CN118995870A - Use of PARP inhibitors Veliparib in screening BRCA1 heterozygous mutant embryos and methods - Google Patents

Abstract

The present invention relates to the application and method of the PARP inhibitor Veliparib in screening BRCA1 heterozygous mutant embryos. The present invention applies Veliparib, a PARP inhibitor with low capture effect, to the screening of BRCA1 heterozygous mutant mouse embryos. Through animal experiments, a new use of 7.5 μM Veliparib is developed to selectively block the development of BRCA1 heterozygous embryos, block the inheritance of the BRCA1 pathogenic gene, and avoid the birth of BRCA1 heterozygotes in the offspring. Compared with the PGT-M technology currently used in clinical practice, the present invention breaks through the idea of using gene sequences to screen mutant embryos, and innovatively and more directly and efficiently screens BRCA1 heterozygous embryos with insufficient homologous recombination function from the protein function level, which has the characteristics of simple operation, high cost performance, low trauma, and high accuracy.

Description

Use of PARP inhibitors Veliparib in screening BRCA1 heterozygous mutant embryos and methods

Technical Field

The invention relates to the field of biotechnology, in particular to an application and a method of a PARP inhibitor Veliparib in screening out BRCA1 heterozygous mutant embryos.

Background

Malignant tumor is used as a major public health event threatening human health, and is the first cause of death in China. Wherein, the hereditary tumor occupies 5-10% of malignant tumor with its unique genetic background and pathogenesis. Among such tumors, hereditary ovarian cancer and breast cancer (HBOC) and BRCA1 genes are widely studied, and pathogenic mutations of BRCA1 are detected in 90% or more of cases in hereditary breast cancer patients. The BRCA1 gene is located on human chromosome 17 and plays a role in the physiological processes of cells such as transcription regulation, DNA repair, cell cycle regulation and the like. Studies have shown that pathogenic variation in BRCA1 can significantly increase the risk of ovarian and breast cancer by up to 65% and 39%, respectively. In addition, such genetic variation increases the risk of patients suffering from other cancers, such as pancreatic cancer, gastric cancer, and male prostate cancer.

In the general population of China, the incidence rate of hereditary BRCA1/2 pathogenic mutation is 0.29% -1.10%. Which pose a great threat to individual and family tumor susceptibility. Fertility decisions are a problem of both urgency and difficulty for women carrying BRCA1 pathogenic mutations. On the one hand, they need to complete fertility as early as possible and reduce the risk of malignancy by preventive surgical fallopian tube ovariectomy; on the other hand, due to the dominant inheritance of BRCA1 mutations, their offspring still have a 50% chance of carrying the same pathogenic mutation. Although prenatal diagnosis (PND) and preembryonic implantation gene testing (PGT) provide a technical means for preventing the spread of genetic diseases, these methods have many drawbacks in practical use. Among other things, female patients undergoing PND may experience recurrent abortion to obtain a healthy fetus. In contrast, while PGT allows a patient to know the genetic mutation status of an embryo prior to pregnancy, decisions can be made in advance; however, it has been found that PGT-M (Preimplantation GENETIC TESTING for Monogenic Diseases, single gene genetic test before implantation) is used only at a rate of 10% to 26% in BRCA1 pathogenic variation carriers, and PGT-M is usually obtained by in vitro fertilization and cultured in vitro, when the embryo develops to four to eight cell stages, one or two cells (called blastomeres) in the embryo are removed by microsurgery, and DNA in the removed cells is detected by probe or sequencing method, and whether the embryo has genetic disease is judged according to the analysis result, and the low use rate is due to more complicated operation and higher detection cost of PGT, and many problems still remain to be solved in the art; for example, there is potential embryo damage during biopsy, and the accuracy of the detection is limited; for example, due to the problems of limited materials, embryo chimerism and the like, and also due to the limitation of single cell gene analysis technology, the false positive rate of FISH detection and the low consistency rate of NGS detection, the final detection result may deviate. Particularly, the human cannot accurately read the functional change of mutation at any site of a certain gene at present, which means that even if the accuracy of PGT-M is sufficiently improved, the data obtained by sequencing cannot represent the influence of the gene function, so that the aim of screening out all gene-defective embryos from the gene function level cannot be achieved.

PARP inhibitors have been found to kill BRCA1 mutated tumor cells since 2005, and have been widely used in maintenance therapy and post-recurrence therapy of ovarian cancer in which homologous recombination repair is defective, the killing effect is mainly achieved by the "synthetic lethal" effect, and simultaneously, the DNA-binding effect, i.e., the effect of firmly binding to the DNA of the cells to assist in preventing DNA repair enzymes, thereby disabling BRCA1 mutated tumor cells from successfully repairing DNA damage and dying. It is also the DNA-binding effect of different PARP inhibitors to varying degrees that also have a certain impact on the physiological function of normal cells, and bone marrow depression, gut toxicity, renal toxicity, etc. can occur after administration to patients. However, there is currently no adequate study or understanding of the specific effects and mechanisms of action on germ cells and embryos. Thus, for Veliparib applications in the field of reproductive health, the present invention needs to be carefully held and its potential impact continues to be explored in depth. In view of this, the use and method of the PARP inhibitors Veliparib of the present invention in screening BRCA1 heterozygous mutant embryos.

Disclosure of Invention

The invention aims to provide an application and a method of PARP inhibitor Veliparib in screening out BRCA1 heterozygous mutant embryo. Aims to screen embryo at protein function level, and realize safer, more effective and more convenient embryo screening before transplantation.

The technical scheme for solving the technical problems is as follows:

in a first aspect, the use of a PARP inhibitor Veliparib in the manufacture of a medicament for screening BRCA1 heterozygous mutant embryos.

Wherein Veliparib is a member of PARP inhibitors, chinese name verapamil, code ABT-888, is a novel effective PARP1 and PARP2 inhibitor, and K _i is 5.2nM and 2.9nM respectively. The molecular formula is C ₁₃H₁₆N₄O_.2C_l H, and the chemical structural formula is: Is used for treating BRCA 1 mutant breast cancer and ovarian cancer.

The beneficial effects of the invention are as follows: the invention applies a PARP inhibitor Veliparib with low capture effect to the screening of BRCA1 heterozygous mutant mouse embryo; by animal experiments: develop a new application of 7.5 mu M Veliparib for selectively blocking the development of BRCA1 heterozygous embryo, block the inheritance of BRCA1 pathogenic genes and avoid the birth of BRCA1 heterozygotes in offspring; compared with the PGT-M technology applied clinically at present, the invention breaks through the thinking of screening mutant embryos by using gene sequences, creatively screens BRCA1 heterozygous embryos with insufficient homologous recombination function more directly and efficiently from the protein function level, and has the characteristics of simple operation, high cost performance, small traumas and high accuracy.

On the basis of the technical scheme, the invention can be improved as follows.

Furthermore, the PARP inhibitor Veliparib utilizes the defect of the homologous recombination repair function of the BRCA1 heterozygote in the BRCA1 heterozygote mutant embryo through the action of synthetic lethality to ensure that the BRCA1 heterozygote mutant embryo accumulates more DNA damage so as to achieve the result of retarding the development of the BRCA1 heterozygote mutant embryo.

Further, the PARP inhibitor Veliparib does not affect the development and implantation of normal embryos, as well as the growth and development of progeny of normal embryo development.

In a second aspect, a medicament for screening BRCA1 heterozygous mutant embryos, said medicament comprising said PARP inhibitor Veliparib.

The actual dosage of the active ingredient (PARP inhibitor Veliparib) in the above-described medicament should be determined according to various relevant factors, including the severity of the disease to be treated, the route of administration, the age, sex, weight of the patient, and therefore, the above-described dosage should not limit the scope of the present invention in any way.

Further, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Further, the medicament also comprises a pharmaceutically acceptable carrier.

Further, the dosage form of the medicine is any one of solution type and microparticle dispersion type (such as microsphere preparation, microcapsule preparation and nanocapsule preparation).

In a third aspect, a screening method for in vitro screening of BRCA1 heterozygous mutant embryos using a PARP inhibitor Veliparib, said screening method for non-diagnostic purposes comprising the steps of: the embryo in vitro fertilisation is placed in a medium containing said PARP inhibitor Veliparib and the culture is continued for at least 3 days until the embryo develops to the blastocyst stage.

Further, the embryo revealing stage of the in vitro fertilization culture is a two-cell stage mouse embryo of the in vitro fertilization culture.

Further, the concentration of PARP inhibitor Veliparib in the medium is 7.5. Mu.M.

Further, a screening method for screening BRCA1 heterozygous mutant embryos in vitro using PARP inhibitor Veliparib comprises the following specific steps: (1) A BRCA1 heterozygous deletion C57BL/6 mouse model is constructed and In Vitro Fertilization (IVF) is carried out to simulate the in vitro fertilization embryo transfer process of a BRCA1 mutant female carrying patient, the obtained mouse embryo is subjected to screening treatment in embryo culture solution containing Veliparib with optimal concentration obtained by screening until the embryo reaches the blastula stage, so that embryo development with the BRCA1 heterozygous mutation is inhibited due to DNA repair dysfunction, and a wild-type embryo can continue normal development through homologous recombination repair.

Compared with PGT-M, the invention also carries out screening of embryos in IVF-ET process by the medicament, the medicament has the action time from embryo 2 cell stage to blastula stage, and the mouse embryo is planted in the uterine cavity of the mouse by the operation which is completely the same as the human embryo transfer process, so that the killing effect of the scheme on mutant embryo and the safety on wild embryo are verified, the biopsy step in the PGT-M method can be effectively avoided, and the potential damage of the embryo caused by biopsy is effectively avoided while the operation is simplified.

In nature, in the embryo obtained by in vitro fertilization of BRCA1 heterozygous female mice and wild male mice, the proportion of BRCA1 heterozygous embryo should be 50% which is the same as the proportion of BRCA1 heterozygote in offspring mice. And (3) identifying the genotype of the embryo successfully developed to the blastula stage after 7.5 mu M Veliparib drug screening by a nested PCR method, and judging that the drug treatment can effectively inhibit the development of the BRCA1 heterozygous embryo so that the embryo cannot reach the blastula stage.

Further, the clinical in vitro fertilization-embryo transfer process is simulated, the blastula treated by the 7.5 mu M Veliparib is transferred into the uterus of a surrogate ICR mouse, the offspring is born after pregnancy, the postnatal genotype of the subsequent offspring mouse is further identified by a PCR method, and the aim of effectively avoiding the birth of the BRCA1 heterozygous mouse is realized by adopting the scheme of drug screening treatment.

Further, a method for verifying Veliparib the physiological functions of wild-type embryos after screening treatment and the safety of offspring mice developed from the embryos is provided. Specific: the detection of the embryo physiological function comprises indexes of embryo proliferation capacity, blastocyst differentiation capacity and embryo invasion capacity; offspring safety includes verification of growth, neural reflex, onset of puberty, fertility, learning and memory; to ensure that the drug treatment has no obvious damage to the physiological function of the wild embryo and the healthy development of the offspring mice.

Drawings

FIG. 1 is a chemical formula Veliparib of the present invention;

FIG. 2 is a schematic diagram of the operation flow of the present invention;

FIG. 3 is a graph showing the effect of Veliparib of the present invention on the in vitro development of each group of embryos; wherein A is the picture of each stage (2-cell/4-cell/8-cell/mulberries/blastocyst) of the embryo of the WT group (WT. Times. WT. Male) and the BRCA1 group (Brca 1 ^+/-. Times. WT) under different Veliparib concentrations; b is a comparison of embryo survival curves between WT and BRCA1 groups at different Veliparib concentrations;

FIG. 4 shows the genotype fractions of blasts of each group after Veliparib treatment in accordance with the present invention; wherein, A is the result of genotype detection electrophoresis of blastula of the control group and Veliparib treatment group by using a semi-nested PCR method, wherein the white number in the figure represents a wild embryo, the yellow number represents a BRCA1 heterozygous embryo, and the green "+" represents a positive control; b is the detection and identification result of two groups of embryo genotypes;

FIG. 5 is a genotype of mice in the blastocyst transfer progeny after treatment of Veliparib of the present invention;

FIG. 6 is a graph showing the detection of the index related to the growth and development of offspring after embryo transfer in a Veliparib-treated group and a control group according to the present invention; wherein, A is the comparison of the weight change of offspring mice in a treatment group and a control group of a plurality of time nodes Veliparib; b is the comparison of the average percentage of open auricles on both sides of two groups of mice; c is a comparison of average percent of mandibular teeth eruption in two groups of mice; d is a comparison of the average percentages of eyes open for two groups of mice;

FIG. 7 is a graph showing the detection of the index of the neural reflex of offspring after embryo transfer according to the present invention; wherein, A is Veliparib treatment group and control group offspring mice are compared by the reverse reflection; b is cliff avoidance test comparison between two groups of mice; c is the negative geotropic experimental comparison between the two groups of mice;

FIG. 8 is a graph showing post-embryo transfer offspring pubertal initiation related index detection according to the present invention; wherein, A is the testicle drop comparison of Veliparib treatment group and control group offspring female mice; b is the comparison of the vaginal opening of the offspring male mice between the two groups;

FIG. 9 is a graph showing the detection of the fertility relevant indicators of offspring after two groups of embryo transfer according to the present invention; wherein, A is the comparison of sperm concentration of the progeny male mice of Veliparib treatment group and control group; b is the comparison of the sperm motility of two groups of mice; c is a comparison of the percentage of forward motile sperm in two groups of mice; d is the comparison of pregnancy rates after natural mating of Veliparib treated and control offspring female mice; e is the comparison of average parity of two groups of female mice; f is a comparison of live yields of two groups of female mice;

FIG. 10 is a graph showing the detection of the learning and memory associated indices of offspring after embryo transfer in two groups according to the present invention; wherein, A is Veliparib representative track images of offspring mice in the treatment group and the control group; b is the comparison of the times of the two groups of offspring mice passing through the platform position; c is a comparison of the percentage of residence time of the two groups of offspring mice in the quadrant where the platform is located.

Detailed Description

The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Examples

1. Experimental reagents and materials

1.1 Major reagents

Veliparib (FIG. 1, selleck), mHTF embryo culture (cosmo bio), KSOM embryo culture (cosmo bio), gestational serum gonadotropins (Beijing Soy Bao), chorionic gonadotrophin for injection (Anhui Prodrug Co.), ready-to-use normal goat serum (Boston organism), triton X-100 (Beijing Soy Bao Co.), 75% ethanol (West Long science), bovine serum albumin (Saxiville organism), DAPI staining reagent (Saxiville organism), red nucleic acid gel dye 10,000× (MCE), sodium pentobarbital (Sigma-Aldrich), 4% paraformaldehyde (Saxiville organism), agarose (Beijing hundred crystal organism), mineral oil for culture (Sigma-Aldrich).

1.2 Major instrumentation

Three gas incubator (COOK), electronic balance (Mettler Toledo), electrophoresis apparatus (Bio-Rad), oral pipette (Drummond scientific), microscope hotplate (Ming and Mei technology Co., guangzhou), front-facing optical microscope (Olympus Co.), fluorescence inverted microscope (Zeiss), high-speed low-temperature centrifuge (Heal Force Co.), PCR apparatus (Bio-Rad), etc.

1.3 Laboratory animals

The experimental study object is a BRCA1 heterozygous deletion C57BL/6 mouse, and the transgenic mouse is constructed by Saikovia Biotechnology Co. The specific method comprises the following steps:

A donor vector containing the "WPRE-rBGpA" cassette, gRNA targeting the mouse BRCA1 gene (CATTTTGTACTTCTTGAATTTGG, SEQ ID NO: 1), and Cas9 mRNA were co-injected into the fertilized eggs of C57BL/6 mice, resulting in offspring mice with a loss of BRCA1 function. The genotype of the F0 primary mice was identified by PCR and subjected to sequencing analysis. F0 primary mice were crossed with wild type C57 mice and germline passaged and follow-up animal experiments were performed. C57BL/6 mice, female mice 4 weeks old (w) and male mice 8 weeks old (w) for in vitro fertilization experiments.

All mice used in the research of the invention are fed into SPF-class animal houses of animal experiment centers of buildings under the subsidiary of the same medical college of China university of science and technology, the fixed light and dark cycle period is kept, the daily rhythms of the mice are met, and the temperature and the humidity are proper. The examination and approval of the ethical committee of the same medical department attached to the university of science and technology, china (ethical number: TJH-202110009) has been passed.

2. Experimental method

The schematic diagram is shown in fig. 2.

2.1 Experimental grouping

40 4-Week-old BRCA1 heterozygous females and 10-week-old wild-type (WT) males were obtained by in vitro fertilization, and the 2-cell embryos were randomly divided into 2 groups, namely Veliparib-treated group and blank control group, wherein the Veliparib drug concentration in the culture broth in Veliparib-treated group was 7.5. Mu.M.

2.2 Genotyping of mice

DNA extraction: the tail or toe of the mice was cut 1-2mm, placed in 1.5mL EP tube, DNA extracted from animal tissue was extracted using DNA extraction lysate, 100. Mu. L Lysis Buffer for PCR and 1. Mu. LProteinase K were added per tube according to instructions, and thoroughly mixed. The reaction was carried out in a metal bath at 60℃for 5 minutes and then at 98℃for 2 minutes, after which the supernatant was transferred to a fresh centrifuge tube and placed on ice.

And (2) PCR: 2. Mu.L of the supernatant was used as a DNA template for PCR reaction. Corresponding PCR mixed solution is prepared in the eight connecting pipes, and the PCR reaction system is as follows:

TABLE 1PCR reaction System

TABLE 2PCR primer sequences are as follows

Mixing, and centrifuging. The PCR reaction was performed in a PCR apparatus as follows:

TABLE 3PCR reaction scheme

Agarose gel electrophoresis: 2g of agarose powder was dissolved in 100mL of 0.5 XTBE running buffer and heated to complete dissolution. Add 10. Mu.L Red nucleic acid gum dye (10,000×), pour into gum making mold and leave for 30 minutes at room temperature. The PCR product and Marker were added to the agarose gel loading wells. Each well typically receives 5-10. Mu.L of sample. The power is turned on, the voltage is adjusted to 100V for 30 minutes, and the DNA fragment moves to the positive direction under the action of an electric field.

Observation results: after electrophoresis, the gel was placed in a gel imaging system for observation and recording of the results.

2.3 In Vitro Fertilization (IVF) and embryo Veliparib treatment:

Female mice promote ovulation: BRCA1 heterozygote C57BL/6 female mice of 4 weeks of age were used for ovulation promotion. Each mouse was sacrificed by intraperitoneal injection of pregnant horse serum gonadotropin (PMSG) 5IU,48h later, injection of chorionic gonadotrophin (HCG) 5 IU/mouse, 14-16h later, cervical amputation and bilateral oviduct removal, a transparent enlargement of the bilateral oviduct of the mouse was found under a split microscope, and Cumulus-oocyte complex (Cumulus-Oocyte Complex, COC) was obtained by tearing with forceps and transferred into mHTF;

sperm acquisition and capacitation: the C57BL/6 male mice at 10 weeks of age were sacrificed, bilateral epididymal tails were taken, sperm were extruded from vas deferens under a microscope and transferred into mHTF culture medium, and after 1h of capacitation, used for fertilization.

In vitro fertilization: adding 3-4 mu L of capacitation sperm into each mHTF liquid drop, and putting into a three-gas incubator for fertilization for 4-6h. Transferring the embryo into KSOM embryo culture solution after fertilization;

embryo Veliparib treatment: the following day, two-cell embryos (24 h after fertilization) were collected and randomly divided into control and Veliparib treated groups and transferred into KSOM broth and KSOM broth containing 7.5 μ MVeliparib, respectively, for further culture.

2.4 Blastocyst-uterine transplants

Preparing pseudopregnant female mice: selecting ICR female mice with a history of fertility and a better mother, wherein the ICR female mice with a size of 8 weeks and the ICR female mice after ligation are in a cage (female: male=2:1), observing the day of the morning, wherein the female mice with vaginal pessaries are pseudopregnant mice, and randomly dividing the mice into a control group and an experimental group for transplanting for pregnancy;

mice were anesthetized: on day 3.5 after pessary, preparing 1% pentobarbital sodium solution as anesthetic by using normal saline, performing intraperitoneal injection at a dose of 50mg/kg according to the weight of the mice, standing for 10 minutes after injection, and ensuring that the mice are subjected to subsequent operation after complete anesthesia;

disinfection and local infiltration anesthesia: the back of the anesthetized female mouse is shaved, the anesthetized female mouse is prone to be fixed on an operating table, and the head of the anesthetized female mouse is wrapped by articles such as towels or cloth strips, so that the female mouse is prevented from waking up in the operation process. Sterilizing the back of female mice with iodine and alcohol;

Incision: cutting a small opening from one side of the back and the spine of the mouse, cutting off the peritoneum, exposing the ovary and the uterus of the surrogate pregnant mouse, wherein the uterus is usually positioned in the deep part of the abdominal cavity and is close to the spine;

Embryo preparation: embryos are removed from the KSOM embryo culture medium and examined for quality and developmental stage using a microscope. Selecting a high-quality blastula which has obvious blastula cavity and is hatched as a transplanting object, and transferring the blastula into a MII culture solution which is balanced in advance;

Transplanting embryo: the embryo transfer process requires two persons to cooperate. One person pulls the ovary, the oviduct and the uterus out of the peritoneum together by using forceps, precisely finds the joint of the uterus and the oviduct under a split microscope to obtain the uterine horn, and pricks a small hole at the uterine horn by using an insulin syringe. Another person uses the oral suction tube to suck the blastula into the capillary glass tube under the microscope, so as to suck the liquid as little as possible, the capillary glass tube loaded with the blastula is inserted into the small hole of the uterine horn, and the blastula in the capillary glass tube is gently blown into the uterus. Care is taken to avoid bringing excessive culture solution or bubbles as much as possible, and the capillary glass tube is not suitable to be inserted too deeply, so that the endometrium is prevented from being damaged or the tube orifice is prevented from being blocked. After completion, the capillary glass tube was checked for remaining embryos.

Suturing the wound: after the implantation is completed, the ovary, oviduct and uterus are carefully placed back into the peritoneum, and the peritoneum and skin are sutured layer by layer using surgical sutures to ensure that the wound is neat and tight to promote postoperative recovery.

Postoperative care: the mice were placed in a warm, dry environment after surgery and returned to the animal room after waiting for the mice to fully wake up. The state and the reaction condition of the mice are observed, the operation part is kept clean and dry, and the occurrence of infection and other complications is avoided. And proper nutrition and water supplement are simultaneously given, so that wound healing and physical recovery are promoted.

2.5 Experimental observations index

2.5.1IVF embryo in vitro developmental Capacity

And (5) observing and recording the result: after 48, 60, 72 and 96 hours of fertilization, the embryos are photographed by using ZEN 2012 software and counted, and the embryo reaching four cells, eight cells, morula and blastocyst development conditions are observed and counted respectively, and the blastocyst cavity is observed under a microscope to determine that the embryo reaches the blastocyst stage. The main parameter calculation method comprises the following steps:

blastula rate = blastula number/number of two-cell embryos x 100%;

Fertilization rate = number of two-cell embryos/number of oocytes x 100%;

embryo viability = number of embryos at this stage (four-cell, eight-cell, mulberries stage)/number of two-cell embryos x 100%;

2.5.2 observation and recording of birth conditions in offspring mice

Starting from the 14 th day after the transplantation, the gestation and delivery conditions of the surrogate female mice are observed every day, the total birth mice, the live birth number and the dead birth number are counted on the birth date of the offspring mice, the weight of each mouse is weighed, and the sex is checked. Post-transplantation offspring mice were evaluated for birth outcome parameters as follows:

pregnancy rate (%) = number of pregnant mice after transplantation/total number of transplanted mice x 100%;

live yield (%) =live number/total number of parity×100%;

birth rate (%) = total number of parity/number of transferred embryos x 100%;

2.5.3 identification of blastula genotype by semi-nested PCR method

And transferring the mixed embryo into Veliparib for treatment for 72 hours according to an experimental plan, and collecting the embryo successfully reaching the blastula stage for genotyping so as to judge the killing effect of the in vitro drug treatment on the BRCA1 heterozygous embryo. However, since blasts are small in amount and low in DNA content, a semi-nested PCR method was selected for detecting their genotypes. The method needs to carry out two rounds of PCR amplification, designs 3 primers, firstly carries out the first round of amplification on blastula DNA by using a first pair of primers F3R6, then carries out the second round of PCR amplification by taking a first round of PCR product as a template, and the second round of primers F3R5. The primer sequences were as follows:

TABLE 4 primer sequences

DNA extraction: extracting embryo DNA by using DNA extraction lysate, adding 10 mu L Lysis Buffer for PCR and 0.1 mu L of protease K into each tube according to the description, fully and uniformly mixing, placing into eight connecting tubes, sucking single blasts into the liquid by using a mouth suction tube, heating in a PCR instrument for 5 minutes after slightly centrifuging, and then reacting for 2 minutes at 98 ℃, wherein the reaction is completed to be used as a first round of PCR template;

F3R6 is used as a first round PCR primer, the reaction conditions are consistent with those of the conventional PCR, and the reaction system is as follows;

TABLE 5 reaction system

After the first round of PCR was completed, 5 μlpcr product was taken as the second round PCR template, F3R5 was used as the second round PCR primer, the reaction conditions were identical to conventional PCR, the reaction system was as follows, and BRCA1 heterozygous mouse rat tail DNA was added as positive control:

TABLE 6 reaction system

Agarose gel electrophoresis: after the reaction is finished, the second round of PCR products are loaded to agarose gel electrophoresis, the experimental operation and conditions are consistent with those of the conventional PCR, and after the electrophoresis is finished, the gel is placed in a gel imaging system to observe and record the result.

2.5.4 Progeny mice safety detection

(1) Female mouse fertility detection

Control and Veliparib treated groups of females were treated with 2:1 and 10w male mice with normal fertility are bred in cages, and after mating for 1 week, the male mice are singly bred in separate cages. Female mice were checked for gestation by visual and touch at day 14 after cage closure, and daily observations were continued for parity, and parity, dead and live parity were recorded for each female mouse on the parity day.

(2) Cliff avoidance test

Cliff avoidance testing is a classical method of assessing rodent stereoscopic vision and sensory ability, which operates specifically as follows: the experimenter places the mice on the day 8 of birth on an acrylic plate with the height of about 30cm from the ground, so that the heads and forelimbs of the mice are exposed at the edge of the plate, the experimenter starts timing when the experimenter loosens hands, if the mice turn around by themselves within 30 seconds more than 180 degrees, the cliff avoidance experiment is positive, and if the mice fall off or do not turn around from a platform, the cliff avoidance experiment is negative.

(3) Negative going geotropic assay

Rodents (such as rats and mice) naturally have a capability to rotate on an inclined plane from a head-down position to a head-up position, a reaction known as negative geodesic movement. This natural reflex is provided in the second week of normal pups immediately after birth. Negative geotropic tests are a key test to evaluate rodent cub reflex, motor coordination, vestibular system and cerebellar functional integrity. Through this experiment, researchers were able to learn more about the characteristics of rodents in terms of neural development and performance. The specific operation method of the negative geotropic experiment is as follows: on the 9 th day after the birth of the mice, an experimenter uses an acrylic plate to build an inclined plane with an angle of 45 degrees, the young mice are placed on the plate downwards, the experimenter starts timing when the experimenter loosens hands, if the mice turn around automatically within 30 seconds to exceed 180 degrees, the experimenter is regarded as positive in a negative geotropic experiment, and if the mice slide down from a platform or do not turn around, the experimenter is regarded as negative.

(4) Specular reflection

The positive reflex is one of classical experiments for checking whether the vision, vestibular system, baroreceptors or tactors of limbs and position sensory functions of experimental animals are normal, and the operation method thereof is as follows: on day 10 after birth, the experimenter first gently held the mouse limbs supine upward with both hands on a smooth and stable plane, ensuring that the body of the mouse does not slip or move. The experimenter then releases both hands while starting the timing. The experimenter uses two hands to fix the four limbs of the mouse on the plane upwards in a supine way, starts timing when the hands are loosened, and if the mouse can quickly adjust the body position of the mouse within 2 seconds, the mouse turns over from a supine state to a normal crawling posture, the mouse is considered to be positive in the turning-over experiment, and otherwise, the mouse is considered to be negative.

(5) Double ear opening of mouse

The double-ear opening condition of the mice is observed from the 4 th day to the 7 th day after the birth of the mice, when the clear separation of auricles at two sides of the mice from scalp is observed, the mice are considered to be positive in double-ear opening, the mice can be carefully observed by using a magnifying glass, and the occupation ratio of one nest of double-ear opening positive mice is recorded every day. During the observation, the mind is kept, the action is gentle, and the overstimulation or the interference to the mice is avoided.

(6) Mandibular tooth eruption of mice

Daily observations from 11 th to 14 th post-natal day of mice record the positive mouse occupancy of mandibular dental eruption in offspring mice. The operation method is as follows: the experimenter gently fixed the mouse with one hand with its head facing upwards and gently poked the mandible of the young mouse with the other hand, and carefully observed that the mandibular incisors of the mice completely erupted from the gums was considered positive for mandibular eruption.

(7) Opening eyes of mice

The mice were observed for open eyes starting from day 16 after birth until day 20, with the following criteria: in an environment with quietness, brightness and no direct irradiation of a strong light source, the eyelids at two sides of a mouse are completely separated in a natural state, eyeballs are clearly visible, no secretion or adhesion is considered as positive when the eyes of the mouse are open, and the ratio of positive mice when the eyes of the mouse are open is recorded every day.

(8) Female mouse vagina opens

Vaginal opening is an indication of the onset of puberty in female mice. The female mice are observed for vaginal opening at the 28 th day after birth, and the specific operation method is as follows: the experimenter gently lifts the mouse tail with one hand and lightly dials the vaginal orifice with a cotton swab with the other hand, observes whether the vaginal orifice is completely separated from the surrounding skin without adhesion, and records the vaginal opening positive mouse duty ratio every day.

(9) Testis descent in Male mice

The male mice are observed and recorded after 22 days of birth, and the testis decline condition of the male mice is recorded, and the specific operation method is as follows: the experimenter grabs the mouse with one hand and lightly presses and touches the scrotum part of the male mouse with the other hand, senses whether the testis of the male mouse falls into the scrotum completely from the abdominal cavity, notices that the testis of the male mouse can not be pressed downwards, and records the proportion of the positive mouse with testicle falling every day.

(10) Morris water maze

Setting up experimental facilities: draining water in a circular bathtub, adding skimmed milk powder into water to make the platform invisible, placing the water level to a level which is about 0.5cm-1cm lower than the water surface, and sticking azimuth prompts in four quadrants of the pool. Ensuring that all devices (such as a pool, a platform, a camera, a computer and the like) work normally, and adjusting the water temperature to 22-25 ℃;

Acquisition training phase: the first five days are training periods, the mice are lightly placed in water facing the water tank wall, each mouse is placed in 4 quadrants respectively every day, the experiment is repeatedly carried out for 4 times, the mice are free to explore in the water for 1 minute, if the mice find the platform within 1 minute, the mice are led out after the mice learn to stay on the platform for 15 seconds, if the mice do not find the platform within 1 minute, the mice are led to the platform manually, and the mice are led out after the mice learn to stay on the platform for 15 seconds;

Formally testing: and the sixth day is a formal test, the platform is removed, the mouse is put into water from the quadrant opposite to the platform, the mouse freely moves in the water maze for 1 minute, the moving track of the mouse is monitored by using the ANY-maze system and a camera, and the performance of the mouse in the test stage is recorded, wherein the performance comprises parameters such as the passing times of the position of the platform, the moving time of the mouse staying in the quadrant region of the original platform and the like.

(11) Mouse body weight curve monitoring

The weights of the mice were weighed on the 1 st, 4 th, 7 th and 14 th days after birth, respectively, and the following procedures were followed: and (3) plugging the electronic balance into a power supply, starting up, and placing a weighing box and zeroing after the reading of the electronic balance is stable. Gently grabbing each group of offspring mice, putting the offspring mice into a weighing box, and reading and recording the numerical values after the numerical values are stable.

(12) Sperm detection in male mice

A37℃water bath was prepared, 1.5mL EP tubes were prepared, 1000. Mu.L PBS was added to each tube after labeling, and the tubes were placed in the water bath and preheated for 30 minutes. About 8w of male mice are taken, abdominal cavities are cut after neck breaking and killing, epididymal tails on two sides of the male mice are taken, the male mice are placed into PBS preheated at 37 ℃, clean ophthalmology scissors are used for extending into an EP tube, and tissues are sufficiently sheared. The EP tube was placed in a water bath and incubated at 37℃for 30min. 100. Mu.L of the supernatant was taken and added in a 1:1 ratio to a fresh ep tube pre-filled with 100. Mu.L of paraformaldehyde, and the mixture was fixed at room temperature for 30min, and the sperm concentration was recorded using an automatic cytometer. And (3) taking 10 mul of supernatant smear, dripping the supernatant smear to the center of a glass slide, lightly covering the glass slide, observing the glass slide under a microscope, selecting a plurality of field counts to respectively count the numbers of forward motile sperms, non-forward motile sperms and motile sperms, counting 200 mice each, and taking an average value after counting twice to calculate the proportion of various sperms.

2.6 Analysis of experimental data

All assays were run in triplicate or more independent replicates and the results were expressed as mean ± SD (standard deviation). Statistical analysis was performed using GRAPHPAD PRISM 9.0.0 software (San Diego, CA, USA). Mean comparison between Veliparib treated and control groups used the independent sample Student's t-test. Significance of the difference in rate between the two groups was statistically significant using chi-square test, P < 0.05.

3. Experimental results

3.1Veliparib Effect on the in vitro development of embryos of groups

To verify Veliparib that they were able to screen for embryos carrying BRCA1 heterozygous mutations, the present invention utilizes In Vitro Fertilization (IVF) techniques to collect wild-type (WT) and BRCA1 mutated mouse embryos. These embryos are then placed in KSOM medium with different concentration gradients Veliparib (0. Mu.M, 2.5. Mu.M, 5. Mu.M, 7.5. Mu.M, 10. Mu.M,). During the course of the experiment, the present invention set a number of time points for observing and recording the development of WT group and BRCA1 group embryos. The present invention closely focuses on the development process of each embryo in the hope of finding the different effects of Veliparib on the two groups of embryos.

As shown in FIG. 3 (A and B), the experimental results show that Veliparib has a certain adverse effect on the early development of both wild-type and BRCA1 group embryos. And Veliparib has a concentration-dependent inhibitory effect on embryo development. Notably, however, embryos of BRCA1 group showed higher sensitivity at the same concentration Veliparib compared to WT group. In particular, at the same Veliparib concentration, more embryo development in the BRCA1 group was blocked and normal development was not continued.

To find the appropriate screening concentrations to distinguish BRCA1 heterozygous embryos from wild-type embryos, the present invention plots survival curves for embryo development by further statistical analysis (B in fig. 3). Without Veliparib, there was no significant difference in survival curves for BRCA1 group embryos and wild-type embryos (fig. 3A and B, p= 0.0702). However, when treated with Veliparib at the same concentration, more embryos in the BRCA1 group delayed or stopped developing, and the difference in survival curves was more pronounced with increasing Veliparib concentration. When Veliparib concentration reached 7.5 μm, significant differences occurred in the survival curves of the two groups (P < 0.001).

3.2Veliparib in vitro Screen of Pre-and post-embryo genotype Condition

Oocytes from BRCA1 heterozygous females were first collected and fertilized in vitro with wild type male sperm, all two-cell embryos were randomly divided into control and Veliparib treated groups (7.5 μm), and embryos reaching blastocyst stage were collected and genotyping by semi-nested PCR at 96h post fertilization, with the results shown in fig. 4a and B. The present invention found that 45.2% (14/31) of blastula in the control group without Veliparib treatment were BRCA1 heterozygous embryos. This distribution was expected and confirmed that heterozygous female oocytes contained the same number of wild type and mutant oocytes and had similar oocyte quality and developmental capacity. In the Veliparib treated group, only 3.6% (1/28) of the blasts were BRCA1 heterozygous embryos, and the remaining blasts were wild-type. These results demonstrate that treatment with 7.5 μ M Veliparib was effective in preventing blastocyst formation in BRCA1 heterozygous embryos.

3.3Veliparib in vitro Screen of the genotype ratio of offspring after embryo transfer

After verifying Veliparib that the development of the BRCA1 heterozygous embryo can be effectively blocked in vitro, the invention further researches whether the screening scheme can effectively avoid the birth of the BRCA1 heterozygous mouse on an individual level through blastocyst-uterus transplantation. The invention uses ICR mice as surrogate mice to conduct blastocyst-uterus transplantation experiments to explore the screening efficacy of Veliparib scheme. The Control group totally comprises 32 pregnant ICR mice for embryo transfer operation, wherein 8 pregnant ICR mice smoothly produce offspring, the Veliparib treatment group totally comprises 35 pregnant ICR mice for embryo transfer operation, and 8 pregnant ICR mice smoothly produce offspring.

The results of the genotyping of the offspring mice are shown in table 7 and fig. 5. The invention first explores the genotype of the offspring generated under natural mating conditions. Of the progeny of 4 pairs of BRCA1 heterozygous female mice that mate naturally with WT male mice, 18 (47.4%) are BRCA1 heterozygotes (including 1 dead fetus), 20 (52.6%) are wild-type, consistent with the dominant genetic nature of the BRCA1 gene, and about 50% of the progeny carry BRCA1 heterozygous mutations. A total of 33 offspring mice were born from the control group, of which 18 (54.5%) were wild-type (including 1 dead fetus), and 15 (45.5%) were BRCA1 heterozygotes (including 1 dead fetus). The 7.5 μ MVeliparib treatment group had 31 offspring born, all of which were wild-type (including 2 dead fetuses), and no BRCA1 heterozygous mice were born, either live or dead fetuses. These results strongly demonstrate the effectiveness of the screening regimen of the present invention.

TABLE 7 genotyping results of F1 Generation mice produced by transplanting offspring pregnant mice

3.4 Embryo transfer related parameters

To investigate initially whether Veliparib treatments affected embryo implantation and gestation ability, the present invention further counted pregnancy related parameters after Veliparib treatment and Control group transplants, as shown in table 8. The invention found that there was no significant difference in average parity (4.13 vs3.88, p=0.23) and average gestation time (18.00 vs18.38, p=0.42) between the two groups, whereas the parity was higher than Veliparib treated group (41.91% vs34.91%, P < 0.05) with the assumption that the number of embryo transferred was similar in the two groups (9.63 vs10.13, p=0.13). In combination with the in vitro genotyping results, it is reasonably explained that, when embryos are treated after the implantation Veliparib, although most of the implanted blasts are wild type, part of blasts carrying BRCA1 heterozygous mutations are transplanted into the uterus, and the subsequent implantation and embryo growth and development cannot be completed normally due to the defect of DNA damage repair function, so that the number of formed offspring is reduced compared with that of the prior art. No significant difference was found between the two groups in terms of live yield (94.38% vs92.71%, p=0.79). These results initially confirm that Veliparib treatment did not affect implantation and normal pregnancy processes of wild-type embryos after screening.

TABLE 8 embryo transfer and pregnancy outcome-related parameter comparison

Note that: each set of data is expressed as mean ± standard deviation, ^* the difference compared to the Control set being statistically significant, ^* P <0.05.

3.5Veliparib post-treatment F1 progeny safety detection

Regarding the safety of offspring, further safety evaluation was performed on offspring mice produced after the Veliparib treatment group and Control group transplantation, and whether or not the indexes such as physical development (weight change, auricle opening, open eyes, eruption of lower incisors), puberty maturation (female mouse vaginal opening, male mouse testis lowering), reproductive function (female mouse natural mating, male mouse sperm detection), neural development (cliff avoidance, negative refraction, regular reflection), learning and memory (Morris water maze) and the like were normal was detected. The results are shown in fig. 6 a-D, fig. 7a and B, fig. 8 a-B, fig. 9 a-F, and fig. 10 a-C, with the exception that the incisor sprouting rate was slightly lower in the treatment group at 13 th postnatal day Veliparib than in the Control group (p=0.0244), and there was no significant difference in the child safety indices (P > 0.05) for the physical development, puberty maturation, reproductive function, neural development, learning memory, etc. of the two groups of mice. Overall, veliparib treatment did not significantly affect the physiological function of the wild-type blastula and the behavioral performance of the offspring mice, which provided a powerful support for the safety of this regimen in clinical applications.

In summary, the invention constructs a C57BL/6 mouse model with the BRCA1 heterozygous deletion, carries out In Vitro Fertilization (IVF) to obtain mouse embryos, and carries out screening treatment in embryo culture solution containing Veliparib; veliparib, by virtue of the unique chemical structure and action mechanism, can effectively inhibit the development of the BRCA1 heterozygous mutant embryo without affecting the wild embryo; the invention can realize screening and blocking of BRCA1 heterozygous mutant embryo in vitro, avoids inheriting pathogenic genes to offspring, provides a new possibility for preventing inheritance pathogenic genes to offspring for females carrying BRCA1 mutation, and has important clinical application value.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (10)

1. Application of PARP inhibitor Veliparib in the preparation of drugs for screening out BRCA1 heterozygous mutation embryos. 2. The use according to claim 1 is characterized in that the PARP inhibitor Veliparib, at a specific dose and during a specific embryonic development period, acts through a synergistic lethal effect, utilizing the insufficient homologous recombination repair function of BRCA1 heterozygous mutant embryos in BRCA1 heterozygous mutant embryos to cause the BRCA1 heterozygous mutant embryos to accumulate more DNA damage, thereby achieving the result of blocking the development of the BRCA1 heterozygous mutant embryos. 3. The use according to claim 1, characterized in that the direct effect of the PARP inhibitor Veliparib at a specific dose in the in vitro culture of embryos at a specific developmental period does not affect the development and implantation of normal embryos, as well as the growth and development of offspring of normal embryonic development. 4. A drug for screening out BRCA1 heterozygous mutation embryos, characterized in that the drug comprises the PARP inhibitor Veliparib according to any one of claims 1 to 3. 5. A drug for screening out BRCA1 heterozygous mutation embryos according to claim 4, characterized in that the drug further comprises a pharmaceutically acceptable excipient. 6. The drug for screening out BRCA1 heterozygous mutation embryos according to claim 4, characterized in that the drug further comprises a pharmaceutically acceptable carrier. 7. A drug for screening out BRCA1 heterozygous mutation embryos according to claim 4, characterized in that the drug is in the form of a solution or a microparticle dispersion. 8. A method for screening BRCA1 heterozygous mutant embryos in vitro using the PARP inhibitor Veliparib, wherein the screening method is used for non-diagnostic purposes and comprises the following steps: placing embryos cultured in vitro fertilization in a culture medium containing the PARP inhibitor Veliparib at a specific concentration as described in any one of claims 1 to 3, and continuing the culture for at least 3 days until the embryos develop to the blastocyst stage. 9. A method for screening BRCA1 heterozygous mutant embryos in vitro using the PARP inhibitor Veliparib according to claim 8, characterized in that the initial drug action time of the embryos cultured in vitro fertilization is a two-cell stage mouse embryo cultured in vitro fertilization. 10 . The method for screening BRCA1 heterozygous mutant embryos in vitro using the PARP inhibitor Veliparib according to claim 8 , wherein the specific concentration of the PARP inhibitor Veliparib in the culture medium is 7.5 μM.