KR20180131545A - Transposon systems and methods of use - Google Patents

Abstract

A method for genetically modifying an immune cell in vitro comprising the steps of (a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzyme, and (b) a recombinant comprising a DNA sequence encoding a transposon And delivering the non-naturally occurring DNA sequence to the immune cell.

Description

Transposon systems and methods of use

Related application

This application claims priority to U.S. Provisional Patent Application No. 62 / 300,387, filed February 26, 2016, the content of which is incorporated herein by reference in its entirety.

Introduction of Sequence Listing

The content of a text file named " POTH-007001WO_SeqList.txt ", which was created on February 24, 2017 and is 26 KB in size, is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to compositions and methods for targeted gene modification.

It has been very difficult to genetically transform untranslated primary human T lymphocytes in vitro using a non-viral vector-based gene transfer delivery system. As a result, the majority of researchers have used viral vector-based transduction, including retroviruses, which typically involve lentiviruses. Many non-viral methods have been tested and include antibody targeted liposomes, nanoparticles, aptamer siRNA chimeras, electroporation, nucleofection, lipid infections and peptide transduction. Taken together, these methods have resulted in poor transfection efficiency, direct cytotoxicity or lack of experimental throughput.

The use of plasmid vectors for genetic modification of human lymphocytes has been limited by the low efficiency of use of currently available plasmid transfection systems and by the toxicity of many plasmid transfection reagents to these cells. There is a long and unmet need for a method of non-viral genetic modification in immune cells.

The transfer of transgene through DNA transposons, such as piggyBac and Sleeping Beauty, when compared to viral transduction of immune cells, such as T lymphocytes, may be beneficial in terms of ease of use, ability to deliver much larger loads , Clinical use rate and production cost. In particular, the piggyBac DNA transposon provides additional advantages in that it provides long-term, high-level and stable expression of the transgene and has a significantly lower mutagenicity than retrovirus and is not carcinogenic and is entirely reversible. Prior art methods have been inefficient and conventional attempts to transfer the transgene to T cells using DNA transposons have not been successful in producing commercially viable products or manufacturing methods. For example, the poor efficiency demonstrated by conventional methods of transferring a transgene to a T cell using a DNA transposon has led to the need for extended amplification in vitro. Previously unsuccessful attempts by other researchers to address this problem have focused on increasing the amount of DNA transposons delivered to immune cells, a well-established strategy for non-immune cells. This disclosure demonstrates that increasing the amount of DNA transposons further exacerbates the efficiency problem in immune cells by increasing DNA mediated toxicity. To solve this problem, semi-intuitively, the method of the present disclosure reduces the amount of DNA transferred to immune cells. Using the methods of this disclosure, the data provided herein suggests that not only reducing the amount of DNA transposons introduced into cells increases the survival rate, but also increases the percentage of cells with transposition events, Results in viable commercial processes and viable commercial products. Thus, the method of the present disclosure demonstrates success, unlike other methods that have failed.

(A) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzyme; and (b) a DNA sequence encoding a transposon. In another aspect, the present invention provides a non-viral method for genetically altering immune cells in vitro. Lt; RTI ID = 0.0 > recombinant < / RTI > and non-naturally occurring DNA sequences to the immune cells. In some embodiments, the method further comprises stimulating the immune cells with one or more cytokine (s).

In some embodiments of the methods of this disclosure, the sequence encoding the transposase enzyme is an mRNA sequence. The mRNA sequence encoding the transposase enzyme can be generated in vitro.

In some embodiments of the methods of this disclosure, the sequence encoding the transposase enzyme is a DNA sequence. DNA sequences coding for transposase enzymes can be generated in vitro. The DNA sequence may be a cDNA sequence.

In some embodiments of the methods of this disclosure, the sequence encoding the transposon enzyme is an amino acid sequence. The amino acid sequence encoding the enzyme can be produced in vitro. After preincubation with the transposon DNA, the protein sPBo can be delivered.

In some embodiments of the methods of the disclosure, the delivery step comprises electroporation or nucleofection of the immune cell.

In some embodiments of the methods of the disclosure, the step of stimulating the immune cells with one or more cytokine (s) occurs after the delivery step. Alternatively or additionally, in some embodiments, the step of stimulating the immune cells with one or more cytokine (s) occurs prior to the delivery step. In some embodiments, the at least one cytokine (s) comprises IL-2, IL-21, IL-7 and / or IL-15.

In some embodiments of the methods of this disclosure, the immune cell is an autoimmune cell. The immune cells may be human immune cells and / or autoimmune cells. Immunocytes may be derived from a source of primary, including, but not limited to, primary cells, cultured cells or cell lines, embryonic or adult stem cells, derived differentiated pluripotent stem cells or transformed differentiated cells. Immune cells may have been previously genetically modified or may be derived from genetically modified cells or cell lines. An immune cell may be modified to inhibit one or more apoptotic pathways, or may be derived from a cell or cell line modified to inhibit such pathway. Immune cells may be modified to have " universally " homology, for example, by the majority of recipients in the context of a therapy involving adoptive cell transfer, or may be derived from cells or cell lines transformed to have such homogeneity have.

In some embodiments of the methods of this disclosure, the immune cell is an activated immune cell.

In some embodiments of the methods of this disclosure, the immune cell is a dormant immune cell.

In some embodiments of the methods of the disclosure, the immune cell is a T lymphocyte. In some embodiments, the T lymphocyte is an activated T lymphocyte. In some embodiments, the T lymphocyte is a dormant T lymphocyte.

In some embodiments of the methods of this disclosure, the immune cell is a natural killer (NK) cell.

In some embodiments of the methods of the disclosure, the immune cell is a cytokine-induced killer (CIK) cell.

In some embodiments of the methods of the disclosure, the immune cell is a natural killer T (NKT) cell.

In some embodiments of the methods of the disclosure, the immune cells are isolated or derived from a human.

In some embodiments of the methods of the disclosure, the immune cells are isolated or derived from a non-human mammal. In some embodiments, the non-human mammal is a rodent, rabbit, cat, dog, pig, horse, cattle or camel. In some embodiments, the immune cells are isolated or derived from non-human primates.

In some embodiments of the methods of this disclosure, the transposase enzyme is a Super piggyBac (TM) transposase enzyme. The super piggyBac (PB) transporter enzyme may comprise or consist of the amino acid sequence at least 75% identical to the following sequence:

In some embodiments of the methods of this disclosure, the transposase enzyme is a Sleeping Beauty transporter enzyme (see, for example, U.S. Patent No. 9,228,180, the disclosure of which is incorporated herein by reference in its entirety). In some embodiments, the Sleeping Beauty transporter is an overactive Sleeping Beauty SB100X transporter. In some embodiments, the Sleeping Beauty transporter enzyme comprises an amino acid sequence that is at least 75% identical to the following sequence:

In some embodiments, including the Sleeping Beauty transposase and the active Sleeping Beauty SB100X transposon gene embodiments, the Sleeping Beauty transposase enzyme comprises an amino acid sequence that is at least 75% identical to the following sequence:

In some embodiments of the methods of the present disclosure, the recombinant and non-naturally occurring DNA sequences comprising the DNA sequence encoding the transposon may be circular DNA sequences. As a non-limiting example, the DNA sequence encoding the transposon may be a plasmid vector. As a non-limiting example, the DNA sequence encoding the transposon may be a minicircle DNA vector.

In some embodiments of the methods of this disclosure, the recombinant and non-naturally occurring DNA sequences encoding the transposon may be linear DNA sequences. Linear recombinant and non-naturally occurring DNA sequences encoding a transposon can be generated in vitro. The linear recombinant and non-naturally occurring DNA sequences of this disclosure may be products of restriction digestion of circular DNA. In some embodiments, the circular DNA is a plasmid vector or a mini-circle DNA vector. The linear recombinant and non-naturally occurring DNA sequences of this disclosure may be products of polymerase chain reaction (PCR). The linear recombinant and non-naturally occurring DNA sequences of this disclosure may be double-stranded doggybone (TM) DNA sequences. The doggybone (TM) DNA sequences of the present disclosure can be generated by an enzymatic process that encodes only antigen-expressing cassettes containing antigens, promoters, poly-A tail and telomeric ends.

In some embodiments of the methods of the disclosure, the recombinant and non-naturally occurring DNA sequences encoding the transposon further comprise a sequence or portion thereof encoding a chimeric antigen receptor. The chimeric antigen receptor (CAR) of the present disclosure may comprise an endo domain comprising (a) an ectodomain comprising an antigen recognition region, (b) a transmembrane domain, and (c) at least one complementary stimulatory domain. In some embodiments, the ectodomain may further comprise a signal peptide. Alternatively or additionally, in some embodiments, the ecto domain may further comprise a hinge between the antigen recognition region and the transmembrane domain. In some embodiments of CARs of the disclosure, the signal peptide may comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide . In some embodiments of CARs of the disclosure, the signal peptide may comprise a sequence encoding a human CD8a signal peptide. In some embodiments, the transmembrane domain may comprise a sequence encoding human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In some embodiments of CARs of the disclosure, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain. In some embodiments of the CARs of the present disclosure, the endo domain may comprise a human CD3ζ endo domain. In some embodiments of CARs of the disclosure, the at least one complement stimulatory domain may comprise human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segments, or any combination thereof. In some embodiments of the CARs of the present disclosure, the at least one complement stimulus domain may comprise a CD28 and / or 4-1BB complement stimulus domain. In some embodiments of the CARs of the present disclosure, the hinge may comprise sequences derived from human CD8a, IgG4 and / or CD4 sequences. In some embodiments of the CARs of the present disclosure, the hinge may comprise a sequence derived from a human CD8 α sequence.

In some embodiments of the methods of the disclosure, the recombinant and non-naturally occurring DNA sequences encoding the transposon further comprise a sequence or portion thereof encoding a chimeric antigen receptor. A portion of the sequence encoding the chimeric antigen receptor may encode an antigen recognition region. The antigen recognition region may comprise one or more complementary determining region (s). The antigen recognition region may comprise an antibody, an antibody mimetic, a protein scaffold or a fragment thereof. In some embodiments, the antibody is a chimeric antibody, a recombinant antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is an affinity-adjusted antibody. Non-limiting examples of antibodies of the disclosure include single chain variable fragment (scFv), VHH, single domain antibody (sdAB), small modular immune drug (SMIP) molecule or nanobody. In some embodiments, the VHH is camelid VHH. Alternatively or additionally, in some embodiments, the VHH is a humanized VHH. Non-limiting examples of antibody fragments of the present disclosure include complementarity determining regions, variable regions, heavy chains, light chains, or any combination thereof. Non-limiting examples of antibody mimetics of the present disclosure include, but are not limited to, affibody, afflilin, affimer, affitin, alphabody, anticalin, For example, avimer, DARPin, Fynomer, Kunitz domain peptide, or monobody. A non-limiting example of a protein scaffold of this disclosure is Centyrin.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; RTI ID = 0.0 > 10 < / RTI > In some embodiments, the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than 100 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; / RTI > per μl of the fection reaction. In some embodiments, the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than 75 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; / RTI > per μl of the fission reaction. In some embodiments, the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than 60 [mu] g / ml. In some embodiments, the transporter is a Sleeping Beauty transporter. In some embodiments, the Sleeping Beauty transporter is a Sleeping Beauty 100X (SB100X) transporter.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; RTI ID = 0.0 > 5 < / RTI > In some embodiments, the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than 50 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; RTI ID = 0.0 > pg < / RTI > In some embodiments, the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than 25 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; RTI ID = 0.0 > pg / 100 < / RTI > In some embodiments, the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 16.7 [mu] g / ml. In some embodiments, the transposon agent is a Super piggyBac (PB) transposase agent.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; RTI ID = 0.0 > u. ≪ / RTI > In some embodiments, the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 5.5 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; RTI ID = 0.0 > pg / 100 < / RTI > In some embodiments, the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 1.9 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is a DNA sequence, the amount of the DNA sequence encoding the transposase enzyme, and the amount of DNA sequence encoding the transposon is selected from electroporation or nucleobase Lt; / RTI > or less per 100 mu l of the fission reaction. In some embodiments, the concentration of the DNA sequence encoding the transposon enzyme and the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than 1.0 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is less than or equal to 10.0 μg per 100 μl of electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 100 μg / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is not more than 7.5 [mu] g per 100 [mu] l electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 75 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposase enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is 6.0 쨉 g or less per 100 袖 l electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 60 [mu] g / ml. In some embodiments, the transporter is a Sleeping Beauty transporter. In some embodiments, the Sleeping Beauty transporter is a Sleeping Beauty 100X (SB100X) transporter.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is less than or equal to 5.0 μg per 100 μl of electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 50 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposase enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is less than or equal to 2.5 μg per 100 μl electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 25 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is less than or equal to 1.67 μg per 100 μl electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 16.7 μg / ml. In some embodiments, the transposon agent is a Super piggyBac (PB) transposase agent.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposase enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is less than or equal to 0.55 μg per 100 μl electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 5.5 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposase enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is less than or equal to 0.19 micrograms per 100 μl electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 1.9 [mu] g / ml.

In some embodiments of the methods of this disclosure, the nucleic acid sequence encoding the transposon enzyme is an RNA sequence and the amount of DNA sequence encoding the transposon is less than or equal to 1.0 μg per 100 μl of electroporation or nucleofection reaction. In some embodiments, the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 1.0 [mu] g / ml.

This disclosure provides immune cells modified according to the methods of this disclosure. Immune cells can be T lymphocytes, natural killer (NK) cells, cytokine-induced killer (CIK) cells or natural killer T (NKT) cells. Immune cells may be further modified by a second gene editing tool, including, but not limited to, gene editing tools, including endonuclease operably linked to Cas9 or TALE sequences. In some embodiments of the second gene editing tool, the endonuclease is covalently operably linked to a Cas9 or TALE sequence. In some embodiments of the second gene editing tool, the endonuclease is operatively linked to the Cas9 or TALE sequence non-cognitionally. In some embodiments, Cas9 is inactivated Cas9 (dCas9). In some embodiments, the inactivated Cas9 comprises D10A and N580A within the catalytic site. In some embodiments, Cas9 is a small inactivated Cas9 (dSaCas9). In some embodiments, dSaCas9 comprises the amino acid sequence:

This disclosure provides immune cells modified according to the methods of this disclosure. Immune cells can be T lymphocytes, natural killer (NK) cells, cytokine-induced killer (CIK) cells or natural killer T (NKT) cells. Immune cells may be further modified by a second gene editing tool, including, but not limited to, gene editing tools, including endogenous nucleases operably linked to Cas9 or TALE sequences. Alternatively or additionally, the second gene editing tool may comprise a resection-only piggyBac transporter to rescue the inserted sequence or any portion thereof. For example, a resection-only piggyBac transporter can be used to "resecrate" the transposon.

This disclosure provides compositions comprising the immune cells of this disclosure.

This disclosure provides a use of a composition comprising an immune cell of the present disclosure for treating a disease or disorder in a subject in need thereof. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is an infectious disease. For example, infectious diseases can be caused by viruses, bacteria, yeast, microorganisms, or any combination thereof. In some embodiments, the immune cells of the composition are autoimmune cells. In some embodiments, the immune cells of the composition are allogeneic immune cells.

The present disclosure provides a culture medium that enhances the survival rate of modified immune cells, including IL-2, IL-21, IL-7, IL-15 or any combination thereof. Modified immune cells can be T lymphocytes, natural killer (NK) cells, cytokine induced killer (CIK) cells or natural killer T (NKT) cells. Modified immune cells may contain one or more exogenous DNA sequences. Modified immune cells may comprise one or more exogenous RNA sequences. Modified immune cells may have been electroporated or have been nucleofected.

Figure 1 is a series of graphs showing transfection efficiency and cell viability after plasmid DNA nucleofection in primary human T lymphocytes.
Figure 2 is a series of graphs showing DNA cytotoxicity against T cells.
Figure 3 is a series of graphs showing that DNA-mediated cytotoxicity in T cells is dose-dependent.
Figure 4 is a series of graphs showing that extracellular plasmid DNA does not exhibit cytotoxicity.
Figure 5 is a series of graphs showing the efficient dislocation using sPBo mRNA in Jurkat cells.
Figure 6 is a series of graphs showing the efficient potentials in T lymphocytes using sPBo mRNA.
Figure 7 is a series of graphs showing the efficient delivery of the linearized DNA transposon product.
Figure 8 is a series of graphs showing the addition of IL-7 and IL-15 after nucleofection and immediate stimulation of T cells to improve cell survival.
Figure 9 is a series of graphs showing that IL-7 and IL-15 rescue T cells from DNA-mediated toxicity.
Figure 10 is a series of graphs showing that immediate stimulation of T cells after nucleofection improves cell viability.
Figures 11a-11c are a series of graphs showing T cell potential using various amounts of DNA. Primary human T cells were incubated with various amounts of DNA using piggyBac ™. T cells were subjected to nucleofection with the indicated amount of transposon and 5 [mu] g sPBO mRNA. Cells were then stimulated via CD3 and CD28 on day 2 post-nucleofection. As expected, high amounts of DNA-nucleated T cells showed high episomal expression on day 1 after nucleofection, whereas episomal expression was rarely observed at low DNA doses. In contrast, the greatest percentage of transgenic positive cells reaching apex at 1.67 [mu] g for this transposon, after amplification on day 21 after nucleofection, were observed at lower DNA amounts. (FIG. 11A) Flow analysis on day 1 and day 21 transition metastatic gene-positive cells. (Figure 11b) Percent of transgene-positive T cells. (Figure 11c) Percent of viable T cells on day 1 and day 21. For all of the graphs shown in this figure, the Y axis increases from 0% to 100%, increasing by 20%, and the X axis is from 0 to 10 ^{5 in} 10 powers.
Figures 12a and 12b are a series of graphs showing T cell potential using Sleeping Beauty ™ 100X (SB100X) transporter with low DNA amount. Primary human T cells were subjected to nucleofection with GFP plasmid encoding piggyBac ™ (PB) or Sleeping Beauty ™ ( SB ) ITR. (Fig. 12A). The cells were subjected to nucleofection with indicated amounts of SB transposon and 1 mu g SB transporter mRNA. (Fig. 12b). Cells were then subjected to nucleofection with indicated amounts of SB transporter and 0.75 占SB transposon. Flow analysis was performed on all samples on day 14 post-nucleofection. For all the graphs shown in this figure, the Y-axis increases from 50K to 50K with increments of 50K, and the X-axis extends from 0 to 10 ⁵ as a power of 10.

A composition and method for genetically modifying immune cells in vitro comprising: (a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzyme; and (b) a recombinant and non-natural product comprising a DNA sequence encoding the transposon A composition and method comprising the step of delivering a DNA sequence to an immune cell is disclosed. In some embodiments, the method further comprises stimulating the immune cells with one or more cytokine (s).

Sentinel of the present disclosure may comprise a protein scaffold that is capable of specifically binding to an antigen. Sentinel of the disclosure may comprise a protein scaffold comprising a consensus sequence of at least one fibronectin type III (FN3) domain, wherein the scaffold may specifically bind to an antigen. At least one fibronectin type III (FN3) domain can be derived from a human protein. The human protein may be tenascin-C. The consensus sequence is

또는

을 포함할 수 있다. 컨센서스 서열은

또는

or

. ≪ / RTI > The consensus sequence is

or

과 적어도 74% 동일한 아미노산 서열을 포함할 수 있다. 컨센서스 서열은 하기 서열을 포함하는 핵산 서열에 의해 코딩될 수 있다:

Lt; RTI ID = 0.0 > 74% < / RTI > The consensus sequence may be encoded by a nucleic acid sequence comprising the sequence:

컨센서스 서열은 (a) 컨센서스 서열의 위치 13 내지 16에서 아미노산 잔기 TEDS(서열번호 8)를 포함하거나 이들로 구성된 A-B 루프; (b) 컨센서스 서열의 위치 22 내지 28에서 아미노산 잔기 TAPDAAF(서열번호 9)를 포함하거나 이들로 구성된 B-C 루프; (c) 컨센서스 서열의 위치 38 내지 43에서 아미노산 잔기 SEKVGE(서열번호 10)를 포함하거나 이들로 구성된 C-D 루프; (d) 컨센서스 서열의 위치 51 내지 54에서 아미노산 잔기 GSER(서열번호 11)을 포함하거나 이들로 구성된 D-E 루프; (e) 컨센서스 서열의 위치 60 내지 64에서 아미노산 잔기 GLKPG(서열번호 12)를 포함하거나 이들로 구성된 E-F 루프; (f) 컨센서스 서열의 위치 75 내지 81에서 아미노산 잔기 KGGHRSN(서열번호 13)을 포함하거나 이들로 구성된 F-G 루프; 또는 (g) (a) 내지 (f)의 임의의 조합 내의 하나 이상의 위치에서 변형될 수 있다. 본 개시의 센티린은 적어도 5개의 피브로넥틴 III형(FN3) 도메인, 적어도 10개의 피브로넥틴 III형(FN3) 도메인 또는 적어도 15개의 피브로넥틴 III형(FN3) 도메인의 컨센서스 서열을 포함할 수 있다. 스캐폴드는 10^-9 M 이하의 K_D, 10^-10 M 이하의 K_D, 10^-11 M 이하의 K_D, 10^-12 M 이하의 K_D, 10^-13 M 이하의 K_D, 10^-14 M 이하의 K_D 및 10^-15 M 이하의 K_D로부터 선택된 적어도 하나의 친화성으로 항원에 결합할 수 있다. K_D는 표면 플라스몬 공명에 의해 측정될 수 있다.

The consensus sequence comprises (a) an AB loop comprising or consisting of the amino acid residue TEDS (SEQ ID NO: 8) at positions 13 to 16 of the consensus sequence; (b) a BC loop comprising or consisting of the amino acid residue TAPDAAF (SEQ ID NO: 9) at positions 22 to 28 of the consensus sequence; (c) a CD loop comprising or consisting of the amino acid residue SEKVGE (SEQ ID NO: 10) at positions 38 to 43 of the consensus sequence; (d) a DE loop comprising or consisting of the amino acid residue GSER (SEQ ID NO: 11) at positions 51 to 54 of the consensus sequence; (e) an EF loop comprising or consisting of the amino acid residue GLKPG (SEQ ID NO: 12) at positions 60 to 64 of the consensus sequence; (f) an FG loop comprising or consisting of the amino acid residue KGGHRSN (SEQ ID NO: 13) at positions 75 to 81 of the consensus sequence; Or (g) at least one position within any combination of (a) to (f). The sentinel of the present disclosure may comprise consensus sequences of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type III (FN3) domains or at least 15 fibronectin type III (FN3) domains. The scaffold is 10 ^-9 M or less for K _D, 10 ^-10 M or less for K _D, 10 ^-11 M or less for K _D, 10 ^-12 M or less for K _D, 10 ^-13 M or less for K _D, 10 ^{- At} least one affinity selected from a K _D of ¹⁴ M or less and a K _D of 10 ^-15 M or less. K _D can be measured by surface plasmon resonance.

The term " antibody mimetic " is intended to describe an organic compound that specifically binds to a target sequence and has a different structure than a naturally occurring antibody. Antibody mimetics may comprise proteins, nucleic acids or small molecules. The target sequence to which the antibody mimetic of the present disclosure specifically binds may be an antigen. Antibody mimetics have properties that are superior to antibodies, including, but not limited to, good solubility, tissue penetration, stability to heat and enzymes (e.g., resistance to enzymatic degradation), and lower production costs . Exemplary antibody mimetics include, but are not limited to, Apiva, Apriline, Apim, Apitin, Alpha Bodies, Anticalines and Avimers (also known as avidity multimers), DARPin (Designated Ankyrin Repeat Protein) But are not limited to, carnitine domain peptides and monobodies.

The apical molecule of the disclosure includes one or more alpha helices without any disulfide bridges or includes a protein scaffold comprised of such alpha helices. Preferably, the apical molecule of the present disclosure comprises or consists of three alpha helices. For example, the epithelial molecule of the present disclosure may comprise an immunoglobulin binding domain. The apical molecule of the present disclosure may comprise the Z domain of protein A.

The ampicillin molecules of the present disclosure include, for example, protein scaffolds produced by the modification of the exposed amino acids of gamma-B crystallin or ubiquitin. The ampulin molecules functionally mimic the affinity of the antibody for the antigen, but do not mimic the antibody structurally. In any of the protein scaffolds used to make ampicillin, amino acids that are accessible to solvents or possible binding partners in appropriately folded protein molecules are considered as exposed amino acids. Any one or more of the amino acids of the exposed amino acids may be modified to specifically bind to the target sequence or antigen.

The apimeric molecule of the present disclosure includes a protein scaffold comprising a highly stable protein engineered to display a peptide loop that provides a high affinity binding site for a particular target sequence. Exemplary apimeric molecules of the disclosure include a cystatin protein or a protein scaffold based on its tertiary structure. Exemplary apimeric molecules of the present disclosure may share a common tertiary structure comprising an alpha helix on top of an antiparallel beta sheet.

The apitin molecule of the present disclosure includes, for example, an artificial protein scaffold having a structure that can be derived from a DNA binding protein (e. G., The DNA binding protein Sac7d). The apitin of the disclosure selectively binds to a target sequence which may be whole or part of an antigen. Exemplary apitins of the disclosure are prepared by randomizing one or more amino acid sequences on the binding surface of a DNA binding protein and performing ribosomal labeling and selection on the resulting protein. Target sequences for apitin of the present disclosure can be found, for example, on the genome or surface of a peptide, protein, virus or bacteria. In some embodiments of this disclosure, the apitin molecule can be used as a specific inhibitor of the enzyme. The apitin molecule of the present disclosure may comprise a heat-resistant protein or derivative thereof.

The alpha body molecule of the present disclosure may also be referred to as a cell penetration alpha body (CPAB). The alpha-body molecules of the disclosure include small proteins (typically less than 10 kDa) that bind to a variety of target sequences (including antigens). Alpha body molecules can reach and bind intracellular target sequences. Structurally, the alpha body molecules of the present disclosure include artificial sequences that form single-stranded alpha helices (similar to naturally occurring coiled-coil structures). The alpha body molecules of the disclosure may comprise a protein scaffold comprising one or more amino acids modified to specifically bind to a target protein. Regardless of the binding specificity of the molecule, the alpha body molecules of this disclosure maintain accurate folding and thermal stability.

The anticarin molecules of the present disclosure include artificial proteins that bind to a target sequence or site in a protein or small molecule. The anticaline molecules of the present disclosure may comprise an artificial protein derived from human lipocalin. An anticaline molecule of the disclosure may be used, for example, in place of a monoclonal antibody or fragment thereof. Anticlin molecules may exhibit better tissue penetration and thermal stability than monoclonal antibodies or fragments thereof. Exemplary anticalyne molecules of the present disclosure may comprise about 180 amino acids, with a mass of about 20 kDa. Structurally, the anticarlyne molecules of the present disclosure comprise a barrel structure comprising an antiparallel beta strand connected in pairs by loop and attached alpha helices. In a preferred embodiment, the anticarlyne molecules of the present disclosure comprise a barrel structure comprising eight loops and eight antiparallel beta strands connected in pairs by an attached alpha helical.

The abiomeric molecules of the present disclosure include artificial proteins that specifically bind to a target sequence (which may be an antigen). The avimers of the present disclosure may recognize multiple binding sites within the same target or different target. When the avimer of the present disclosure recognizes more than one target, the avimers mimic the function of the bispecific antibody. Artificial protein avimers may comprise two or more peptide sequences each having about 30 to 35 amino acids. These peptides can be linked via one or more linker peptides. The amino acid sequence of one or more peptides of the avimer may be derived from the A domain of the membrane receptor. Avimers optionally have a rigid structure which may contain disulfide bonds and / or calcium. Abimers of the present disclosure may exhibit greater thermal stability than antibodies.

The DARPins (Designated Ankyrin Repeat Proteins) of the present disclosure include genetically engineered, recombinant or chimeric proteins with high specificity and high affinity for the target sequence. In some embodiments, the DARPins of the present disclosure are derived from an ankyrin protein and optionally comprise at least three repeating sub-motifs of the ankyrin protein (also referred to as repeating structural units). The ankyrin protein mediates high affinity protein-protein interactions. The DARPins of this disclosure include large target interaction surfaces.

The finomers of this disclosure comprise a small binding protein (about 7 kDa) derived from the human Fyn SH3 domain and engineered to bind the target sequence and molecules with affinity and equivalent specificity equivalent to that of the antibody.

The Kunitz domain of the present disclosure comprises a protein scaffold comprising a Kunitz domain. The Kunitz domain contains an active site for inhibiting protease activity. Structurally, the Kunitz domain of the present disclosure comprises a disulfide rich alpha + betafold. This structure is exemplified as a small pancreatic trypsin inhibitor. Kunitz domain peptides recognize specific protein structures and are used as competitive protease inhibitors. The Kunitz domain of the present disclosure may comprise a calantide (derived from a human lipoprotein-associated coagulation inhibitor (LACI)).

The monobody of the present disclosure is a small protein with a size comparable to a single chain antibody (containing about 94 amino acids and having a mass of about 10 kDa). This genetically engineered protein specifically binds to a target sequence, including an antigen. The monobodies of the present disclosure can specifically target one or more different proteins or target sequences. In a preferred embodiment, the monobodies of the present disclosure mimic the structure of human fibronectin, and more preferably comprise a protein scaffold that mimics the structure of the tenth extracellular < RTI ID = 0.0 > type III < / RTI > domain of fibronectin. As well as the tenth extracellular < RTI ID = 0.0 > type III < / RTI > domain of fibronectin, its monobody mimetics also contain seven beta sheets forming the three exposed loops corresponding to the barrels in each plane and three complementarity determining regions (CDRs) . In contrast to the structure of the variable domain of the antibody, the monobody lacks any binding site for the metal ion as well as the central disulfide bond. The multispecific monobody can be optimized by modifying the loops BC and FG. The monobodies of this disclosure may include adnectin.

As used throughout this disclosure, unless the context clearly requires otherwise, singular terms include plural referents. Thus, for example, reference to " method " includes a plurality of such methods, and reference to " dose " includes reference to one or more doses or equivalents thereof known to those skilled in the art.

The term " about " or " approximately " refers to a method in which a value is measured or determined in part, such as an allowable error for a particular value determined by a person having ordinary skill in the art, Range. ≪ / RTI > For example, " about " may mean that it is within one or more standard deviations. Alternatively, " about " may mean a range of 20% or less, 10% or less, 5% or less, or 1% or less of a predetermined value. Alternatively, particularly with respect to biological systems or processes, this term may mean that the term is within one digit of the value, preferably within 5 times, and more preferably within 2 times. Where a particular value is recited herein and in the claims, the term " about " should be assumed, which means that it is within an acceptable tolerance range for a particular value unless otherwise stated.

The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. &Quot; Isolated " or " purified " polynucleotide or protein, or biologically active portion thereof, when normally found in its natural production environment, is capable of binding a component that normally accompanies or interacts with the polynucleotide or protein ≪ / RTI > Thus, an isolated or purified polynucleotide or protein, when produced by recombinant techniques, has substantially no other cellular material or cell culture medium, or substantially no chemical precursors or other chemicals when chemically synthesized. Optimally, an " isolated " polynucleotide refers to a sequence (optimally protein coding sequence) that naturally flanks the polynucleotide in the genomic DNA of the organism from which it is derived (i.e., the 5 ' end of the polynucleotide, 3 ' end). For example, in various embodiments, the isolated polynucleotide can be about 5kb, 4kb, 3kb, 2kb, 1kb that naturally flanking the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. , 0.5 kb or less than 0.1 kb of nucleotide sequence. Proteins that are substantially free of cellular material include preparations of proteins with less than about 30%, 20%, 10%, 5% or 1% (dry weight) contaminating proteins. When the protein of the present invention or a biologically active portion thereof is recombinantly produced, the culture medium optimally contains less than about 30%, 20%, 10%, 5% or 1% (dry weight) of chemical precursor, Chemical substances.

This disclosure provides the disclosed DNA sequences and fragments and variants of the proteins encoded by the DNA sequences. As used throughout this disclosure, the term " fragment " means a portion of a DNA sequence or a portion of an amino acid sequence and a protein encoded thereby. A fragment of the DNA sequence comprising the coding sequence retains the biological activity of the native protein and therefore can encode a protein fragment that retains DNA recognition or binding activity to the target DNA sequence as described herein. Alternatively, fragments of DNA sequences useful as hybridization probes generally do not encode proteins that retain biological activity or that do not possess promoter activity. Thus, fragments of the DNA sequence may have at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and the full length polynucleotides of the invention.

The nucleic acids or proteins of the present disclosure can be quashed by a modular method that includes pre-assembling monomer units and / or repeating unit units in a target vector that can later be assembled into the final destination vector. The polypeptides of the present disclosure may comprise repeat moieties of the present disclosure and may be constructed by a modular method by preassembling the repeat moieties in a target vector that may later be assembled into the final destination vector. The present disclosure provides not only the polypeptide produced by this method, but also the nucleic acid sequence encoding the polypeptide. The present disclosure provides host organisms and cells comprising a nucleic acid sequence encoding a polypeptide produced by this modular method.

The term " antibody " is used in its broadest sense and specifically covers monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitope specificity. It is also within the scope of this disclosure to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (collectively referred to herein as "analogs") of the antibodies herein. Thus, according to one embodiment herein, the term " antibody of the present " in its broadest sense covers also such analogs. In general, one or more amino acid residues in such analogs may have been replaced and / or deleted and / or added relative to the subject antibodies defined herein.

As used herein, " antibody fragment " and all grammatical derivatives thereof are defined as part of an intact antibody comprising an antigen-binding site or variable region of the intact antibody, wherein the moiety is the constant heavy chain domain of the Fc region of the intact antibody, CH2, CH3 and CH4 depending on the antibody isotype. Examples of antibody fragments include Fab, Fab ', Fab'-SH, F (ab') ₂ and Fv fragments; Diabody; (1) a single chain Fv (scFv) molecule, (2) a single chain polypeptide containing only one light chain variable domain, or a fragment thereof comprising three CDRs of said light chain variable domain without an associated heavy chain moiety, and (3) a single chain polypeptide containing only one heavy chain variable region, or a fragment thereof comprising three CDRs of said heavy chain variable region without an associated light chain moiety, one non-interrupted sequence of adjacent amino acid residues Any antibody fragment (referred to herein as a "single chain antibody fragment" or "single chain polypeptide") that is a polypeptide having a constructed primary structure; And antibody specific fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain (s) may contain any constant domain sequence found in the non-Fc region of the intact antibody (CH1 in the IgG isotype) and / Or may include a hinge region sequence of the heavy chain (s) or a leucine zip sequence fused to or located in the constant domain sequence. The term also includes single domain antibodies (" sdAB "), which generally refers to antibody fragments with a single monomeric variable antibody domain (e.g., from camelid). Such antibody fragment types will be readily understood by those of ordinary skill in the art.

&Quot; Binding " refers to sequence specific non-covalent interactions between macromolecules (e.g., between proteins and nucleic acids). As long as the interaction is sequence-specific as a whole, it is not necessary that all components of the binding interaction are sequence-specific (e. G., In contact with phosphate residues in the DNA backbone).

The term " comprising " is intended to mean that the compositions and methods include the recited elements but not the others. &Quot; consisting essentially of " when used in defining the compositions and methods, will mean excluding any other essentially meaningful elements from the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude minor amounts of contaminants or inert carriers. &Quot; consisting of " will mean excluding the elements of minor components in the other components and the actual process steps. Embodiments defined by each of these transition terms are within the scope of the present invention.

The term " epitope " refers to an antigenic determinant of a polypeptide. The epitope may comprise three amino acids in an epitope-specific spatial configuration. In general, an epitope consists of at least 4, 5, 6 or 7 such amino acids, more usually at least 8, 9 or 10 such amino acids. Methods for identifying the spatial configuration of amino acids are well known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.

As used herein, " expression " refers to the process by which the polynucleotide is transcribed into mRNA and / or the transcribed mRNA is later translated into a peptide, polypeptide or protein. When the polynucleotide is derived from genomic DNA, expression may involve splicing of mRNA in eukaryotic cells.

&Quot; Gene expression " means that information contained in a gene is converted into a gene product. The gene product may be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA or any other type of RNA) Lt; / RTI > The gene product may be modified by RNA modified by procedures such as capping, polyadenylation, methylation and editing, and by modifications such as by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation and glycosylation Protein.

&Quot; Modulation " or " control " of gene expression refers to a change in the activity of a gene. Modulation of expression may include, but is not limited to, gene activation and gene suppression.

The term " operably linked " or an equivalent term thereof (e.g., " operably linked ") means that two or more molecules interact to form one or both of these molecules, It is positioned relative to each other so as to affect the functions that can be attributed.

A non-covalently linked component, and a non-covalently linked component, are prepared and used. The various components may take a variety of different forms as described herein. For example, proteins that are non-covalently linked (i.e., operably linked) can be used to allow transient interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and disassociate makes functional associations possible in environments where such association is required solely or primarily for the desired activity. The connection may be a connection of a duration sufficient to allow the desired effect.

A method for directing a protein to a particular locus in the genome of an organism is disclosed. The method may include providing a DNA localization component and providing the effector molecule, wherein the DNA localization component and the effector molecule may be operably linked through a non-covalent linkage.

The term " scFv " means a single chain variable fragment. scFv is a fusion protein of the variable region of the heavy chain (VH) and the light chain (VL) of immunoglobulin linked by a linker peptide. The linker peptide may be about 5 to 40 amino acids in length or about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35 or 40 amino acids in length . Single chain variable fragments lack a constant binding region (e. G., Protein G) used to purify antibodies as they lack the constant Fc region found in intact antibody molecules. The term is an intrabody which is a stable antibody in the cytoplasm of a cell and also includes an scFv capable of binding to intracellular proteins.

The term " single domain antibody " means an antibody fragment having a single monomeric variable antibody domain capable of selectively binding to a particular antigen. A single domain antibody is a peptide chain of about 110 amino acids in length, typically comprising a heavy chain antibody or one variable domain (VH) of a common IgG, and the peptide chain generally has affinity for the antigen But has stronger heat resistance and stability against detergents and high concentration of urea. An example is a single domain antibody derived from a camelid or a fish antibody. Alternatively, single domain antibodies can be made from conventional murine or human IgG with four chains.

As used herein, the terms " specifically bind " and " specific binding " refer to the ability of an antibody, antibody fragment or nanobody to preferentially bind to a particular antigen present in a homogeneous mixture of different antigens. In some embodiments, specific binding interactions distinguish undesirable antigens from desirable antigens in a sample by a factor of about 10-fold to 100-fold or greater in some embodiments (e. G., About 1000-fold or more than 10,000-fold) something to do. &Quot; Specificity " means the ability of an immunoglobulin or immunoglobulin fragment, such as a nanobody, to bind preferentially to an antigenic target relative to a different antigenic target, and does not necessarily imply high affinity.

A " target site " or " target sequence " is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided that sufficient conditions exist for binding.

The term " nucleic acid " or " oligonucleotide " or " polynucleotide " means at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of a complementary strand. Thus, the nucleic acid can also encompass the depicted single stranded complementary strand. Nucleic acids of the present disclosure also encompass substantially the same nucleic acid that retains the same structure or encodes the same protein and its complement.

Probes of the present disclosure may comprise single-stranded nucleic acids that are capable of hybridizing to a target sequence under stringent hybridization conditions. Thus, the nucleic acids of the present disclosure can refer to probes that hybridize under stringent hybridization conditions.

The nucleic acids of the disclosure may be single or double stranded. The nucleic acids of this disclosure may comprise double-stranded sequences even when the majority of the molecules are single-stranded. The nucleic acids of the present disclosure may comprise single-stranded sequences even when the majority of the molecules are double-stranded. The nucleic acids of the disclosure may comprise genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of this disclosure may contain combinations of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may contain a combination of bases including uracil, adenine, tyamine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. The nucleic acids of this disclosure can be synthesized to include unnatural amino acids and modifications. The nucleic acids of the present disclosure can be obtained by a chemical synthesis method or a recombinant method.

The nucleic acid of the disclosure may be a non-naturally occurring nucleic acid, whether it is the entire sequence or any portion thereof. The nucleic acids of this disclosure are not naturally occurring, and may contain one or more mutations, substitutions, deletions or insertions that make the entire nucleic acid sequence a non-naturally occurring nucleic acid sequence. The nucleic acids of this disclosure may contain one or more overlapping, inversed or repeated sequences, and the resulting sequences are not naturally produced, making the entire nucleic acid sequence a non-naturally occurring nucleic acid sequence. The nucleic acids of the present disclosure are not naturally occurring, and may contain modified, artificial or synthetic nucleotides that make the entire nucleic acid sequence a non-naturally occurring nucleic acid sequence.

When considering redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotide sequences are contemplated herein.

As used throughout this disclosure, the term " operably linked " refers to the expression of a gene under the control of a spatially linked promoter. The promoter may be located 5 '(upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene controlled by the promoter in the gene from which the promoter is derived. Changes in the distance between the promoter and the gene can be tolerated without loss of promoter function.

As used throughout this disclosure, the term " promoter " means a synthetic or naturally occurring molecule capable of conferring, activating or enhancing expression of a nucleic acid in a cell. Promoters may include one or more specific transcription control sequences to further enhance expression and / or alter spatial expression and / or temporal expression thereof. The promoter may also include a distal enhancer and inhibitor element that can be located as many as several thousand base pairs from the start site of the transcription. The promoter may be derived from a source comprising viruses, bacteria, fungi, plants, insects and animals. Promoters can be used for the expression of a gene component at any time, for a cell, tissue or organ in which expression occurs, for a developmental stage in which expression occurs, or for external stimuli such as physiological stress, Or differentially. Typical examples of the promoter include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF- SV40 early promoter or SV40 late promoter and CMV IE promoter.

The term " substantially complementary ", as used throughout this disclosure, refers to any of the following: " substantially complementary ", such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 65, 90, 95, 100, 180, 270, 360, 450 or 540 or more nucleotides over the region of SEQ ID NO: Means a first sequence which is identical to the first sequence in terms of%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%, or means that two sequences hybridize under stringent hybridization conditions do.

As used throughout this disclosure, the term " substantially identical " means that the first and second sequences are 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 At least 60%, at least 65%, at least 70%, at least 75%, at least 70%, at least 75%, at least 70%, at least 75% Or the first sequence is substantially complementary to the complement of the second sequence with respect to the nucleic acid, or the first sequence is substantially complementary to the complement of the second sequence .

As used throughout this disclosure, the term " variant " when used to describe a nucleic acid refers to (i) a portion or fragment of the mentioned nucleotide sequence; (ii) the complement of the nucleotide sequence mentioned or a portion thereof; (iii) a nucleic acid substantially identical to said nucleic acid or a complement thereof; Or (iv) a nucleic acid that hybridizes under stringent conditions to a nucleic acid, its complement or substantially the same sequence as said.

As used throughout this disclosure, the term " vector " means a nucleic acid sequence containing a replication origin. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be an autonomously replicating extrachromosomal vector, preferably a DNA plasmid. The vector may comprise an amino acid and a DNA sequence, an RNA sequence, or a combination of both DNA and RNA sequences.

As used throughout this disclosure, the term " variant " when used to describe a peptide or polypeptide refers to a peptide or amino acid sequence that has at least one biological activity that has a different amino acid sequence by insertion, deletion, ≪ / RTI > polypeptide. Variants may also refer to proteins having substantially the same amino acid sequence as the mentioned protein with an amino acid sequence having at least one biological activity.

Conservative substitution of amino acids, i. E. Replacing amino acids with different amino acids of similar properties (e. G., Hydrophilicity, degree and distribution of charged regions) is typically recognized in the art as involving slight variations. This slight change can be confirmed by considering the hydropathic index of the amino acid in part as understood in the art (Kyte et al., J. MoI. Biol. 157: 105-132 (1982) ). The hydrophobic index of an amino acid is based on its hydrophobicity and charge considerations. Amino acids of similar hydropathic indices can be substituted and still retain protein function. In one embodiment, the amino acid with a hydrotropic index of 占 2 is substituted. The hydrophilicity of an amino acid may be used to show the substitution resulting in a protein having biological functions. Consideration of the hydrophilicity of amino acids with respect to peptides makes possible the calculation of the greatest local average hydrophilicity of the peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity (see, U.S. Patent No. 4,554,101).

Substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, e. G., Immunogenicity. Substitutions can be made by amino acids having hydrophilicity values within ± 2 of each other. Both the hydrophobicity index and the hydrophilic value of the amino acid are affected by the specific side chain of the amino acid. Consistent with the observations, amino acid substitutions that are compatible with biological functions are understood to depend on the relative similarity of the amino acids and, in particular, the side chains of the amino acids, as determined by their hydrophobicity, hydrophilicity, charge, size and other properties.

As used herein, " conservative " amino acid substitutions may be defined as described in the following Tables A, B, In some embodiments, the fusion polypeptide and / or the nucleic acid encoding such fusion polypeptide comprises conservative substitutions introduced by modification of the polynucleotide encoding the polypeptide of the present invention. Amino acids can be classified according to their physical properties and their contribution to secondary and tertiary protein structure. Conservative substitutions are substitutions of one amino acid by another amino acid of similar nature. Exemplary conservative substitutions are set forth in Table A below.

[Table A]

Alternatively, conservative amino acids listed in Table B below can be separated as described in Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77.

[Table B]

Alternatively, exemplary conservative substitutions are set forth in Table C. < tb > < TABLE >

[Table C]

The polypeptides of the present disclosure are intended to include polypeptides having one or more insertions, deletions or substitutions of amino acid residues, or any combination thereof, as well as polypeptides having modifications other than insertions, deletions or substitutions of amino acid residues It should be understood. The polypeptides or nucleic acids of the present disclosure may contain one or more conservative substitutions.

As used throughout this disclosure, the term " more than one " of the above-mentioned amino acid substitutions is intended to encompass amino acid substitutions of two, three, four, five, six, seven, eight, nine, ten, , 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more of the stated amino acid substitutions. The term " more than one " may mean two, three, four or five mentioned amino acid substitutions.

The polypeptides and proteins of the present disclosure may be non-naturally occurring polypeptides and proteins regardless of their entire sequence or any portion thereof. The polypeptides and proteins of this disclosure are not naturally occurring, and may contain one or more mutations, substitutions, deletions or insertions that make the entire amino acid sequence a non-naturally occurring amino acid sequence. The polypeptides and proteins of the present disclosure may contain one or more overlapping, inversed or repeated sequences, and the resulting sequences are not naturally occurring, making the entire amino acid sequence a non-naturally occurring amino acid sequence. Polypeptides and proteins of the present disclosure may contain modified, artificial or synthetic amino acids that are not naturally occurring and make the entire amino acid sequence a non-naturally occurring amino acid sequence.

As used throughout this disclosure, " sequence identity " refers to a standalone executable BLAST engine program (bl2seq) (which can be retrieved from the National Biotechnology Information Center (NCBI) ftp site) that blasts two sequences, (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250, incorporated herein by reference in its entirety). The term " same " or " identity " when used in connection with two or more nucleic acid or polypeptide sequences means a specified percentage of identical residues throughout a specified region of each sequence. This percentage optimally aligns the two sequences, compares the two sequences over a specified region, identifies the number of positions where the same residues are present in both of the sequences, and obtains the number of consecutive positions , Dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percent of sequence identity. Where two sequences have different lengths or the alignment produces one or more staggered ends and the specified comparison region comprises only a single sequence, the residue of a single sequence is included in the denominator of the calculation but not in the molecule . When comparing DNA and RNA, the tyramine (T) and uracil (U) can be regarded as equivalent. The identity can be performed manually or by using a computer sequence algorithm, such as BLAST or BLAST 2.0.

As used throughout this disclosure, the term " endogenous " means that the nucleic acid or protein sequence is naturally associated with the target gene or host cell into which it is introduced.

As used throughout this disclosure, the term " exogenous " refers to a naturally occurring nucleic acid, such as a non-naturally occurring multiple copy of a DNA sequence, or a nucleic acid comprising a naturally occurring nucleic acid sequence located at a non- Quot; means that the protein sequence is not naturally associated with the target gene or host cell into which it is introduced.

The present disclosure provides a method for introducing a polynucleotide construct comprising a DNA sequence into a host cell. &Quot; Introducing " means providing a polynucleotide construct to a plant in such a way that the construct enters the interior of the host cell. The method of the present invention does not rely on any particular method of introducing a polynucleotide construct into a host cell, except that the polynucleotide construct enters the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals, including, but not limited to, stable transformation methods, transient transformation methods, and viral mediated methods are well known in the art.

&Quot; Stable transformation " means that a polynucleotide construct introduced into a plant can be inserted into the host's genome and be inherited by its offspring. By " transient transformation " is meant that the polynucleotide construct introduced into the host is not inserted into the host's genome.

As used throughout this disclosure, the term " genetically modified plant (or transgenic plant) " refers to a plant comprising an exogenous polynucleotide in its genome. Generally and preferably, the exogenous polynucleotide is stably inserted into the genome such that the polynucleotide is transferred to the next generation. Exogenous polynucleotides can be inserted into the genome alone or as part of a recombinant expression cassette. &Quot; Transformation " is used herein to encompass any cell, cell line, union tissue, tissue, plant part or plant with a genotype altered by the presence of exogenous nucleic acid, and may be used herein as an initial transformant Lt; RTI ID = 0.0 > transgenic < / RTI > As used herein, the term " transformation " is intended to encompass all types of infectious diseases caused by conventional plant breeding methods, or by natural occurrence events such as random mating-modification, non-recombinant viral infection, non- recombinant bacterial transformation, non- recombinant dislocations or spontaneous mutations (Chromosomal or non-chromosomal) genomic alterations.

As used throughout this disclosure, the term " modified " is intended to mean that the sequence is considered merely modified by the binding of the polypeptide. The term is not intended to imply that the sequence of the nucleotide is altered, but such changes may occur following binding of the polypeptide with the nucleic acid of interest. In some embodiments, the nucleic acid sequence is DNA. Modifications of the nucleic acid of interest (in the sense of binding to it by the modified polypeptide to contain the modular repeating unit) can be accomplished by any of a number of methods (e. G., Gel mobility variability assay, labeled polypeptide The use of the de-label may be detected by radioactive, fluorescent, enzymatic or biotin / streptavidin labeling. Variations of the nucleic acid sequence of interest (and detection thereof) may be all that is required (e.g., in the diagnosis of a disease). However, preferably further processing of the sample is performed. Conveniently, the polypeptide (and nucleic acid sequence specifically bound thereto) is separated from the remainder of the sample. Advantageously, the polypeptide-DNA complex is bound to a solid phase support to facilitate such separation. For example, the polypeptide may be present in an acrylamide or agarose gel matrix, or more preferably, on the surface of a membrane or in a well of a microtiter plate.

Unless otherwise indicated, all percentages and ratios are calculated by weight.

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

All maximum numerical limitations provided throughout this disclosure are inclusive of all lower limit numerical limitations as are expressly recited in this disclosure. All minimum numerical limitations provided throughout this disclosure will include all upper limit numerical limitations as defined by the upper limit numerical limitation herein. All numerical ranges provided throughout this disclosure will include all narrower numerical ranges falling within these broader numerical ranges, all of which are expressly recited herein.

The values disclosed herein are not to be understood as being strictly limited to the exact numerical values mentioned. Instead, each such value, unless otherwise specified, is intended to mean both the recited value and the functionally equivalent range surrounding the value. For example, a value disclosed as " 20 占 퐉 " is intended to mean " about 20 占 퐉.

All references cited herein, including any cross-referenced or related patents or applications, are expressly incorporated herein by reference in their entirety, unless expressly excluded or otherwise limited. The citation of any document is intended to be merely illustrative of the fact that this document is prior art to any invention disclosed or claimed herein, or that such document, either alone or in any combination with any other reference or reference, But does not admit that it teaches, suggests or discloses the present invention. In addition, if any meaning or definition of a term in this document is inconsistent with any meaning or definition of the same term in the referenced literature, the meaning or definition assigned to that term in that document will prevail.

While certain embodiments of the disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the disclosure. The appended claims include all such variations and modifications that are within the scope of this disclosure.

Example

In order that the invention disclosed herein may be more efficiently understood, embodiments are provided below. It is to be understood that this embodiment is for illustrative purposes only and is not to be construed as limiting the invention in any way. (Maniatis et al., Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989)), using commercially available reagents throughout this example, except where otherwise stated. Molecular cloning reactions and other standard recombinant DNA techniques were performed according to methods.

Example 1: In vitro transformation of T cells

For the generation of self-CAR-T immunotherapy and other applications, the piggyBac ™ (PB) transposon system was used to genetically modify human lymphocytes. Purified T lymphocytes from patient blood or isolated export products were electroporated into plasmid DNA transposons and transposons. Including the Neon (Thermo Fisher), BTX ECM 830 (Harvard Apparatus), Gene Pulser (BioRad), MaxCyte PulseAgile (MaxCyte), and Amaxa 2B and Amaxa 4D (Lonza) for T cell delivery of the transposon system. . Some were tested using in-house developed buffers as well as electroporation buffers provided or recommended by the manufacturer. The results show that dormant T lymphocytes specifically exhibit refractory to DNA transfection and appear to be inversely related to cell viability and electroporation efficiency as measured by GFP expression from electroporated plasmids (dogma ). Figure 1 shows an example of an experiment to test a plurality of electroporation systems and nucleofection programs.

To further test whether plasmid DNA exhibits toxicity to T cells during nucleofection, primary human T lymphocytes were electroporated with two different DNA plasmids. The first plasmid was the pmaxGFP ™ plasmid provided as a control plasmid in the Lonza Amaxa nucleofection kit. It is highly purified by HPLC and does not contain endotoxin at detectable levels. The second plasmid was a PB transposon produced in-house by the present inventors which encodes a human EF1 alpha promoter that induces GFP. Transfection efficiency and cell viability measured by GFP expression from electroporated plasmids were evaluated by FACS on day 2, day 3 and day 6 after electroporation. The data is shown in Fig. Simulated electroporated cells (plasmid DNA member) showed relatively high cell viability (54%) until day 6 after electroporation whereas T cells electroporated with either plasmid showed only 1.4% The survival rate was 2.6%. This data shows that the plasmid DNA showed cytotoxicity against T lymphocytes. In addition, this data shows that DNA-mediated toxicity was not due to a transposon element, such as an ITR region or a core insulator, because the pmaxGFP ™ plasmid lacked elements and was similarly cytotoxic at the same DNA concentration to be. The two plasmids have approximately the same size, which means that a similar amount of DNA has been electroporated into T cells.

To test whether DNA-mediated toxicity in T cells is dose-dependent, we performed titration of our PB-GFP plasmids. Figure 3 shows the results obtained when the dose of plasmid DNA added to the nucleofection reaction was gradually increased (1.3, 2.5, 5.0, 10.0 and 20.0 ug of plasmid DNA), measured on day 1 and day 5 after nucleofection Lt; RTI ID = 0.0 > cell viability. ≪ / RTI > Even 1.3 μg of plasmid DNA was responsible for a 2.4-fold reduction in T cell survival by day 4.

Since it was apparent that the plasmid DNA exhibited toxicity to T cells during nucleofection, the present inventors examined whether extracellular plasmid DNA contributes to apoptosis. Figure 4 shows that extracellular plasmid DNA did not show cytotoxicity against T cells. In the experiment, 45 袖 g to 5 ㎍ of plasmid DNA was added to the cells after electroporation, and almost no apoptosis was observed on day 1 or day 4. Similarly, when 5 의 of plasmid DNA was added to the nucleofection reaction in the absence of electroporation, little apoptosis was observed. However, when plasmid DNA was added before the electroporation reaction, the cells showed a 2.0-fold decrease in cell survival rate on day 1 and a 13.2-fold decrease in cell survival on day 4.

Since DNA-mediated toxicity is dose-dependent, the present inventors have now focused their attention on a method for reducing the total amount of DNA transferred to T cells, which is required for disposition. One relatively simple way to accomplish this would be to deliver the transposon encoded in the mRNA instead of the transposase encoded in the DNA. MRNA delivery to primary human T cells is highly efficient resulting in high transfection efficiency and high survival rates. The present inventors subcloned Super piggyBac (TM) transposase enzyme into the in-house mRNA producing vector of the present inventors and produced high quality sPBo mRNA. Simultaneous transfer of PB-GFP transposon with various doses of sPBo mRNA (30, 10, 3.3, 3, 1, 0.33 ug mRNA) in Jurkat cells demonstrated a strong dislocation in all tested doses (Fig. 5). This data shows that sPBo transporter can be delivered and is equally effective with plasmid DNA or mRNA. In addition, the amount of sPBo mRNA makes little difference in the overall potential efficiency in Jurkat, the total putter of GFP + cells or the MFI of GFP expression. To see if this was also valid for T lymphocytes, we delivered PB-GFP with sBPo plasmid DNA or 5 μg of sPBo mRNA at a ratio of 3: 1. GFP potentials were evaluated on day 7 after the nucleofection reaction and addition of IL7 and IL15. Figure 6 shows that sPBo mRNA efficiently mediated the translocation of GFP transposons into T lymphocytes. Significantly, in contrast to pDNA, the co-delivery of sPBo as mRNA improved T cell survival; 32.4% vs. 25.4% respectively. This data suggests that the simultaneous delivery of sPBo as mRNA will result in lower cytotoxicity as it will save capacity in the total amount of plasmid DNA delivered to T cells.

Since the current plasmid transposon also contains the skeleton required for plasmid amplification in bacteria, it is possible to significantly reduce the total amount of DNA by excluding this sequence. This can be achieved by restriction cleavage of the plasmid transposon prior to the nucleofection reaction. In addition, this can be accomplished by administering the transposon as a PCR product or doggybone (TM) DNA that is double-stranded DNA produced in vitro by a mechanism that eliminates the early framework elements required for bacterial replication of the plasmid.

We conducted pilot experiments to see whether plasmid transposons need to be circular plasmid transposons or can be delivered to cells in a linear fashion. To test this, the transposon was incubated with restriction enzyme (ApaLI) overnight to linearize the plasmid. Uncut or linearized plasmids were electroporated into primary T lymphocytes and GFP expression was assessed after 2 days. Figure 7 shows that the linearized plasmid was also efficiently delivered to the cell nucleus. This data demonstrates that linear transposon products can also be efficiently electroporated into primary human T cells.

We have shown earlier that plasmid DNA shows toxicity in primary T lymphocytes, but we have observed that this toxic effect is not as pronounced in tumor cell lines and other transformed cells. Based on these observations, the inventors hypothesized that primary T lymphocytes may exhibit refractory to plasmid DNA transfection due to enhanced DNA sensing pathways that protect immune cells from infection by viruses and bacteria. If this data is the result of an enhanced DNA sensing mechanism, it may be possible to enhance the plasmid transfection efficiency and / or cell survival rate by adding a DNA sense pathway inhibitor to the post-nucleofection reaction. Thus, the present inventors have tested a number of different reagents that inhibit the pathway involved in TLR-9 pathway, caspase pathway, or cytoplasmic double stranded DNA sensing. These reagents include Bafilomycin A1, an autophagic inhibitor that interferes with endosomalisation and blocks NFkB signaling by TLR9, Chloroquine, a TLR9 antagonist, TLR9 antagonist and Quinacrine, a cGAS antagonist, AC-YVAD-CMK, which is a caspase-1 inhibitor that targets the AIM2 pathway, Z-VAD-FMK, a pan-caspase inhibitor, and Z-IETD-FMK, a caspase-8 inhibitor induced by the TLR9 pathway. In addition, we also tested the stimulation of electroporated T cells by addition of cytokines IL7 and IL15, and addition of anti-CD3 anti-CD28 Dynabeads ^® human T-amplification CD3 / CD28 beads. The results are shown in FIG. We have found that some compounds or caspase inhibitors have had any positive effect on cell survival at day 4 after the nucleofection at the tested dose. However, the present inventors recognize that additional dosing studies may be required to better test these reagents. It may be more effective to genetically suppress these pathways. The conditions after the two kinds of nucleofection did not improve the survival rate of T cells. The addition of IL7 and IL15 improved the survival rate more than three times when compared to the introduction of plasmid transposon alone without further treatment whether added at 1 hour after electroporation or at day 1 after electroporation. Moreover, stimulating T cells after nucleofection using activator or amplification beads significantly enhanced T cell survival; The stimulation was better when the beads were added at 1 hour or 1 day after nucleofection as compared to when they were added at 2 days after nucleofection. Finally, we also tested ROCK inhibitors and tested the removal of killed cells from cultures using a killed cell removal kit from Miltenyi, but failed to confirm an improvement in cell survival.

To further extend this finding, which demonstrates that stimulation of T cells after nucleofection improves survival, we repeated the study using the addition of cytokines IL7 and IL15. Figure 9 shows that the addition of these cytokines at a dose of 20 ng / ml each immediately after nucleofection or up to 1 hour after nucleofection enhanced the cell survival rate to 2.9-fold compared to the treatment members. The addition of these cytokines up to day 1 after nucleofection improved the survival rate, but was not as strong as before.

Since the inventors have discovered that immediate stimulation of T cells post-nucleofection can increase cell viability, we hypothesize that stimulating cells prior to nucleofection can also improve survival and transfection efficiency I built it. To test this, we stimulated primary T lymphocytes 2, 3 or 4 days before transposononucleoprotein. Figure 10 shows that a certain level of potential occurs when T cells are stimulated prior to the nucleofection reaction and then transposons and transposons are co-transferred. The efficacy of the full stimulation may be influenced by the kinetics of the stimulation and thus may depend on the exact type of amplification technique selected.

                         SEQUENCE LISTING <110> Poseida Therapeutics, Inc.   <120> TRANSPOSON SYSTEM AND METHODS OF USE <130> POTH-007001WO &Lt; 150 > US 62 / 300,387 <151> 2016-02-26 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 594 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 1 Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln 1 5 10 15 Ser Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp             20 25 30 His Val Ser Glu Asp Asp Val Glu Ser Asp Thr Glu Glu Ala Phe Ile         35 40 45 Asp Glu Val His Glu Val Gln Pro Thr 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 Ser 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 Ser 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 <211> 340 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 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 His 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 <211> 340 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 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 His 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> Synthetic Construct <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 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 Ser Ser Ser Ser Ser Ser Tyr Glu Thr Phe Lys Lys Ser         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 <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 Gly 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 <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 <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 <400> 8 Thr Glu Asp Ser One <210> 9 <211> 7 <212> PRT <213> Homo sapiens <400> 9 Thr Ala Pro Asp Ala Ala Phe 1 5 <210> 10 <211> 6 <212> PRT <213> Homo sapiens <400> 10 Ser Glu Lys Val Gly Glu 1 5 <210> 11 <211> 4 <212> PRT <213> Homo sapiens <400> 11 Gly Ser Glu Arg One <210> 12 <211> 5 <212> PRT <213> Homo sapiens <400> 12 Gly Leu Lys Pro Gly 1 5 <210> 13 <211> 7 <212> PRT <213> Homo sapiens <400> 13 Lys Gly Gly His Arg Ser Asn 1 5

Claims (109)

As a method for genetically transforming immune cells in vitro,
(a) a nucleic acid or amino acid sequence comprising a sequence encoding a transposase enzyme, and
(b) a recombinant and non-naturally occurring DNA sequence comprising a DNA sequence encoding a transposon
Lt; RTI ID = 0.0 &gt; immune &lt; / RTI &gt; cells. 2. The method of claim 1, wherein the sequence encoding the transposase enzyme is an mRNA sequence. The method of claim 1, wherein the sequence encoding the transposase enzyme is a DNA sequence. The method of claim 1, wherein the sequence encoding the transposase enzyme is an amino acid sequence. 5. The method of any one of claims 1 to 4, wherein the delivery step comprises electroporation or nucleofection of immune cells. 6. The method according to any one of claims 1 to 5, further comprising the step of stimulating the immune cells with one or more cytokine (s). 7. The method of claim 6, wherein stimulating the immune cells with one or more cytokine (s) occurs after the delivery step. 7. The method of claim 6, wherein stimulating the immune cells with one or more cytokine (s) occurs prior to the delivery step. 9. A method according to any one of claims 6 to 8, wherein the at least one cytokine (s) comprises IL-2, IL-21, IL-7 and / or IL-15. 10. The method according to any one of claims 1 to 9, wherein the immune cell is an autoimmune cell. 11. The method according to any one of claims 1 to 10, wherein the immune cell is a T lymphocyte. 11. The method according to any one of claims 1 to 10, wherein the immune cells are natural killer (NK) cells. 11. The method according to any one of claims 1 to 10, wherein the immune cell is a cytokine-induced killer (CIK) cell. 11. The method according to any one of claims 1 to 10, wherein the immune cells are natural killer T (NKT) cells. 15. A method according to any one of claims 2 and 14, wherein the mRNA sequence encoding the transposase enzyme is produced in vitro. 16. The method according to any one of claims 1 to 15, wherein the transposase enzyme is Super piggyBac (TM) (sPBo) transposase enzyme. 17. The method of claim 16, wherein the Super piggyBac (PB) transporter enzyme comprises at least 75% identical amino acid sequence to the following amino acid sequence:

16. The method according to any one of claims 1 to 15, wherein the transposase enzyme is a Sleeping Beauty transposase enzyme. 19. The method of claim 18, wherein the Sleeping Beauty transporter is an active Sleeping Beauty SB100X transporter. 20. The method of claim 18 or 19, wherein the Sleeping Beauty transporter enzyme comprises at least 75% identical amino acid sequence to the following amino acid sequence:

20. The method of claim 18 or 19, wherein the Sleeping Beauty transporter enzyme comprises at least 75% identical amino acid sequence to the following amino acid sequence:

22. The method according to any one of claims 1 to 21, wherein the recombinant and non-naturally occurring DNA sequences comprising a DNA sequence encoding a transposon are annular. 23. The method of claim 22, wherein recombinant and non-naturally occurring DNA sequences encoding a transposon are included in the plasmid vector. 23. The method of claim 22, wherein recombinant and non-naturally occurring DNA sequences encoding a transposon are included in a minicircle DNA vector. 22. The method according to any one of claims 1 to 21, wherein the recombinant and non-naturally occurring DNA sequences encoding the transposon are linear. 26. The method according to any one of claims 1 to 25, wherein recombinant and non-naturally occurring DNA sequences encoding a transposon are generated in vitro. 26. The method of claim 25 or 26, wherein the recombinant and non-naturally occurring DNA sequences encoding the transposon are the restricted degradation products of the circular DNA. 28. The method of claim 27, wherein the circular DNA is a plasmid vector or a mini-circle DNA vector. 26. The method of claim 25 or 26 wherein the recombinant and non-naturally occurring DNA sequences encoding the transposon are products of a polymerase chain reaction (PCR). 26. The method of claim 25 or 26, wherein the recombinant and non-naturally occurring DNA sequences encoding the transposon are double-stranded doggybone (TM) DNA sequences. 31. The method of claim 30, wherein the doggybone (TM) DNA sequence is produced by an enzymatic process that encodes only an antigen expression cassette comprising an antigen, a promoter, a poly-A tail and a telomeric end. 32. The method according to any one of claims 1 to 31, wherein the immune cells are isolated or derived from a human. 32. The method according to any one of claims 1 to 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, cattle, camel or primate. 35. The method according to any one of claims 1 to 34, wherein the recombinant and non-naturally occurring DNA sequences encoding the transposon further comprise a sequence or a portion thereof encoding a chimeric antigen receptor. 36. The method of claim 35, wherein a portion of the sequence encoding the chimeric antigen receptor encodes an antigen recognition region. 36. The method of claim 35 or 36 wherein the antigen recognition region comprises at least one complementarity determining region (s). 38. The method of claim 35 or 36 wherein the antigen recognition region comprises an antibody, an antibody mimetic, a protein scaffold or a fragment thereof. 39. The method of claim 38, wherein the antibody is a chimeric antibody, a recombinant antibody, a humanized antibody, or a human antibody. 40. The method of claim 39, wherein the antibody is affinity adjusted. 39. The method of claim 38, wherein the antibody comprises or consists of a single chain variable fragment (scFv), VHH, a single domain antibody (sdAB), a small modular immune drug (SMIP) molecule or a nanobody. 42. The method of claim 41, wherein the VHH is camelid. 43. The method of claim 41 or 42 wherein the VHH is humanized. 39. 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. 39. The composition of claim 38, wherein the antibody mimetic is selected from the group consisting of affibody, afflilin, affimer, affitin, alphabody, anticalin, avimer, ), DARPin, a Fynomer, a Kunitz domain peptide or a monobody. 39. The method of claim 38, wherein the protein scaffold comprises or consists of Centyrin. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is not more than 10 μg per 100 μl of electroporation or nucleofection reaction. 49. The method of claim 48, wherein the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 100 [mu] g / ml in the electroporation or nucleofection reaction. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of DNA sequence encoding the transposon is not more than 7.5 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 50. The method of claim 49, wherein the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 75 [mu] g / ml in the electroporation or nucleofection reaction. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is 6.0 [micro] g or less per 100 μl of the electroporation or nucleofection reaction. 52. The method of claim 51, wherein the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 60 [mu] g / ml in the electroporation or nucleofection reaction. 52. The method of claim 50 or 51 wherein the transposase is Sleeping Beauty Transposase. 57. The method of claim 53, wherein the Sleeping Beauty transposon is a Sleeping Beauty 100X (SB100X) transposon. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is 5.0 [micro] g or less per 100 [mu] l of electroporation or nucleofection reaction. 56. The method of claim 55, wherein the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 50 占 퐂 / ml in the electroporation or nucleofection reaction. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 2.5 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 58. The method of claim 57, wherein the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 25 [mu] g / ml in the electroporation or nucleofection reaction. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is not more than 1.67 쨉 g per 100 쨉 l of electroporation or nucleofection reaction. 60. The method of claim 59, wherein the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 16.7 g / ml in the electroporation or nucleofection reaction. 60. The method of claim 59 or 60 wherein the transporter is a Super piggyBac (PB) transposase. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is not more than 0.55 쨉 g per 100 쨉 l of electroporation or nucleofection reaction. 63. The method of claim 62, wherein the concentration of the DNA sequence encoding the transposon enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 5.5 [mu] g / ml in the electroporation or nucleofection reaction. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is not more than 0.19 쨉 g per 100 쨉 l of electroporation or nucleofection reaction. 63. The method of claim 62, wherein the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 1.9 [mu] g / ml in the electroporation or nucleofection reaction. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is a DNA sequence,
(b) the amount of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is not more than 0.1 쨉 g per 100 쨉 l of electroporation or nucleofection reaction. 67. The method of claim 66, wherein the concentration of the DNA sequence encoding the transposase enzyme and the amount of the DNA sequence encoding the transposon is less than or equal to 1.0 [mu] g / ml in the electroporation or nucleofection reaction. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is less than or equal to 10 μg per 100 μl of electroporation or nucleofection reaction. 69. The method of claim 68, wherein the concentration of the amount of DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than 100 [mu] g / ml. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is not more than 7.5 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 70. The method of claim 70, wherein the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 75 [mu] g / ml. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is not more than 6.0 [mu] g per 100 [mu] l electroporation or nucleofection reaction. 72. The method of claim 72, wherein the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 60 [mu] g / ml. 74. The method of claim 72 or 73 wherein the transposase is Sleeping Beauty Transposase. 74. The method of claim 74, wherein the Sleeping Beauty transporter is a Sleeping Beauty 100X (SB100X) transposon. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is not more than 5.0 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 80. The method of claim 76, wherein the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is no more than 50 [mu] g / ml. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is not more than 2.5 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 79. The method of claim 78, wherein the concentration of the amount of the DNA sequence coding for the transposon in the electroporation or nucleofection reaction is less than or equal to 25 [mu] g / ml. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is not more than 1.67 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 79. The method of claim 80, wherein the concentration of the amount of the DNA sequence coding for the transposon in the electroporation or nucleofection reaction is less than or equal to 16.7 g / ml. 83. The method of claim 80 or 81 wherein the transporter is Super piggyBac (PB) transposase. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is not more than 0.55 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 83. The method of claim 83, wherein the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than 5.5 [mu] g / ml. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is not more than 0.19 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 86. The method of claim 85, wherein the concentration of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 1.9 [mu] g / ml. 46. The method according to any one of claims 5 to 46,
(a) the nucleic acid sequence encoding the transposase enzyme is an RNA sequence,
(b) the DNA sequence encoding the transposon is not more than 0.1 [mu] g per 100 [mu] l of electroporation or nucleofection reaction. 87. The method of claim 87, wherein the concentration of the amount of the DNA sequence encoding the transposon in the electroporation or nucleofection reaction is less than or equal to 1.0 [mu] g / ml. 88. Immune cells modified according to the method of any one of claims 1 to 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. 90. The immune cell according to claim 89 or 90, further modified by 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. 92. The immune cell of claim 92, wherein the endonuclease is covalently operably linked to a Cas9 or TALE sequence. 92. The immune cell of claim 92, wherein the endonuclease is operably linked to Cas9 or TALE sequences non-cognitionally. The immune cell according to any one of claims 89 to 94, wherein the Cas9 is Cas9 inactivated (dCas9). 95. The immune cell of claim 95, wherein the inactivated Cas9 comprises D10A and N580A within the catalytic region. 96. The immune cell according to claim 95 or 96, wherein Cas9 is a small inactivated Cas9 (dSaCas9). 98. The immune cell of claim 97, wherein dSaCas9 comprises the amino acid sequence:

98. A composition comprising an immune cell according to any one of claims 89 to 98. 98. Use of a composition according to claim 99 for the treatment of a disease or disorder in a subject in need thereof. 102. The use according to claim 100, wherein the disease or disorder is cancer. 104. The use according to claim 100, wherein the disease or disorder is an infectious disease. 103. Use according to any one of claims 99 to 102, wherein the immune cell is autologous. 102. The use according to any one of claims 99 to 102, wherein the immune cell is homologous. A culture medium for enhancing the survival rate of modified immune cells, comprising IL-2, IL-21, IL-7, IL-15 or any combination thereof. 105. The culture medium of claim 105, wherein the modified immune cell is a T lymphocyte, a natural killer (NK) cell, a cytokine induced killer (CIK) cell or a natural killer T (NKT) cell. 106. The culture medium of claim 105 or 106, wherein the modified immune cells comprise at least one exogenous DNA sequence. 106. The culture medium of claim 105 or 106, wherein the modified immune cells comprise at least one exogenous RNA sequence. 108. The culture medium according to any one of claims 105 to 108, wherein the modified immune cells are electroporated or nucleocamped.

Images