEISQDLRKKIVDLHKSGSSLGAISKRLKVPRSSVQTIVRKYKHHGTTQPSYRSGRRRYLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGRSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMRKENYVDILKQHLKTSVRKLKLGRKWVFQMDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPTYCGKLVEGYPKRLTQVKQFKGNATKY(SEQ ID NO:2)。

21. The method of claim 18 or 19, wherein the Sleeping Beauty transposase comprises an amino acid sequence that is at least 75% identical to:

MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRSGRRRYLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY(SEQ ID NO:3)。

22. The method of any one of the preceding claims, wherein the recombination and non-naturally occurring DNA sequence comprising the DNA sequence encoding the transposon is circular.

23. The method of claim 22, wherein the recombinant non-naturally occurring DNA sequence encoding the transposon is contained in a plasmid vector.

24. The method of claim 22, wherein the recombinant non-naturally occurring DNA sequence encoding the transposon is contained in a minicircle DNA vector.

25. The method of any one of claims 1-21, wherein the recombinant, non-naturally occurring DNA sequence encoding the transposon is linear.

26. The method of any preceding claim, wherein the recombinant non-naturally occurring DNA sequence encoding the transposon is generated in vitro.

27. The method of claim 25 or 26, wherein the recombinant non-naturally occurring DNA sequence encoding the transposon is the product of restriction digestion of circular DNA.

28. The method of claim 27, wherein the circular DNA is a plasmid vector or a minicircular DNA vector.

29. The method of claim 25 or 26, wherein the recombinant non-naturally occurring DNA sequence encoding the transposon is the product of a polymerase chain reaction (PCR).

30. The method of claim 25 or 26, wherein the recombinant, non-naturally occurring DNA sequence encoding the transposon is a double-stranded doggybone ^™ DNA sequence.

31. The method of claim 30, wherein the doggybone ^(TM) DNA sequence is produced enzymatically by encoding only an antigen expression cassette comprising an antigen, a promoter, a poly A tail and telomeric ends.

32. The method of any preceding claim, wherein the immune cells are isolated or derived from a human.

33. The method of any one of claims 1-31, wherein the immune cells are isolated or derived from a non-human mammal.

34. The method of claim 33, wherein the non-human mammal is a rodent, rabbit, cat, dog, pig, horse, cow, camel or primate.

35. The method of any preceding claim, wherein the recombinant non-naturally occurring DNA sequence encoding the transposon further comprises a sequence encoding a chimeric antigen receptor or a portion thereof.

36. The method of claim 35, wherein the portion of the sequence encoding the chimeric antigen receptor encodes an antigen recognition region.

37. The method of claim 35 or 36, wherein the antigen recognition region comprises one or more complementarity determining regions.

38. The method of claim 35 or 36, wherein the antigen recognition region comprises an antibody, antibody mimetic, protein scaffold or fragment thereof.

39. The method of claim 38, wherein the antibody is a chimeric, recombinant, humanized, or human antibody.

40. The method of claim 39, wherein the antibody is affinity modulated.

41. The method of claim 38, wherein the antibody comprises a single chain variable fragment (scFv), VHH, single domain antibody (sdAB), small modular immunopharmaceutical (SMIP) molecule or nanobody, or is composed of composition.

42. The method of claim 41, wherein the VHH is camelid.

43. The method of claim 41 or 42, wherein the VHH is humanized.

44. The method of claim 38, wherein the antibody fragment comprises or consists of a complementarity determining region, a variable region, a heavy chain, a light chain, or any combination thereof.

45. The method of claim 38, wherein the antibody mimetic comprises Affimer, Afflilin molecule, Adhesin, Affitin molecule, Alphabody, Anticalin, Avimer molecule, DARPin, Fynomer, Kunitz domain Peptides or monomers, or consisting of them.

46. The method of claim 38, wherein the protein scaffold comprises, or consists of, Centyrin.

47. The method of any one of claims 5-46,

(a) wherein the nucleic acid sequence encoding the transposase is a DNA sequence, and

(b) An electroporation or nucleofection reaction in which the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon are equal to or less than 10 μg/100 μL.

48. The method of claim 48, wherein, in the electroporation or nucleofection reaction, the concentration of the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon is equal to or less than 100 μg/ mL.

49. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction wherein the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence is equal to or less than 7.5 μg/100 μL.

50. The method of claim 49, wherein, in the electroporation or nucleofection reaction, the concentration of the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon is equal to or less than 75 μg/ mL.

51. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction wherein the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence is equal to or less than 6.0 μg/100 μL.

52. The method of claim 51, wherein, in the electroporation or nucleofection reaction, the concentration of the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon is equal to or less than 60 μg/ mL.

53. The method of claim 50 or 51, wherein the transposase is a Sleeping Beauty transposase.

54. The method of claim 53, wherein the Sleeping Beauty transposase is Sleeping Beauty 100X (SB100X) transposase.

55. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction wherein the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence is equal to or less than 5.0 μg/100 μL.

56. The method of claim 55, wherein, in the electroporation or nucleofection reaction, the concentration of the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon is equal to or less than 50 μg/ mL.

57. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction wherein the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence is equal to or less than 2.5 μg/100 μL.

58. The method of claim 57, wherein, in the electroporation or nucleofection reaction, the concentration of the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon is equal to or less than 25 μg/ mL.

59. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction wherein the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence is equal to or less than 1.67 μg/100 μL.

60. The method of claim 59, wherein, in the electroporation or nucleofection reaction, the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence are at a concentration equal to or less than 16.7 μg /mL.

61. The method of claim 59 or 60, wherein the transposase is a Super piggyBac (PB) transposase.

62. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction wherein the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence is equal to or less than 0.55 μg/100 μL.

63. The method of claim 62, wherein, in the electroporation or nucleofection reaction, the concentration of the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon is equal to or less than 5.5 μg /mL.

64. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction wherein the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence is equal to or less than 0.19 μg/100 μL.

65. The method of claim 64, wherein, in the electroporation or nucleofection reaction, the concentration of the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon is equal to or less than 1.9 μg /mL.

66. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction wherein the amount of transposase-encoding DNA sequence and the transposon-encoding DNA sequence is equal to or less than 0.1 μg/100 μL.

67. The method of claim 66, wherein, in the electroporation or nucleofection reaction, the concentration of the amount of DNA sequence encoding transposase and the amount of DNA sequence encoding transposon is equal to or less than 1.0 μg /mL.

68. The method of any one of claims 5-46,

(a) wherein the nucleic acid sequence encoding the transposase is an RNA sequence, and

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 10 μg/100 μL.

69. The method of claim 68, wherein the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 100 μg/mL in an electroporation or nucleofection reaction.

70. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 7.5 μg/100 μL.

71. The method of claim 70, wherein in an electroporation or nucleofection reaction, the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 75 μg/mL.

72. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 6.0 μg/100 μL.

73. The method of claim 72, wherein in the electroporation or nucleofection reaction, the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 60 μg/mL.

74. The method of claim 72 or 73, wherein the transposase is a Sleeping Beauty transposase.

75. The method of claim 74, wherein the Sleeping Beauty transposase is Sleeping Beauty 100X (SB100X) transposase.

76. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 5.0 μg/100 μL.

77. The method of claim 76, wherein in the electroporation or nucleofection reaction, the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 50 μg/mL.

78. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 2.5 μg/100 μL.

79. The method of claim 78, wherein in an electroporation or nucleofection reaction, the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 25 μg/mL.

80. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 1.67 μg/100 μL.

81. The method of claim 80, wherein in an electroporation or nucleofection reaction, the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 16.7 μg/mL.

82. The method of claim 80 or 81, wherein the transposase is a Super piggyBac (PB) transposase.

83. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 0.55 μg/100 μL.

84. The method of claim 83, wherein in an electroporation or nucleofection reaction, the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 5.5 μg/mL.

85. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 0.19 μg/100 μL.

86. The method of claim 85, wherein the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 1.9 μg/mL in an electroporation or nucleofection reaction.

87. The method of any one of claims 5-46,

(b) An electroporation or nucleofection reaction in which the DNA sequence encoding the transposon is equal to or less than 0.1 μg/100 μL.

88. The method of claim 87, wherein in an electroporation or nucleofection reaction, the amount of DNA sequence encoding the transposon is at a concentration equal to or less than 1.0 μg/mL.

89. An immune cell modified by the method of any one of claims 1-88.

90. The immune cell of claim 89, wherein the immune cell is a T-lymphocyte, a natural killer (NK) cell, a cytokine-induced killer (CIK) cell, or a natural killer T (NKT) cell.

91. The immune cell of claim 89 or 90, further modified with a second gene editing tool.

92. The immune cell of claim 91, wherein the second gene editing tool comprises an endonuclease operably linked to a Cas9 or TALE sequence.

93. The immune cell of claim 92, wherein the endonuclease is covalently operably linked to a Cas9 or TALE sequence.

94. The immune cell of claim 92, wherein the endonuclease is operably linked to a Cas9 or TALE sequence in a non-covalent manner.

95. The immune cell of any one of claims 89-94, wherein the Cas9 is inactivated Cas9 (dCas9).

96. The immune cell of claim 95, wherein the inactivated Cas9 comprises D10A and N580A in the catalytic site.

97. The immune cell of claim 95 or 96, wherein the Cas9 is small and inactive Cas9 (dSaCas9).

98. The immune cell of claim 97, wherein the dSaCas9 comprises the amino acid sequence:

mkrnyilglaigitsvgygiidyetrdvidagvrlfkeanvennegrrskrgarrlkrrrrhriqrvkkllfdynlltdhselsginpyearvkglsqklseeefsaallhlakrrgvhnvneveedtgnelstkeqisrnskaleekyvaelqlerlkkdgevrgsinrfktsdyvkeakqllkvqkayhqldqsfidtyidlletrrtyyegpgegspfgwkdikewyemlmghctyfpeelrsvkyaynadlynalndlnnlvitrdenekleyyekfqiienvfkqkkkptlkqiakeilvneedikgyrvtstgkpeftnlkvyhdikditarkeiienaelldqiakiltiyqssediqeeltnlnseltqeeieqisnlkgytgthnlslkainlildelwhtndnqiaifnrlklvpkkvdlsqqkeipttlvddfilspvvkrsfiqsikvinaiikkyglpndiiielareknskdaqkminemqkrnrqtnerieeiirttgkenakyliekiklhdmqegkclysleaipledllnnpfnyevdhiiprsvsfdnsfnnkvlvkqeeaskkgnrtpfqylsssdskisyetfkkhilnlakgkgrisktkkeylleerdinrfsvqkdfinrnlvdtryatrglmnllrsyfrvnnldvkvksinggftsflrrkwkfkkernkgykhhaedaliianadfifkewkkldkakkvmenqmfeekqaesmpeieteqeykeifitphqikhikdfkdykyshrvdkkpnrelindtlystrkddkgntlivnnlnglydkdndklkklinkspekllmyhhdpqtyqklklimeqygdeknplykyyeetgnyltkyskkdngpvikkikyygnklnahlditddypnsrnkvvklslkpyrfdvyldngvykfvtvknldvikkenyyevnskcyeeakklkkisnqaefiasfynndlikingelyrvigvnndllnrievnmidit yreylenmndkrppriiktiasktqsikkystdilgnlyevkskkhpqiikkg (SEQ ID NO: 4).

99. A composition comprising the immune cell of any one of claims 89-98.

100. Use of the composition of claim 99 for treating a disease or disorder in a subject in need thereof.

101. The use of claim 100, wherein the disease or disorder is cancer.

102. The use of claim 100, wherein the disease or disorder is an infectious disease.

103. The use of any one of claims 99-102, wherein the immune cells are autologous.

104. The use of any one of claims 99-102, wherein the immune cells are allogeneic.

105. A medium for enhancing the viability of modified immune cells comprising IL-2, IL-21, IL-7, IL-15, or any combination thereof.

106. The culture medium of claim 105, wherein the modified immune cells are T-lymphocytes, natural killer (NK) cells, cytokine-induced killer (CIK) cells or natural killer T (NKT) cells cell.

107. The medium of claim 105 or 106, wherein the modified immune cells comprise one or more exogenous DNA sequences.

108. The medium of claim 105 or 106, wherein the modified immune cells comprise one or more exogenous RNA sequences.

109. The medium of any one of claims 105-108, wherein the modified immune cells have been electroporated or nucleofected.