Construction method and application of human TLT-1 transgenic mouse model
Through CRISPR-Cas9 gene editing technology, a mouse model that only expresses human TLT-1 was constructed, which solved the problem of insufficient homology between mouse TLT-1 and human TLT-1 in the existing technology, and realized an effective research and development platform for targeted human TLT-1 drugs.
Patent Information
- Application Number
- CN202411710756.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art is difficult to effectively use mouse models to develop targeted drugs for human TLT-1, and mouse TLT-1 has only about 70% homology with human TLT-1, resulting in difficulty in R&D.
Through CRISPR-Cas9 gene editing technology, two gRNAs targeting the gene encoding murine TLT-1 were designed, gene edited, and stable human TLT-1 homozygous transgenic mice were constructed to ensure that their platelets only express human TLT-1 and do not express murine TLT-1.
The construction of a stable human TLT-1 transgenic mouse model is achieved, providing an effective platform for the research and development of human TLT-1 targeted drugs with high specificity and low off-target risk.
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Figure CN119709878A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of gene editing, and specifically relates to a method for constructing a humanized TLT-1 transgenic mouse model and application thereof. Background Art
[0002] Platelets usually circulate in the bloodstream in a resting (non-activated) state. When the vascular endothelium is damaged, the interaction between platelet surface receptors, such as glycoproteins (GP) Ib-IX-V and IIb / IIIa, and von Willebrand factor (VWF) exposed at the site of vascular damage triggers platelet adhesion, aggregation, and activation at the site of injury, as well as changes in platelet structure leading to phosphatidylserine (PS) externalization, which provides the necessary conditions for the formation of coagulation factor complexes and activation of thrombin. Platelet activation leads to changes in the conformation of GPIIb / IIIa membrane proteins, which further leads to the release of platelet α-granules and dense granules, the release of a series of procoagulant factors, and the transfer of some adhesion receptors (such as P-Selectin and TLT-1) from the inside to the outside. Studies have shown that TLT-1 receptors are only expressed on the surface of activated platelets (Washington AV, Quigley L, McVicar DW. Initial characterization of TREM-like transcript (TLT)–1: a putative inhibitory receptor within the TREMcluster. Blood 2002;100 (10): 3822–3824), and activated platelets are basically only present in sites such as bleeding, thrombosis, inflammation and tumor microenvironment. Many drugs, including small molecule drugs and large molecule protein drugs, are effective in treating diseases such as bleeding, thrombosis, inflammation and tumors. However, due to the lack of targeting, their side effects are obvious. For example, coagulation factors such as factor VII cause thrombosis when treating bleeding events, and tPA causes bleeding adverse reactions when treating thrombotic events. Therefore, there is an urgent need for therapeutic drugs with targeted advantages in clinical practice. In view of the above characteristics of activated platelets and their surface receptor TLT-1, using platelet TLT-1 receptor as a therapeutic target for biomacromolecule drugs has the advantages of high specificity and few side effects, and is expected to stand out in the treatment of diseases such as bleeding, thrombosis, inflammation and tumors.
[0003] Since the physiological, anatomical, biochemical and immune characteristics of experimental animals are very similar to those of humans, the progress of biomedical research often relies on the use of animal models as the experimental basis for experimental hypotheses and clinical hypotheses to explore the mechanisms of human physiology and disease. In the process of drug development, experimental animals can be used as models for drug efficacy and safety evaluation. Through experiments, we can understand the mechanism, pharmacokinetics, toxicity and efficacy of drugs. The mouse genome is highly homologous to the human genome. There are about 2.5 billion base pairs on the 20 pairs of chromosomes of mice, which are similar to the 2.9 billion base pairs on the 23 pairs of chromosomes of humans. The number of genes in the two species is comparable, so the research results obtained using mice are believed to be extrapolated to humans. However, since mouse TLT-1 and human TLT-1 have only about 70% homology, it is still difficult to develop drugs targeting human TLT-1 using mouse models. 1) Obtain high-affinity molecules (antibodies, polyclonal antibodies or single-domain antibodies) that can target both human and mouse TLT-1 targets; 2) The copy number of mouse platelet TLT-1 receptors is much higher than that of human TLT-1. The use of mice to develop drugs targeting TLT-1 may not truly reflect the actual efficacy. Therefore, constructing transgenic mice that can stably express human TLT-1 is of great significance for studying this target in the development of new disease therapies.
[0004] The CRISPR-Cas9 system was originally discovered in bacteria as an immune mechanism to defend against foreign viruses and plasmids. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a series of repeated DNA sequences separated by unique spacer sequences. These spacer sequences are viral DNA fragments acquired by bacteria when they were previously attacked by viruses. Cas9 (CRISPR-associated protein 9) is a nuclease that can cut double-stranded DNA at a specific DNA sequence. In gene editing, researchers design a single-stranded RNA guide molecule (sgRNA) that is complementary to the target DNA sequence and introduce it into the target cell together with the Cas9 protein complex. The sgRNA guides Cas9 to locate the target DNA sequence and prompts Cas9 to cut the DNA double strand at that location. When the cell repairs the DNA breaks produced by the cutting, mutations can be introduced to achieve gene knockout, insertion or replacement. CRISPR / Cas9 technology consists of a guide RNA (sgRNA or gRNA) responsible for targeting and a Cas9 nuclease responsible for cutting. Compared with traditional ZFNs and TALENs technologies, it is simpler and only requires the design and construction of the corresponding sgRNA for a specific target sequence to achieve efficient DNA cutting and thus mediate effective gene editing. There are two main applications of CRISPR-Cas9 gene editing technology: 1) Gene knockout: Double-strand breaks are introduced into the target gene through CRISPR-Cas9, and the cell repairs the breaks through a non-homologous end joining repair mechanism. Insertion or deletion mutations may be introduced during the process, resulting in loss of function of the target gene; 2) Gene insertion or replacement: Using the homologous recombination mechanism, specific mutations or exogenous sequences are introduced into the target gene. This requires the provision of a homologous template DNA, which the cell uses to repair double-strand breaks through homologous recombination to achieve precise gene editing. Summary of the invention
[0005] In view of these problems existing in the prior art, the present invention provides a nucleic acid combination and method for constructing a human TLT-1 transgenic mouse model, and combines CRISPR-Cas9 gene editing technology to obtain a stable human TLT-1 homozygous transgenic mouse, whose platelet surface only expresses human TLT-1 and does not express mouse TLT-1. This lays a foundation for the development of new drugs for this target in bleeding, thrombosis, inflammation and tumors.
[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a method for constructing a humanized TLT-1 transgenic mouse model.
[0007] The construction method includes gene editing through gRNA1, gRNA2, Donor DNA and Cas9 endonuclease; the gRNA1 includes the sequence shown in SEQ ID NO.1-6, and the gRNA2 includes the sequence shown in SEQ ID NO.7-8; the Donor DNA includes the human TLT-1 gene.
[0008] SEQ ID NO.1: GCAGTTTTAGAGAGGAGG; SEQ ID NO.2: CAGAACCTACTACTGCCC; SEQ ID NO.3: ACGTGACTGTATGAGACA; SEQ ID NO.4: CGATGGATAGCTGGAGCG; SEQ ID NO.5: CGAGAGACCGGTAGGCAA; SEQ ID NO.6: TTGGTGGGGTATCAGACC; SEQ ID NO.7:GCATAGGGTCTCCCTGTT; SEQ ID NO. 8: GGACTGAGTGGCAACGGA.
[0009] The human TLT-1 gene has a nucleotide sequence as shown in SEQ ID NO.9.
[0010] SEQ ID NO.9: .
[0011] The Donor DNA also includes homology arms, the remaining sequence after the target sequence is cut, the stop codon, and polyA.
[0012] Preferably, the Donor DNA has a nucleotide sequence as shown in SEQ ID NO.10.
[0013] SEQ ID NO.10:
[0014] Specifically, the gene editing is completed in a fertilized egg cell.
[0015] Preferably, the fertilized egg cells include but are not limited to those from C57BL / 6 mice.
[0016] Specifically, the humanized TLT-1 transgenic mouse model includes the F0 generation and its offspring.
[0017] Preferably, the offspring include but are not limited to offspring bred from F1 generation positive mice.
[0018] On the other hand, the present invention provides an application of a humanized TLT-1 transgenic mouse model in the quality control of TLT-1 targeted drugs, wherein the humanized TLT-1 transgenic mouse model is constructed by the aforementioned construction method.
[0019] The TLT-1 targeted drugs include, but are not limited to, any one or more of antibodies, polypeptides, fusion proteins, and small molecule drugs targeting TLT-1.
[0020] The quality control includes but is not limited to intra-batch quality control, inter-batch quality control, etc.
[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a CRISPR-Cas9 system for constructing a human TLT-1 transgenic mouse model. The system comprises two gRNAs targeting mouse TLT-1 coding genes. The gRNAs have high cutting efficiency, high specificity and low off-target risk. The system can be used not only to construct a human TLT-1 transgenic mouse model, but also to knock out the mouse TLT-1 gene.
[0022] The present invention provides a nucleic acid combination for constructing a human TLT-1 transgenic mouse model, a method and an application thereof, and obtains a stable human TLT-1 homozygous transgenic mouse that only expresses human TLT-1 and does not express mouse TLT-1.
[0023] The mouse model constructed in the present application can be used for the research related to the targeted drugs of human TLT-1, which is beneficial to the development and research of new anti-thrombotic drugs and drugs for the treatment of acute myocardial infarction and / or cerebral infarction.
[0024] The mouse model constructed in the present application can be further used for a series of studies on tumors, promoting coagulation, etc., including but not limited to screening antibodies, polyclonal antibodies or single-domain antibodies, fusion proteins, small molecules, etc. targeting TLT-1. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 :Gene editing design ideas.
[0026] Figure 2 : Activity detection results of gRNA target (M: BM2000 Marker; NC, blank control).
[0027] Figure 3 :Activity detection results of gRNA targets.
[0028] Figure 4 :Agarose gel electrophoresis results of M-KI-G21-N6(1-26) amplified with TG-F / TG-R (17# is the positive result).
[0029] Figure 5 : Agarose gel electrophoresis results of M-KI-G21-N6 (17, WT, NC) amplified with F1 / R1 (17# is the positive result; M: BM2000 Marker; WT, wild type; NC, blank control).
[0030] Figure 6 : Agarose gel electrophoresis results of M-KI-G21-N6 (17, WT, NC) amplified with F2 / R2 (17# is the positive result; M: BM2000 Marker; WT, wild type; NC, blank control).
[0031] Figure 7 : Amplification detection of hTreml1 gene L-terminal insertion electrophoresis results (2# and 14# are positive results; PCR product size: 1214bp; M: BM2000 Marker; WT, wild type; NC, blank control).
[0032] Figure 8 : Amplification detection of hTreml1 gene R-terminal insertion electrophoresis results (2# and 14# are positive results; PCR product size: 736bp; M: BM2000 Marker; WT, wild type; NC, blank control).
[0033] Fig. 9 :Flow cytometry was used to detect the expression of TLT-1 molecules in mouse platelets.
[0034] Fig.10 : Flow cytometry was used to detect the expression of TLT-1 molecules in human and mouse platelets. DETAILED DESCRIPTION
[0035] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.
[0036] Example 1: Obtaining transgenic mice by gene editing The human Treml1 gene (hTreml1, Gene ID: 340205), mouse Treml1 gene (mTreml1, Genbank: 71326) and the upstream and downstream homologous arm sequence information of mTreml1 in the mouse genome were obtained from the database (NCBI). According to the principle of CRISPR gene editing, E1 and E2 were knocked out after the ATG of mTreml1, and hTreml1 was knocked in. See the schematic diagram Figure 1 . Mouse Treml1 gene transcript: ENSMUST00000224001.2.
[0037] Specifically, using CRISPR / Cas9 technology, the Cas9 / gRNA target site in the mouse genome is designed, and the Cas9 / gRNA target site is reverse transcribed into gRNA. The Donor sequence with the target gene is designed. The designed Donor, gRNA, and Cas9 are then microinjected into the fertilized egg.
[0038] The following steps are involved: 1) Design gRNA target: According to the mouse genome sequence of hTreml1 site-directed knock-in and the knock-in position (upstream near the ATG of mTreml1 and downstream after Exon2 of mTreml1), the gRNA target was designed, see Table 1.
[0039] Table 1 gRNA targets
[0040] 2) In vitro activity detection of gRNA targets The activity detection of gRNA target in vitro was performed using the spCas9-gRNA target efficiency detection kit (manufacturer: Beijing Weishanglide, catalog number: VK007), which includes the following: a. Prepare a genomic sequence (DNA fragment) including the gRNA target to verify the enzyme activity; b. Using the genomic DNA of the target gene as a template, PCR amplifies the DNA fragment with the gRNA target site; c. The gRNA target (including the PAM sequence) can be constructed into the PCR product by bridge PCR; d. Reverse transcribe gRNA using spCas9-gRNA target efficiency detection kit; e. DNA was verified by in vitro enzyme digestion with spCas9 and gRNA.
[0041] The results of enzyme digestion verification are shown in Figure 2 , and the gRNA target activity was calculated based on the ratio of DNA fragments digested by agarose gel electrophoresis. The results are shown in Tables 2 and Figure 3 .
[0042] Table 2 Activity analysis of gRNA targets
[0043] In this embodiment, it can be seen from the target activity data that the two targets Treml1-L2 and Treml1-R3 are selected for gene knockout, and Treml1-L2 and Treml1-R3 are transcribed in vitro into gRNA1 and gRNA2, respectively.
[0044] 3) Design Donor sequence according to gRNA target The target gene knocked in is the CDS sequence of the human Treml1 gene (see SEQ ID NO.9), and then the Donor sequence (see SEQ ID NO.10) is designed based on the CRISPR gene editing principle.
[0045] SEQ ID NO.9: ATGGGCCTCACCCTGCTCTTGCTGCTGCTCCTGGGACTAGAAGGTCAGGGCATAGTTGGCAGCCTCCCTGAGGTGCTGCAGGCACCCGTGGGAAGCTCCATTCTGGTGCAGTGCCACTACAGGCTCCAGGATGTCAAAGCTCAGAAGGTGTGGTGCCGGTTCTTGCCGGAGGGGTGCCAGCCCCTGGTGTCCTCAGCTGTGGATCGCAGAGCTCCAGCGGGCAGGCGTACGTTTCTCACAGACCTGGGTGGGGGCCTGCTGCAGGTGGAAATGGTTACCCTGCAGGAAGAGGATGCTGGCGAGTATGGCTGCATGGTGGATGGGGCCAGGGGGCCCCAGATTTTGCACAGAGTCTCTCTGAACATACTGCCCCCAGAGGAAGAAGAAGAGACCCATAAGATTGGCAGTCTGGCTGAGAACGCATTCTCAGACCCTGCAGGCAGTGCCAACCCTTTGGAACCCAGCCAGGATGAGAAGAGCATCCCCTTGATCTGGGGTGCTGTGCTCCTGGTAGGTCTGCTGGTGGCAGCGGTGGTGCTGTTTGCTGTGATGGCCAAGAGGAAACAAGAATCCCTCCTCAGTGGTCCACCACGTCAGTGA; SEQ ID NO.10:
[0046] 4) Construction of human TLT-1 transgenic mice According to the method in the Mouse Embryo Operation Manual (3rd Edition) (Andras Nagy, Chemical Industry Press, 2006), gRNA1, gRNA2, Donor DNA and Cas9 nickase were microinjected into the fertilized eggs of pregnant mice (C57BL / 6). The injected fertilized eggs were transferred to the culture medium for a short culture, and then transplanted into the oviduct of the recipient mother mouse for development. Two weeks after the birth of the mice (F0 generation), the mouse tail or toes were cut to obtain the genome, and the genotype was identified by PCR to confirm the knockout of the target gene.
[0047] 4.1. Identification of F0 generation mouse genotype: The specific information of F0 generation mice is shown in Table 3. After the genome was extracted, PCR was performed using the identification primers shown in Table 4. The results of agarose gel electrophoresis are shown in Table 3. Figure 4-6 The M-KI-G21-N6-17#(♀) mouse strain was confirmed by sequencing to be an F0 generation positive mouse with site-specific knock-in of the human Treml1 gene.
[0048] Table 3 Information of F0 generation mice
[0049] Table 4 Primer sequences
[0050] 4.2. Genotype identification of F1 mice: M-KI-G21-N6-17#(♀) was crossed with C57♂ mice (Beijing Weishanglide) to obtain F1 mice. The specific information is shown in Table 5. After the genome was extracted, PCR was performed using the identification primers shown in Table 6: 17♀xC57♂: 1-3♂, 4-8♀ and 9-12♂, 13-16♀ to amplify and detect the insertion of the L end of the hTreml1 gene. The results of agarose gel electrophoresis are shown in Figure 7 ; 17♀xC57♂: 1-3♂, 4-8♀ and 9-12♂, 13-16♀ amplification detection of hTreml1 gene R-terminal insertion, agarose gel electrophoresis results are shown in Figure 8 ; The M-KI-G21-N6-17-2 (♂) and M-KI-G21-N6-17-14 (♀) mouse strains were confirmed by sequencing to be F1 generation positive mice with site-specific knock-in of the human TLT-1 gene. The offspring bred from the F1 generation positive mice are stable human TLT-1 homozygous transgenic mice.
[0051] Table 5 F1 generation mouse information
[0052] Table 6 Primer sequences
[0053] Example 2: Flow cytometry detection 1) Detection of TLT-1 expression in platelets of transgenic mice by flow cytometry In order to detect the expression of TLT-1 molecules in platelets of transgenic mice, flow cytometry (BD, Accuri TM C6 Plus) and the accompanying instructions were used to detect the binding of hTLT-1 flow cytometry antibody (R&D, FAB2394G), 12006 antibody (self-made, sequence as shown in SEQ ID NO.37: QVKLEESGGGLVQTGGSLRLSCAASGIIFGLSAMGWYRQGPGNQRELVATITTSYYTKYLDSVKGRFTISRDNTKNTVDLQMNSLKPEDTGVYYCHAEPTFSSGEFWGQGTLVTVSS, only binding to mTLT-1 but not hTLT-1), NX06-8 antibody (self-made, neither binding to mTLT-1 nor hTLT-1), isotype control antibody (R&D, IC013G), negative control (fluorescence value of platelets themselves) to the platelets of transgenic mice obtained in Example 1, and the experimental results are shown in Tables 7 and 8. Fig. 9 , hTLT-1 flow cytometry antibody can bind to transgenic mouse platelets, indicating that transgenic mouse platelets express hTLT-1; while 12006, NX06-8 and isotype control antibodies did not bind to transgenic mouse platelets, indicating that transgenic mice did not express mTLT-1.
[0054] Table 7 Expression of TLT-1 molecules in mouse platelets detected by flow cytometry
[0055] 2) Detection of the expression of TLT-1 molecules in human and transgenic mouse platelets by flow cytometry In order to evaluate the expression of hTLT-1 molecules in transgenic mouse platelets, flow cytometry was used to detect the binding of hTLT-1 flow antibody, isotype control antibody and negative control to human platelets and transgenic mouse platelets obtained in Example 1. The experimental results are shown in Tables 8 and Fig.10 , indicating that hTLT-1 flow cytometry antibody can bind to human and transgenic mouse platelets.
[0056] In summary, Example 1 obtained stable human TLT-1 homozygous transgenic mice. The transgenic mice only expressed human TLT-1 but not mouse TLT-1, and can be used for related research on human TLT-1.
[0057] Table 8 Expression of TLT-1 molecules in human and mouse platelets detected by flow cytometry
[0058] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.
Claims
1. A method for constructing a human TLT-1 transgenic mouse model, characterized in that: The method comprises performing gene editing through gRNA1, gRNA2, Donor DNA and Cas9 endonuclease; the gRNA1 comprises a sequence as shown in SEQ ID NO.1-6, the gRNA2 comprises a sequence as shown in SEQ ID NO.7-8; and the Donor DNA comprises a human TLT-1 gene.
2. The construction method according to claim 1, characterized in that: The human TLT-1 gene has a nucleotide sequence as shown in SEQ ID NO.
9.
3. The construction method according to claim 2, characterized in that: The Donor DNA also includes homology arms, the remaining sequence after the target sequence is cut, the stop codon, and polyA.
4. The construction method according to claim 3, characterized in that: The Donor DNA has a nucleotide sequence as shown in SEQ ID NO.
10.
5. The construction method according to any one of claims 1 to 4, characterized in that: The gene editing is completed in the fertilized egg cell.
6. The construction method according to claim 5, characterized in that: The fertilized egg cell is derived from mice.
7. The construction method according to claim 1, characterized in that: The humanized TLT-1 transgenic mouse model includes the F0 generation and its offspring.
8. The construction method according to claim 7, characterized in that: The offspring are the offspring bred from the F1 generation positive mice.
9. Application of a human TLT-1 transgenic mouse model in quality control of TLT-1 targeted drugs, characterized in that: The humanized TLT-1 transgenic mouse model is constructed by the construction method described in any one of claims 1 to 8.
10. The use according to claim 9, characterized in that: The TLT-1 targeted drug includes at least one of an antibody, a polypeptide, a fusion protein, and a small molecule drug targeting TLT-1.