A method for tracking and assessing the organ metabolic distribution of platelets based on mitochondrial DNA.

Abstract

This invention belongs to the field of biotechnology and relates to a method for tracking and assessing the metabolic distribution of platelets in organs based on mitochondrial DNA. Specifically, this invention constructs a humanized mouse model, injects exogenous platelets into the humanized mouse model, and sequences the specific SNVs of the exogenous platelet mitochondrial DNA to trace the metabolic distribution of human platelets or human drug-loaded platelets. The safety is assessed by analyzing the metabolic distribution when human platelets are used as a delivery system.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for tracking and assessing the organ metabolic distribution of platelets based on mitochondrial DNA. Background Technology

[0002] Platelets are a key biomaterial in clinical transfusion therapy, thrombosis prevention, and drug delivery. Their metabolic distribution and safety in vivo directly affect clinical application outcomes. Traditional platelet tracking technologies have significant drawbacks: radioactive labeling methods (such as indium-111 and chromium-51 labeling) pose radiation exposure risks and cannot accurately locate organ distribution; biotin labeling methods require pretreatment of platelets, which may destroy their biological activity; and HLA phenotyping methods are limited by individual genetic differences and are difficult to apply to subjects who have received multiple transfusions.

[0003] Therefore, developing a specific tracking method to accurately quantify the metabolic distribution characteristics of platelets in key organs is of great significance for the quality control of platelet delivery and the safety evaluation of drug carriers. Summary of the Invention

[0004] This invention provides a highly specific and sensitive method for tracking platelet organ metabolism distribution, providing a scientific basis for clinical applications.

[0005] In a first aspect of the invention, a testing system for the organ distribution level of test platelets is provided, the system comprising: (Z1) Platelet delivery module, the platelet delivery module being configured to administer test platelets into a test subject, wherein the test subject is a non-human mammal; (Z2) Nucleic acid extraction module, wherein the nucleic acid extraction module is configured to: collect organ tissue samples from the test subject, thereby providing mitochondrial DNA samples from the organ tissue; (Z3) Test module, the test module is configured to: provide a platelet-specific SNV sequence and test the platelet-specific SNV level in a mitochondrial DNA sample of the organ tissue; (Z4) Analysis module, which is configured to provide the distribution level of the test platelets in the organ tissue based on the test platelet-specific SNV level.

[0006] In another preferred embodiment, the organ distribution level of the test platelets includes: the absolute level of the test platelets and / or the relative level of the test platelets.

[0007] In another preferred embodiment, the test is a qualitative test and / or a quantitative test.

[0008] In another preferred embodiment, the nucleic acid extraction module is configured to collect organ tissue samples from the test subject at different times, thereby providing mitochondrial DNA samples from the organ tissue at different times; The testing module is configured to: provide a platelet-specific SNV sequence and test the platelet-specific SNV level in mitochondrial DNA samples of the organ tissue at different time points; The analysis module is configured to provide the distribution of the test platelets in the organ tissue at different time points based on the test platelet-specific SNV levels in the organ tissue at different time points.

[0009] In another preferred embodiment, the metabolic half-life of the test platelets in the organ tissue is provided based on the distribution of the test platelets in the organ tissue at different times.

[0010] In another preferred embodiment, the nucleic acid extraction module is configured to collect different organ tissue samples from the test subject, thereby providing mitochondrial DNA samples from each organ tissue; The testing module is configured to: provide platelet-specific SNV sequences and test the platelet-specific SNV levels in mitochondrial DNA samples from various organs and tissues; The analysis module is configured to provide the distribution of the test platelets in each organ tissue based on the specific SNV levels of the test platelets in each organ tissue.

[0011] In another preferred embodiment, the system further includes a result output module configured to provide a safety evaluation result of the test platelets by measuring the test platelet-specific SNV levels in different organs at the same time and / or by statistically analyzing the test platelet-specific SNV levels in the same organ at different times.

[0012] In another preferred embodiment, the test platelets comprise wild-type human platelets or human platelets loaded with a drug.

[0013] In another preferred embodiment, the human platelets comprise: platelets directly derived from the human body and / or platelets obtained through in vitro differentiation.

[0014] In another preferred embodiment, the human platelets are drug-loaded platelets obtained by directly incubating the drug with platelets.

[0015] In another preferred embodiment, the drug-loaded human platelets are drug-loaded platelets differentiated in vitro from platelet precursor cells expressing the drug.

[0016] In another preferred embodiment, the drug-loaded platelets are prepared by: genetically engineering platelet precursor cells to obtain drug-expressing platelet precursor cells; and differentiating the drug-expressing platelet precursor cells into platelets to obtain drug-loaded platelets.

[0017] In another preferred embodiment, the SNV level of the human mitochondrial DNA includes: absolute level and / or relative level.

[0018] In another preferred embodiment, the non-human mammal includes: mice, rats, rabbits, dogs, pigs, or non-human primates.

[0019] In another preferred embodiment, the distribution levels of wild-type human platelets and drug-loaded human platelets in different organ tissues are compared to provide a safety evaluation of drug-loaded platelets.

[0020] In another preferred embodiment, the distribution levels of wild-type human platelets and the metabolic half-life of drug-loaded human platelets are compared to provide a safety evaluation of drug-loaded platelets.

[0021] In another preferred embodiment, the platelet-specific SNV sequence referred to is an SNV sequence that is specifically present in human platelets but not in platelets from non-human mammals.

[0022] In another preferred embodiment, the non-human mammal is a humanized non-human mammal; The platelet-specific SNV sequence refers to an SNV sequence that is specifically present in human platelets but not in the humanized non-human mammalian donor platelets.

[0023] In another preferred embodiment, the humanized non-human mammal is a non-human mammal with a humanized blood system and / or immune system.

[0024] In another preferred embodiment, it further includes a result output module (Z5) configured to provide a safety evaluation result of the test platelets based on the distribution level of the test platelets in organ tissues.

[0025] In another preferred embodiment, the security evaluation result includes evaluating one or more features selected from the group consisting of: (i) Risk of abnormal platelet aggregation in the test organs; (ii) Risk of abnormal platelet clearance in the test subjects; (iii) Risk of tissue toxicity to the tested platelets; (iv) Thrombosis risk in subjects.

[0026] In another preferred embodiment, the different organs and tissues include: brain, quadriceps femoris muscle, ovary, testis, bladder, jejunum, stomach, pancreas, inguinal lymph nodes, kidney, adrenal gland, femoral bone marrow, heart, liver, spinal cord (thoracic vertebrae), lung, spleen, or combinations thereof.

[0027] In another preferred embodiment, the test comprises sequencing, digital PCR, or a combination thereof.

[0028] In another preferred embodiment, the test refers to high-throughput sequencing.

[0029] In another preferred embodiment, the different times refer to collection at 30 min (±5 min), 2 h (±10 min), 6 h (±10 min), 12 h (±15 min), 24 h (±30 min), 72 h (±30 min), and 168 h (±30 min) after human platelets are administered to the subject.

[0030] In a second aspect of the invention, a method for evaluating the safety of exogenous platelets in non-human mammals for non-diagnostic and non-therapeutic purposes is provided, comprising the following steps: (S1) The test platelets were administered to non-human mammals; (S2) Collect organ and tissue samples from non-human mammals to provide mitochondrial DNA samples from said organ and tissue; (S3) Provide a platelet-specific SNV sequence and test the platelet-specific SNV level in a mitochondrial DNA sample of the organ tissue; (S4) Provide the distribution level of the test platelets in the organ tissue based on the test platelet-specific SNV level; (S5) Provide the safety assessment results of the test platelets based on their distribution level in the organ tissue.

[0031] In another preferred embodiment, the test platelet is a drug-loaded platelet.

[0032] In another preferred embodiment, in step (S2), organ tissue samples of the subject are collected at different times to provide mitochondrial DNA samples of the organ tissue at different times; In step (S3), a platelet-specific SNV sequence is provided, and the platelet-specific SNV level in mitochondrial DNA samples of the organ tissue is tested at different time points. In step (S4), the distribution of the test platelets in the organ tissue at different time points is provided based on the test platelet-specific SNV level in the organ tissue at different time points.

[0033] In another preferred embodiment, in step (S2), different organ tissue samples of the test subject are collected respectively, thereby providing mitochondrial DNA samples of each organ tissue; In step (S3), a platelet-specific SNV sequence is provided, and the platelet-specific SNV level in mitochondrial DNA samples from various organs and tissues is tested. In step (S4), the distribution of the test platelets in each organ tissue is provided based on the test platelet-specific SNV level in each organ tissue.

[0034] In another preferred embodiment, the safety evaluation results are the platelet-specific SNV levels of the test platelets in different organs at the same time, and / or the platelet-specific SNV levels of the test platelets in the same organ at different times.

[0035] In another preferred embodiment, the distribution level of the drug-loaded test platelets in the organ tissue is provided, as well as the distribution level of wild-type platelets in the organ tissue, thereby providing a safety evaluation result of the drug-loaded platelets relative to wild-type human platelets.

[0036] In another preferred embodiment, the administration comprises: intravenous injection.

[0037] In another preferred embodiment, the non-human mammal is a humanized non-human mammal.

[0038] In another preferred embodiment, the humanized non-human mammal is a non-human mammal with a humanized blood system and / or immune system.

[0039] In another preferred embodiment, the platelet-specific SNV sequence refers to an SNV sequence that is specifically present in human platelets but not in the donor platelets of the humanized non-human mammal.

[0040] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0041] Figure 1 The reconstitution effects of the two selected donors (0336 and 0178) in an immunodeficient mouse model are shown. The hCD45 ratio represents the proportion of human blood cells in the peripheral blood of mice.

[0042] Figure 2 The results showed that drug-loaded platelets could still be detected in a mouse model of leukemia more than 24 hours after infusion. G1 was injected with control platelets, and G2 was injected with drug-loaded platelets.

[0043] Figure 3 The specific SNV sequences of platelets from different sources are shown.

[0044] Figure 4 The results of in vitro validation of the specific SNV sequence are shown.

[0045] Figure 5 The results showed that drug-loaded platelets did not cause changes in ALT and AST after infusion into a humanized mouse tumor model, indicating that drug-loaded platelets did not cause CRS-induced liver damage.

[0046] Figure 6 The results showed that after infusion of drug-loaded platelets into a humanized mouse tumor model, the release of cytokines remained at a low level, indicating that drug-loaded platelets did not cause CRS. Detailed Implementation

[0047] Through extensive and in-depth research, the inventors have unexpectedly discovered, for the first time, a method for tracking and assessing the metabolic distribution of platelets in organs based on mitochondrial DNA. Specifically, this invention involves constructing a humanized mouse model and, after injection of exogenous platelets, sequencing specific SNVs of exogenous platelet mitochondrial DNA to trace the metabolic distribution of exogenous unprocessed platelets or drug-loaded platelets. This invention was completed based on this discovery.

[0048] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0049] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0050] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0051] As used in this article, the term "or a combination thereof" means "or any combination thereof".

[0052] As used herein, "at different times" refers to collecting organ and tissue samples from the subject at regular intervals. In specific implementations, the time interval between any two collections may be equal or unequal. In specific implementations, a total of 5-15 collections are performed, preferably 5-10, and optimally 7. In specific implementations, the collection frequency is higher in the early stages and lower in the later stages. In specific implementations, samples are collected at 30 min (±5 min), 2 h (±10 min), 6 h (±10 min), 12 h (±15 min), 24 h (±30 min), 72 h (±30 min), and 168 h (±30 min) after platelet administration.

[0053] Drug-loaded platelets and their safety evaluation As used herein, the term drug-loaded platelet refers to a platelet loaded with a drug. In specific embodiments, the platelet is a directly isolated platelet or a platelet obtained through in vitro differentiation. In specific embodiments, the drug-loaded platelet is obtained by incubating the platelet with a drug. In specific embodiments, the drug-loaded platelet is obtained by expressing a drug in platelet precursor cells, followed by differentiating the drug-expressing platelet precursor cells.

[0054] In specific implementations, compared to directly modifying platelets (e.g., through click chemistry), the strategy of gene transduction at the iPSC stage followed by differentiation to obtain engineered platelets avoids protein shedding or instability issues that may arise from exogenous modifications, resulting in more persistent and reliable drug expression. Simultaneously, since genetic modification occurs at the iPSC cell origin stage, after monoclonal selection or the establishment of stable lines, a cell population with relatively consistent expression levels can be obtained, significantly reducing drug loading fluctuations between different batches. This approach is independent of later conjugation efficiency or reaction condition control, improving batch-to-batch consistency and reproducibility. Furthermore, after obtaining platelets, this strategy only requires routine purification and collection steps, avoiding potential impacts on platelet membrane structure, membrane protein spatial conformation, and integrin functional status that may result from cross-linking reactions, surface modifications, or exposure of active groups.

[0055] The platelet organ distribution tracking method of the present invention is applicable to any platelet, preferably drug-loaded platelets, and more preferably drug-loaded platelets differentiated in vitro from drug-expressing platelet precursor cells.

[0056] Platelet organ distribution tracking The core of platelet organ distribution tracking is to locate the distribution, aggregation, and clearance sites of exogenous platelets in the body. It has important value in five major fields: basic research, clinical diagnosis, treatment evaluation, drug development, and transfusion medicine. It directly serves the precision diagnosis and treatment and mechanism analysis of platelet delivery, bleeding / thrombosis, immunity, tumors, and transplantation.

[0057] Platelet organ distribution tracking is of core guiding significance in platelet-targeted delivery systems. Due to their natural tendency to damage blood vessels, thrombi, inflammation, and tumor microenvironments, platelets are regarded as ideal in vivo drug carriers. Tracking technology can intuitively reveal the targeted homing, organ enrichment, and retention patterns of drug-loaded platelets in vivo, providing the most direct experimental basis for carrier design and drug loading methods, and is a fundamental prerequisite for achieving precise delivery.

[0058] By dynamically tracking the organ distribution of platelets, we can clarify the targeting efficiency and off-target risk of drug delivery, determine whether drug-loaded platelets are truly enriched in the target lesion (such as tumors or thrombus sites) or remain in large quantities in organs such as the liver and spleen, thereby quantifying targeting, optimizing surface modification and drug loading strategies, increasing local drug concentration at the lesion, reducing systemic toxicity, and significantly improving the safety and effectiveness of the delivery system.

[0059] Furthermore, platelet distribution tracking can evaluate the in vivo stability and therapeutic efficacy of delivery systems, monitor the lifespan, aggregation behavior, and drug release patterns of drug-loaded platelets in circulation in real time, assess delivery differences under different disease models, and provide key data support for the clinical translation of novel platelet-targeted drugs, anti-tumor drug delivery systems, and thrombolytic agents. It promotes the research and application of precision and intelligent drug delivery and is an indispensable and important technology in the field of biomaterials and nanomedicine.

[0060] Platelet mitochondrial DNA Platelet mitochondrial DNA and other cellular mitochondrial DNA are both double-stranded circular 16.6 kb genomes encoding two rRNAs, 22 tRNAs, and 13 oxidative phosphorylation core proteins. They lack histone protection, are maternally inherited, exhibit multiple copies and heterogeneity, and rely on nuclear gene-encoded proteins for replication and repair. However, platelets generally suffer from short lifespans, instability after drug loading, and susceptibility to activation / lysis. Therefore, while some studies have used mtDNA sequencing to track the overall recovery rate of transfused platelets, current technologies have failed to achieve targeted tracking of key metabolic organs such as the heart and liver, and lack a safety assessment system based on organ metabolic distribution.

[0061] Humanized mice Humanized mice best mimic the human circulatory and immune systems, and are highly valuable for assessing the actual organ distribution of exogenous platelets after they enter the human body. However, during the humanization process, both the introduced platelets and the officially administered platelets are human platelets, making biological differentiation difficult. This limits the assessment of organ distribution of exogenous platelets in animal models.

[0062] The tracking method of the present invention This invention first identifies exogenous platelet-specific single nucleotide variants (SNVs): DNA was extracted from exogenous platelets and donor cells used for humanization of mice, quantified, and used to construct libraries. Mitochondrial DNA probes were captured using a Human Mitochondrial Panel, and the captured libraries were then sequenced using high-throughput sequencing to identify SNVs. By comparing the platelet mitochondrial DNA data obtained through NGS with a standard reference genome, and using tools such as GATK, single nucleotide variant (SNV) sites that could distinguish exogenous platelets from human cells used for humanization of mice were identified. Error-prone regions and low-frequency variants were excluded from these sites.

[0063] Optionally, exogenous platelets and platelets from donors used to construct humanized mice are mixed in vitro at a certain ratio, and then sequenced to see if the proportion of the selected SNV sequence in the mixed sample is consistent with the mixing ratio.

[0064] Finally, the distribution of exogenous platelets in tissues was detected: exogenous platelets were infused into humanized mice, and samples at different time points were subjected to DNA extraction and sequencing according to the method in the first step. This allowed us to obtain the distribution information of exogenous platelets in different tissues at different time points.

[0065] This invention provides a humanized mouse model as a test animal. In this humanized mouse model, the human blood and immune systems are reconstructed.

[0066] After platelet administration, test animals were euthanized at different time points, and tissues from various organs were collected. Total DNA was extracted from the tissue samples, and nucleic acid concentration was measured. In a specific implementation, genomic DNA was extracted from whole blood and various tissues using a magnetic bead method. In another specific implementation, the OD value of the nucleic acid was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the concentration was calculated to determine whether the sample extraction was normal.

[0067] Subsequently, using the exogenous platelet mitochondrial DNA-specific SNV search method provided by this invention, high-throughput sequencers were used for detection, raw data were output using relevant software, and the content of the test sample (exogenous platelets) was calculated.

[0068] Finally, it provides the distribution of platelets in different organs and tissues at the same time; and / or provides the distribution of platelets in the same organ at different times, thereby providing the metabolic half-life (t1 / 2) of platelets in each organ.

[0069] The main advantages of this invention include: (a) Based on the tracing method of the present invention, donor cells of humanized mice and exogenous (or test) platelets can be effectively distinguished, without being limited by low organ mitochondrial DNA concentration or easy platelet rupture, and the organ distribution tracking of exogenous mitochondria can be realized.

[0070] (b) The tracing method of the present invention provides a comprehensive evaluation. Through the analysis of metabolic parameters at multiple time points and in multiple organs, it can provide early warning of safety issues such as abnormal accumulation of platelets in organs and metabolic disorders, and provide a basis for clinical decision-making.

[0071] (c) The tracing method of the present invention is based on multiple SNV sequences, which eliminates the error-proneness of using a single gene for tracing and has high accuracy.

[0072] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0073] Example 1: Recovery and counting of frozen platelets.

[0074] Remove the platelet cryopreservation tubes from the liquid nitrogen tank. Immediately use hemostatic forceps to gently clamp the base of the pressure buffer tube, keeping the rubber stopper facing down (do not invert). Completely submerge the cryopreservation tubes below the water surface in the water bath and continuously shake in a circular motion to ensure even heating. After approximately 30 seconds, briefly remove the tubes every 5-10 seconds to observe the ice crystal size. Stop the water bath when the ice crystals are slightly smaller than a mung bean. Wipe dry with sterile gauze, spray with 75% ethanol, and transfer to a biosafety cabinet. Remove the aluminum foil from the blue rubber stopper at the bottom of the tube, cut the pressure buffer tube, and use a 1 mL syringe to puncture the blue rubber stopper at the bottom of the tube. While shaking, draw up all the samples and transfer them to a new 1.5 mL centrifuge tube. After thoroughly mixing with a 1000 μL pipette, immediately draw up as much of the sample as possible and transfer it to a 15 mL centrifuge tube. Then add frozen platelet diluent so that the volume ratio of the drawn frozen platelet stock solution to the frozen platelet diluent is 1:1.5.

[0075] Thoroughly mix the platelet suspension using a 1000 μL pipette, and immediately take two 30 μL samples from the middle layer, transferring each sample to a 0.5 mL centrifuge tube for formulation analysis (cell counting by flow cytometry). Store the remaining platelet suspension at room temperature.

[0076] The principle of platelet counting is as follows: when flow cytometry antibody (PE-CD41) is added to the test sample, the fluorescently labeled antibody specifically binds to the platelet surface antigen. Before testing, an equal volume of absolute counting microspheres as the test sample is added and thoroughly mixed. The number of cells in a given volume of sample can be calculated based on the known number of absolute counting microspheres. Combined with the flow cytometry antibody clustering method, the absolute number of a certain group of cells within a given volume can be calculated. Specific experimental method: Platelets to be tested are sampled into a 1.5 mL sterile centrifuge tube and tested immediately after sampling. Using reverse pipetting, 50 μL of platelet sample and 20 μL of diluted PE-CD41 antibody are added to the top of the stainless steel holder of the absolute counting tube (Note: Do not touch the microspheres or the tube wall). The tube is capped, gently vortexed to mix, and incubated at room temperature in the dark for 15 min. Then, 4.3.5. 400 μL of diluent is added to each tube, the tube is capped, gently vortexed to mix, and incubated at room temperature in the dark for 15 min. Prepared samples should be stored in the dark at 4-10°C before analysis and should be analyzed within 1 hour. Before running the flow cytometer, the counting tube should be vortexed at low speed to resuspend the microspheres and reduce cell aggregation.

[0077] After counting, thoroughly pipette the platelet suspension, accurately aspirate a certain volume (as much as possible), and centrifuge at 1000 g for 10 min at room temperature, with an ascending speed of 5 and a descending speed of 4. After disinfection by spraying with 75% ethanol, transfer to a biosafety cabinet. Based on the results of the formulation analysis, carefully aspirate a certain volume of supernatant using a pipette, leaving a volume sufficient to achieve the desired platelet concentration after resuspending.

[0078] Example 2: Construction of a humanized mouse Nalm6 tumor model and platelet drug delivery.

[0079] NPG-dKO mice are severely immunodeficient mice. Six-week-old NPG-dKO mice were injected via tail vein with 1E7 human peripheral blood mononuclear cells (PBMCs), thereby implanting human immune cells (mainly human T cells, monocytes, and B cells) into the mice; thus, the mice had human immune surveillance / immune response, simulating the immune environment in the human body.

[0080] NALM6-Luc tumor cells were revived and passaged. Eight days after NPG-dKO mice were inoculated with huPBMCs, NALM6-Luc cells in the logarithmic growth phase were collected. The culture medium was removed and the cells were washed twice with DPBS. Then, 5E5 tumor cells were injected into each mouse via tail vein injection.

[0081] On day 13 post-inoculation with huPBMCs, the humanization reconstitution rate was assessed by flow cytometry, and the results were as follows: Figure 1 As shown, the reconstitution effects of PBMCs from different donors (0336 and 0178) transplanted into immunodeficient mouse models via tail vein injection (IV) with 5M or 10M cells. The hCD45 ratio represents the proportion of human blood cells in the mouse peripheral blood. 5M and 10M represent 5E6 and 10E6 PBMCs injected, respectively, and iv indicates intravenous injection. This figure shows that, in testing different PBMC injection doses, the 10E6 cell number resulted in better reconstitution. Furthermore, the reconstitution effect varied among different donors after PBMC injection into mice, but all were above 10%.

[0082] On day 15 after huPBMC inoculation (i.e. day 7 after NALM6-Luc inoculation), the average tumor photon count measured by in vivo imaging reached 2.80E+04.

[0083] Nineteen mice were randomly assigned to groups according to the experimental design based on reconstruction rate and tumor photon count. The day of grouping was defined as D0. Clodronate liposomes were injected intraperitoneally on D-1, D3, D6, and D10 to clear residual macrophages in the mice. Platelets (drug-loaded platelets and control platelets) were injected on D0, D4, D7, and D11 at a dose of 1E7 platelets per mouse.

[0084] The results are as follows Figure 2 As shown in the figure. G1 represents control platelets injected without the drug, and G2 represents platelets injected with the drug. Drug-loaded platelets can still be detected in the leukemia mouse model more than 24 hours after transfusion.

[0085] Example 3: SNV sequences that specifically distinguish exogenous platelet DNA from humanized mouse donor platelet DNA.

[0086] Exogenous platelet DNA (named Sample A) was extracted, and mitochondrial DNA from platelets derived from donor cells used for the humanization of immunodeficient mice (named Sample B) was extracted. Both samples were subjected to NGS sequencing to identify SNV sequences specifically expressed in different donors.

[0087] like Figure 3 As shown in the figure. The red-marked sequences are some of the SNV sequences specific to sample B.

[0088] Samples A and B were mixed in different ratios (1:1, 1:5, 1:10, 1:20, 1:50), and then DNA was extracted and sequenced. Selected sample-specific SNVs were used for fitting.

[0089] like Figure 4 As shown, the proportions of A and B specific SNV sequences in different mixed samples are basically consistent with the mixing ratio of the sample, indicating that the method has high accuracy.

[0090] Example 4: Platelet tracing and distribution detection.

[0091] This embodiment prepared drug-loaded platelets differentiated in vitro (hereinafter referred to as drug-loaded platelets). Specifically, the drug was expressed in iPSCs through gene editing during their precursor cell stage (such as the iPSC stage), so that the platelets differentiated from iPSCs would carry the drug. For example, the platelets were simultaneously loaded with 4-1BBL and IL-12. Following the method in Example 2, humanized mice were constructed using donor peripheral blood mononuclear cells (PBMCs). Drug-loaded platelets were transplanted into the mice via tail vein injection. The mice were euthanized at 30 min (±5 min), 2 h (±10 min), 6 h (±10 min), 12 h (±15 min), 24 h (±30 min), 72 h (±30 min), and 168 h (±30 min) after drug administration. Whole blood and tissues (brain, quadriceps femoris muscle, ovary, testis, bladder, jejunum, stomach, pancreas, inguinal lymph nodes, kidney, adrenal gland, femoral bone marrow, heart, liver, spinal cord (thoracic vertebrae), lung, and spleen) were collected.

[0092] Genomic DNA was then extracted from whole blood and various tissues using a magnetic bead method. The OD value of the nucleic acid was measured and the concentration was calculated using an enzyme-linked immunosorbent assay (ELISA) reader to determine whether the sample extraction was normal.

[0093] Mitochondria were enriched using a mitochondrial capture probe. Subsequently, mitochondrial DNA was sequenced, and the distribution information of exogenous platelets in different tissues and at different time points was obtained based on the proportion of specific SNVs, thereby assessing the safety of platelet drug delivery.

[0094] This embodiment also detected cytokine release and blood biochemical ALT / AST levels in mouse peripheral blood, and the results are as follows: Figure 5 and Figure 6 As shown, the risk of platelet drug delivery without cytokine release syndrome (CRS) has been preliminarily demonstrated.

[0095] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A test system for organ distribution levels of platelets in a subject, characterized in that, The system includes: (Z1) Platelet delivery module, the platelet delivery module being configured to administer test platelets into a test subject, wherein the test subject is a non-human mammal; (Z2) Nucleic acid extraction module, wherein the nucleic acid extraction module is configured to: collect organ tissue samples from the test subject, thereby providing mitochondrial DNA samples from the organ tissue; (Z3) Test module, the test module is configured to: provide a platelet-specific SNV sequence and test the platelet-specific SNV level in a mitochondrial DNA sample of the organ tissue; (Z4) Analysis module, which is configured to provide the distribution level of the test platelets in the organ tissue based on the test platelet-specific SNV level.

2. The system of claim 1, wherein, The organ distribution level of the test platelets includes: the absolute level of the test platelets and / or the relative level of the test platelets.

3. The system as described in claim 1, characterized in that, The nucleic acid extraction module is configured to collect organ tissue samples from the test subjects at different times, thereby providing mitochondrial DNA samples from the organ tissues at different times; The testing module is configured to: provide a platelet-specific SNV sequence and test the platelet-specific SNV level in mitochondrial DNA samples of the organ tissue at different time points; The analysis module is configured to provide the distribution of the test platelets in the organ tissue at different time points based on the test platelet-specific SNV levels in the organ tissue at different time points.

4. The system as described in claim 1, characterized in that, The nucleic acid extraction module is configured to collect different organ and tissue samples from the test subjects to provide mitochondrial DNA samples from each organ and tissue. The testing module is configured to: provide platelet-specific SNV sequences and test the platelet-specific SNV levels in mitochondrial DNA samples from various organs and tissues. The analysis module is configured to provide the distribution of the test platelets in each organ tissue based on the test platelet-specific SNV levels in each organ tissue.

5. The system of claim 1, wherein, The test platelets comprise either wild-type human platelets or drug-loaded human platelets.

6. The evaluation system of claim 5, wherein, The drug-loaded human platelets are drug-loaded platelets differentiated in vitro from drug-expressing platelet precursor cells.

7. The evaluation system of claim 1, wherein, The platelet-specific SNV sequence refers to an SNV sequence that is specifically present in human platelets but not in platelets from non-human mammals.

8. The evaluation system of claim 1, wherein, The non-human mammals mentioned are humanized non-human mammals; The platelet-specific SNV sequence refers to an SNV sequence that is specifically present in human platelets but not in the humanized non-human mammalian donor platelets.

9. The system of claim 1, wherein, It also includes a (Z5) result output module, which is configured to provide a safety evaluation result of the test platelets based on the distribution level of the test platelets in organ tissues.

10. A method for evaluating the safety of exogenous platelets in a non-human mammal for non-diagnostic, non-therapeutic purposes, characterized in that, Includes the following steps: (S1) The test platelets were administered to non-human mammals; (S2) Collect organ and tissue samples from non-human mammals to provide mitochondrial DNA samples from said organ and tissue; (S3) Provide a platelet-specific SNV sequence and test the platelet-specific SNV level in a mitochondrial DNA sample of the organ tissue; (S4) Provide the distribution level of the test platelets in the organ tissue based on the test platelet-specific SNV level; (S5) Provide the safety assessment results of the test platelets based on their distribution level in the organ tissue.

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