CN112080522A

CN112080522A - Construction method of CD47 humanized mouse model

Info

Publication number: CN112080522A
Application number: CN202010967642.4A
Authority: CN
Inventors: 王韬; 琚存祥; 李松; 张明坤
Original assignee: Guangdong Yaokang Biotechnology Co ltd
Current assignee: Guangdong Yaokang Biotechnology Co ltd
Priority date: 2020-09-15
Filing date: 2020-09-15
Publication date: 2020-12-15

Abstract

The invention discloses a method for constructing a CD47 humanized mouse model, which is characterized in that a mouse model capable of expressing human CD47 is constructed by replacing a mouse CD47 gene with human CD47 on mouse fertilized egg cells with a C57BL/6 background and a BALB/C background by utilizing homologous recombination and an embryonic stem cell technology. According to the invention, a gene modification method is adopted, a human coding gene is substituted for a corresponding gene of a mouse, and the prepared humanized mouse model of the immune checkpoint has human functional genes and can be used for screening and evaluating human drugs of the immune checkpoint.

Description

Construction method of CD47 humanized mouse model

Technical Field

The invention relates to the technical field of animal genetic engineering and genetic modification, in particular to a method for constructing a CD47 humanized mouse model.

Background

Cancer immunotherapy is a therapeutic method for attacking cancer cells by means of the human body's own immune system. The balance between the immune system and cancer cells is a dynamic process of long-term gambling, both positive and interwoven. Immune cells of a healthy body can discover and kill most of cancerated cells, but under the induction of various congenital or acquired factors, the immune system loses absolute advantages and is even 'countered' by cancer cells, so that the immune system becomes a key member in the occurrence and development of cancers.

Many types of tumor immunotherapy play an anti-tumor role mostly through T cells, and in the process of cancer deterioration, tumor cells often block the 'immune monitoring' role of T cells on tumors, so that the phenomenon of immune escape is caused.

CD47, also known as integrin-associated protein, is widely expressed on the cell surface and interacts with inhibitory receptor signaling regulatory protein α (sirpa), thrombospondin (TSP1) and Integrins (Integrins) to mediate a series of responses including apoptosis, proliferation, immunity, etc. The CD47 molecule is proved to be in an over-expression state in many malignant tumors, such as Acute Myeloid Leukemia (AML), B cell and T cell acute leukemia, non-Hodgkin lymphoma, etc., and the expression level is shown to be in a negative correlation with the prognosis of the disease. Tumor cells can escape immune surveillance by macrophages through the CD 47-sirpa signaling pathway. Therefore, the CD47 antibody can block the combination of CD47 and SIRP alpha, activate the phagocytosis of macrophages to tumors and the antigen presenting effect of DC cells, and can be used for combined treatment with other tumor killing path medicines, such as immunotherapy, small molecule targeted medicines, chemotherapy, radiotherapy and the like.

Screening of immune checkpoint drugs and preclinical testing require evaluation in animal models, and rodents, as the most widely used experimental small animal models, have become indispensable surrogate models in the study of human tumor therapy. However, due to differences in species attributes, immune checkpoint antibodies screened on mice are not fully tried in humans.

Chinese patent 201510310055.7 discloses a humanized mouse capable of expressing a mouse-human fusion CD47 gene, the extracellular portion being divided into mouse CD47 amino acid sequence and the intracellular portion being a human CD47 amino acid sequence. Chinese patent application 201810295709.7 discloses a preparation method and application of a humanized modified animal model of CD47 gene, humanized CD47 gene encodes extracellular region, transmembrane domain and intracellular region of humanized CD47 protein, wherein the transmembrane domain and intracellular region are animal sources, all or part of the extracellular region is encoded by a human source part, and the animal source part and the human source part are connected with the CD47 promoter endogenous to the animal model through sequence splicing.

Although some CD47 humanized mice already exist in the prior art, all of them have certain limitations, and the construction of more different CD47 humanized mice is still an important requirement of immune checkpoint drug screening, and particularly, an efficient CD47 humanized mouse construction method is still pursued in the field.

Disclosure of Invention

The invention aims to provide a method for constructing a humanized mouse model of CD47, which is characterized in that a coding gene of humanized CD47 replaces a corresponding gene of a mouse to prepare the humanized mouse model of an immune checkpoint, has human functional genes, can be used for screening and evaluating human immune checkpoint drugs, and solves the problem that an immune checkpoint antibody screened on the mouse is not completely tried on the human due to the difference of species attributes of rodents; the invention also designs the sgRNA with high activity, which has high targeting property and cutting activity and is beneficial to improving the positive rate of results.

In order to achieve the purpose, the invention provides the following technical scheme:

a method of constructing a humanized mouse model of CD47, the method comprising: replacing a corresponding mouse-derived CD47 gene segment with a human-derived CD47 gene segment on a background mouse fertilized egg cell by using homologous recombination and an embryonic stem cell technology, expressing a human-derived CD47 gene in a background mouse cell and generating humanized CD47 protein, and reducing or eliminating the expression of an endogenous CD47 gene of a mouse, thereby obtaining a mouse model capable of expressing humanized CD 47; the humanized CD47 gene encodes the extracellular region of humanized CD47 protein; the intracellular region of the humanized CD47 protein was expressed from the mouse endogenous CD47 gene.

Furthermore, the amino acid sequence of the extracellular region coded by the human CD47 gene fragment is SEQ No. 1; namely 14 th to 141 th amino acids of the human CD47 protein.

SEQ No.1：

CCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNE。

Further, the humanized CD47 gene of the CD47 humanized mouse encodes an amino acid sequence of SEQ No. 2; among them, the amino acid sequence encoded by the human CD47 gene is not underlined, and the amino acid sequence encoded by the murine CD47 gene is underlined.

SEQ No.2：

MWPLAAALLLGSCCCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNEQGS ACSYEEEKGGCK。

In a specific embodiment, the method comprises the steps of:

(1) designing and constructing a targeting vector: designing sgRNA, and constructing an sgRNA expression vector;

(2) microinjecting the Cas9/sgRNA system and the targeting vector into mouse fertilized eggs, transplanting the mouse fertilized eggs into a pseudopregnant female mouse, and waiting for the birth of the mouse;

(3) identification and screening of F0 mouse.

In one embodiment, the sgRNA sequence of step (1) is selected from: hCD47-5S1(SEQ No.3), hCD47-5S2(SEQ No.4), hCD47-3S1(SEQ No.5), hCD47-3S2(SEQ No. 6).

Preferably, the sgRNA designed in step (1) is a pair of sgRNAs consisting of SEQ No.4 and SEQ No. 6.

Further, the step (1) further comprises sgRNA cleavage activity identification, wherein the sequence of a PCR amplification primer in the identification process is as follows: identifying the 5' end KO as SEQ No.7 and SEQ No. 8; 3' end KO identification: SEQ No.9 and SEQ No. 10.

CD47-sgRNA-In5F1 tgaaaatggataagcgcgatgc(SEQ No.7)；

CD47-sgRNA-In5R1 aacatgaggtttcctgttgccca(SEQ No.8)；

CD47-sgRNA-In3F1 agaaccagaaaccaagttaagctg(SEQ No.9)；

CD47-sgRNA-In3R1 aaagcaagaacagatgggaagtag(SEQ No.10)。

In one embodiment, the strain of background mice is selected from C57BL/6 or BALB/C.

The invention also provides a sgRNA sequence specifically targeting the mouse CD47 gene, which is characterized in that the sgRNA sequence is selected from: SEQ No.3, SEQ No.4, SEQ No.5 and SEQ No. 6.

hCD47-5S1 ggaaccttgcagaagtcact(SEQ No.3)；

hCD47-5S2 gcatcgtccgtaatgtgg(SEQ No.4)；

hCD47-3S1 aatccctgcttcccagttgc(SEQ No.5)；

hCD47-3S2 gtaactcacttacagagtcc(SEQ No.6)。

Compared with the prior art, the invention has the following beneficial effects: 1) on a mouse with a sound immune system, a mouse model capable of interacting with the anti-human CD47 monoclonal antibody is constructed by replacing a mouse-derived CD47 gene with a human-derived CD47 gene. Compared with the common mouse, the model realizes the humanized modification of key target molecules, reserves the complete immune system, can be used for screening and evaluating drugs aiming at human genes, and is a highly ideal preclinical drug test model. The prepared humanized mouse model of the immune checkpoint has human functional genes and can be used for screening and evaluating human drugs of the immune checkpoint; 2) the high-activity sgRNA is designed, and the efficient cutting activity is realized, so that the construction method of the CD47 humanized mouse is more efficient, the positive rate of the result is improved, and the efficient sgRNA is provided for the further research of the construction of the CD47 humanized mouse.

Drawings

FIG. 1 is a graph of the first embryo targeting efficiency test, in which numbers 1-36 are embryo targeting identifications of combinations of hCD47-5S2 and hCD47-3S 2; the number of the embryo is 37-112, the embryo targeting identification of the combination of hCD47-5S1 and hCD47-3S 1;

FIG. 2 is a second embryo targeting efficiency test chart; the serial numbers 1-53 in the first step are the embryo targeting identification of the combination of hCD47-5S2 and hCD47-3S 2; ② the number 1-51 is the embryo targeting identification of the combination of hCD47-5S1 and hCD47-3S 1;

FIG. 3 hCD47-BALB/c mouse CD47-KI-target5 end and 3 end identification electrophoretograms;

FIG. 4 shows the identification of electrophoretograms at the CD47-KI-target5 and 3 ends of hCD47-C57BL/6 mice.

Detailed Description

The technical solution of the present invention will be clearly and completely described and illustrated in the following embodiments.

The invention utilizes homologous recombination and embryonic stem cell technology to replace the mouse CD47 gene with the human CD47 gene on mouse fertilized egg cells with C57BL/6 background and BALB/C background, thereby constructing a mouse model capable of expressing the human CD 47.

In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front", "rear", "both ends", "one end", "the other end", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "connected," and the like are to be construed broadly, such as "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

Example 1 determination of the human sequence of the human CD47 Gene fragment substitution region and insertion

According to the structure and the function of the humanized CD47, an extracellular region coded by the humanized CD47 gene is selected to replace an extracellular region coded by the murine CD47 gene, an intracellular region of a mouse is reserved, and the amino acid sequence (Aa: 14-141) of the extracellular region coded by the selected humanized CD47 gene is shown as SEQ No. 1;

SEQ No.1：

determining the splicing sequence of the humanized CD47 protein:

the extracellular region coded by the humanized CD47 gene is replaced by the extracellular region coded by BALB/C and C57BL/6 murine CD47 gene by homologous recombination, the intracellular region sequences of BALB/C and C57BL/6 mice are reserved, and the amino acid sequence coded by the successfully targeted CD47 humanized mouse gene is shown as SEQ No. 2; among them, the amino acid sequence encoded by the human CD47 gene is not underlined, and the amino acid sequence encoded by the murine CD47 gene is underlined.

SEQ No.2：

Example 2

Selecting two ends of an extracellular region of a mouse CD47 gene as targeting sites, and designing a homologous DNA donor containing an extracellular region of a human-derived CD47 gene and an identification scheme;

preparing Cas9 or an expression vector thereof based on CRISPR/Cas9 technology; sgrnas were designed in the humanized replacement regions against murine sequences (see table 1). Designing and synthesizing and recognizing a 5 'end target site and a 3' end target site, and constructing a sgRNA expression vector. Two sgRNA recognition sites are respectively positioned at two ends of an extracellular region of a mouse CD47 gene, and the target site sequence of each sgRNA on CD47 is shown in Table 1. Each sgRNA sequence was cloned into a pUC57kan-T7-delG vector to construct a pUC57-sgRNA plasmid.

Table 1 sgRNA sequences

sgRNA name	sgRNA sequence (5 '→ 3')	PAM
			hCD47-5S1	ggaaccttgcagaagtcact(SEQ No.3)	AGG
hCD47-5S2	gcatcgtccgtaatgtgg(SEQ No.4)	AGG
			hCD47-3S1	aatccctgcttcccagttgc(SEQ No.5)	TGG
hCD47-3S2	gtaactcacttacagagtcc(SEQ No.6)	AGG

Example 3

sgRNA transcription method: PCR was performed using PrimerStar or PrimerStarMax system, sgRNA-F, sgRNA-R as primers, and a correctly sequenced puc57-sgRNA plasmid as a template, and the PCR product was purified (see Table 2 and Table 3 for PCR reaction system and conditions) to prepare a sgRNA transcription template. Transcription of sgRNA was performed using T7-ShortScript in vitro transcription kit (AM 1354).

TABLE 2 PCR reaction System

TABLE 3 PCR reaction conditions

sgRNA screening: after the sgrnas at the 5 'end and the 3' end are respectively incubated with the Cas9 protein, the mixed solution is injected into a fertilized egg for 0.5 day, after the fertilized egg is cultured to a blastocyst stage, the KO positive rate of the mouse CD47 gene is identified, so that the sgrnas with high cleavage activity are screened, and the sgrnas with the highest cleavage activity at the 5 'end and the 3' end are respectively selected to pair into a sgRNA pair.

The sgRNA cleavage identification method comprises the following steps: the collected blastocysts were subjected to PCR amplification (the PCR protocol is shown In Table 4), the amplified bands were subjected to secondary sequencing (5S1-5S2 sequencing primer: CD47-sgRNA-In5F1, 3S1-3S2 sequencing primer: CD47-sgRNA-In3F1), compared with wt bands, the probability of mutation was counted (the identification results are shown In Table 5), and finally hCD47-5S2 and hCD47-3S2 with the highest cleavage activity were selected.

Table 4 sgRNA cleavage PCR identification protocol

TABLE 5 sgRNA cleavage Activity

sgRNA name	Cutting efficiency
		hCD47-5S1	11/14＝78.5％
hCD47-5S2	20/20＝100％
		hCD47-3S1	11/15＝73％
hCD47-3S2	20/20＝100％

Example 4

The hCD47-5S2 and hCD47-3S2 were selected to be paired with high cleavage efficiency, and the hCD47-5S1 and hCD47-3S1 were selected to be relatively low in cleavage efficiency. The Cas9/sgRNA system and the targeting vector are injected into a mouse fertilized egg of 0.5 day (the extracellular region of a mouse CD47 gene is knocked out and the extracellular region of a human CD47 gene is inserted into a genome at the same time), and a single cell is taken after the culture till the blastocyst stage for embryo targeting efficiency identification, wherein the identification scheme is shown in the following table 6. The results of the identification of the two embryo targeting efficiency tests show (table 7, fig. 1, fig. 2): the embryo targeting efficiency of the combination of hCD47-5S2 and hCD47-3S2 is high, and the positive numbers of the two embryos which are combined by specific hCD47-5S2 and hCD47-3S2 are respectively 2#, 12#, 13#, 14# -16#, 25#, 26#, 36# and 1#, 4#, 9#, 16#, 26# -29#, 31#, 32#, 37#, 38#, 45#, 47#, 48#, and 49 #; the specific two-embryo targeting positive number of the combination of hCD47-5S1 and hCD47-3S1 is # 38.

TABLE 6 identification of embryo targeting efficiency primers

Note: KI is an on-target genotype; WT is wild type

TABLE 7 efficiency of two embryo targeting

sgRNA combination name	Efficiency of first embryo targeting test	Efficiency of secondary embryo targeting test
			hCD47-5S1and3S1	1/77＝1.3％	0/51＝0％
hCD47-5S2and3S2	9/35＝25.7％	16/53＝30.2％

Example 5

hCD47-5S2 and hCD47-3S2 with high cleavage efficiency were selected as sgRNAs at the 5 'end and the 3' end. The Cas9/sgRNA system and the targeting vector are injected into a mouse fertilized egg of 0.5 day (the extracellular region of a mouse CD47 gene is knocked out and the extracellular region of a human CD47 gene is inserted into a genome), the mouse fertilized egg is transplanted into a pseudopregnant female mouse of 0.5 day, and after the mouse is born, a midget mouse is screened out through gene identification (F0).

Genotyping of humanized mouse F1 generation: breeding a targeted F0 mouse screened by gene identification with a background mouse, wherein the progeny mouse is F1, carrying out PCR identification on the obtained mouse tail genomic DNA of the F1 mouse after targeting by using two pairs of primers respectively, wherein the primers hCD47-geno-5F1/hCD47-geno-5R1 are respectively positioned outside a 5 'homology arm and in a human fragment of a targeting vector, and if the pair of primers is amplified to generate a PCR product, the target vector is effectively inserted into the mouse genome 5'; hCD47-geno-3F1/hCD47-geno-3R1 are located in the human fragment and outside the 3 'homology arm of the targeting vector respectively, and PCR products are generated by the amplification of the pair of primers, which indicates that the target vector is effectively inserted in the 3' of the mouse genome. And (3) carrying out sequencing verification on the mouse clone with positive PCR identification at two ends, and identifying the clone with correct sequencing as a positive mouse after the homologous DNA donor replaces the corresponding sequence of the mouse in the mouse genome. Wherein the F0 and F1 identifying primers are shown in Table 8.

TABLE 8F 0 and F1 identifying primers

Note: KI is an on-target genotype; WT is wild type

Example 5

Using BALB/c as background mouse, processing according to the above method to obtain 4 positive F1 mice, as shown in the hCD47F1 rat tail DNA identification electrophoresis chart of figure 3, 5 'and 3' identifying the object bands are positive, which indicates that the mouse is positive mouse with correct target; the rest identified no band were all negative, mice were treated.

BALB/c background F1 generation mice identification:

identification of the CD47-KI-target5 'end and the 3' end:

the B6 negative control is genomic DNA;

n is blank control, no template control; TRANS2KPLUSII band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp;

BALB/c background F1 mouse tails are subjected to CD47 gene identification, F1 mouse PCR experiment results are shown in figure 3, and human CD47 gene 5 'and 3' identification of 20#, 21#, 23# and 24# mice are positive, which indicates that the mice are positive mice correctly subjected to gene recombination.

Example 6

Mice with C57BL/6 as background were treated as described above to obtain 4 positive F1 mice. As shown in fig. 4, hCD47F1 rat tail DNA identified the electrophoretogram, and 5 'and 3' identified that the target bands were positive, indicating that the mice were positive for correct gene recombination; the rest identified no band were all negative, mice were treated.

Identification of C57BL/6 background F1 generation mice:

identification of the CD47-KI-target5 'end and the 3' end:

p is a positive control;

WT negative control was genomic DNA;

n is blank control, no template control; m is the TRANS2KPLUSII band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp;

the CD47 gene identification is carried out on the F1 rat tail under the C57BL/6 background, the PCR experiment result of the F1 mouse is shown in figure 4, and the 5 'and 3' identifications of the humanized CD47 genes of 1#, 4#, 5#, and 7# mice are positive, which indicates that the mouse is a positive mouse which is correctly subjected to gene recombination.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Sequence listing

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Claims

1. A method of constructing a humanized mouse model of CD47, the method comprising: replacing a corresponding murine CD47 gene segment with a humanized CD47 gene segment on a background mouse fertilized egg cell, expressing a humanized CD47 gene in the background mouse cell to generate humanized CD47 protein, and reducing or eliminating the expression of an endogenous CD47 gene of the mouse, thereby obtaining a mouse model capable of expressing humanized CD 47; the humanized CD47 gene fragment encodes the extracellular region of humanized CD47 protein; the intracellular region of the humanized CD47 protein was expressed from the mouse endogenous CD47 gene.

2. The method for constructing a humanized mouse model of CD47, according to claim 1, wherein the humanized mouse model of CD47 comprises the following steps: the amino acid sequence of the extracellular region coded by the humanized CD47 gene fragment is SEQ No. 1; namely 14 th to 141 th amino acids of the human CD47 protein.

3. The method for constructing a humanized mouse model of CD47 according to claim 1 or 2, wherein: the humanized CD47 gene of the CD47 humanized mouse encodes an amino acid sequence shown in SEQ No. 2.

4. The method for constructing the humanized mouse model of CD47 according to any one of claims 1 to 3, wherein the method comprises the following steps:

(3) identification and screening of F0 mouse.

5. The method for constructing a humanized mouse model of CD47, according to claim 4, wherein the sgRNA sequence of step (1) is selected from the group consisting of: hCD47-5S1(SEQ No.3), hCD47-5S2(SEQ No.4), hCD47-3S1(SEQ No.5), hCD47-3S2(SEQ No. 6).

6. The method for constructing the humanized mouse model of CD47 according to any one of claims 4 or 5, wherein the sgRNA designed in step (1) is a pair of sgRNAs consisting of SEQ No.4 and SEQ No. 6.

7. The method for constructing the humanized mouse model of CD47 according to any one of claims 4 to 6, wherein step (1) further comprises sgRNA cleavage activity identification, wherein the PCR amplification primer sequence during identification is as follows: identifying the 5' end KO as SEQ No.7 and SEQ No. 8; 3' end KO identification: SEQ No.9 and SEQ No. 10.

8. The method for constructing a humanized mouse model of CD47 as claimed in any one of claims 1 to 7, wherein the background mouse is selected from C57BL/6 or BALB/C.

9. A sgRNA sequence specifically targeting the mouse CD47 gene, wherein the sgRNA sequence is selected from the group consisting of: SEQ No.4 and SEQ No. 6.

10. Use of the sgRNA sequence specifically targeting the mouse CD47 gene according to claim 9 for the preparation of a humanized CD47 mouse model.