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CAR-TOr TCR-T is discouraged? Repeated infusion of specific CAR or TCR mRNA- nanoparticles -T cells can alleviate the disease.

Engineering chimeric antigen receptor (CAR) or T cell receptor (TCR) is helpful to create disease-specific T cells for targeted therapy, but the cost and rigor of manufacturing engineering T cells in vitro may be prohibitive, so programming T cells in vivo may be a feasible alternative. An injectable nanocarrier is reported here, which delivers in vitro transcribed (IVT) CAR or TCR mRNA for instantaneous reprogramming of T cells to recognize disease-related antigens. In mouse models of human leukemia, prostate cancer and hepatitis B-induced hepatocellular carcinoma, repeated infusion of these polymer nanocarriers can induce enough host T cells to express tumor-specific CAR or virus-specific TCR, thus leading to disease regression, and the level is similar to that of in vitro engineered lymphocytes. Considering that they are easy to manufacture, distribute and manage, these nanocarriers and related platforms may become treatments for many diseases.

Adoptive T cell therapy is an effective method to treat cancer or infectious pathogens by genetic modification of T cells obtained from patients or donors, which has been supported by a large number of clinical trials and showed impressive results. However, the complexity and high cost of making customized T cell products for each patient, rather than preparing drugs in batches in a standardized form, make it difficult to compete with first-line treatment schemes such as small molecule drugs or monoclonal antibodies. At present, most CAR-T and TCR-T cells are manufactured through complicated processes, including: (i) white blood cell separation, and T cells are extracted from patients who are connected to the separator through two venous catheters for several hours. This is uncomfortable for patients, will generate a lot of money costs, and may eventually limit the large-scale adoption of autologous T cells; (ii) activation and transduction of T cells; (iii) expanding the transduced T cells in a tissue culture medium supplemented with cytokines for about 2 weeks; (iv) T cells are washed and concentrated before administration. For T cell products produced in central facilities and transported to remote treatment centers, cells must be frozen; (v) Every batch of CAR-T products needs to be tested for quality control and release. The whole process must be carried out under GMP-compliant environmental control conditions, and the maintenance and operation costs are very high. Because every CAR-T product is made of the starting material (T cells) of the patient to be treated, there is no economies of scale.

In vitro transcription (IVT)mRNA has become a subversive new drug, which can be used to directly encode protein related to treatment in vivo. The synthesized mRNA molecules can be designed and operated quickly, and mass-produced relatively economically and efficiently. In the past few decades, scientists have learned how to optimize mRNA from pharmacology and immunology, so that it can be used in clinical applications more like drugs.

Here, we explore using mRNA as an injectable drug to reprogram circulating T lymphocytes to express disease-specific receptors instantaneously, thus bypassing the need to extract and culture lymphocytes from patients (Figure 1). In order to protect the therapeutic load and accurately target it to T cells, biodegradable polymer nanocarriers were developed. First, it was proved in vitro that the application of a single nanoparticle can use CD19-specific 1928z CAR(FDA approved for the treatment of B-cell lymphoma) or HBcore18-27 TCR for HBV core antigen (currently in the phase I study for the treatment of patients with HBV-related hepatocellular carcinoma; NCT03634683) routinely transfected more than 70% of cultured T cells. T cells transfected with nanoparticles instantaneously express these CAR transgenes or TCR transgenes on their surfaces for an average of 7 days. In-situ xenotransplantation mouse models of lymphoma, prostate cancer and HBV-induced hepatocellular carcinoma have proved that mRNA particles encoding CAR or TCR can genetically reprogram circulating T cells when given regularly, so as to induce similar therapeutic effects to traditional adoptive transfer T cells transduced by virus in vitro.

Fig. 1 is a schematic diagram of how to reprogram T cells in situ with IVT mRNA carried by polymer nanoparticles to express disease-specific CARs or TCRs. The surface of these particles is covered with ligands targeting cytotoxic T cells, so once they are injected into the circulation of patients, the transgene they carry can be transferred to lymphocytes, and the cells can be instantly programmed to express their disease-specific CAR or TCR on their surfaces.

At present, the insertion of CAR or TCR into lymphocytes by gene transfer is carried out in a special manufacturing workshop outside the patient’s body, but the process of transferring cells to and from the clean room and the gene transfer procedure itself are labor-intensive, costly and time-consuming. If engineered T-cell therapy reaches its promise of expanding to different populations of various cancer types, the economic and manufacturing challenges may increase.

Here, it is proved that mRNA nano-drugs can achieve effective cell therapy without side effects through the convenience of ready-made drugs. Just like traditional medicine, with this new treatment, patients can easily re-administer medicine as long as they need it.

CAROr TCR gene mRNA nanocarrier transfecting T cells.

In order to deliver IVT mRNA encoding disease-specific receptor gene to human lymphocytes, a biodegradable poly-β-amino ester (PBAE) polymer preparation was used as the carrier matrix (Figure 2a). The PBAE-447 polymer used in the research to concentrate mRNA into nanoparticles was originally developed by Jordan Green Laboratory of Johns Hopkins University. In the past ten years, the key features of PBAE have been widely described. PBAEs escape endosomes by protonation at low pH value, and osmotic pressure accumulates due to buffering, which leads to endosomes destruction. Qualcomm combinatorial library screening of PBAEs for nucleic acid delivery showed that the existence of tertiary amine improved the buffering capacity at low pH and promoted endosome escape. The ester bond in the main chain structure of PBAEs is hydrolyzed in aqueous solution, which makes the toxicity of PBAEs lower than that of other non-degradable cationic polymers, such as PEI, which is widely studied as a nucleic acid delivery carrier. Cationic PBAE self-assembled with anionic nucleic acid into nano-complex through electrostatic interaction (Figure 2b). By coupling anti-CD8 antibody to polyglutamic acid (PGA), the particles were electrostatically adsorbed to form a conjugate, which made the particles have cell targeting. The mRNA nanocarriers thus obtained can be freeze-dried for long-term storage. Before use, the granules were hydrated within a few seconds after adding sterile water to restore their original concentration. Particle tracking analysis (NanoSight NS300, Malven Panalytical) to characterize particles produced in ten independent batches (fig. 2c). The results show that the average particle size of PbAE/PGA-anti-CD8 nanoparticles is 106.9±7.2nm. The zeta potential is 4 2, and the encapsulation efficiency of mRNA detected by Qubit RNA HS kit is 90.9 6.2%. When the used nanoparticle formula PBAE: mRNA is 60: 1,

Fig. 2 Design and manufacture of lymphocyte programming nanoparticles. A: Schematic diagram of T cell targeting IVT mRNA nanocarriers used in the experiment. In order to create a reagent that can modify primary T lymphocytes simply by contact (which is difficult to achieve by non-viral infection methods), polymer nanoparticles composed of four functional components are designed: (i) surface-anchored targeting ligands, which selectively bind nanoparticles to T cells and initiate rapid receptor-induced endocytosis to internalize them. Anti-CD8 antibody was used in the experiment. (ii) negatively charged coating, which shields the nanoparticles by reducing the surface charge of the nanoparticles, so as to minimize off-target binding. Because it has been widely used in drug delivery platform, PGA was chosen to realize this. (iii) a carrier matrix, which aggregates and prevents nucleic acids from being degraded by enzymes when they are in the endocytosis, but releases them once the particles are transported into the cytoplasm, thus enabling the translation of the encoded protein. Therefore, a biodegradable poly (β-amino ester) (PBAE) polymer formula is used, and its half-life in water is between 1 and 7 hours. (iv) Nucleic acid (IVT mRNA) wrapped in a vector and producing transient expression of disease-specific CAR or TCR. B: Describe how to make nanoparticles. C: particle size distribution, measured by NanoSight NS300 instrument.

Firstly, it is determined whether adding targeted IVT mRNA nanocarriers in human lymphocyte culture can stably transfect cells. In order to test this technique in clinical related systems, the nanoparticles were loaded with IVT mRNA encoding leukemia-specific 1928z CAR (Figure 3a-e). CD19 targeting receptor is the most researched CAR-T cell product. In the second example, IVT mRNA encoding high affinity HBV-specific TCR is provided here (Figure 3f-j). T cell therapy for chronic hepatitis B is a new method to restore antiviral immunity and cure infection. HBcore18-27 TCR against HBV core antigen was isolated from a HLA-A02.01 donor who had solved HBV infection. For 1928z CAR and HBcore18-27 TCR constructs, real-time quantitative PCR and flow cytometry were used to measure their expression levels in human T cells after transfection with single nanoparticles. It was found that the transgenic expression reached its peak at 24 hours after nanoparticles were exposed, and then gradually decreased (Figure 3a,f). It is worth noting that only nanoparticles functionalized with T-cell-specific antibodies (anti-CD8 or anti-CD3) can effectively deliver transgenes, while the gene expression produced by isotype control functionalized nanoparticles is close to the background level (Figure S1). This translates into a high level of expression on the surface of CAR or TCR, reaching a maximum on the second day.(75% 11% T cells express 1928z CAR, Figure 3b, C; On average, 89 4% of T cells expressed HBcore18-27 TCR (fig. 3g,h). As expected, the receptor expression was transient. After 8 days of culture, the receptor expression of CAR and TCR decreased to 28 6% and 26 9% respectively. Next, the virus method was used to compare the functions (killing and cytokine production) of T cells transfected with nanoparticles and T cells designed with these receptors. In order to prove the specificity of tumor antigen, T cells transduced with CAR gene (P28z, targeting prostate specific membrane antigen) or TCR gene (MSLN-TCR, mesothelin specific) unrelated to tumor were used as control group. Using real-time IncuCyte? living cell analysis, it is impossible to measure the significant difference in the ability of T cells transduced by IVT mRNA to selectively lyse antigen-positive target cells (Raji lymphoma cells in 1928z CAR and HepG2 hepatoma cells in HBcAg stably transduced HBcAg for HBcore18-27 TCR) (Figure 3d,i). In addition, similar levels of effector cytokines secreted by T cells were also detected in the nanoparticle transfection group and the virus transduction group (Figure 3e,j).

Fig. 3 IVT mRNA nanocarriers effectively transfect human T cells by CAR or TCR transgene. Isolated human CD8+T cells were stimulated by beads coated with antibodies against TCR/CD3 and CD28 receptor. After 24 hours, beads were removed, and CD8 targeted nanoparticles (NPs) containing mRNA encoding leukemia-specific 1928zCAR(a-e) or HBcore18-27 TCR(f-j) were mixed into the cell suspension at a concentration of 3 μ 3μg mRNA/10^6 cells. A: QCPR was used to detect the relative 1928z CAR mRNA expression of T cells exposed to 1928z CAR NPs over time. B: T cells were detected by flow cytometry at different time points after incubation with NPs carrying 1928z encoded mRNA. C: Summary chart of gene transfer efficiency in vitro. D: To compare the killing activity of T cells of nanoparticles and retrovirus transfection group on Raji lymphoma cells in vitro. T cells and Raji tumor cells were co-cultured in a ratio of 5:1. The IncuCyte living cell analysis system was used to quantify the immune cell killing of Raji NucLight red blood cells by T cells transfected with 1928z-CAR or control (P28z-CAR) over time. E: The secretion of IL-2(24h), TNF-α and IFN-γ(48h) was detected by e：ELISA.The HBcore18-27 TCR mRNA of f-j is the same.

Fig. S1 CD3-targeted mRNA nanoparticles selectively transfect human T cells.

Recognition of leukemia by reprogramming host T cells with nanoparticles

Next, it is studied whether IVT mRNA NPs targeting lymphocytes can reprogram the circulating T cells, and the number is large enough to achieve tumor regression with similar effects as traditional methods. As the first in vivo test system, 1× 10 6 CD19+Raji cells expressing firefly luciferase were inoculated into immunocompromised NOD. CG-PRKDCSCID IL 2RGT M1WJL/SZJ (NSG) mice to establish leukemia model. Five days later, mice were recombined into 10× 10 6 CD3+human T cells, and nanoparticles loaded with the gene encoding 1928z CAR (50μg/ dose) were infused six times a week to produce leukemia-specific or control particles loaded with the mRNA encoding GFP (Figure 4a). According to the kinetics of in vitro measurement of CAR surface expression with IVT mRNA nanoparticles, a weekly administration regimen of nanoparticles was selected, which showed the expression of related receptors for 8 days (Figure 3b,c). In order to compare the efficacy of nanoparticle infusion with traditional adoptive T cell therapy, 5×10^6 T cells were transduced into another group of mice by lentivirus encoding 1928z CAR in vitro. This amount is equivalent to the higher dose of CAR-T cells used in clinical research at present. In clinical research, patients were treated with CAR-T cells weighing as high as 1.2×10^7 CAR-T kilogram. In another dosage regimen,The adoptive transfer of lentivirus-transduced CAR-T cells was combined with systemic injection of nanoparticles carrying control GFP mRNA to determine whether nanoparticle-mediated transfection damaged the anti-tumor function of T cells. The mice in the control group were either not treated or infused with untransformed human effector T cells. Tumor growth was continuously quantified by bioluminescence imaging and the difference of survival rate was monitored. It was found that the adoptive transfer of 1928z CAR-T cells engineered in vitro significantly improved the survival rate. Of the 10 mice, 6 tumors were eradicated, and the tumors of the other mice subsided, and the average 32-day survival rate increased (Figure 4b,c). This therapeutic benefit obtained by traditional adoptive T cell therapy is similar to the treatment of IVT mRNA nanoparticles which programmed the same CAR into lymphocytes in vivo, which achieved the eradication of 7 tumors in 10 mice and the average survival time of recurrent animals was 37 days (Figure 4c). Flow cytometry analysis of peripheral blood 2 days after the first administration showed that nanoparticles carrying 1928z effectively reprogrammed circulating T cells to identify leukemia cells (average 10% 4.3% CAR+CD8+, Figure 4d,e). As expected, the transient expression of these CARs lasted for one week (0.8 0.4% of CAR+CD8+T cells on the 7th day). It is worth noting that repeated doses of nanoparticles are as effective as the first injection, with an average of 10.7 3.6% gene transfer to host T cells (Figure 4e). This shows that,Although IVT mRNA is transient, it can be used as a platform for continuous in situ CAR expression in host lymphocytes.

Fig. 4 CAR lymphocytes programmed with nanoparticles can lead to leukemia regression, and its curative effect is similar to adoptive t cell therapy. A: Timeline and administration scheme of nanoparticles (NP). B: sequential biological imaging of Raji lymphoma cells expressing firefly luciferase injected into NSG mice. C: The survival rate of animals after treatment is depicted as Kaplan-Meier curve. D: Flow cytometry of peripheral T cells before and after injection of nanoparticles carrying IVT mRNA encoding 1928z CAR. E: shows the percentage of CD8+T cells transfected by CAR after repeated infusion of 1928z CAR NPs. Each line represents an animal. The average transfection rate (SD) at each time point is displayed at the top.

Therapeutic response of hosts with normal immune function

In order to study how exclusive targeting limits the interaction of nanoparticles to circulating T cells and how it affects their fate, Ai14 reporter mice with complete immune activity were used. In this transgenic model, all cells contain a termination box flanking loxP, which prevents the transcription of tdTomato protein driven by CAG promoter. Only the cells that successfully transduced the mRNA encoding Cre recombinase (Cre) would cut off the termination cassette flanking loxP, resulting in permanent tdTomato transcription and then strongly amplified tdTomato expression. Firstly, the fluorescence of the whole organ in Ai14 mice was measured after injecting CD3-targeted (or isotype-controlled functionalized) nanoparticles carrying Cre mRNA. The gene expression mediated by non-targeted particles is the highest in the liver, while the gene transfer induced by lymphocyte-targeted nanocarriers is mainly in the spleen, lymph nodes and thymus (Figure 5a,b). Detailed flow cytometry analysis of spleen (Figure 5c) showed that CD3-targeted nanoparticles preferentially transfected T cells (8.1 1.9%) without affecting the activity. Other CD45+ subtypes, such as macrophages (3.2 1.5%), B cells (1.1 0.9%), neutrophils (0.3 0.2%) and DC cells (1.9 0.8%), have lower dtTomato signal levels.

Fig. 5 Effective T cell targeting in mice with normal immune function. B6. CG-GT (Rosa) 26Sortm14 (CAG-TDtomato) Hze/J (AI Reporter) Mice were injected with 3 doses of nanoparticles containing 15μg mRNA encoding nuclear localization signal (NLS)-Cre intravenously every day. Nanoparticles were targeted to mouse T cells using full-length anti-CD3 MuIgG2a or IgG2a isotype control. Both antibodies were designed as LALAPG variants to eliminate Fc receptor binding and complement activation. 48h after the last injection, the organs were collected and the dtTomato fluorescence of the whole organs was measured by fluorescence IVIS imaging. Single cell suspensions of spleen and blood were labeled with antibodies against various immune cell subtypes and analyzed by flow cytometry. A: The representative dtTomato expression in organs under fluorescence IVIS imaging. B: Quantify the fluorescence signal of each organ. C: It shows the average SD percentage of immune CD45+dtTomato+ cell types in spleen. Macrophages (CD45+, CD11b+, MHCII+, CD11c?, Ly6C?/Low, Ly6G?), B cells (CD45+, B220+), T cells [CD4+T cells (CD45+, TCRβ+, CD4+, CD8-) and CD8+T cells (CD45) were detected.(CD45+, CD11b+, MHCII+, CD11c, Ly6G+) and DC cells (CD45+, CD11c+, CD11b, MHCII+).

Based on these distribution studies, it is then tested whether the measured number of mRNA nanoparticles redirected T cells is enough to reduce established cancers in fully immune hosts. To this end, Eμ-ALL01 leukemia cells expressing luciferase were injected into albino C57BL/6 mice (a model of B-cell acute lymphoblastic leukemia was established in mice with normal immune function), and bioluminescence imaging was used to quantify the difference of tumor progression between treatment groups (Figure 6a). Mice either received CD3-targeted nanoparticles to deliver mRNA encoding 1928z CAR of the whole mouse, or received GFP control (see Figure S2 for methods). The third group did not receive treatment. It was found that only infusion of nanocarriers encoding 1928z CAR could effectively control the progress of leukemia (Figure 6b,c). Compared with GFP control group, the tumor load was reduced by an average of 26 times after three weeks of treatment.

Fig. 6 Anti-leukemia response of mice with normal immune function. A: Timeline and administration plan. B: signal intensity diagram of e-all 01 luciferase after nanoparticle injection. Each line represents an animal, and each point reflects the photon count of the whole animal. C: Sequential biological imaging of Eμ-ALL01 leukemia cells expressing firefly luciferase by systemic injection in albino C57BL/6 mice. Five representative mice from each group (n=10) are shown.

Fig. S2 anti-CD3 mouse IgG2a LALA-PG and non-conjugator control.

Therapeutic response of solid tumor

In order to formalize that this technology is not only related to the treatment of hematological malignant tumors, but also related to the treatment of solid tumors, the ability of nanoparticles aimed at introducing prostate cancer-specific CAR gene into circulating host T cells to induce prostate cancer regression in mice was studied. Unlike leukemia cells, leukemia cells express high levels of CD19 antigen and are easily accessed by circulating lymphocytes, while solid malignant tumors are heterogeneous and protected. This means that some tumor cells will escape the recognition of targeted CAR and will be surrounded by immune suppression defense system, which may lead to T cell dysfunction. In fact, the whole genome/transcription analysis of 140 cases of prostate cancer metastasis was used to determine that prostate cancer lesions showed heterogeneous expression of three key cell surface proteins [prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA) and receptor tyrosine kinase-like orphan receptor 1(ROR1)] in patients (Figure 7a). In order to summarize human diseases, LNCaP C42 prostate cancer cells were transplanted into the prostate dorsal lobe of NSG mice in situ (Figure 7c), and the cells showed heterogeneous expression of these key cell surface proteins (Figure 7b). In order to continuously monitor the tumor load by bioluminescence imaging, firefly luciferase (Fluc) was used to mark the tumor cells. After orthotopic transplantation, all the mice developed pathological changes repeatedly within three weeks (Figure 7c, right), and were reconstructed with human 10× 10 6 CD3+human T cells.And randomly assigned to different treatment groups or control groups (fig. 7d). Firstly, the therapeutic effect of systemic injection of 10 6 CAR+T cells transduced in vitro against tumor antigen ROR1 on tumor-bearing mice was tested. It was found that although anti-ROR1 CAR-T cells did not achieve tumor clearance, the survival rate of treated mice more than doubled (69 days compared with 32 days in untreated control group; Fig. 7d). In order to determine whether "ready-made" nano-preparations can achieve similar therapeutic effects, ROR1 CAR transgenic nanoparticles (50μg mRNA/ dose; Fig. 7e). Compared with the untreated control group, particle-induced CAR programming prolongs the survival time by an average of 40 days, which is similar to the survival benefit obtained by traditional adoptive T cell therapy (Figure 7d,f). Proper localization and persistence of T cells is a prerequisite for anti-solid tumor activity, so the frequency of ROR1 CAR-T cells infiltrating into prostate cancer over time was evaluated. The flow cytometry analysis of LNCaP C42 prostate cancer resected on the 4th, 7th and 11th days after T cell metastasis showed that T cells were infused into the tumor site by intravenous infusion (average 892 295 car+T cells /mg tumor tissue), but they could not grow (only 1.04 times the overall expansion between the 4th and 11th days; Fig. 7g,h). In addition, in-situ programmed CAR-T cells effectively penetrated into the tumor (average 648 240 CAR+T cells /mg tumor tissue)., and maintain a high level of CAR transgene (average 91 7 CAR+T cells /mg tumor tissue, Figure 7h) before downregulating the receptor. On the same day, the tumor lesions were infiltrated by freshly reprogrammed peripheral T cells after intravenous injection of ROR1 CAR-encoded mRNA nanoparticles (on the 11th day, the average was 1066 225 car+T cells /mg tumor; Fig. 7h), which summarizes the oscillation dynamics of T cell reprogramming induced by mRNA nanoparticles that have been observed in leukemia research (fig. 4e).

In order to determine the reason why adoptive transferred T cells and injected mRNA nanocarriers failed to completely eliminate the disease, the antigenic phenotype of recurrent prostate cancer was identified by flow cytometry. One of the most common escape strategies in cancer is to reduce the expression of target antigen, because CARs produces selective pressure. In preclinical and clinical studies, this phenomenon is reported as the cause of failure when adoptive T cells targeting only a single antigen are used to treat heterogeneous tumors (such as metastatic prostate cancer). It was found that compared with untreated LNCaP C42 prostate cancer expressing ROR1 tumor antigen at different levels, two treatment groups (adoptive transferred T cells or nanoparticle programmed T cells) finally produced ROR1 low/negative immune escape variants (Figure 7i).

Fig. 7 IVT-mRNA nanocarriers encoding prostate cancer-specific CAR can improve the survival rate of mice with existing diseases. C: Three weeks after transplantation, LNCaP C42 prostate cancer was imaged by bioluminescence in vivo. The picture on the right is a representative photo of prostate dorsal lobe tumor (white arrow). D: Sequential biological imaging of LNCaP C42 prostate cancer cells expressing firefly luciferase transplanted into the prostate of NGS mice in situ. E: Timeline and nanoparticle administration scheme. F: the survival rate of animals after treatment, depicted as Kaplan-Meier curve. G: On the 11th day after treatment, the recovered cells of patients with prostate cancer were detected by multicolor flow cytometry. ROR1 CAR+T cells with adoptive transfer or in situ programming were identified by CD45 and the positive marker of c-myc labeled in the receptor. H: The absolute number of ROR1-CAR+T cells in the tumors isolated on the 4th, 7th and 11th day after the start of treatment. The total number of living cells (trypan blue negative) multiplied by the percentage of ROR1-CAR and CD45 positive. I: Flow cytometry was used to quantitatively detect the expression of ROR1 antigen on LNCaP C42 prostate tumor cells treated with CAR-T cells or ROR1 4-1BBz CAR NP.

HBVIn situ programming of specific T cells

The gene transfer of CARs encoding IVT mRNA can only target T cells to antigens located on the cell surface, so many tumor antigens or virus antigens in cells cannot reach these receptors. It has been proved in vitro that lymphocyte-targeted IVT mRNA nanoparticles can reprogram T cells with engineered TCR, which recognize intracellular HBV core antigen (HBcAg) in HLA background (Figure 3f–J). In view of the fact that more than 300 million people in the world are chronically infected with HBV, and a large number of them develop cirrhosis and liver cancer, it is obviously not feasible to tailor T cell products for each patient. As the first step to treat this disease with IVT mRNA technology, a mouse model of hepatocellular carcinoma (HCC) induced by HBV was established. After laparotomy, 1 million HepG2 cells stably transduced with HBcAg and luciferase were injected into the liver. All mice developed multifocal lesions repeatedly within 7 days (fig. 8a), when they were reconstructed with unstimulated 10× 10 6 CD3+human HLA-A*02:01 T cells, and received twice weekly infusion of nanoparticles loaded with mRNA encoding HBcore18-27 TCR (50μ g/dose) to produce HCC-specific or control particles loaded with mRNA encoding GFP. The mice in the third group were treated with a single dose of 10×10^6 CD3 T cells (HLA-A*02:01).The cells were transduced in vitro with retrovirus vector encoding HBcore18-27 TCR, and the control mice did not receive any treatment. Four days after the second nanoparticle administration (18th day), the liver was isolated to directly quantify the tumor load by bioluminescence imaging, and the single cell suspension was labeled with HBV C18-27 MHC I Pentamer to quantify the percentage of TCR-T cells expressing HBCore 18-27. It was found that enough HBcore antigen-specific T cells were programmed by nanoparticle injection to induce disease regression, and similar therapeutic effects could be achieved compared with engineered lymphocytes in vitro (compared with untreated controls, the photon count was reduced by 13 times and 18.9 times, respectively, Figure 8b,c). Flow cytometry of dissecting liver confirmed that the density of HBcore18-27 TCR-T cells in animals treated with engineered T cells in vitro was equal to that of in-situ programmed nanoparticles (Figure 8d,e).

Fig. 8 In-situ programming of HBV-specific T cells using nanoparticles loaded with TCR transgenes. A: A xenograft tumor model of HCC mice induced by HBV was established. HepG2 cells stably transduced with HBcAg and luciferase were injected into the liver of NSG mice reconstructed with human T cells by surgery. Three weeks after implantation, HepG2 tumor was observed by bioluminescence imaging in vivo, and it was assigned to the nanoparticle group (50μg of mRNA encoding HBcore18-17 TCR six times a week) or the T cell treatment group (5×10^6 T cells were transduced with lentivirus encoding HBcore18-17 TCR mRNA vitro). B, c: quantification of bioluminescent liver signal after 6 weeks of treatment. D: Multicolor flow cytometry for recovering cells from the liver 18 days after the start of treatment. The adoptive transfer or in situ programming of HBcore18-27 TCR+ T cells were identified by the positive markers of CD45, CD8 and MHC Pentamer. The absolute number is shown in E. The total cell count of living cells (trypan blue negative) is multiplied by the percentage of HBcore18-27 TCR++,CD8+ and CD45+.

In a word, the results show that repeated infusion of T cell targeting polymer nanocarriers can deliver tumor-specific CAR or virus-specific TCR transgenes to a sufficient number of host T cells, and induce disease regression at a level similar to that of in vitro engineering lymphocyte mass injection.

TCell-programmed nanoparticles are biocompatible

Systematic administration of nano-drugs may cause infusion reaction in patients, which usually delays or stops clinical transformation. These reactions can be manifested as fever, chills, stiffness, rash, chest pain or dyspnea, and in rare cases, they can be fatal. Identifying the risk of infusion reaction early in the process of drug development is helpful to alleviate potential safety concerns after the product enters clinical trials, save time and money for developers, and avoid potential dangerous complications for patients.

The detection cascade scheme developed by NCL is used here, which can indicate the infusion reaction. Specifically, the influence of nanoparticles on complement activation (NCL method ITA-5.2), its hemolysis characteristics (ITA-1) and its influence on T cell oxidative stress (ITA-32) were analyzed. In order to study these effects of nanoparticle concentration in clinical correlation, the theoretical plasma concentration (TPC) was first calculated, which is an effective mouse dose (in the experiment: 50μg mRNA/ dose), and scaled to the equivalent human dose (=2.03μg mRNA/mL blood; Fig. 9a). In order to evaluate the effect of T-cell-targeted mRNA nanoparticles on red blood cells, the hemolysis test was carried out by measuring the release of hemoglobin by spectrophotometry after exposure to different concentrations of particles. The performance of hemolysis test was tested by negative (PBS) and positive (Triton-X) controls. It was found that the hemolysis rate of TPC particles was lower than 2% (the average was 1.21 0.26%, while that of PBS control was 0.7 0.11%; Fig. 9b), which is defined as non-hemolytic. Nano-particles also did not induce the activation of complement iC3b or Bb, while C4d was slightly higher than twice the determination threshold at TPC concentration (average 2.3 0.13%, compared with 1 0.003% in PBS control; Fig. 9c). Finally, mitochondrial oxidative stress was measured as the key determinant of nanoparticle-induced damage, because reactive oxygen species (ROS)Over-production will cause damage to organelles and DNA, and eventually lead to cell death. In addition, another consequence of ROS overproduction is the activation of cell signaling pathways that stimulate the expression of pro-inflammatory and fibrotic cytokines. Compared with PBS control, T-cell-programmed nanoparticles only induced a very mild increase in oxidative stress (average 3.6 0.2 times) (Figure 9d).

Fig. 9 In vitro analysis of possible infusion reactions. A: calculation of theoretical plasma concentration. Hemolytic activity of b: t cells targeting mRNA nanoparticles. C: quantitative determination of complement activation by enzyme immunoassay. The 2-fold change relative to the negative PBS control is defined as the determination threshold (dotted line). Study on oxidative stress response of lymphocyte mitochondria after d: NP transfection.

Under the guidance of in vitro evaluation of the possible infusion reaction caused by T cell programming mRNA nanoparticles, a comprehensive toxicity assessment was conducted for rodents. Rats are the first rodent species to predict the toxicity of nucleic acid-based molecular therapy to human health, because their metabolic physiology (especially kidney and liver functions) is closer to humans than mice. SD rats (6-8 weeks old) were injected with a dose of nanoparticles carrying 100μg mRNA, which is equivalent to the rat dose of 50μg mRNA in mice, based on the standardization of body surface dose. These experiments were carried out using 1928z CAR nanoparticles. 1928z CAR recognizes human CD19, but does not cross-react with rat CD19 to ensure that the change of measured parameters can be attributed to nanoparticles rather than their reprogramming activities. The control group was injected with 25mM sodium acetate buffer (carrier control group) or not. After 48 hours, the animals were killed, blood samples were taken to determine clinical biochemical indexes, and gross anatomy was carried out. The following tissues were evaluated by pathologists certified by the Committee: lung, liver, heart, brain, kidney, spleen, bone marrow and duodenum. There were no histological changes attributable to nanoparticle drug therapy (fig. 10a). The few lesions noted are mild to mild, which are considered accidental and have nothing to do with the study. Two of the five rats in all groups had the least inflammatory infiltration in the liver. These tiny infiltrations are considered as background lesions and have nothing to do with treatment. Similarly, all groups have individuals with mild to mild chronic inflammation of renal pelvis.The chronic nature of the lesion is inconsistent with the acute treatment effect, so it is considered accidental. Compared with the control group, the whole blood platelet count of the animals in the nanoparticle treatment group was slightly lower (407 115 k/μ l and 290.4 56.3 k/μ l respectively; Fig. 10b). Compared with the control group, the animals in the nanoparticle group also had slight hypoglycemia (average 45.4 26 mg/dl and 78.3 69.8 mg/dl, respectively; Fig. 10c). All other serum chemical indexes (including liver function and renal function) of the rats treated with nanoparticles were similar to those of the control group, indicating that no systemic toxicity occurred. Serum IL-6 level moderately increased to an average of 16.5 pg/ml 5.9 pg/ml (Figure 10d), which can be considered as safe according to previous reports.

Fig. 10 The infusion of nanocarriers has nothing to do with acute systemic toxicity. Female SD rats were intravenously injected with CD8-targeted IVT mRNA encoding 1928z CAR. After 48 hours, a pathologist certified by the Committee made a comprehensive histopathological evaluation and serum chemical analysis in a blind way. A: representative H&E stained sections of various organs isolated from control or nanoparticle-treated animals. B: blood cell count. C: serum chemistry. D: Detection of serum TNF-α, IL-1β and IL-6 cytokines.

summary

Although T cells modified by CAR and TCR have transformed a few hematologic cancer, it is obvious that their current clinical application only represents a small part of the possibility that this technology may provide. Theoretically, therapeutic T cells can treat malignant tumors and chronic infections with targeted antigens, as proved by a large number of preclinical reports. However, at present, the method of producing disease-specific T cells in vitro is very complicated and cannot support the treatment of a large number of patients, because a new lymphocyte cluster must be produced for each patient. In order to make it easier for patients to obtain T cell products, the field has turned to allogeneic technology to provide a larger scale and lower cost. Several clinical T cell companies have begun to test CAR-T cells, which are "ready-made" products made by healthy unrelated donors rather than patients. Although this method can treat cancer patients who can’t make autologous T cells because of their low lymphocyte count or poor T cell quality, it requires several extra cell engineering steps to prevent donor cells from attacking the host, which in turn prevents patients’ own T cells from rejecting the infusion products. This is usually done by multiple gene editing to remove natural TCR and HLA molecules from T cell products. However, these extra operations increase the complexity, time and cost of the manufacturing process, while reducing the cell yield and vitality. In order to prevent rejection, patients who receive universal CAR-T cells first severely suppress immunity through lymph depletion chemotherapy.This takes time and exposes patients to additional toxicity. Therefore, in vitro engineering of allogeneic cell products is unlikely to significantly increase the number of patients receiving T cell therapy, especially those patients with infectious diseases who need rapid intervention to keep the endogenous immune system intact.

Previously, an injectable DNA-based nano-reagent was described, which can program circulating T cells with leukemia-specific CAR transgene. In order to overcome the inherent low gene transfer of plasmid DNA, plasmid DNA must enter the nucleus to be transcribed into mRNA. The transposon/transposase system encoding CAR was loaded on nanoparticles, and the system was randomly inserted into the genome of the target cell. Although this study provides proof of concept that it is possible to program CAR-T cells in situ with injectable nano-reagents, it will be challenging to transform this DNA nano-drug into clinic for the following reasons: ① Unpredictable genotoxicity and expression kinetics. These nanoparticles stably integrate their therapeutic CAR transgenes into target cells, resulting in permanent genomic changes and unpredictable genotoxicity of various cell types. In addition, once nanoparticles are injected into patients, doctors cannot control the kinetics of CAR expression in vivo; ② The copy number of CAR gene related to each nanoparticle is low. The number of CAR genes that can be loaded into these DNA nanoparticles is limited by the size of the vector skeleton and promoter sequence, and the requirement of stable integration of transposase expression vectors. This greatly limits the efficiency of in-situ gene transfer, especially when trying to deliver large transgenes encoding TCRα and β chains; ③ Abundant tumor antigens are needed to expand the small population of in situ transfected CAR-T cells to the number related to treatment. This amplification period takes time,This is a disadvantage for patients with rapidly progressive diseases or definite solid tumors.

Here, we explore the use of IVT mRNA to quickly and specifically program antigen recognition ability into circulating T cells as a strategy to treat cancer and infectious diseases. Compared with DNA nanocarriers, the synthesized mRNA molecules can be directly translated into therapeutic target proteins without entering the nucleus, thus ensuring high transfection rate and rapid therapeutic effect. Their tailoring size (the actual CAR coding or TCR coding sequence in this study +276 bases of 5′ UTR and polyA region) leads to a high copy number per nanoparticle. In addition, because the delivered mRNA plays its role in cytoplasm, uncontrolled insertion mutation and promoter dependence are avoided. It is proved here that simply injecting well-designed mRNA nanocarriers can selectively introduce CAR gene or TCR gene into host T cells, and program them to make the number sufficient to cause disease regression, which is similar to the adoption method. Several ongoing clinical trials are testing the repeated infusion of in vitro engineered mRNA CAR-T cells (NCT01355965, NCT01897415, NCT02277522 and NCT02624258) in cancer patients, and the first data shows that the instantaneous CAR expression after cell infusion is enough to trigger an anti-tumor response.

Three important reasons why IVT mRNA has rapidly become a new adoptive T cell therapy tool are its inherent safety, efficient translation of recombinant protein and its ability to control the pharmacokinetics of treatment, which is similar to traditional small molecule drugs. In fact, the kinetics of T cells expressing CAR measured in mice after multiple doses is similar to the profile of drugs with a definite half-life (Figure 4e). This is in sharp contrast to the rather unpredictable T cell dynamics after the adoptive transfer of engineering T cells. In this case, the cell concentration in the blood rises to the highest, and then decreases within a variable period of several days to several months. Although in-situ programming has obtained the ability to control the pharmacokinetic characteristics of the therapy and reprogram the fresh population of host lymphocytes regularly, thus potentially bypassing some major obstacles in the wide application of T cell therapy (such as T cell failure and dysfunction, and long-term toxicity), this technology still has some limitations: (1) It depends on the existence of a sufficient number of functional T cells in patients. Lymphocytosis is common in patients with advanced cancer who receive a large number of chemotherapy drugs. Therefore, the patient’s blood is likely to need to be pre-screened before the clinical trial of ready-made nano-reagents. (2) The efficacy of drugs can be passivated by immune response. Because T-cell programmed nanoparticles are regularly injected into patients with intact immunity, anti-drug antibodies may be formed. For the clinical transformation of this technology, it is important to select a fully humanized CD8 targeting ligand.To provide CAR/TCR structure with low immune risk, and synthesize mRNA with pseudouridine (or N1- methyl-pseudouridine described recently) and 5- methylcytosine to reduce innate immune response.

In order to redirect circulating T cells to resident tumor cells in situ, several biotechnological drug manufacturers have developed bispecific antibodies, including BiTEs, DART and diabodies. Among them, blinatumomab (a CD19-specific BiTE) has shown encouraging results in the clinical study of patients with hematological malignancies. However, BiTEs must be continuously infused, which will produce systemic toxicity. In addition, like traditional monoclonal antibodies, BiTE does not undergo active biological distribution or self-expansion after infusion. In contrast, the gene modification system based on nanoparticles described here can produce new tumor-specific T cells, which, as a "living drug", actively locate at the target, increase in number and continuously destroy cancer cells. People’s interest in CAR-T cell therapy is still strong, so it is time to introduce ready-made nano-reagents as a competitive technology, which can quickly reprogram T cells, identify and destroy tumors without laboratory operation.

Before conducting clinical research on humans, we will refer to the FDA’s regulations on nano-drugs and expand cooperation with NCL to confirm the safety of nano-particles in large animal species. Different from the treatment methods established in clinic (such as small molecules or antibodies), CAR-programmed nanoparticles are multi-component three-dimensional structures, which require repeatable manufacturing processes to reliably realize the expected physical and chemical characteristics, biological behavior and pharmacological characteristics. The safety and effectiveness of this nano-drug may be affected by small changes in many parameters, which need to be carefully monitored, especially in the case of targeting unexpected sites and potential toxicity. In addition, compared with traditional drugs, nano-drugs need additional development and regulatory considerations.

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Mueller, K. T. et al. Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia. Blood 130, 2317–2325 (2017).

Beatty, G. L. et al. Activity of mesothelin-specific chimeric antigen receptor T cells against pancreatic carcinoma metastases in a phase 1 trial. Gastroenterology 155, 29–32 (2018).

Foster, J. B., et al. The emerging role of in vitro-transcribed mRNA in adoptive T cell immunotherapy. Mol. Ther. 27, 747–756 (2019).

Calmes-Miller, J. FDA approves second CAR T-cell therapy. Cancer Discov. 8, 5–6 (2018).

Parayath, N.N., et al. In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo. Nat Commun 11, 6080 (2020).

Smith, T. T. et al. In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers. Nat. Nanotechnol. 12, 813–820 (2017).

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Order of Beijing Municipal People’s Government

No.13

　　The Interim Measures of Beijing Municipality on Invoice Management are hereby promulgated and shall come into force as of June 1, 1989.

Interim Measures of Beijing Municipality on Invoice Management

　　Article 1 In order to strengthen the administration of tax collection and maintain the order of socialist commodity economy, these Measures are formulated in accordance with the Provisional Regulations on the Administration of Tax Collection in People’s Republic of China (PRC) and the Interim Measures for the Administration of National Invoices, combined with the specific conditions of this Municipality.

　　Article 2 All units, individual industrial and commercial households (including private enterprises, the same below) and invoice printing units that use invoices within the administrative area of this Municipality must fully implement the Interim Measures for the Administration of National Invoices and these Measures.

　　These measures are not applicable to invoices used by foreign-invested enterprises, foreigners and overseas Chinese, Hong Kong, Macao and Taiwan personnel in this Municipality, receipts of money used by administrative institutions and internal settlement vouchers used by independent accounting units, and other non-operating special bills, which are managed according to the relevant provisions of the state and this Municipality.

　　Article 3 The Municipal Taxation Bureau and its branches are the invoice management organs of this Municipality, and are responsible for organizing and supervising the implementation of these Measures.

　　Fourth units and individual industrial and commercial households in the city to sell goods (products) to receive payment, provide labor services or engage in other business to obtain operational income, must truthfully issue invoices to the payer.

　　Commercial and service industries may not issue invoices when they retail small commodities or charge small service fees to consumers, but consumers may not refuse to issue invoices if they ask for them.

　　Units and individual industrial and commercial households in the city to buy goods (products), accept labor services or other services to pay, must obtain invoices from the payee.

　　Article 5 The main contents of an invoice include: invoice name, track number, serial number and its use, name of payer, name of commodity (product) or business (service) item, unit of measurement, quantity, unit price, amount, cumulative amount in figures and words, drawer, date of invoice, and name of tax authority that printed or approved printing.

　　Article 6 The invoices of this Municipality shall be printed or approved for printing by the Municipal Taxation Bureau, and the "Seal of Invoice Producer of Beijing Taxation Bureau" shall be overprinted.

　　The "Seal of Invoice Producer of Beijing Municipal Taxation Bureau" is the legal symbol of this Municipality’s invoices, which is uniformly made by the Municipal Taxation Bureau, and no unit or individual is allowed to forge it.

　　Article 7 The invoices of this Municipality shall be used exclusively according to the following categories:

　　1. Special invoices for the industry;

　　2. Special invoices for individual businesses;

　　3. Special invoices for temporary operation;

　　4. Special vouchers for payment of personal income;

　　Five, industrial and commercial enterprises to allocate materials special invoices;

　　6. Special invoices for capital transactions of industrial and commercial enterprises;

　　Seven, approved by the Municipal Taxation Bureau overprint "Beijing Municipal Taxation Bureau invoice producer seal" of other invoices.

　　The specific scope of application of the above invoices shall be stipulated by the Municipal Taxation Bureau.

　　Article 8 For the time being, the following professional bills shall be determined and printed by the units using the bills themselves, and it is not necessary to overprint the invoice producer seal of Beijing Municipal Taxation Bureau:

　　A, finance, insurance, posts and telecommunications, water supply, power supply, gas supply, heating, real estate management, medical care and other industries to use professional bills;

　　Two, railway, highway, aviation, water transport and other departments of the car tickets, air tickets, baggage tickets;

　　Tickets for theaters, stadiums (gymnasiums), clubs, parks, playgrounds, museums and exhibition halls;

　　4. Special vouchers for collecting money from abroad;

　　Five, approved by the Municipal Taxation Bureau can not overprint "Beijing Municipal Taxation Bureau invoice producer seal" of other professional bills.

　　Article 9 The invoices of this Municipality are only used within the administrative area of this Municipality. Units and individual industrial and commercial households in this Municipality who engage in business activities in other places shall apply to the local tax authorities for the use of local invoices with the Certificate of Tax Administration for Outdoor Operation of Fixed Industry and Commerce issued by the local tax authorities in this Municipality.

　　Foreign units and individual industrial and commercial households to engage in business activities in the city, should hold the foreign local tax authorities issued the "fixed industrial and commercial outdoor business tax management certificate", to the city business tax authorities to apply for the use of the city invoice; Temporary operators shall apply to the local tax authorities for the use of zero copies of local invoices.

　　Tenth units and individual industrial and commercial households need invoices, by units or individual industrial and commercial households to apply to the local tax authorities, with the local tax authorities issued the "invoice purchase voucher", to the designated tax authorities to buy.

　　Units that use a large number of invoices or have special business operations may, with the approval of the Municipal Taxation Bureau, print invoices in the approved style at the designated printing house with the Notice of Printing Invoices issued by the local tax authorities. If it is necessary to overprint the seal of the unit on the invoice, it must be approved by the tax authorities. Without approval, overprinting is not allowed.

　　Article 11 Units and individual industrial and commercial households should strengthen the management of invoices, establish a system, assign special personnel to be responsible for them, keep them properly, strictly implement the procedures for collecting invoices, truthfully register the Invoice Use Register, and submit the Report on the Use of Invoices by Enterprises to the local tax authorities quarterly.

　　Twelfth units and individual industrial and commercial households should check the whole invoice one by one before using it; If it is found that the invoice is missing, the whole invoice should be submitted to the local tax authorities for identification before processing. Invoices must be used in numerical order, copied in each copy, truthfully filled out column by column, and stamped with the special financial seal or invoice seal of the unit. Incorrectly filled invoices should be stamped with a void stamp, affixed to their stubs and saved.

　　Invoice stubs issued by units and individual industrial and commercial households shall be kept for at least 5 years. To destroy invoice stubs, a register must be made, a written application must be submitted to the local tax authorities, and the stub can be destroyed only after it is approved by the tax authorities.

　　Article 13 Where an invoice is filled out by a computer, it must be approved by the Municipal Taxation Bureau to print an off-machine invoice with the stamp of "Invoice Producer Seal of Beijing Taxation Bureau". Off-board invoices shall be entered into the computer in numerical order, and the stubs shall be bound and kept in numerical order.

　　Article 14 Units and individual industrial and commercial households that need to cancel or replace invoices due to job change, reorganization, division, merger, joint venture, relocation, closure, bankruptcy and other reasons shall, within 30 days from the next day, go to the local tax authorities to cancel or apply for replacement, and shall not handle it without authorization.

　　Fifteenth printing units shall print invoices with the "Beijing Municipal Taxation Bureau Invoice Certificate" issued by the tax authorities.

　　Without the approval of the tax authorities, no unit or individual may print invoices.

　　Article 16 Units that have been approved by the tax authorities and obtained the Invoice Certificate of Beijing Taxation Bureau for printing invoices must abide by the following provisions:

　　First, the establishment of invoice printing management and quality inspection system, designate a person to be responsible, and set up a special inventory to put invoices;

　　Two, according to the tax authorities issued the "notice of printing invoices" limited number and style of printing invoices;

　　Three, timely destruction of all disabled pages in the process of printing invoices;

　　Four, the printing invoice shall not be transferred to other units for printing;

　　Five, designate a person to manage the "Beijing Municipal Taxation Bureau invoice producer seal", to prevent loss, prohibit lending, transfer, destruction, and report to the tax authorities on a quarterly basis.

　　Article 17 It is forbidden to lend, transfer, resell, open, tear up, alter or forge invoices, to practise fraud when filling out invoices, to sell or use or destroy invoices without authorization. If an invoice is lost, it must be reported to the local tax authorities within 3 days.

　　Eighteenth units that use invoices, individual industrial and commercial households and units that print invoices must accept the supervision and inspection of the tax authorities.

　　In violation of the Interim Measures for the Administration of National Invoices and these Measures, any unit or individual may report and expose to the tax authorities. The tax authorities shall reward the whistleblower according to regulations and keep it confidential.

　　Article 19 Anyone who violates these measures and commits any of the following acts shall be confiscated by the tax authorities, ordered to make corrections within a time limit, and may, according to the seriousness of the case, impose a fine of less than 5,000 yuan on the unit or individual industrial and commercial households, and impose a fine of less than 1000 yuan on the person who is directly responsible and the person who instigates or harbors them; Refuses to correct, the tax authorities may revoke its tax registration certificate and collect its invoices and other tax management certificates:

　　1. Failing to purchase, keep or use invoices according to the provisions of these Measures;

　　2. Lend, transfer, resell, open, tear up, alter, forge invoices and practise fraud when filling out invoices;

　　3. Failing to report to the tax authorities within 3 days after losing the invoice;

　　Four, without the approval of the tax authorities, unauthorized printing, sale, use and destruction of invoices;

　　Five, forged "Beijing Municipal Taxation Bureau invoice producer seal";

　　Six, the invoice making unit lost, transferred, destroyed "Beijing Municipal Taxation Bureau invoice producer seal" or not according to the provisions of these measures to print invoices;

　　Seven, refused to accept the supervision and inspection of the tax authorities.

　　Tax evasion, tax evasion and refusal to pay taxes due to the above acts shall be punished in accordance with the relevant provisions of the Provisional Regulations on the Administration of Tax Collection in People’s Republic of China (PRC), in addition to the provisions of these measures; If the case constitutes a crime, the responsible person shall be investigated for criminal responsibility according to law.

　　Twentieth Municipal Taxation Bureau may formulate detailed rules for implementation according to these measures, and be responsible for the interpretation of specific issues in the implementation of these measures.

　　Article 21 These Measures shall come into force as of June 1, 1989. In 1984, the Municipal People’s Government approved the "Trial Measures for the Administration of the Use of Uniform Invoices and Uniform Receipts of Money in Beijing" issued by the Municipal Taxation Bureau, which was abolished at the same time.

　　 　"According to the previous survey of school-age children in the jurisdiction, some schools have a high degree shortage due to the dense population distribution within the service scope and the growing population of new residential quarters. In 2023, they will face a situation beyond the existing degree acceptance capacity." In mid-June, the Education Bureau of Anning District, Lanzhou City, Gansu Province issued such a "degree warning", indicating that the scope of primary and secondary school enrollment will be appropriately adjusted. "School-age children in the district will be admitted in strict accordance with the batch order, and unacceptable students will be arranged by the District Education Bureau to enroll in other schools."

　　 　Entering the "enrollment season", school enrollment in the compulsory education stage affects the hearts of thousands of parents of prospective first-year students. With the seventh year of the implementation of the "two-child policy", the conduction effect brought by the "baby boom" is concentrated in this year’s primary school entrance. According to media reports, since the beginning of this year, Beijing, Guangzhou, Jinan, Dalian, Changchun, Qingdao and Chengdu have issued degree warnings for primary and secondary schools. Will this year be the "hardest year to enter school"? Behind the degree warning, how can all localities ensure that they are "close to school" and "eager to learn"? The reporter conducted an interview.

　　Large increment of school-age population and regional imbalance cause "degree shortage"

　　 　For some primary and secondary school students in Beilun District, Ningbo City, Zhejiang Province, going to physical education class should be "upstairs" rather than "downstairs" — — Their playground was built on the roof.

　　 　"Not only the playground was built with fences on the roof, but also a runway was opened around the teaching building and next to the fence. Almost all the available open spaces have been developed into sports venues. " Liu Jie, chief of the Basic Education Section of the Education Bureau of this district, told the reporter that in order to increase the per capita exercise area, some schools had to "look around for space".

　　 　Behind the emergency move is the increasing number of local students and students. Liu Jie told reporters, "This year’s increment is the biggest. At least 1,000 new degrees should be added in the whole region. According to the maximum class size of no more than 45 students, more than 20 new classes have to be added."

　　 　The "peak" of the degree demand increase is not only reflected in the first-and second-tier cities in coastal developed areas. According to a material provided by the Education and Sports Bureau of Xihu District, Nanchang, Jiangxi Province, with the implementation of the comprehensive two-child policy in 2016, the number of students enrolled in compulsory education in this district has shown a significant growth trend in 2022, and the number of students enrolled in 2023 will come from the year with the highest birth rate in the whole region, and the number of school-age children will reach a new high.

　　 　"The data shows that in September 2022, there were 6,652 students enrolled in primary schools and 2,240 students enrolled in junior high schools in Xihu District. The total number of students enrolled increased by 972 compared with 2021, and the number of students enrolled increased by 3,120 compared with 2021 (excluding municipal and private schools). Through the bottom-up analysis, the number of bottom-ups in 2023 increased by 1,282 compared with that in 2022 (excluding municipal and private schools). " According to the relevant person in charge, "according to the pre-judgment, there will be 7671 primary school-age children in this district this year."

　　 　In addition to the large increase of school-age students, the regional distribution of required degrees is uneven, which is another feature of the data. The reporter learned that the growth of school-age children and adolescents in Xihu District of Nanchang City in 2023 was bounded by Hongcheng Road, and the total amount of the old city and Chaoyangzhou area increased slightly, with the main growth concentrated in Hongcheng area and Chaoyang New City area south of Hongcheng Road. It can be seen that for the new city area, the degree shortage situation is more obvious.

　　 　"It is estimated that the school-age population of basic education in Ganzhou will gradually fall back after reaching the peak of 1.8 million in 2022, but the school-age population will quickly concentrate in cities," said Lai Zhengwen, deputy mayor of Ganzhou Municipal People’s Government of Jiangxi Province. "The proportion of students in county towns is 68.5%, which has exceeded the urbanization rate of 56.4%, and the gap in urban compulsory education degrees is about 200,000."

　　 　"Some areas appear ‘ Degree shortage ’ The problem may be related to the local education authorities and other relevant departments’ inaccurate prediction of the population birth rate and the demand for educational resources. " Wang Jianmin, a professor and doctoral supervisor at Beijing Normal University, said that due to factors such as economic and social development, new situations and new features such as increased mobility of school-age children have emerged, and the education authorities should make predictions and actively respond to changes in the demand for teaching resources.

　　Take multiple measures to dig up the stock and promote the increment, so as to ensure that we should live within our means.

　　 　"The situation was predicted a few years ago. This new wave was reflected in the number of kindergarten students three years ago, and then it will be transmitted to secondary schools step by step." Liu Jie said, "In this case, we will definitely give an early warning, or we must ensure that we should make every effort and enter the school nearby."

　　 　"According to estimates, Xihu District of Nanchang City can actually provide 7,965 degrees (according to 45 students/class) in 177 primary schools in 2023, which can generally meet the increase in the enrollment demand of school-age children and adolescents in the whole region in 2023. We are trying our best to recruit students at the peak of the enrollment population to ensure that every school-age child and teenager in the region can enter school normally." The relevant person in charge of Xihu District said.

　　 　Faced with the demand for new degrees with a large total amount and uneven geographical distribution, local initiatives are different. The common measure is to make full use of the new degree resources by properly adjusting the division of school districts. The reporter learned that there is a new campus west of Ziyu Road and south of Shuichang Road in Nanchang — — Ziyu Campus of Nanchang Zhanqian Road School Education Group will be delivered in the near future, and it will officially open in September. There are 36 classes in the campus, including 24 classes in primary schools, which can accommodate 1080 people. There are 12 classes in junior high school, which can accommodate 600 people. According to the arrangement, another 26 classrooms can be rebuilt and expanded in the campus, with the maximum capacity of 62 classes. It is estimated that 1,890 new degrees can be added in primary schools and 1,050 new degrees can be added in junior high schools.

　　 　"Through the previous field research, the campus has drawn up the corresponding school district division plan according to the degree it can provide and the degree demand. According to the specific geographical location of the campus and the principle of taking the main road as the boundary, the surrounding areas east of Taohua South Road, south of Shuichang Road, west of Zhenjun Road and north of Jiuzhou Street are designated as the school district scope, which can alleviate the degree shortage of Taohua Campus (formerly Taohua School) of Railway No.1 School Education Group." Yan Qianqian, deputy head of the Compulsory Education Unit of Xihu District Education and Sports Bureau, introduced.

　　 　Liu Jie said that private schools will take advantage of the opportunity of "changing from private schools to public schools" to allocate new degree resources to areas with tight degrees. "Private schools were originally enrolled for the whole region, and now the policy has been adjusted to block-oriented recruitment, which is supplemented to some public schools with tight resources in the school district. These schools have a good standard of running schools and ordinary people are willing to go. "

　　 　In addition to making good use of new resources, it is also the choice of many regions to fully tap the "stock". Faced with the increasing enrollment demand year by year, some schools in Chaoyang New Town in Nanchang have checked the existing teaching space and invited professionals to make planning and accounting. The results show that the increase in enrollment demand can be met by transforming the existing classrooms.

　　 　"The campus renovation project of OCT Campus of Zhanqian Road School Education Group is expected to renovate 27 classrooms and 14 offices, and 725 new degrees can be added; The whole campus classroom renovation project of Songbai School Education Group Chaoyang Campus is expected to renovate 29 classrooms, which can add 1,240 degrees. Relevant work will be implemented from July to August to ensure timely completion and ensure the smooth enrollment of the two schools in 2023. " The relevant person in charge said.

　　 　"Generally speaking, if local government departments do a good job in demand forecasting, they can solve it by increasing teaching classes without building new schools. For the substantial increase in the demand for educational resources, we can consider setting up a new school to deal with it. " Wang Jianmin analyzed.

　　Strengthen the forward-looking policy and reasonably respond to the changes in the demand for educational resources.

　　 　Liu Jie said that by recruiting more than 200 teachers in various subjects and conducting pre-job training in time, there are still ways to think about "software", "‘ Hardware ’ Short-term supply shortage is more realistic. The design scale of a school is certain. After increasing the number of classes, the area of playgrounds, toilets and canteens per capita will become tense. " Liu Jie said that at present, some schools can only improve the use efficiency by means of multiple rounds of dining, and alleviate the problem of insufficient space per capita.

　　 　"In recent years, Beilun District has made a comprehensive plan for the construction of basic education resources and laid out many schools. However, after all, a school has a long use cycle from new construction to investment, which may not keep up with the increase of students, and the configuration is still slightly lagging behind. " Liu Jie said that if the three large-scale schools can be put into use in succession in 2024-2025, the per capita tension will be fully relieved from next year to the year after. "A grade can add at least 1,000 degrees."

　　 　"While ensuring the supply of classrooms and ensuring the demand for admission, Xihu District will also do a good job in upgrading the hardware of each campus in the region, and strive to improve the conditions for running schools and build a quality campus." The person in charge said that the district adopts the strategy of "one chess game" in the whole district, ensures the scientific flow of teachers in the region through the two-way communication mechanism within and outside the group, reasonably selects new teachers for posts through personnel agents, increases teacher training, and ensures that full-time teachers in new degree schools are adequately equipped.

　　 　Wang Jianmin believes that in the short term, the problem of degree shortage in areas may have a certain impact on the teaching quality due to the expansion of class size, the addition of new classes and the introduction of new teachers. Various forces should be mobilized to solve it in time, with government departments leading, social forces supporting and parents actively cooperating. "Classrooms, multimedia, laboratories and other resources will have an impact, but the key is the shortage of high-quality teachers. Teachers’ teaching level and classroom management experience are scarce resources, which cannot be achieved overnight, and it takes time to accumulate and precipitate."

　　 　Then, with the decline of the number of people born after the "peak", will the new education and teaching resources be "idle"?

　　 　"From the data of kindergarten enrollment, we predict that there will definitely be a wave of decline in the next few years, but it will not drop anywhere." Liu Jie said that Beilun District of Ningbo is located in the coastal area, and its industrial base is mainly manufacturing, which is attractive to foreign population and its base will not drop too obviously. "Then with ‘ Peak ’ As time goes by, junior and senior high schools will have to supplement many resources; Now the state promotes the construction of a high-quality balanced area for compulsory education, and the number of primary school classes can be appropriately reduced; In addition, faculty and staff also retire naturally every year, which can basically maintain a relative balance. "

　　 　The reporter noted that the Opinions on Building a High-quality and Balanced Basic Public Education Service System issued by the Central Office and the State Council not long ago proposed that all provinces (autonomous regions and municipalities directly under the Central Government) should formulate standards for the allocation of urban compulsory education degrees, and cities and counties should rationally plan and guarantee sufficient construction land, strictly implement the regulations on the allocation of schools in new residential quarters, and speed up the expansion and expansion of urban schools.

　　 　"First, we must establish an early warning mechanism for resource allocation risks, and strengthen information integration, monitoring and early warning of population and education data; Second, we should optimize the distribution of educational resources according to the changes of school-age population and urban economic and social development, and make up for the gap in resource supply as a whole; Third, we should optimize the allocation of teachers’ resources and fully implement ‘ District Management School Appointment ’ Reform, through the combination of teacher preparation and government procurement services, timely supplement the shortage of subject teachers, strengthen the construction of high-school and consistent schools, and vigorously cultivate teachers with high-school and backwards compatibility ability to enhance the overall deployment ability. " Meng Hui, deputy mayor of Wuhan Municipal People’s Government, said.

　　 　"In short, the large-scale emigration or return of the population and the changes in the birth rate of newborns should be taken as an important basis for the education authorities to evaluate the changes in the demand for educational resources, and they should make judgments and plans and make timely adjustments to avoid them as much as possible ‘ Degree shortage ’ Or ‘ Excess degree ’ Situation. " Wang Jianmin proposed.

　　 　(Reporter Zhou Shixiang)

　　CCTV News:On the afternoon of August 2nd, the Ministry of Foreign Affairs of China released a 12-page document, which disclosed in detail the details of Indian border guards illegally crossing the border into the Donglang area of China. As of the end of July, more than 40 Indian border guards and a bulldozer were still illegally stranded in the territory of China. China solemnly declares that no country should underestimate China’s determination to defend its territorial sovereignty.

　　Donglang area is located in Yadong County, Xizang Autonomous Region City, China, and there is no sovereignty dispute.

　　On August 2nd, Beijing time, the Ministry of Foreign Affairs of China announced the fact that Indian border guards crossed into Indian territory in Sikkim section of China-border and China’s position. In this 12-page document, the Chinese side explained the sovereignty of Donglang area, the truth that Indian troops illegally crossed the border, the hypocrisy of Indian excuses and China’s position on handling this issue with words and pictures.

　　First of all, China once again made it clear that there is no sovereignty dispute in Donglang area. According to the document, Donglang area is located in Yadong County, Xizang Autonomous Region, China, bordering Indian Sikkim State in the west and Bhutan Kingdom in the south. In 1890, China and Britain signed the Sino-British Treaty on Tibet and India, which demarcated the border between Tibet and Sikkim in China. According to the provisions of the treaty, Donglang area is located on the China side of the border line and is undisputed territory of China. For a long time, Chinese border guards and herders have been patrolling and grazing in this area. At present, the border between Donglang area and Sikkim is a part of the Sikkim section of the Sino-Indian border.

　　Ministry of Foreign Affairs: When the number of Indian troops crossing the border was the highest, more than 400 people crossed the border more than 180 meters deep.

　　The document also announced the details of the illegal crossing of the Indian army: on June 16, 2017, the Chinese side carried out road construction in Donglang area; On June 18th, more than 270 Indian border guards, armed with two bulldozers, crossed the border line of Sikkim section at Dokala Pass for more than 100 meters and entered China to obstruct China’s road construction activities, which caused tension. When the Indian border guards crossed the border, the number of people reached more than 400. Together with two bulldozers and three tents, the depth of crossing the border reached more than 180 meters. By the end of July, more than 40 Indian border guards and a bulldozer were still illegally stranded in the territory of China.

　　The Ministry of Foreign Affairs of China stressed that this incident occurred in the demarcated area with clear boundary lines, which is essentially different from the friction between the border guards of both sides in the undefined area in the past. Indian border guards crossed the established border and violated China’s sovereignty and territorial integrity, the 1890 Treaty and the Charter of the United Nations. It was a gross violation of the basic principles of international law and the basic norms of international relations, and it was very serious in nature!

　　Ye Hailin, a special commentator, said that the information released by the Ministry of Foreign Affairs this time is actually our explanation of the Indian army’s invasion of China territory in the past month.If this incident has any consequences, it is all caused by the illegal entry of Indian troops.Since the person in charge is an Indian armed man, even the explanation of past actions is to prepare for the actions we will take.

　　After the Indian army illegally crossed the border, India made a fuss about "national security"

　　Since the Indian army illegally crossed the border in June, India has given a big excuse: China’s road construction in Donglang area will endanger India’s so-called "national security." Against the background of exaggerating the threat of China, Indian officials and the media keep making tough remarks.

　　On July 25th, Chand, Deputy Chief of Staff of the Indian Army, said, "China’s economic aggregate is five times that of India, and it also has a strong army, which will be a" threat "to India for many years to come. Chande also stressed that under the current situation, India needs to pay more attention to its own security.

　　In this regard, the Ministry of Foreign Affairs of China pointed out that resolution 3314 adopted by the United Nations General Assembly on December 14, 1974 stipulated that the armed forces of one country should not be excused for invading or attacking the territory of another country for any reason, whether political, economic, military or other. Crossing the established border into the territory of neighboring countries on the grounds of so-called "security concerns" violates the basic principles of international law and the basic norms of international relations, and will not be tolerated by any sovereign country, let alone the normal way for China and India to get along with each other.

　　Where does India’s so-called "security concerns" come from? What is the essence behind it?

　　Ye Hailin, a special commentator, said that the Indian invasion in Donglang did have his anxiety, and he felt that his territory and the northeast region were threatened. The communication between any countries is principled, and your feelings cannot be used as a legitimate basis for invading other countries, as the Indian side calls it.Concern is also his own imagination.

　　The Ministry of Foreign Affairs of China further stated on the 2nd that for a long time, Indian troops have built a lot of infrastructure such as roads on the Indian side of the border line at Dokala Pass and its vicinity, and even built military facilities such as bunkers on the border line. On the contrary, only a small amount of infrastructure construction has been carried out in China. In recent years, Indian border guards have also prevented Chinese border guards from patrolling normally along the border line and attempted to build military facilities across the border. Chinese border guards have repeatedly protested against this and dismantled Indian border facilities according to law. In fact, it is India’s attempt to constantly change the status quo of the Sikkim section of the Sino-Indian border, posing a serious security threat to China.

　　Ministry of National Defense: Remind India not to take any chances.

　　It has been more than a month since the Indian army illegally invaded the Donglang area of China — — During this period, China’s foreign affairs and national defense departments constantly clarified their positions to India through various occasions.

Wu Qian, spokesman of the Ministry of National Defense of China

　　Wu Qian, spokesman of China’s Ministry of National Defense, said on July 24th that China’s determination and will to defend the country’s territorial sovereignty are unshakable, and it will safeguard its territorial sovereignty and security interests at all costs. Chinese border guards have taken emergency measures on the spot and will further strengthen targeted deployment and training. We want to remind China not to take chances and hold unrealistic illusions. The 90-year history of the founding of the Indian People’s Liberation Army has proved that our ability and means to defend national sovereignty and territorial integrity have been constantly strengthened, and our determination and determination have been unshakable to shake the mountains and ease the difficulties of the People’s Liberation Army.

Wang Yi, Foreign Minister of China

　　China’s Foreign Minister, Wang Yi, also said, "The right and wrong of the problem is very clear. Even senior Indian officials have publicly stated that China soldiers have not entered Indian territory. In other words, the Indian side admitted that it had entered the territory of China. It is also very simple to solve this problem, that is, to quit honestly. "

　　In the position paper released on the 2nd, the Ministry of Foreign Affairs of China once again stressed that since the incident, China has exercised utmost restraint with the utmost goodwill and made efforts to communicate with India through diplomatic channels to resolve the incident. But no country should underestimate the determination of the government and people of China to defend its territorial sovereignty. China will take all necessary measures to safeguard its legitimate rights and interests.

The next luxury car market has a good show! Because on November 8, "Land Rover Tiantuan" completed the last piece of "puzzle"-the 2025 model was officially listed. This means that following the launch of the 25-year model in September this year, the latest lineup of Land Rover’s core luxury SUV is complete.

Why do you say "there is a good show"? In fact, friends who have recently paid attention to the luxury car market must know that Jaguar Land Rover has won "half the country" in the high-end luxury SUV market.

From January to September this year, the sales volume of Range Rover reached 11,548 units. In the SUV market above 1.5 million, Range Rover almost took over half of the market share. So far, Range Rover has won the sales champion in this market segment for 21 consecutive months. In the SUV market of millions and above, Jaguar Land Rover has a market share of over 30%, among which Land Rover Defender ranks second and Range Rover Sport Edition ranks sixth.

You know, from January to September, the three major models of Land Rover have not yet completed the model change, but they have such dominance in the high-end luxury SUV market. After they complete the update and upgrade, they will become Jaguar Land Rover’s solid backing in the high-end SUV market.

Let’s look back to the 25-year Range Rover Sport and see if there is anything exciting about the Range Rover Sport Edition after the 1990 s change.

First of all, the price range of the new car is 950,000-1,360,000 yuan. Compared with the 24 Range Rover sports, the new car is lowered by 18,000 yuan. As the most sporty model in the Land Rover family, the new Range Rover not only has a comprehensive upgrade in configuration, but also enters the market at a more attractive price, which can be said to once again enhance its competitiveness in the high-end luxury SUV market.

The 2025 Range Rover Sport continues the classic design of the family. The new car adopts a three-layer design grille, smart numbers and satin bronze details, showing a fashionable and dynamic appearance. The addition of black appearance kit makes the texture of Dynamic HSE and above more sharp.

In terms of interiors, the new Range Rover Sport pursues simplicity and luxury, providing windsor and semi-aniline leather interiors, 22-way adjustable seats, flying wing headrest and intelligent steering wheel. The new car is also equipped with an e+ entertainment interconnection system, which supports applications such as WeChat, Tencent Map, Himalayan, OTA upgrade and provides a convenient interconnection experience.

In addition, there are more decentralized configurations, such as in-car air purification system Pro, British treasure audio system such as wireless charging, 23-inch 10-spoke aluminum alloy wheels, etc., which bring all-round luxury experience.

In terms of power configuration, the 2025 Range Rover Sport offers three power versions to meet diverse driving needs. The basic model is equipped with a 3.0-liter in-line 6-cylinder 360-horsepower twin-supercharged engine, with an acceleration of 6 seconds per 100 kilometers; The acceleration time of 100 kilometers with 400 horsepower model is 5.5 seconds; The P550e plug-in hybrid version has a powerful torque of 550 HP and 800 Nm, and the acceleration of 100 km takes only 4.9 seconds.

In addition, the second generation Terrain Response? intelligent all-terrain feedback system supports multi-mode switching and off-road road condition adaptation, with a maximum wading depth of 900 mm, which greatly improves the stability and safety of driving.

Generally speaking, the 25-year Range Rover Sport with reduced price and increased allocation has once again improved its own gold content. It is the "Almighty King" and has become more impeccable after the upgrade iteration. It is no wonder that Jaguar Land Rover’s high-end SUV sales are so good, because it seems that every time it is upgraded, it can firmly hold the hearts of consumers.