CN109321587A - An ACAT1 gene-interfered chimeric antigen receptor T cell - Google Patents

Abstract

The invention discloses the construction and application of a chimeric antigen receptor T cell with ACAT1 gene interference. Specifically, the present invention provides an interfering RNA that inhibits the expression of the ACAT1 gene, and a nucleic acid construct comprising the interfering RNA and a chimeric antigen receptor coding sequence, and an enhanced shRNA and nucleic acid construct are prepared based on the CAR-T cells. The interfering RNA of the present invention can effectively inhibit the expression of ACAT1 gene, and significantly enhance the killing efficiency of CAR-T cells to tumor cells.

Description

A kind of Chimeric antigen receptor T cell of ACAT1 gene interference

Technical field

The present invention relates to field of biotechnology, relate more specifically to a kind of Chimeric antigen receptor T of ACAT1 gene interference Cell.

Background technique

Cholesterol is the required lipid molecular being present in mammalian cell membrane, the cell of most of organs and tissue The demand to Membrane cholesterol is met by endogenous cholesterol biosynthesis.However, abnormal high cholesterol levels, especially It is free (no esterification) cholesterol, to cell nocuousness.Cell can by inhibit endogenous cholesterol biosynthesis and LDL by Body surface reaches, to cope with cholesterol feeding.In addition, these cells have been developed that other mechanism also to prevent.Pass through cholesterol acyl Based transferase (ACAT), converts cholesteryl ester for free cholesterol, to prevent the free cholesterol on cell membrane excessively long-pending It is tired.Two key genes for encoding cholesterol Esterified Enzyme are Acat1 and Acat2.Acat1 wide expression and various intracellular, and Acat2 is mainly expressed in liver and small intestine.

Largely research shows that film rouge can direct regulatory T-cell signal transduction and immune function.Cholesterol is as cell The key components of film, have proved to be to be formed T cell receptor (TCR) polymerization and formed T cell immunological synapse institute it is required 's.

Chimeric antigen receptor T cell (CART) immunotherapy is the process gene modification by integrating Chimeric antigen receptor T cell resist the therapy of tumour cell.Chimeric antigen receptor can be special with specific recognition tumor associated antigen or tumour Activation and proliferation signal are transferred in T cell by Specific Antigen, identification after combining, and are caused t cell activation, are proliferated, discharge cell The factor, thus killing tumor cell.The killing-efficiency of CART determines its antitumous effect in vivo, therefore, how to improve The tumor-killing efficiency of CART has critical significance.

Summary of the invention

The purpose of the present invention is to provide a kind of Chimeric antigen receptor T cells of ACAT1 gene interference.Specifically, this hair It is bright that cholesterol Esterified Enzyme ACAT1 gene is interfered by way of RNAi, to be obviously improved the tumor-killing efficiency of CART.

In the first aspect of the present invention, a kind of nucleic acid constructs, the structure of the nucleic acid constructs such as following formula A are provided Or shown in formula B:

B1-Z-B2 (A)

B2-Z-B1 (B)

In formula,

Each "-" independently is key or nucleotide catenation sequence；

B1 is the first expression cassette, and first expression cassette is used for the RNA interfering of expression inhibiting ACAT1 gene expression；

B2 is the second expression cassette, and second expression cassette is for expressing Chimeric antigen receptor；

Z is optional intervening sequence.

In another preferred example, second expression cassette contains the nucleic acid of the coding Chimeric antigen receptor (CAR) Sequence.

In another preferred example, the target sequence that the RNA interfering is directed to is as shown in SEQ ID NO.:1.

In another preferred example, the RNA interfering is selected from the group: shRNA, miRNA, piRNA and siRNA.

In another preferred example, the RNA interfering is shRNA.

In another preferred example, the sequence of the shRNA is as shown in SEQ ID NO.:2.

In another preferred example, the sequence of the RNA interfering is as shown in SEQ ID NO.:14.

In another preferred example, first expression cassette also includes the first promoter sequence.

In another preferred example, first promoter includes U6 promoter.

In another preferred example, the length of the nucleotide catenation sequence be m nucleotide, wherein m for 1-20 (compared with Good ground 1-10) positive integer.

In another preferred example, the length of the intervening sequence is 3-30bp, preferably 4-20bp.

In another preferred example, the structure of the Chimeric antigen receptor is as shown in following formula C:

L-scFv-H-TM-C-CD3 ζ (C)

In formula,

Each "-" independently is link peptide or peptide bond

L is optional boot sequence (Leader sequence, i.e. signal peptide sequence)；

ScFv is antigen-binding domains；

H is hinge region；

TM is transmembrane domain；

C is that costimulatory signal receptor tyrosine activates motif (costimulatory molecules)；

CD3 ζ is the endochylema signal transduction sequence derived from CD3 ζ.

In another preferred example, the L be albumen selected from the group below signal peptide: CD8, GM-CSF, CD4, CD137, Or combinations thereof.

In another preferred example, the sequence of L is as shown in 1-25 of SEQ ID NO.:12.

In another preferred example, the scFv is the single chain antibody variable region sequences of target tumor antigen.

In another preferred example, the scFv is the single chain antibody variable region sequences that targeting is selected from the group antigen: CD19, CD20、CD22、CD123、CD47、CD138、CD33、CD30、mesothelin、EGFR、GPC3、BCMA、 ErbB2、NKG2D Ligands, LMP1, EpCAM, VEGFR-1, Lewis-Y, ROR1, Claudin18.2, or combinations thereof.

In another preferred example, scFv includes the antigen-binding domains for targeting CD19.

In another preferred example, scFv is the antigen-binding domains for targeting CD19, the preferably sequence of scFv such as SEQ Shown in the position 26-810 of ID NO.:12.

In another preferred example, the H1 be albumen selected from the group below hinge area: CD8, CD28, CD137 or its Combination.

In another preferred example, the sequence of hinge region is as shown in the position 811-945 of SEQ ID NO.:12.

In another preferred example, the TM is the transmembrane region of albumen selected from the group below: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or A combination thereof.

In another preferred example, TM is the transmembrane region derived from CD8, the preferably sequence of TM such as SEQ ID NO.:12's Shown in 946-1017.

In another preferred example, the C is that the costimulatory signal receptor tyrosine of albumen selected from the group below activates base Sequence: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134,4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

In another preferred example, C is that the costimulatory signal receptor tyrosine in the source 4-1BB activates motif, preferably C's Sequence is as shown in the position 1018-1143 of SEQ ID NO.:12.

In another preferred example, the sequence of CD3 ζ is as shown in the position 1144-1482 of SEQ ID NO.:12.

In another preferred example, the sequence of the Chimeric antigen receptor is as shown in SEQ ID NO.:12.

In another preferred example, the of the nucleotide sequence such as SEQ ID NO.:13 of the Chimeric antigen receptor is encoded Shown in 3675-5156.

In another preferred example, second expression cassette also includes the second promoter sequence.

In another preferred example, second promoter is selected from the group: constitutive promoter or inducible promoter.

In another preferred example, second promoter includes EF1 α promoter.

In the second aspect of the present invention, a kind of carrier is provided, the carrier contains described in the first aspect of the present invention Nucleic acid constructs.

In another preferred example, the carrier is plasmid, viral vectors.

In another preferred example, the carrier is slow virus carrier, adenovirus vector.

In another preferred example, the carrier is PLL3.7 carrier.

In the third aspect of the present invention, a kind of host cell is provided, of the invention the is contained in the host cell Nucleic acid constructs described in the first aspect of the present invention of external source is integrated in carrier or chromosome described in two aspects.

In another preferred example, the cell is isolated cell and/or the cell is genetically engineered cell.

In another preferred example, the cell behaviour or inhuman mammalian cell.

In another preferred example, the cell is T cell.

In another preferred example, the expression of the ACAT1 gene of the cell is suppressed.

In the fourth aspect of the present invention, a kind of cell preparation is provided, it is thin that the cell preparation contains Chimeric antigen receptor Born of the same parents, the Chimeric antigen receptor cell, which contains in carrier described in the second aspect of the present invention or chromosome, is integrated with external source The first aspect of the present invention described in nucleic acid constructs.

In another preferred example, the ACAT1 gene expression of the Chimeric antigen receptor cell is suppressed.

In another preferred example, the cell preparation is liquid.

In another preferred example, the cell concentration of the cell preparation is 1 × 10⁴-1×10¹⁰A cell/ml, preferably It is 1 × 10⁵-1×10⁹A cell/ml.

In another preferred example, the dosage form of the cell preparation includes injection.

In the fifth aspect of the invention, a kind of pharmaceutical composition is provided, the composition contains pharmaceutically acceptable Nucleic acid constructs described in carrier and the first aspect of the present invention, carrier or the present invention described in the second aspect of the present invention The third aspect described in cell.

In the sixth aspect of the present invention, nucleic acid constructs, the present invention described in a kind of the first aspect of the present invention are provided Second aspect described in it is thin described in cell or the fourth aspect of the present invention described in carrier or the third aspect of the present invention The purposes of pharmaceutical composition described in born of the same parents' preparation or the fifth aspect of the present invention, be used to prepare inhibit tumour cell drug or Preparation.

In another preferred example, the tumour cell is selected from the group: leukaemia cell, liver cancer cells, prostate cancer are thin Born of the same parents, ovarian cancer cell, myeloma cell.

In the seventh aspect of the present invention, a kind of RNA interfering for inhibiting ACAT1 gene expression, the RNA interfering are provided For target sequence as shown in SEQ ID NO.:1.

In the eighth aspect of the present invention, a kind of method for treating disease is provided, including is applied to object in need for the treatment of Cell preparation described in cell described in suitable the third aspect of the present invention or the fourth aspect of the present invention is of the invention Pharmaceutical composition described in 5th aspect.

In another preferred example, the disease is tumour.

In the ninth aspect of the present invention, a kind of method for preparing CAR-T cell (T cell of CAR- modification), institute are provided Method is stated comprising steps of by load described in nucleic acid constructs or the second aspect of the present invention described in the first aspect of the present invention Body is transduceed in T cell, to obtain the CAR-T cell.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, Not repeated them here.

Detailed description of the invention

Fig. 1 shows pLL3.7- (shRNA-A/NC)-EGFP carrier structure schematic diagram.

Fig. 2 shows recombinant plasmid EcoR I digestion qualification result.

Fig. 3 shows interference effect testing result of the ACAT1-shRNA in Jurkat cell.Wherein, Fig. 3 A is shown Efficiency of infection of the two kinds of viruses of pLL3.7-shRNA-A-EGFP and pLL3.7-shRNA-NC-EGFP in Jurkat cell connects Nearly 100%.Under Fig. 3 B shows that Jurkat cell its ACAT1 genetic transcription through pLL3.7-shRNA-A-EGFP infection is obvious It adjusts.Fig. 3 C shows that its ACAT1 protein level is significantly lowered through the Jurkat cell of pLL3.7-shRNA-A-EGFP infection.Figure 3D display can persistently be reduced through its ACAT1mRNA level of the Jurkat cell of shRNA interference.

Fig. 4 shows three kinds of pLL3.7-CAR19, pLL3.7-shRNA-A-CAR19, pLL3.7-shRNA-NC-CAR19 The structural schematic diagram of plasmid.

Fig. 5 shows ACAT1-shRNA-CART killing-efficiency result (E:T=0.5:1).

Wherein, Fig. 5 A and Fig. 5 B shows each group flow cytomery as a result, Fig. 5 C shows each group pair The killing-efficiency of Nalm6.Specifically, target cell Nalm6 cell co-cultures after the dyeing of eFluor 670 with effector cell, 0h (upper row) and 6h (lower row) select the channel FL4 to carry out the detection of eFluor 670 on flow cytometer respectively, iris out all EFluor 670 positive cell, as a result as shown in Figure 5 B；After the positive circle door of eFluor 670, the channel FL2 is selected to carry out PI dye Color, the cell of PI stained positive are apoptotic cell.According to streaming as a result, calculating each group to the killing-efficiency of Nalm6, such as Shown in Fig. 5 C.

Fig. 6 shows pLL3.7- (shRNA-A/B/C/NC)-EGFP recombinant plasmid EcoR I digestion qualification result.

Fig. 7 A shows that the shRNA of three kinds of different sequences (shRNA-A/B/C/NC) of design is horizontal in ACAT1 mRNA On interference effect.

Fig. 7 B shows the shRNA of three kinds of different sequences (shRNA-A/B/C/NC) of design in ACAT1 genetic transcription Interference effect.

Fig. 8 shows the structure of typical shRNA a kind of.

Specific embodiment

The present inventor is surprised to find that a kind of doing for inhibition ACAT1 gene expression by depth studying extensively for the first time RNA is disturbed, and the nucleic acid constructs comprising the RNA interfering and Chimeric antigen receptor coded sequence, and is based on the shRNA Enhanced CAR-T cell is prepared for nucleic acid constructs.Experiment shows that RNA interfering of the invention can effectively inhibit The expression of ACAT1 gene significantly increases the killing-efficiency of CAR-T cells against tumor cells.This hair is completed on this basis It is bright.

Chimeric antigen receptor

Chimeric antigen receptor (CAR) of the invention includes extracellular domain, transmembrane domain and intracellular domain. Extracellular domain includes target-specific binding members (also referred to as antigen-binding domains).Intracellular domain includes costimulation Signal transduction area and ζ chain part.Costimulatory signal conducting region refers to a part of the intracellular domain including costimulatory molecules. Costimulatory molecules be effective response of the lymphocyte to antigen required for cell surface molecule, rather than antigen receptor or it Ligand.

Between the extracellular domain and transmembrane domain of CAR, or CAR cytoplasmic domain and transmembrane domain it Between, it may be incorporated into connector.As used herein, term " connector " is often referred to play the born of the same parents that transmembrane domain is connected to polypeptide chain Extracellular portion or any oligopeptides or polypeptide of cytoplasmic domain effect.Connector may include 0-300 amino acid, preferably 2 to 100 amino acid and most preferably 3 to 50 amino acid.

In of the invention one preferable embodiment, the extracellular domain of CAR provided by the invention includes targeting The antigen-binding domains of CD19.CAR of the invention can be carried out when expressing in T cell based on antigen-binding specificity Antigen recognizing.When its combine its associated antigen when, influence tumour cell, cause tumour cell not grow, be prompted to death or with Other modes are affected, and the tumor load of patient is caused to reduce or eliminate.Antigen-binding domains are preferably and from costimulation The intracellular domain fusion of one or more of molecule and ζ chain.Preferably, antigen-binding domains and 4-1BB signal pass The intracellular domain fusion of transduction domain and the combination of CD3 ζ signal domain.

In one embodiment, CAR of the invention includes target-specific binding of referred to as antigen-binding domains Element.The antigen-binding domains of CAR of the present invention are the specific binding members for targeting CD19.

For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include the extracellular structure for being fused to CAR The transmembrane domain in domain.In one embodiment, using naturally with the associated transmembrane structure of one of structural domain in CAR Domain.In some instances, transmembrane domain may be selected, or modified by amino acid replacement, to avoid by such structure Domain is bound to the transmembrane domain of identical or different surface membrane protein, to minimize other members with receptor complex Interaction.

Intracellular domain in CAR of the invention includes the signal transduction structural domain of 4-1BB and the signal transduction knot of CD3 ζ Structure domain.

Nucleic acid constructs

The present invention provides a kind of RNA interfering for inhibiting ACAT1 gene expression, the target sequence of the RNA interfering such as SEQ Shown in ID NO.:1, preferred RNA interfering is shRNA, and preferably the sequence of shRNA is as shown in SEQ ID NO.:2.

The present invention also provides the nucleic acid constructs comprising the RNA interfering and Chimeric antigen receptor coded sequence.Specifically Ground, the nucleic acid constructs include the first expression cassette and the second expression cassette, wherein first expression cassette includes to inhibit The RNA interfering of ACAT1 gene expression, second expression cassette include the nucleic acid sequence of encoding chimeric antigen receptor.

The nucleic acid sequence of coding expectation molecule is obtained using the recombination method being known in the art, such as logical It crosses and screens library from the cell of expressing gene, by obtaining the gene from the known carrier including the gene, or pass through benefit With the technology of standard, it is directly separated from cell and tissue comprising the gene.Optionally, interested gene can be synthesized Production.

Present invention provides the carriers for being wherein inserted into nucleic acid constructs of the invention.It is such as slow derived from retrovirus The carrier of virus is the suitable tools for realizing long-term gene transfer because they allow long-term, the stable integration of transgenosis and It is proliferated in daughter cell.It is more than the load from oncogenic retrovirus such as murine leukemia virus that slow virus carrier, which has, The advantages of body, because of their transducible non-proliferative cells, such as liver cell.They also have the advantages that low immunogenicity.

Simplified summary as the first expression cassette, and can usually be operated by the RNA interfering that is operably connected to promoter The nucleic acid of ground connection coding CAR polypeptide or part thereof is to promoter, as the second expression cassette, by the first expression cassette and the second table It is incorporated to expression vector up to box, realize RNA interfering and encodes the coexpression of the natural or synthetic nucleic acid of CAR.The carrier is suitable for Replicate and integrate eukaryocyte.Typical cloning vector includes the transcription and translation that can be used for adjusting desired nucleic acid sequence expression Terminator, initiation sequence and promoter.

It is a kind of preferably comprising nucleic acid constructs of the present invention carrier sequence as shown in SEQ ID NO.:14.

The gene delivery protocols of standard can also be used in expression construct of the invention, are used for nucleic acid immunization and gene therapy. The method of gene delivery is well known in the art.See such as U.S. Patent number 5,399,346,5,580,859,5,589, 466, it is incorporated to by reference of text herein.In another embodiment, the present invention provides gene therapy vectors.

The nucleic acid can be cloned into the carrier of many types.For example, the nucleic acid can be cloned into such carrier comprising But it is not limited to plasmid, phasmid, phage-derived object, animal virus and clay.Specific carrier interested includes that expression carries Body, replicating vector, probe generation vectors and sequencing vector.

Further, expression vector can be supplied to cell in the form of viral vectors.Viral vector technology is in the art It is well known and in (2001, Molecular Cloning:A the Laboratory Manual, Cold such as such as Sambrook Spring Harbor Laboratory, New York) and other virology and molecular biology manual in be described. The virus that can be used as carrier includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpesviral and slow virus.It is logical Often, suitable carrier includes the replication orgin to work at least one organism, promoter sequence, convenient restriction enzyme Site and one or more selectable labels are (for example, WO01/96584；WO01/29058；With U.S. Patent number 6,326, 193)。

Many systems based on virus are developed, for gene transfer to be entered mammalian cell.For example, reverse transcription Virus provides the convenient platform for gene delivery system.Using the technology that is known in the art by the base of selection Because being inserted into carrier and being packaged into retroviral particle.The recombinant virus then can be separated and be transferred to internal or external Subject cell.Many retroviral systems are well known in the art.In some embodiments, it is carried using adenovirus Body.Many adenovirus vectors are well known in the art.In one embodiment, using slow virus carrier.

Additional promoter element, such as enhancer, the frequency that adjustable transcription starts.Normally, these are located at In the region 30-110bp of beginning site upstream, although having shown that many promoters recently also and including the function in initiation site downstream It can element.Interval between promoter element is often flexible, to be squeezed or move relative to another when element When, keep promoter function.In thymidine kinase (tk) promoter, the interval between promoter element, which can be increased, to be separated 50bp, activity are just begun to decline.Depending on promoter, showing discrete component can cooperate or independently work, to play turn Record.

One example of suitable promoter is instant early stage cytomegalovirus (CMV) promoter sequence.The promoter sequence The strong constitutive promoter sequence for being operably coupled to any polynucleotide sequence high level expression thereon can be driven by being classified as Column.Another example of suitable promoter is -1 α of the elongation growth factor (EF-1 α).It is also possible, however, to use other composing types Promoter sequence, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer viral (MMTV), people exempt from The long end of epidemic disease defective virus (HIV) repeats (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Ai Bai Immediately early promoter, Rous sarcoma virus promoter and people's gene open Si Tan-Ba Er (Epstein-Barr) virus Mover, such as, but not limited to actin promoter, Myosin promoter, ferroheme promoter and creatine kinase promoter. Further, the present invention should not limited to the application of constitutive promoter.Inducible promoter is also contemplated as of the invention one Part.The use of inducible promoter provides molecular switch, can be when such expression is desired, and opening can be grasped Make the expression of the polynucleotide sequence of ground connection inducible promoter, or closes expression when expression is undesirable.Induction type The example of promoter includes but is not limited to metallothionein promoter, Glucocorticoid promoter, progesterone promoter and tetracycline Promoter.

In order to assess the expression of CAR polypeptide or part thereof, the expression vector for being introduced into cell also may include selectable mark Any of gene or reporter or both are remembered, in order to from the cell for seeking to be transfected or infect by viral vectors Expression cell is identified and selected in group.In other respects, selectable label can be carried on independent section of DNA and be used for Cotransfection program.The flank of selectable label and both reporters can all have adjusting sequence appropriate, so as to It is expressed in host cell.Useful selectable marker includes such as antibiotics resistance gene, such as neo etc..

Reporter is used to identify the cell of potential transfection and for evaluating the functionality for adjusting sequence.Normally, it reports Gene is following gene: it is not present in recipient organism or tissue or is expressed by recipient organism or tissue, and its Polypeptide is encoded, the expression of the polypeptide is clearly showed that by some property such as enzymatic activitys for being easy detection.Drawn in DNA After entering recipient cell, the expression of reporter is measured under the suitable time.Suitable reporter may include that coding is glimmering Light element enzyme, beta galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or Green Fluorescent Protein gene base Because of (for example, Ui-Tei etc., 2000FEBS Letters479:79-82).Suitable expression system is well known and using Know technology preparation or commercially obtains.In general, the reporter expression of display highest level has minimum 5 flanking regions Construct be accredited as promoter.Such promoter region can be connected to reporter and open for evaluating reagent adjusting Mover-driving transcription ability.

Gene is introduced into cell and is well known in the art the method that gene expression enters cell.In expression vector Content in, carrier can be easily introduced into host cell by any method in the art, for example, mammal, thin Bacterium, yeast or insect cell.For example, expression vector can be transferred to host cell by physics, chemistry or biological means.

By polynucleotides introduce host cell physical method include calcium phosphate precipitation, lipofection, particle bombardment, Microinjection, electroporation etc..Production includes that the method for the cell of carrier and/or exogenous nucleic acid is well known in the present art.See (2001, Molecular Cloning:A Laboratory Manual, Cold the Spring Harbor such as such as Sambrook Laboratory,New York).It is calcium phosphate transfection by the preferred method that polynucleotides introduce host cell.

It include using DNA and RNA carrier by the biological method that interested polynucleotides introduce host cell.Virus Carrier, especially retroviral vector have become most widely used by gene insertion mammal such as people's cell Method.Other viral vectors may originate from slow virus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus etc..See Such as U.S. Patent number 5,350,674 and 5,585,362.

It include dispersion system of colloid by the chemical means that polynucleotides introduce host cell, such as macromolecular complex is received Rice glue capsule, microballoon, pearl；With the system based on lipid, including oil in water emulsion, micella, mixed micelle and liposome.As body Outer and internal tool for transmitting (delivery vehicle) exemplary colloid system is liposome (for example, artificial membrane vesicle).

Using non-viral delivery system, exemplary tool for transmitting is liposome.Consider to use lipid system Nucleic acid is introduced host cell (external, in vitro (ex vivo) or in vivo) by agent.On the other hand, which can be with lipid It is associated.Nucleic acid associated with lipid can be encapsulated into the aqueous interior of liposome, be dispersed in the lipid bilayer of liposome It is interior, through both associated connection molecule is attached to liposome with liposome and oligonucleotides, liposome is fallen into, with lipid Bluk recombination is dispersed in the solution comprising lipid, mixes with lipid, combine with lipid, is included in lipid as suspension, It is compound or otherwise associated with lipid included in micella or with micella.Lipid associated with composition, lipid/ DNA or lipid/expression vector are not limited to any specific structure in solution.For example, they may be present in bilayer structure In, as micella or there is " (collapsed) of collapse " structure.They can also simply be distributed in the solution, may Form size or the inhomogenous aggregation of shape.Lipid is fatty material, can be the natural lipid occurred or synthesize.For example, Lipid includes lipid droplet, natural that the derivative such as rouge in cytoplasm and comprising long-chain fat race hydrocarbon and they occurs Fat acid, alcohols, amine, alkamine and aldehydes such compound in.

In a preferred embodiment of the invention, the carrier is slow virus carrier.

RNA interferes (RNAi)

As used herein, term " RNA interference (RNA interference, RNAi) " refers to: some small double-stranded RNAs The expression that can efficiently, specifically block internal specific gene, promotes mRNA to degrade, and lures that cells show goes out specific gene and lacks into The phenotype of mistake is also referred to as RNA intervention or RNA interference.RNA interference is that the gene in mRNA level in-site of high special is heavy Silent mechanism.

As used herein, term " siRNA (small interfering RNA, siRNA) ", " RNA interfering " are Refer to a kind of short-movie section double stranded rna molecule, can be using the mRNA of homologous complementary sequence as the target specific mRNA of degradation, this Process is exactly RNA interference channel (RNA interference pathway).

In the present invention, RNA interfering includes siRNA, shRNA and corresponding construction.

A kind of typical construction is double-strand, and its normal chain or minus strand contain structure shown in formula AI:

Seq_{It is positive}-X-Seq_ReverselyFormula AI

In formula,

Seq_{It is positive}For ACAT1 gene or the nucleotide sequence of segment；

Seq_ReverselyFor with Seq_{It is positive}The nucleotide sequence being substantially complementary；

X is positioned at Seq_{It is positive}And Seq_ReverselyBetween intervening sequence, and the intervening sequence and Seq_{It is positive}And Seq_ReverselyNot mutually It mends.

In a preference of the invention, Seq_{It is positive}、Seq_ReverselyLength be 19-30bp, preferably 20-25bp.

In the present invention, a kind of typical shRNA is as shown in the formula AI I in Fig. 8,

In formula,

Seq’_{It is positive}For Seq_{It is positive}The corresponding RNA sequence of sequence or sequence fragment；

Seq’_ReverselyFor with Seq '_{It is positive}The sequence being substantially complementary；

X ' is nothing；Or for positioned at Seq '_{It is positive}And Seq '_ReverselyBetween intervening sequence, and the intervening sequence and Seq '_{It is positive} And Seq '_ReverselyIt is complementary,

| | it indicates in Seq_{It is positive}And Seq_ReverselyBetween the hydrogen bond that is formed.

In a kind of preferred embodiment, the length of the intervening sequence X is 3-30bp, preferably 4-20bp.

In a kind of preferred embodiment, the shRNA can be folded into a kind of stable stem ring (hair clip) structure, institute The loop-stem structure length stated is generally between 50-100bp.The shRNA can be folded into stable loop-stem structure, stem ring knot The stem two sides of structure include the two sequences being substantially complementary.

By RNAi mechanism identical with the processing of miRNA, shRNA can be processed to siRNA.Use bacterium or disease Poisonous carrier, shRNA are imported into the nucleus of target cell, and in some cases, carrier can be stably integrated into genome In.According to the difference of the promoter of driving expression, shRNA can be by rna plymerase ii or III catalysis transcription.In quilt Before Exportin-5 transporte to cells matter, these initial front body structures need to be combined with Drosha and its double-stranded RNA first Companion DGCR8 processes to form pre-shRNA.Pre-shRNA then by Dicer and TRBP/PACT digestion, removes hairpin structure, At two 3, ' there are two the double-strand siRNA of the 20-25nt of free base for end band for generation.The subsequent quilt of this active siRNA It is integrated into silencing complex up.

In a kind of preferred embodiment, the shRNA is sheared the siRNA to form interference ACAT1 gene expression, Preferably, the sequence of the siRNA is as shown in SEQ ID NO.:14.

In a kind of preferred embodiment, the sequence of the shRNA is as shown in SEQ ID NO.:2.

As used herein, it " is substantially complementary " and refers to that the sequence of nucleotide is enough complementations, can be contemplated that with one kind Mode interact, such as formed secondary structure (such as loop-stem structure).In general, the nucleotide of two " being substantially complementary " Sequence from each other at least 70% nucleotide be complementary；Preferably, at least 80% nucleotide is complementary；More Preferably, at least 90% nucleotide is complementary.Generally, most 8 be can have between two molecules complementary enough A unmatched nucleotide, it is preferable that there is 1,2,3,4,5 unmatched nucleotide.

As used in this application, " stem ring " structure is also referred to as " hair clip " structure, refers to a kind of nucleic acid molecule, can shape At it is a kind of include double-stranded region (stem) secondary structure, the double-stranded region by the nucleic acid molecule two regions (position In on same molecule) it is formed, the two sides of column double stranded section are divided in two regions；It further includes at least one " ring " structure, including non- Complementary nucleic acid molecule, i.e. single-stranded regions.Even if two regions of the nucleic acid molecule are not complete complementary, nucleotide Double stranded section can also keep double-stranded state.For example, insertion, missing, substitution etc. can lead to the not complementary of a zonule or should Zonule itself forms the secondary structure of loop-stem structure or other forms, however, two regions can be still substantially complementary, and It interacts in foreseeable mode, forms the double-stranded region of loop-stem structure.Loop-stem structure is those skilled in the art Known to member, usually after the nucleic acid for obtaining a nucleotide sequence with primary structure, those skilled in the art's energy Enough determine whether the nucleic acid can form loop-stem structure.

In the present invention, " loop-stem structure " may be present in the end of shRNA shown in formula AII, such as due to Seq '_{It is positive}With Seq’_ReverselyAfter forming basic complementation, X ' will form a fixed end loop-stem structure；" loop-stem structure " also may be present in Inside formula AII depicted shRNA, such as due to Seq '_{It is positive}And Seq '_ReverselyBetween not complete complementary, cause complementary combination Seq’_{It is positive}And Seq '_ReverselyBase will form the stem ring (internal loop) inside one.

Therapeutic application

The present invention includes the cell with slow virus carrier (LV) transduction for encoding nucleic acid constructs of the present invention (for example, T is thin Born of the same parents) carry out therapeutic application.The T- cell response that the T cell of transduction can cause CAR- to mediate, and the T cell transduceed ACAT1 gene is suppressed, to significantly improve its killing-efficiency to tumour cell.

Therefore, present invention provides stimulation is immune to the target cell group of mammal or T cell-mediation of tissue The method of response comprising following steps: it is administered to the T cell that mammal expresses nucleic acid constructs of the present invention.

In one embodiment, the present invention includes a kind of cell therapy, and wherein T cell is expressed this by gene modification The nucleic acid constructs of invention obtains the repressed CAR-T cell of ACAT1 gene, and CAR-T cell needs connecing for its by injection In receptor.The cell of injection can kill the tumour cell of recipient.Unlike antibody therapy, CAR-T cell can answer in vivo System generates the long-term persistence that can lead to continued tumor control.

In one embodiment, CAR-T cell of the invention can undergo firm internal T cell to extend and sustainable Extended time quantum.In addition, the immune response that CAR is mediated can be a part of adoptive immunotherapy step, wherein CAR- is modified Immune response of the induced t cell to the antigen-binding domains specificity in CAR.For example, anti-CD19 CAR-T cell causes to resist Express the specific immune response of the cell of CD19.

Although data disclosed herein specifically disclose including anti-CD19scFv, hinge and transmembrane region and 4-1BB and The slow virus carrier of CD3 ζ signal transduction structural domain, but this invention generally should be construed as including to every in construct component part One any amount of variation.

Medicable cancer includes that not by vascularization or substantially there are no by the tumour and vascularization of vascularization Tumour.Cancer may include non-physical knurl (such as haematological tumours, such as leukaemia and lymthoma) or may include solid tumor. It include but is not limited to cancer, enblastoma and sarcoma and certain leukaemia or lymph with the cancer types that CAR of the invention is treated Malignant tumour, benign and malignant tumour and malignant tumor, such as sarcoma, cancer and melanoma.It also include adult lesion/cancer disease and youngster Virgin lesion/cancer disease.

Hematologic cancer is the cancer of blood or marrow.The example of hematology (or hematogenous) cancer includes leukaemia, packet Include acute leukemia (such as acute lymphoblastic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and pulpefaction Cellularity, promyelocyte, grain-monocyte type, monocarpotic cellularity and erythroleukemia), chronic leukemia (such as chronic marrow Cell (granulocytic) leukaemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, Lymthoma, hodgkin's disease, non Hodgkin lymphom (painless and high-grade form), Huppert's disease, Walden this Special Lun Shi macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumor is the abnormal mass of the tissue usually not comprising tumour or fluid zone.Solid tumor can be benign or malignant 's.Different types of solid tumor names (such as sarcoma, cancer and lymthoma) with the cell type for forming them.Solid tumor is such as The example of sarcoma and cancer includes fibrosarcoma, myxosarcoma, embryonal-cell lipoma celiothelioma, lymphoid malignancy, cancer of pancreas ovary Cancer,.

CAR- modification T cell of the invention also is used as the vaccine to mammal Ex vivo immunization and/or in vivo Type.Preferably, mammal is behaved.

For Ex vivo immunization, at least one of the following occurs in vitro before cell application is entered mammal: i) Amplifying cells, ii) nucleic acid that will encode CAR introduces cell and/or iii) Cell Cryopreservation.

In vitro program is well known in the present art, and is being discussed more fully below.Briefly, cell from Separate in mammal (preferably people) and with the carrier of expression CAR disclosed herein carry out gene modification (that is, ex vivo transduction or Transfection).The cell of CAR- modification can be administered to mammalian subject, to provide treatment benefit.Mammalian subject can It can be self relative to recipient for people and the CAR- cell modified.Optionally, cell can be of the same race relative to recipient Allogene, isogenic (syngeneic) or xenogenesis.

Other than for Ex vivo immunization using based on the vaccine of cell, present invention provides vivo immunizations to draw Play the composition and method for the immune response of antigen in patient.

The present invention provides the methods for the treatment of tumour comprising is administered to subject a effective amount of hair for needing it The T cell of bright CAR- modification.

The T cell of CAR- modification of the invention can be administered alone or as pharmaceutical composition and diluent and/or and its His component such as IL-2, IL-17 or other cell factors or cell mass combine application.Briefly, pharmaceutical composition of the invention Object may include target cell group as described herein, with one or more pharmacy or physiologically acceptable carriers, diluent or tax Shape agent combines.Such composition may include buffer such as neutral buffered saline, sulfate buffered saline etc.；Carbon aquation Close object such as glucose, mannose, sucrose or glucan, mannitol；Protein；Polypeptide or amino acid such as glycine；Antioxygen Agent；Chelating agent such as EDTA or glutathione；Adjuvant (for example, aluminium hydroxide)；And preservative.Composition of the invention is excellent Choosing is formulated for intravenously applying.

The mode that pharmaceutical composition of the invention can be suitable for the disease of (or prevention) to be treated is applied.The number of application Amount and frequency will be determined by such factor, although such as the illness of patient and the type of patient disease and severity --- it is appropriate Dosage can be determined by clinical test.

When pointing out " effective quantity in immunology ", " antitumor effective quantity ", " tumour-inhibition effective quantity " or " therapeutic dose ", The precise volume of the present composition to be administered can be determined that the age of consideration patient (object), weight, tumour are big by doctor The individual difference of small, infection or metastasis degree and illness.It can usually point out: the pharmaceutical composition including T cell described herein It can be with 10⁴To 10⁹A cell/kg weight dosage, preferably 10⁵To 10⁶A cell/kg weight dosage (including those models Enclose interior all integer values) application.T cell composition can also be with these dosage multiple applications.Cell can be by using immune Well known injection technique in therapy (see such as Rosenberg etc., NewEng.J.of Med.319:1676,1988) application.It is right It can be by monitoring the disease indication of patient and therefore adjustment for the treatment of is led by medicine in the optimal dose and therapeutic scheme of specific patient Field technique personnel are readily determined.

The application of object composition object can carry out in any convenient manner, including pass through spray-on process, inject, swallow, is defeated Liquid, implantation or transplanting.Compositions described herein can by subcutaneous, intradermal, tumor, in knot, in spinal cord, intramuscular, pass through vein Patient is administered in interior (i.v.) injection or peritonaeum.In one embodiment, T cell composition of the invention passes through intradermal Or subcutaneous injection is administered to patient.In another embodiment, T cell composition of the invention is preferably infused by i.v. Penetrate application.The composition of T cell can be injected directly into tumour, lymph node or infection position.

In some embodiments of the present invention, using method described herein or it is known in the art other by T cell The cell for extending to the method activation and extension of therapeutic level, in conjunction with any amount of related form of therapy (for example, it Before, simultaneously or after) be administered to patient, the form of therapy includes but is not limited to be treated with following reagent: the reagent Such as antiviral therapy, cidofovir and interleukin 2, cytarabine (also being known as ARA-C) or that MS patient His pearl monoclonal antibody treats or to the method pearl monoclonal antibody in distress treatment of psoriatic or to the other treatment of PML patient.Further real Apply in mode, T cell of the invention can with below in conjunction with using: chemotherapy, radiation, immunosuppressor, such as, cyclosporin, sulphur Azoles purine, methopterin, mycophenolate and FK506, antibody or other immunotherapeutic agents.In further embodiment, Cell composition of the invention and bone-marrow transplantation utilize chemotherapeutics such as fludarabine, external beam radiation therapy (XRT), ring Phosphamide is administered to patient in conjunction with (prior to, concurrently with, or after for example).For example, in one embodiment, object can be through The standard care of high dose chemotherapy is gone through, carries out autologous peripheral blood stemcell transplant later.In some embodiments, after the transfer, right Injection as receiving the immunocyte of extension of the invention.In an additional embodiment, the cell of extension is in surgery hand The application of preoperative or surgical site infections.

The dosage for being administered to the above treatment of patient will become with the exact properties for the treatment of illness and the recipient for the treatment of Change.The practice that people's applied dose ratio can receive according to this field is implemented.In general, each treatment or each course for the treatment of, it can be by 1 ×10⁶It is a to 1 × 10¹⁰A modified T cell of the present invention (e.g., CAR-T20 cell), for example, by the mode of venous re-transfusion, It is applied to patient.

Main advantages of the present invention include:

(a) ACAT1 gene is interfered using RNAi for the first time, the tumor-killing efficiency of CART can be promoted；

(b) the RNAi sequence of the designed ACAT1 as shown in Table 1 of this research, can effectively realize to ACAT1 base The interference of cause；

(c) the CD19-CAR carrier based on Pll3.7 of ACAT1RNAi is incorporated designed by the present invention, it was confirmed that energy The killing-efficiency of CD19-CART is significantly improved, but the content of present invention is not limited to be suitble to the CAR carrier of Pl l3.7 building In all other plasmid vectors for being suitable for constructing CART, target spot is also not necessarily limited to CD19-CART, should be suitable for all target spots CART。

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to routine Condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.

Some common materials are as follows in embodiment:

PEI (polyethyleneimine): it is purchased from Thermo；Opti-MEM culture medium: it is purchased from Thermo；Polybrene: it is purchased from Millipore；IL-2: it is purchased from Peprotech；X-VIVO15: it is purchased from Lonza；TransAct: it is purchased from Metyni；PLL3.7 is carried Body: (East China Normal University Li great Li professor's experimental group is gifted).

Embodiment 1

The building of pLL3.7-ACAT1-shRNA interference carrier

The design of 1.1 RNAi target sequences

According to website (https: //rnaidesigner.thermofisher.com/rnaiexpress/des Ign.do ACAT1 gene RNAi target sequence) is designed, as a result shown in table 1.

Table 1.ACAT1 gene RNAi target sequence

Target sequence title	Initial position	The nucleotide sequence of RNAi target sequence	GC%	SEQ ID NO.
					Target sequence A	131	GGTGCAGGAAATAAGATATGT	38.1	SEQ ID NO.:1

1.2 according to targeting sequence design interference sequence:

Based on the target sequence filtered out, following design and determine interference sequence: 5 ends are started with G, set G+C content as 30%~50%.According to the requirement of pll3.7 carrier, U6 promoter l T are rebuild at the end of positive-sense strand 5 ' plus T, interfere target sequence Afterwards plus Loop ring " TTCAAGAGA ", addition invert complementary series and termination signal " TTTTTT ", add EcoR I enzyme at 3 ends Enzyme site GAATTC is to facilitate identification.Filling-in Xho I restriction enzyme site synthesizes 1 pair of complementary fragment again.Resultant interference target sequence The sequence of siRNA is CCACGUCCUUUAUUCUAUACA (SEQ ID NO.:14).

Sequence is upset into design NC (Negative Control) sequence, each sequence is as shown in table 2 below

The oligonucleotide sequence that table 2. is separately designed for target sequence and negative control sequence

1.3 building interference plasmids

It is 0.1mol/L that the interference fragment of synthesis, which is diluted to concentration, and upstream and downstream segment respectively takes 10ul to be added in EP pipe, It mixes gently, cooled to room temperature after 95 DEG C of water-bath 5min.It takes 2ul product to detect through 2.0% agarose gel electrophoresis to close Chain situation.After the annealing of the precursor sequence of interference sequence A and negative control sequence NC, verifies and anneal through acrylamide gel electrophoresis Success.

Unloaded pLL3.7 gives restriction enzyme Xho I, I double digestion of Hpa, connects after glue recycling with the interference fragment for closing chain Take over night, construction recombination plasmid.Recombinant plasmid EcoR I digestion is identified and is sequenced.

Embodiment 2

PLL3.7-shRNA- (A/NC)-(EGFP/CAR19) slow virus packaging

1. plasmid transfection

1) by plasmid, PEI, Opti-MEM culture medium is placed in room temperature 5min；

2) it takes 436 μ l of Opti-MEM in 1.5mlEP pipe, adds 64 μ g PEI mixing, be stored at room temperature 5min；

3) 12 μ g vector plasmids are taken, 8 μ g psPA × 2,4 μ g pMD2.G are added Opti-MEM to 500 μ l, are stored at room temperature 5min；

4) the PEI-Opti-MEM solution prepared is added in the Opti-MEM containing plasmid, is stored at room temperature 20min；

5) 1ml DNA/PEI mixture is slowly instilled in the 293T culture dish that the previous day completes, is mixed gently, 37 DEG C Incubator is incubated for, 6-8h or overnight；

6) fresh culture is replaced, 37 DEG C of incubators is put into and continues to be incubated for.

2. collection virus and concentration

1) after plasmid transfection 48h, after collecting supernatant, addition 10ml fresh culture continues culture to 72h, collects again Supernatant after mixing with the 48h supernatant collected, is placed in 4 DEG C of refrigerators stand-by；

2) 4 DEG C, 4000g is centrifuged 10min, removes cell fragment；

3) supernatant being obtained by filtration with 0.45 μm of filter；

4) filtered viral supernatants are transferred in ultracentrifugation pipe, 25000 turns of centrifugation 2h, with 1/100 supernatant volume PBS is diluted, and is transferred in closed centrifuge tube and is stayed overnight after blowing and beating repeatedly for 4 DEG C；

5) virus liquid is dispensed to suitable volume, is placed in -80 DEG C and saves, and 200 μ l viruses is taken to carry out titre survey It is fixed.

3. virus titer measures

1) 293T cell is digested, is counted after centrifugation, cell suspension is made with serum-containing media, adjustment cell density is 4 ×10⁵Polybrene to 12 μ g/ml is added in/ml, and 0.5ml cell suspension is added into every hole of 24 well culture plates；

2) viral supernatants are diluted in the following proportions with full culture medium: 1:3；1:9；1:27；

3) virus liquid after diluting respectively by 100 μ l virus stock solution useds and by different proportion, is added to the 24 of inoculating cell In orifice plate；

4) infection supernatant is discarded after 16h, adds the fresh full culture medium of 0.5ml；

5) after 48h flow cytometer detection be infected cell destination gene expression；

6) titre, titre=2*10 are calculated⁶* efficiency of infection * extension rate.

As a result as follows:

After collection virus concentration, detected through titre, pLL3.7-shRNA-A-EGFP, pLL3.7-shRNA-NC-EGFP two The titre of kind virus is respectively 1*10⁸、1.2*10⁸。

Embodiment 3

PLL3.7-ACAT1-shRNA-EGFP carrier interferes verification test

Jurkat T is inoculated in 24 orifice plates, every hole 2 × 10⁵A cell, totally 9 hole.It is divided into 3 groups: blank negative control And pLL3.7-shRNA-A-EGFP slow virus group, pLL3.7-shRNA-NC-EGFP, every group of each 3 hole.Two experimental group difference The virus of respective volume is added according to MOI=10:1, and 10 μ g/ml polybrene are added and promote infection.After 10h by cell into Row is collected, and 1000g is centrifuged 10min, discards culture medium, and fresh culture is added.

Cell, flow cytometer detection GFP expression efficiency, i.e. virus infection positive rate are collected after slow-virus infection 48h.As a result such as Shown in Fig. 3 A.Sense of the two kinds of viruses of pLL3.7-shRNA-A-EGFP and pLL3.7-shRNA-NC-EGFP in Jurkat cell Efficiency is contaminated close to 100%.

Three groups of Jurkat T cells after collecting infection 48h.With Trizol lytic cell, extracted total RNA is reversed Record.The interference effect of Q-PCR analysis recombinant plasmid.Energy after pLL3.7-shRNA-A-EGFP slow virus infected cell as the result is shown Interfere significantly with the transcription (Fig. 3 B) of ACAT1.Through its ACAT1 albumen water of the Jurkat cell of pLL3.7-shRNA-A-EGFP infection It is flat also significantly to lower (Fig. 3 C).

It can continue the transcription of inhibition Acat1 to verify pLL3.7-shRNA-A-EGFP slow virus infected cell, point Infection 0d, 2d, 4d, 6d, 8d cell is not collected, detects the transcriptional level of Acat1.It was found that as time went on, shRNA-A sequence Column can continue to interfere the expression of Jurkat T cell ACAT1, and mRNA level in-site can be by lasting reduction (Fig. 3 D).

Embodiment 4

The confirmatory experiment of ACAT1-shRNA-CART promotion CART tumor-killing efficiency

The preparation of 1.ACAT1-shRNA-CART

D0 is immunized after taking the bleeding of the umbilicus in fresh source to separate PBMC by density gradient centrifugation with CD4/CD8 magnetic bead Magnetic bead screening, the positive cell of acquisition is according to 1*10⁶The density of/ml is inoculated with, and addition contains 10%FBS, 100U/ml IL-2 X-vivo15 culture medium, then according to 1:100 ratio be added TransAct, d2 collect T cell, centrifugation change liquid, will Tri- kinds of slow virus of CD19-CAR, pLL3.7-shRNA-A-CAR19, pLL3.7-shRNA-NC-CAR19 are respectively according to MOI= The ratio of 10:1 is added in culture medium, and liquid is changed in centrifugation after 16h, and fresh culture is added and continues to cultivate.D6 collects cell and carries out Flow cytometer detection.As a result as shown in Figure 5.

2.ACAT1-shRNA-CART Cytotoxicity in vitro confirmatory experiment

1) eFluor 670 of Nalm6 cell is dyed:

Prepare single cell suspension, 1*10⁶/ml。

2ml PBS centrifuge washing is added twice, washes serum.

Cell is resuspended with PBS, adjustment cell density is 2*10⁶/ml。

10 μM of isometric 670 reagents of eFluor are added, vortex cell, 37 DEG C are protected from light incubation 10 minutes；

The complete medium of 10% serum of 4-5 times of volume pre-cooling is added, is incubated for 5 minutes on ice；

Complete medium is washed 3 times.

2) mixed culture of Nalm6 cell and CART cell

By the above-mentioned Nalm6 after the dyeing of eFluor 670 respectively according to 1*10⁵The quantity in/hole is seeded in 24 orifice plates；

Do not feel tri- kinds of CART of CD19-CAR, pll3.7-shRNA-A-CAR19, pll3.7-shRNA-NC-CAR19 and Malicious PBMC catch an illness respectively according to 5*10⁴The quantity in/hole is seeded in the hole of inoculation Nalm6 in advance, and every group sets three repetitions, Every hole fluid infusion is to 1ml；

Culture plate after mixing with cells is put and cultivates 6h into 37 DEG C of incubators；

After 6h, all cells in every hole are collected, cell is transferred in streaming pipe, and PI is added and is incubated for, upper machine examination It surveys.

3) killing-efficiency is analyzed

It selects the channel FL4 to carry out the detection of eFluor 670 on flow cytometer, irises out all 670 positives of eFluor Cell, as a result as shown in Figure 5 B；

After the positive circle door of eFluor 670, the channel FL2 is selected to carry out PI dyeing, the cell of PI stained positive is apoptosis Cell.According to streaming as a result, calculating each group to the killing-efficiency of Nalm6, as shown in Figure 5 C.Difference is calculated by PI value The killing-efficiency of tumour cell in processing group, shRNA-A processing group have highest killing-efficiency, be 60%, with without The shRNA-NC group of CD19-CART or ACAT1 the interference blank sequence of ACAT1 interference is compared, and has significant difference.Thus fill Divide and confirms that there is better killing-efficiency through the CD19-CART of ACAT1 interference.

Comparative example C1 and C2

During screening the shRNA-A interference sequence of the application, applicant is also while for multiple and different target sequences Different interference sequences is devised, and has carried out related experiment.Experimental method is identical as embodiment 1-4.

1. other RNAi target sequences filtered out are as shown in table 3, the upstream and downstream segment of the corresponding interference sequence of each target sequence As shown in table 4, wherein shRNA-A is the original sequence of embodiment.

Table 3.ACAT1 gene RNAi target sequence

Embodiment	Target sequence title	Initial position	The nucleotide sequence of RNAi target sequence	GC%	SEQ ID NO.:
						Embodiment 1	Target sequence A	131	GGTGCAGGAAATAAGATATGT	38.1	1
Comparative example C1	Target sequence B	137	GGAAATAAGATATGTGGAACG	38.1	6
						Comparative example C2	Target sequence C	263	GCTTGGTTCCATTGCAATTCA	42.86	7

The oligonucleotide sequence that table 4. is separately designed for target sequence and negative control sequence

Using method same as Example 1, chain is closed to interference fragment upstream and downstream segment, it is normal to close chain situation.It goes forward side by side Row digestion identification, as a result shown in 5.

Using method same as Example 2, slow virus packaging is carried out.

Using method same as Example 3, the interference effect of pLL3.7-shRNA-A/B/C/NC-EGFP plasmid is detected Fruit, as a result as shown in Figure 7.

Fig. 7 A the results show that ACAT1mRNA express multiple from the point of view of, in numerous RNA interferings being screened, shRNA-A Having the effect of best, mean value 0.28, the interference class mean of shRNA-B and shRNA-C are respectively 0.44 and 0.80, because This shRNA-A segment has optimal interference effect.

Fig. 7 B is after the shRNA of different fragments interference, and western blot detects the protein level of ACAT1, shRNA- The band of A is most light, also shows that its interference effect is most strong.

All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

Sequence table

<110>East China Normal University

Shanghai Bang Yao Biotechnology Co., Ltd

<120>a kind of Chimeric antigen receptor T cell of ACAT1 gene interference

<130> P2017-1527

<160> 14

<170> PatentIn version 3.5

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Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

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Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

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

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Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

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Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

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Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

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Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

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Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

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Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

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Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

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Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

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Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

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Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

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Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

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Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Thr Thr

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Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

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Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

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Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

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Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

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Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

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

355 360 365

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

370 375 380

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln

385 390 395 400

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

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Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

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Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

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ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200

ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260

aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320

tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380

caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440

aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500

aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga 1560

gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 1620

ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 1680

ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 1740

tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 1800

aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1860

aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1920

gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1980

tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 2040

gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 2100

gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 2160

aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 2220

ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 2280

ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata 2340

gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca 2400

ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg 2460

ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca 2520

aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga 2580

cagcagagat ccagtttggt tagtaccggg cccgctctag agatccgacg ccgccatctc 2640

taggcccgcg ccggccccct cgcacagact tgtgggagaa gctcggctac tcccctgccc 2700

cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac 2760

agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa 2820

gagatacaaa tactaaatta ttattttaaa aaacagcaca aaaggaaact caccctaact 2880

gtaaagtaat tgtgtgtttt gagactataa atatcccttg gagaaaagcc ttgtttggtg 2940

caggaaataa gatatgtttc aagagaacat atcttatttc ctgcaccttt tttgaattcc 3000

tcgaggtcga cggtatcgat aagctcgctt cacgagattc cagcaggtcg agggacctaa 3060

taacttcgta tagcatacat tatacgaagt tatattaagg gttccaagct taagcggccg 3120

cggatctgcg atcgctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc 3180

gagaagttgg ggggaggggt cggcaattga acgggtgcct agagaaggtg gcgcggggta 3240

aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg 3300

tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca 3360

cagctgaagc ttcgaggggc tcgcatctct ccttcacgcg cccgccgccc tacctgaggc 3420

cgccatccac gccggttgag tcgcgttctg ccgcctcccg cctgtggtgc ctcctgaact 3480

gcgtccgccg tctaggtaag tttaaagctc aggtcgagac cgggcctttg tccggcgctc 3540

ccttggagcc tacctagact cagccggctc tccacgcttt gcctgaccct gcttgctcaa 3600

ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca gatccaagct gtgaccggcg 3660

cctacgctag cgctatgctt ctcctggtga caagccttct gctctgtgag ttaccacacc 3720

cagcattcct cctgatccca gacatccaga tgacacagac tacatcctcc ctgtctgcct 3780

ctctgggaga cagagtcacc atcagttgca gggcaagtca ggacattagt aaatatttaa 3840

attggtatca gcagaaacca gatggaactg ttaaactcct gatctaccat acatcaagat 3900

tacactcagg agtcccatca aggttcagtg gcagtgggtc tggaacagat tattctctca 3960

ccattagcaa cctggagcaa gaagatattg ccacttactt ttgccaacag ggtaatacgc 4020

ttccgtacac gttcggaggg gggactaagt tggaaataac aggctccacc tctggatccg 4080

gcaagcccgg atctggcgag ggatccacca agggcgaggt gaaactgcag gagtcaggac 4140

ctggcctggt ggcgccctca cagagcctgt ccgtcacatg cactgtctca ggggtctcat 4200

tacccgacta tggtgtaagc tggattcgcc agcctccacg aaagggtctg gagtggctgg 4260

gagtaatatg gggtagtgaa accacatact ataattcagc tctcaaatcc agactgacca 4320

tcatcaagga caactccaag agccaagttt tcttaaaaat gaacagtctg caaactgatg 4380

acacagccat ttactactgt gccaaacatt attactacgg tggtagctat gctatggact 4440

actggggtca aggaacctca gtcaccgtct cctcagcggc cgcaaccacg acgccagcgc 4500

cgcgaccacc aacaccggcg cccaccatcg cgtcacagcc cctgtccctg cgcccagagg 4560

cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc gcctgtgata 4620

tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca ctggttatca 4680

ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa ccatttatga 4740

gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca gaagaagaag 4800

aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc gcgtacaagc 4860

agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag tacgatgttt 4920

tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg aagaaccctc 4980

aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac agtgagattg 5040

ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag ggtctcagta 5100

cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct cgctaagaat 5160

tcgtcgaggg acctaataac ttcgtatagc atacattata cgaagttata catgtttaag 5220

ggttccggtt ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt 5280

acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 5340

gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 5400

cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 5460

aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 5520

ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 5580

tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 5640

ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct 5700

ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 5760

cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 5820

cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac 5880

ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc 5940

tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta 6000

agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 6060

ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 6120

acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca 6180

ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc 6240

aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg 6300

gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca catggcccga 6360

gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc 6420

tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa 6480

gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag 6540

tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt 6600

aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 6660

aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 6720

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 6780

tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 6840

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 6900

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 6960

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 7020

acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 7080

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 7140

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 7200

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 7260

aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 7320

ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 7380

agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 7440

atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 7500

cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 7560

aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 7620

cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 7680

aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 7740

ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 7800

gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 7860

ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 7920

tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 7980

tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 8040

ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 8100

tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 8160

agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 8220

acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 8280

ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 8340

gttccgcgca catttccccg aaaagtgcca cctgac 8376

<210> 14

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ccacguccuu uauucuauac a 21

Claims (10)

1. a nucleic acid construct is characterized in that, the structure of the nucleic acid construct is shown in the following formula A or formula B: B1-Z-B2 (A) B2-Z-B1 (B) In the formula, each "-" is independently a bond or a nucleotide linker sequence; B1 is the first expression cassette, and the first expression cassette is used to express the interfering RNA that inhibits the expression of the ACAT1 gene; B2 is the second expression cassette, and the second expression cassette is used to express the chimeric antigen receptor; Z is an optional spacer sequence. 2. nucleic acid construct as claimed in claim 1, is characterized in that, the target sequence that described interfering RNA is aimed at is as shown in SEQ ID NO.:1. 3. nucleic acid construct as claimed in claim 1 is characterized in that, described interfering RNA is selected from lower group: shRNA, miRNA, piRNA and siRNA, preferably, described interfering RNA is shRNA, more preferably Ground, the sequence of described shRNA is shown as SEQID NO.:2. 4. nucleic acid construct as claimed in claim 1, is characterized in that, the structure of described chimeric antigen receptor is shown in following formula C: L-scFv-H-TM-C-CD3ζ (C) In the formula, Each "-" is independently a connecting peptide or peptide bond L is an optional leader sequence (Leader sequence, namely signal peptide sequence); scFv is an antigen binding domain; H is the hinge region; TM is the transmembrane domain; C is a costimulatory signal receptor tyrosine activation motif (costimulatory molecule); CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ. 5. a carrier, it is characterised in that the carrier contains the nucleic acid construct of claim 1. 6. A host cell, characterized in that, the host cell contains the vector described in claim 2 or the nucleic acid construct described in claim 1 integrated in the chromosome. 7. A cell preparation, characterized in that, the cell preparation contains a chimeric antigen receptor cell, and the chimeric antigen receptor cell contains the vector described in claim 2 or has an exogenous chromosome integrated into it. The nucleic acid construct of claim 1. 8. a pharmaceutical composition is characterized in that, described composition contains pharmaceutically acceptable carrier and the described nucleic acid construct of claim 1, the described carrier of claim 2 or the described cell of claim 3 . 9. the described nucleic acid construct of claim 1, the described carrier of claim 2, the described cell of claim 3 or the described cell preparation of claim 4 or the described pharmaceutical composition of claim 5 It is characterized in that it is used for preparing medicines or preparations for inhibiting tumor cells. 10. An interfering RNA for inhibiting the expression of ACAT1 gene, wherein the target sequence for the interfering RNA is as shown in SEQ ID NO.: 1.