WO2021121166A1 - 用于癌症治疗的多靶向siRNA - Google Patents

用于癌症治疗的多靶向siRNA Download PDF

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WO2021121166A1
WO2021121166A1 PCT/CN2020/135814 CN2020135814W WO2021121166A1 WO 2021121166 A1 WO2021121166 A1 WO 2021121166A1 CN 2020135814 W CN2020135814 W CN 2020135814W WO 2021121166 A1 WO2021121166 A1 WO 2021121166A1
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sirna
expression
target gene
plasmid
reduces
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French (fr)
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张辰宇
陈熹
付正
李菁
张翔
梁宏伟
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南京大学
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Priority to EP20902598.0A priority Critical patent/EP4079854A4/en
Priority to US17/798,236 priority patent/US20230193278A1/en
Priority to JP2022561432A priority patent/JP2023518101A/ja
Publication of WO2021121166A1 publication Critical patent/WO2021121166A1/zh

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Definitions

  • the present invention belongs to the field of biotechnology, and relates to multi-targeting siRNA for cancer treatment.
  • siRNA can specifically bind mRNA to interfere with gene expression at the post-transcriptional level. Therefore, in order to directly deliver siRNA drugs to the established target cells, target tissues or target organs, it is inevitable to deliver the drugs through the blood circulatory system. How to safely, effectively and stably deliver siRNA to target cells or target organs in vivo is also the most critical issue for the development of siRNA drugs.
  • the current in vivo delivery strategies of siRNA drugs can be divided into several categories, such as naked siRNA direct delivery, viral vectors, chemical modification, nanoparticles, liposomes, and so on. Take synthetic unmodified naked siRNA as an example. After intravenous injection, the siRNA needs to circulate in the blood until it reaches the target cell. During this period, a large part of siRNA will be filtered out of the body by the kidney, and some will be processed by phagocytes.
  • the delivery methods using chemical modification, nanometer, liposome or viral vector all have their own safety problems.
  • exosomal siRNA delivery technology In recent years, another rapidly progressing delivery method is based on exosomal siRNA delivery technology. Its advantage is that exosomes can encapsulate and protect miRNAs (siRNA analogs) to freely cross cell membranes and biological barriers and reach recipient cells. A natural carrier for the delivery of miRNA between cells and tissues. This method has been widely reported to be successful in a variety of disease models, but these experiments are often regardless of cost and cost. In the actual operation, packaging siRNA into exosomes requires large-scale cell culture, which is time-consuming and time-consuming. In addition, the separation and purification of exosomes also requires a lot of manpower and material resources, so mass production of exosomal siRNA is not realistic. On the other hand, due to the complicated process of producing exosomal siRNA, it has high requirements on cell state, separation process, and personnel operation. It is difficult to ensure consistency between batches. Therefore, it is difficult to meet the requirements of production quality control. Realize industrialized production.
  • Synthetic biology refers to the use of engineering concepts and system design theories to design and create components (parts), devices (devices) or modules (modules), with specific products as the target, assembling various standardized functional components together, and at the same time through the overall Optimizing and regulating the path, forming a new artificial biological system with predetermined functions, and realizing the large-scale application of synthetic biological systems in the fields of chemicals, medicine, diagnosis and treatment of major diseases, agriculture, energy, and environment.
  • synthetic biology provides safe, efficient, and controllable experimental tools and verification methods for the development of new biomedical technologies. It has been used in in vivo library construction, drug discovery, drug synthesis, drug delivery, and drug optimization, etc. aspect.
  • the purpose of the present invention is to provide a method for directly using an artificially designed plasmid system for in vivo treatment.
  • RNA composition including:
  • the first siRNA molecule that reduces the expression of the first target gene
  • a second siRNA molecule that reduces the expression of the second target gene
  • the first target gene is selected from the group consisting of EGFR, KRAS, or a combination thereof, and the siRNA composition reduces the expression of two or more genes.
  • the second target gene is selected from the group consisting of EGFR, TNC, or a combination thereof.
  • the first target gene and the second target gene are different.
  • the first siRNA molecule has a sequence shown in SEQ ID NO.: 1 or 2.
  • sequence of the first siRNA molecule is shown in SEQ ID NO.: 1 or 2.
  • the second siRNA molecule has a sequence shown in SEQ ID NO.:3.
  • sequence of the second siRNA molecule is shown in SEQ ID NO.:3.
  • the targeting peptide element is selected from the following group: RVG, LAMP2B, or a combination thereof.
  • the targeting peptide element is a fusion protein composed of RVG and LAMP2B.
  • sequence of the targeting peptide element is shown in SEQ ID NO.:4.
  • the second aspect of the present invention provides a carrier, including:
  • the first siRNA molecule that reduces the expression of the first target gene
  • a second siRNA molecule that reduces the expression of a second target gene; wherein the first target gene is selected from the group consisting of EGFR, TNC, KRAS, or a combination thereof, and the siRNA molecule reduces two or more The expression of each gene.
  • the carrier has a structure shown in formula I of 5'-3':
  • Z0 is a promoter element
  • Z1 is the coding sequence of an optional targeting peptide element
  • Z2 is the first siRNA molecule that reduces the expression of the first target gene
  • Z3 is an optional second siRNA molecule that reduces the expression of the second target gene.
  • the promoter element includes a constitutive promoter.
  • the promoter element is selected from the group consisting of CMV, U6, or a combination thereof.
  • the second target gene is selected from the group consisting of EGFR, TNC, KRAS, or a combination thereof.
  • the first target gene and the second target gene are different.
  • the first siRNA molecule has a sequence shown in SEQ ID NO.: 1 or 2.
  • the second siRNA molecule has a sequence shown in SEQ ID NO.:3.
  • the targeting peptide element is selected from the following group: RVG, LAMP2B, or a combination thereof.
  • the targeting peptide element is a fusion protein composed of RVG and LAMP2B.
  • sequence of the targeting peptide element is shown in SEQ ID NO.:4.
  • sequence of the vector is shown in SEQ ID NO.:5.
  • the expression vector includes a viral vector and a non-viral vector.
  • the viral vector includes retrovirus, lentivirus, adenovirus, and adeno-associated virus vector.
  • the expression vector is a plasmid.
  • the third aspect of the present invention provides a use of the siRNA composition according to the first aspect of the present invention or the carrier according to the second aspect of the present invention for the preparation of drugs or preparations for the treatment of cancer.
  • the treatment is a treatment performed by directly injecting the siRNA composition according to the first aspect of the present invention or the vector according to the second aspect of the present invention into the body.
  • the cancer is selected from the group consisting of lung cancer, glioblastoma, or a combination thereof.
  • the formulation is a liquid formulation.
  • the concentration of the siRNA composition or the carrier is 0.5 mg/kg to 20 mg/kg, preferably, 1 mg/kg to 10 mg/kg, more preferably Ground, 5mg/kg-10mg/kg.
  • the fourth aspect of the present invention provides a pharmaceutical preparation, which contains:
  • the formulation is a liquid dosage form.
  • the preparation is an injection.
  • the vector includes a plasmid.
  • the vector or plasmid contains a promoter, an origin of replication and a marker gene.
  • the concentration of the carrier is 0.5 mg/kg to 20 mg/kg, preferably, 1 mg/kg to 10 mg/kg, more preferably, 5 mg/kg to 10 mg/kg. kg.
  • the pharmaceutical preparation includes other drugs for treating cancer.
  • the other drugs for treating cancer include gefitinib.
  • the fifth aspect of the present invention provides a method for treating cancer, including:
  • siRNA composition according to the first aspect of the present invention, the carrier according to the second aspect of the present invention, or the pharmaceutical preparation according to the fourth aspect of the present invention are directly injected into a subject in need.
  • the administered dose is 0.5mg/kg-20mg/kg, preferably, 1mg/kg-10mg/kg, more preferably, 5mg/kg-10mg/kg
  • the administration includes injection of plasmids.
  • Figure 1 shows a schematic diagram of the plasmid molecule composed of the genetic elements of the present invention.
  • Figure 2 shows the in vitro interference efficiency test of plasmid molecules. Construct plasmids with different interference sequences according to the method shown in Figure 1, and use cell experiments to verify the interference efficiency.
  • AC CMV-siR E plasmid molecule was transfected into LLC cells, and the expression level of siRNA (A) and its inhibition of EGFR gene mRNA (B) and protein (C) expression were detected;
  • DF CMV-RVG-siR E +T plasmid molecules were transfected into U87MG cells and the expression level of siRNA (D) was detected, as well as its inhibition of EGFR, TNC gene mRNA (E) and protein (F) expression.
  • Figure 3 shows the distribution of siRNA expressed by the CMV-siR E plasmid in various tissues. After injection of plasmid 1, 3, 6, 9, 12, 24, and 48 hours, the mice were sacrificed and mouse tissues were taken: A: detection of siRNA expression elements and mature siRNA levels in liver tissue; B: lung, kidney, kidney, Detect siRNA levels in spleen, brain, heart, pancreas, muscle, CD4 + T cells and other tissues; C: Detect siRNA expression in mouse plasma at the above time points and its content in plasma exosomes.
  • FIG 4 shows the therapeutic effect and survival statistics of the CMV-siR E plasmid molecule on the LLC tumor in situ lung cancer mouse model.
  • the LLC in situ tumor-implanted lung cancer mouse models were equally divided into groups, and PBS, control plasmid (CMV-scrR), gefitinib or CMV-siR E plasmid were injected every two days for a period of 2 weeks, and they were detected by CT scan before and after treatment.
  • the size of mouse tumors, and statistics of survival A: representative CT scan 3D imaging results; B: tumor volume changes in mice; C: survival statistics. Among them, * means p ⁇ 0.05, ** means p ⁇ 0.01, and *** means p ⁇ 0.005.
  • Figure 5 shows the therapeutic effect and survival statistics of CMV-siR K plasmid molecule on KrasG12D; p53fl/fl transgenic lung cancer mouse model.
  • the KrasG12D; p53fl/fl transgenic lung cancer mouse models were equally divided into groups, and PBS, control plasmid (CMV-scrR) or CMV-siR K plasmid was injected every two days for a period of 2 weeks.
  • the tumor size of mice was detected by CT scan before and after treatment. , And statistics of survival.
  • A representative CT scan 3D imaging results
  • B tumor volume changes in mice
  • C survival statistics. Among them, * means p ⁇ 0.05, ** means p ⁇ 0.01, and *** means p ⁇ 0.005.
  • Figure 6 shows the therapeutic effect and survival statistics of the CMV-RVG-siR E+T plasmid molecule on the mouse model of glioblastoma in situ.
  • the mouse models of glioblastoma tumor implantation in situ were divided into groups, and injected with PBS, control plasmid (CMV-scrR), or CMV-RVG-siR E+T plasmid every two days, and verified by detecting siRNA and protein levels
  • the CMV-RVG-siR E+T plasmid can effectively deliver siRNA to the brain and inhibit the expression of EGFR and TNC genes.
  • the mouse model was treated for 2 weeks, and the changes in the size of mouse tumors were detected by in vivo imaging technology, and survival statistics were calculated.
  • B Changes in tumor volume in mice;
  • C Survival statistics. Among them, * means p ⁇ 0.05, ** means p ⁇ 0.01, and *** means p ⁇ 0.005.
  • Figure 7 shows the safety test of plasmid administration.
  • A-F The effect of plasmid administration on the biochemical indicators of alanine aminotransferase, aspartate aminotransferase, total bilirubin, urea, alkaline phosphatase and creatinine in the serum of mice;
  • G the effect of plasmid administration on the tissue structure of mice.
  • siRNA composition or vector for the first time which contains (a) a first siRNA molecule that reduces the expression of the first target gene; (b) optionally, a targeting peptide element And (c) optionally, a second siRNA molecule that reduces the expression of a second target gene; wherein the first target gene is selected from the group consisting of EGFR, KRAS, or a combination thereof, and the second target The gene is selected from the group consisting of EGFR, TNC, or a combination thereof.
  • the siRNA composition can reduce the expression of two or more genes.
  • the present invention also unexpectedly discovered that the siRNA composition or carrier of the present invention can be directly injected into the body to directly form exosomes for the treatment of cancer. On this basis, the inventor completed the present invention.
  • the term "about” may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • the terms "host”, “subject”, and “desired subject” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, such as cows, horses, dogs, cats, pigs, sheep, goats, camels, rats, mice, hares, and rabbits.
  • the first siRNA molecule refers to an siRNA molecule capable of reducing the expression of the first target gene (eg, EGFR, KRAS).
  • sequence of the first siRNA is shown in SEQ ID NO.: 1 or 2.
  • SEQ ID NO. 1 UUGGGCUUCUCUUAACUCCU (EGFR siRNA);
  • SEQ ID NO. 2 GCAAAUACACAAAGAAAGCCC (KRAS siRNA).
  • the second siRNA molecule refers to an siRNA molecule that can reduce the expression of the second target gene (eg, EGFR, TNC).
  • sequence of the second siRNA is shown in SEQ ID NO.:3.
  • SEQ ID NO. 3 CACACAAGCCAUCUACACAUG (TNC siRNA).
  • siRNA composition including:
  • the first siRNA molecule that reduces the expression of the first target gene
  • a second siRNA molecule that reduces the expression of the second target gene
  • the first target gene is selected from the group consisting of EGFR, KRAS, or a combination thereof.
  • the second target gene is selected from the group consisting of EGFR, TNC, or a combination thereof.
  • the first target gene and the second target gene are different.
  • the siRNA composition of the present invention can reduce the expression of two or more genes. Moreover, the siRNA composition of the present invention can be directly injected into the body to directly form exosomes in the body to treat cancer.
  • the present invention also provides a carrier, which contains the siRNA composition of the present invention.
  • the expression vector usually also contains a promoter, an origin of replication, and/or a marker gene. Methods well known to those skilled in the art can be used to construct the expression vector required by the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as calamycin, gentamicin, hygromycin, and ampicillin resistance.
  • representative promoters include (but are not limited to): CMV promoter, U6, T7 promoter, or a combination thereof.
  • the targeting peptide element is selected from the following group including, but not limited to, RVG-, LAMP2B.
  • the targeting peptide element of the present invention includes rabies virus glycoprotein.
  • Rabies virus glycoprotein (RVG) is a neurophilic protein that can bind to acetylcholine receptors expressed by nerve cells.
  • Rabies virus is a single-stranded negative-stranded RNA virus of the Rhabdoviridae family and has an envelope.
  • the virus mainly encodes glycoprotein G.
  • the G protein is anchored on the surface of the virus envelope in the form of a trimer, and can bind to receptors on the cell surface to mediate membrane fusion and allow the virus to invade cells.
  • G protein is the main antigen protein of rabies virus, which stimulates the body to produce neutralizing antibodies.
  • RVG peptide specifically binds to choline bodies expressed by neuronal cells, and RVG targets are expressed outside the cell membrane, guiding exosomes to pass through the blood-brain barrier and transport to nerve cells.
  • the targeting peptide element of the present invention is RVG-LAMP2b, that is, a fusion protein composed of RVG and LAMP2B.
  • the present invention also provides a method for treating cancer, that is, administering a safe and effective amount of the siRNA composition or carrier or pharmaceutical preparation of the present invention to a desired subject, thereby treating cancer.
  • the present invention combines the above-mentioned synthetic biology design concept with the body’s own miRNA secretion and circulation mechanism for the first time, pioneering the use of mammal’s own tissues and organs (mainly liver) as the natural biological cell chassis, directly using artificial The plasmid system is designed for in vivo treatment.
  • the present invention establishes for the first time an in vivo siRNA targeted delivery method, its representative feature is to use replaceable synthetic biological elements to construct a fully functional gene loop.
  • the biological element includes two parts: a core element and an alternative element: the core element is composed of a promoter element and a first siRNA expression element; the alternative element includes a targeting element and a second siRNA expression element.
  • siRNA expression elements can effectively express one or more siRNAs in the body and automatically assemble them into exosomes; targeting elements can express peptides with targeting functions, which can be automatically expressed on the surface of exosomes, making exosomes
  • the body has the ability to target specific cells or tissues; the activation element can simultaneously activate the expression of the above-mentioned targeting peptide and siRNA.
  • the present invention constructs elements with different functions on the plasmid vector backbone.
  • the promoter element and the siRNA expression element are separately connected in series, the exosome-encapsulated siRNA can be expressed in vivo; the promoter element and multiple siRNAs can be expressed
  • elements are connected in series, they can express exosomes wrapped with multiple siRNAs; when promoter elements, targeting elements, and siRNA expression elements are connected in series, they can express exosomes wrapped with siRNA and have the ability to target specific tissues or cells.
  • a plasmid expressing siRNA is injected through the tail vein.
  • siRNA can be detected in multiple tissues such as liver, lung, kidney, spleen, stomach and other tissues, but its precursor molecules can only be detected in liver tissue. It is suggested that plasmid molecules may be expressed in liver cells and secrete siRNA into other tissues; detection of the siRNA content in plasma and plasma exosomes found that almost all siRNA molecules in plasma are concentrated in plasma exosomes.
  • the siRNA can cross the blood-brain barrier to reach the brain tissue, inhibiting the expression of two different genes .
  • the plasmid molecules designed in the present invention can be processed and expressed in vivo to produce siRNA, and then secrete the siRNA molecules to other tissues and organs in the form of exosomes. And it achieves targeted delivery to make it cross the blood-brain barrier to reach the brain tissue to function.
  • siRNA that inhibits EGFR and KRAS genes has achieved good therapeutic effects in lung cancer in situ tumor implantation and transgenic animal tumor models, respectively; through the combined targeted delivery of siRNA that inhibits TNC and EGFR genes, it crosses the blood-brain barrier It reaches the brain tissue and has achieved good therapeutic effects in the glioblastoma orthotopic tumor model.
  • the technical method for realizing siRNA self-production and transportation of the present invention largely solves the current problems of high production cost and easy degradation of siRNA, and is a low-cost and high-efficiency siRNA drug production and delivery method. At the same time, the present invention proves the safety based on the gene therapy model.
  • Glue is recycled, and the skeleton is temporarily stored in a -20 degree refrigerator.
  • Two pairs of synthesized single-stranded oligomeric DNA was dissolved into 100 ⁇ M with ddH2O, and 5 ⁇ l of each complementary single-stranded was mixed in pairs, and the system was annealed according to the system given in Table 2. Heat the two oligo mixtures at 95 degrees for 5 minutes, and then place them at room temperature for 20 minutes to form double-stranded DNA.
  • each 2L conical flask contains 1L LB, 6 bottles of co-shaking bacteria liquid 6L. Shaking time does not exceed 16h
  • Each of the 6 adsorption columns was poured into 10mL buffer ED, centrifuged at 8000rpm, 2min, and the waste liquid was discarded.
  • mice were injected 5 ⁇ 10 6 LLC cells into nude mice through the tail vein vein. After 30 days, the mice were monitored using a non-invasive Micro-CT scan to ensure successful tumor formation in the lungs. Then, the tumor-bearing mice were randomly divided into 4 groups: intravenous injection of PBS or 5mg/kg CMV-scr R or CMV-siR E gene loop every 2 days, 1 group of 5mg/kg gefitinib gavage, co-treatment 7 times. The course of treatment lasts for 2 weeks.
  • mice with successful tumor implantation are randomly grouped and used to assess survival time and tumor progression. Mice used for survival analysis were monitored after treatment without any further treatment. For tumor progression analysis, only the mice that survived after the end of the 2-week treatment period were analyzed using Micro-CT. After the Micro-CT scan, the mice were sacrificed, lung tissues were taken, and histopathological staining and immunohistochemical methods were used for analysis, and then used.
  • Adeno-Cre can be accurately and directed to the lungs of mice without being trapped in the oral cavity and respiratory tract.
  • Micro-CT monitoring was used at different times (30, 40 and 50 days) after inhalation to ensure tumor formation.
  • the mice were randomly divided into two groups and treated with 5 mg/kg CMV-scrR or CMV-siRK via the tail vein for 2 weeks (7 injections). Then, monitor the mice to determine survival time or assess tumor growth.
  • Micro-CT in small animals monitors the progress of lung tumors:
  • This article uses small animal Micro-CT analysis to evaluate lung tumor growth, because even without any contrast agent, the Micro-CT image clearly distinguishes the lung tumor from the surrounding tissues, and the reconstructed 3-D lung image can reflect more intuitively Show the actual location of the tumor in the lung tissue.
  • Use Bruker's SkyScan 1176 Micro-CT analyzer for Micro-CT scanning which scans an area of 180° with a resolution of 35 ⁇ M and a rotation step size of 0.800.
  • the system consists of two cermet tubes, equipped with a fixed 0.5 mm aluminum filter and two 1280 x 1024 pixel digital X-ray cameras. X-ray images were obtained at 50kV and 500 ⁇ A. Scan the mouse in the supine position.
  • the N-Recon program uses the N-Recon program to classify, process and reconstruct the micro-CT data in batches. Then use the DataViewer to image the reconstructed data, and after the tumor location is identified and identified, the CTan program is further used to calculate the tumor volume, and the CTVol program is used to complete the whole lung reconstruction.
  • a venous blood sample was collected from the mouse and placed in a plasma separator tube.
  • the plasma was separated by centrifugation at 800 ⁇ g for 10 minutes at room temperature, and the cell debris was removed by centrifugation at 10,000 ⁇ g for 15 minutes at room temperature.
  • the supernatant plasma was recovered, and the Total Exosome Isolation kit was used to isolate exosomes according to the manufacturer's instructions.
  • Plasmid molecules targeting EGFR and TNC genes were constructed respectively, and the promoter elements and siRNA expression elements were connected in series to construct CMV-siR E and respectively connected to the backbone vector ( Figure 1).
  • the plasmid molecules were transfected into the mouse lung cancer cell line LLC After 36 hours, qRT-PCR and Western blotting were used to detect the mRNA (Figure 2A-B) and protein (Figure 2C) expression levels of the EGFR gene in the cells.
  • the promoter element, targeting element and two siRNA expression elements targeting EGFR and TNC genes were connected in series to construct CMV-RVG-siR E+T ( Figure 1), and the plasmid molecules were transfected into the glioblastoma cell line U87MG After 36 hours, qPCR and western experiments were used to detect the mRNA (Figure 2D-E) and protein ( Figure 2F) expression levels of the EGFR and TNC genes in the cells. The results show that the artificially constructed siRNA expression plasmid molecules can effectively inhibit the expression of their genes in cell lines.
  • siRNA-expressing plasmid was injected into the tail vein of normal mice at a dose of 10 mg/kg; after 1, 3, 6, 9, 12, 24, and 48 hours, the mice were sacrificed and their liver, lung, Kidney, spleen, brain, heart, pancreas, muscle, CD4 + T cells and other tissues were tested for siRNA levels.
  • the results showed that there was a large amount of siRNA distribution in the liver tissue of mice ( Figure 3A), and the elements expressing siRNA could only be detected in the liver.
  • SiRNA can be detected in plasma ( Figure 3C), and it is mainly present in the form of microvesicles.
  • a large amount of siRNA expression can be detected ( Figure 3B), and the expression level in other tissues is low or no signal.
  • Example 3 The therapeutic effect of plasmid molecules on lung tumor models
  • the LLC lung cancer in situ tumor implantation mouse model was used as the experimental object to confirm the therapeutic effect of the CMV-siR E plasmid on lung tumors.
  • CMV-siR K plasmid was studied, and the replaceability of siRNA expression elements was verified.
  • CT imaging was used to verify that the transgenic mice were successfully modeled, and the mice were randomly divided into 3 groups, and were injected with PBS, control plasmid (CMV-scrR) and CMV-siR K plasmid at a dose of 10 mg/kg. It was administered once every two days for a total of two weeks of treatment. CT imaging was used to detect changes in lung tumors before and after treatment (Figure 5A), and the survival of the mice was counted.
  • the elements expressing the rabies virus peptide RVG are integrated into the plasmid vector.
  • the EGFR gene and The siRNA expression elements of the TNC gene were integrated into the plasmid vector, and the CMV-RVG-siR E+T plasmid molecule was constructed.
  • mice were randomly divided into 3 groups, and PBS, control plasmid (CMV-scrR) and CMV-RVG-siR E+T plasmid were injected at a dose of 10 mg/kg, and they were administered once every two days for a total of two weeks of treatment.
  • CMV-scrR control plasmid
  • CMV-RVG-siR E+T plasmid were administered once every two days for a total of two weeks of treatment.
  • Figure 6C In vivo imaging tracking detects tumor volume
  • the results proved that the injection of CMV-RVG-siR E+T plasmid can effectively inhibit plasmoblastoma, the tumor volume of the experimental group was significantly reduced, and the survival time was significantly prolonged (Figure 6D-E).

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Abstract

提供了用于癌症治疗的多靶向siRNA，具体地，提供了一种siRNA组合物，包括：降低第一靶基因的表达的第一siRNA分子；任选的，靶向肽元件的编码序列；和任选的，降低第二靶基因的表达的第二siRNA分子；其中所述第一靶基因选自下组：EGFR、KRAS、或其组合，并且所述siRNA组合物降低两个或更多个基因的表达。提供的siRNA或载体可直接注射入体内治疗癌症。

Description

用于癌症治疗的多靶向siRNA 技术领域
本发明属于生物技术领域，涉及用于癌症治疗的多靶向siRNA。
背景技术
siRNA能够特异性结合mRNA在转录后水平干扰基因的表达。因此为了将siRNA药物直接送达既定的靶细胞、靶组织或者靶器官，不可避免的要通过血液循环系统来进行药物递送。而在体内如何将siRNA安全、有效、稳定地递送到靶细胞或者靶器官，也是开发siRNA药物最为关键的问题。目前siRNA药物的体内递送策略主要可以分为裸siRNA直接递送、病毒性载体、化学修饰、纳米颗粒、脂质体等几大类。以合成的未经修饰的裸siRNA为例，经静脉注射后，siRNA需要随血液循环流动直到抵达靶细胞。这期间，很大一部分siRNA会被肾脏过滤清除出体外，有一部分将被吞噬细胞处理掉。而采用化学修饰、纳米、脂质体或病毒载体的递送方式，均存在其各自的安全性问题。
近年来，另外一项进展迅速的递送方式是基于外泌体的siRNA传输技术，其优势在于外泌体可以包裹和保护miRNA(siRNA类似物)自由穿越细胞膜和生物屏障并达到受体细胞，是在细胞间、组织间传递miRNA的天然载体。该方法已被广泛报道在多种疾病模型上获得成功，但是这些实验往往是不计成本和代价的，而在实际操作过程中，将siRNA包装进入外泌体需要大规模的细胞培养，耗时耗力，而且花费十分昂贵，此外分离纯化外泌体也需要耗费大量的人力物力，因此大批量生产外泌体siRNA并不现实。另一方面，由于生产外泌体siRNA的过程繁复，对细胞状态、分离过程、人员操作都有较高要求，难以保证批次之间的一致性，因此很难满足生产质控的要求，无法实现工业化生产。
合成生物学是指利用工程学概念和系统设计理论设计创建元件(parts)、器件(devices)或模块(modules)，以特定产物为目标，将各种标准化的功能元件组装在一起，同时经过整体路径的优化调控，形成具有预定功能的全新人工生物体系，实现合成生物体系在化学品、医药、重大疾病的诊断与治疗、农业、能源、环境等领域的规模化应用。在药物研发领域，合成生物学为新型生物医药技术的开发提供了安全、高效、可控的实验工具和验证方法，已经被应用于体内文库构建、药物发现、药物合成、药物输送和药物优化等方面。
因此，本领域迫切需要开发一种直接利用人工设计的质粒系统进行体内治疗的方法。
发明内容
本发明的目的在于提供一种直接利用人工设计的质粒系统进行体内治疗的方法。
在本发明的第一方面，提供了一种siRNA组合物，包括：
降低第一靶基因的表达的第一siRNA分子；
任选的，靶向肽元件的编码序列；和
任选的，降低第二靶基因的表达的第二siRNA分子；
其中所述第一靶基因选自下组：EGFR、KRAS、或其组合，并且所述siRNA组合物降低两个或更多个基因的表达。
在另一优选例中，所述第二靶基因选自下组：EGFR、TNC、或其组合。
在另一优选例中，所述第一靶基因和第二靶基因不同。
在另一优选例中，所述第一siRNA分子具有如SEQ ID NO.:1或2所示的序列。
在另一优选例中，所述第一siRNA分子的序列如SEQ ID NO.:1或2所示。
在另一优选例中，所述第二siRNA分子具有如SEQ ID NO.:3所示的序列。
在另一优选例中，所述第二siRNA分子的序列如SEQ ID NO.:3所示。
在另一优选例中，所述靶向肽元件选自下组：RVG、LAMP2B、或其组合。
在另一优选例中，所述靶向肽元件为RVG与LAMP2B组成的融合蛋白。
在另一优选例中，所述靶向肽元件的序列如SEQ ID NO.:4所示。
本发明第二方面提供了一种载体，包括：
启动子元件；
降低第一靶基因的表达的第一siRNA分子；
任选的，靶向肽元件的编码序列；和
任选的，降低第二靶基因的表达的第二siRNA分子；其中所述第一靶基因选自下组：EGFR、TNC、KRAS、或其组合，并且所述siRNA分子降低两个或更多个基因的表达。
在另一优选例中，所述载体具有5’－3’的式I所示的结构：
Z0-Z1-Z2-Z3  (I)
其中，Z0为启动子元件；
Z1为任选的靶向肽元件的编码序列；
Z2为降低第一靶基因的表达的第一siRNA分子；
Z3为任选的降低第二靶基因的表达的第二siRNA分子。
在另一优选例中，所述启动子元件包括组成型启动子。
在另一优选例中，所述启动子元件选自下组：CMV、U6、或其组合。
在另一优选例中，所述第二靶基因选自下组：EGFR、TNC、KRAS、或其组合。
在另一优选例中，所述第一靶基因和第二靶基因不同。
在另一优选例中，所述第一siRNA分子具有如SEQ ID NO.:1或2所示的序列。
在另一优选例中，所述第二siRNA分子具有如SEQ ID NO.:3所示的序列。
在另一优选例中，所述靶向肽元件选自下组：RVG、LAMP2B、或其组合。
在另一优选例中，所述靶向肽元件为RVG与LAMP2B组成的融合蛋白。
在另一优选例中，所述靶向肽元件的序列如SEQ ID NO.:4所示。
在另一优选例中，所述载体的序列如SEQ ID NO.:5所示。
在另一优选例中，所述的表达载体包括病毒载体、非病毒载体。
在另一优选例中，所述病毒载体包括逆转录病毒、慢病毒、腺病毒、腺相关病毒载体。
在另一优选例中，所述的表达载体为质粒。
本发明第三方面提供了一种本发明第一方面所述的siRNA组合物或本发明第二方面所述的载体的用途，用于制备治疗癌症的药物或制剂。
在另一优选例中，所述治疗为通过将本发明第一方面所述的siRNA组合物或本发明第二方面所述的载体直接注射入体内进行的治疗。
在另一优选例中，所述癌症选自下组：肺癌、胶质母细胞瘤、或其组合。在另一优选例中，所述制剂为液体制剂。
在另一优选例中，所述药物或制剂中，所述siRNA组合物或所述的载体的浓度为0.5mg/kg－20mg/kg，较佳地，1mg/kg－10mg/kg，更佳地，5mg/kg—10mg/kg。
本发明第四方面提供了一种药物制剂，所述药物制剂含有：
(a)本发明第二方面所述的载体；和
(b)药学上可接受的载体。
在另一优选例中，所述的制剂为液体剂型。
在另一优选例中，所述的制剂为注射剂。
在另一优选例中，所述的载体包括质粒。
在另一优选例中，所述的载体或质粒含有启动子、复制起点和标记基因。
在另一优选例中，所述药物制剂中，所述载体的浓度为0.5mg/kg－20mg/kg，较佳地，1mg/kg－10mg/kg，更佳地，5mg/kg—10mg/kg。
在另一优选例中，所述药物制剂包括其他用于治疗癌症的药物。
在另一优选例中，所述其他用于治疗癌症的药物包括吉非替尼。
本发明第五方面提供了一种治疗癌症的方法，包括：
向需要的对象直接注射本发明第一方面所述的siRNA组合物、本发明第二方面所述的载体或本发明第四方面所述的药物制剂。
在另一优选例中，所述施用的剂量为0.5mg/kg－20mg/kg，较佳地，1mg/kg－10mg/kg，更佳地，5mg/kg—10mg/kg
在另一优选例中，所述施用包括注射质粒。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明基因元件构成的质粒分子示意图。
图2显示了质粒分子体外干扰效率检测。按照图1所示方法构建不同干扰序列的质粒，利用细胞实验验证干扰效率。A-C：CMV-siR E质粒分子转染LLC细胞，并检测siRNA的表达水平(A)，以及其对EGFR基因mRNA(B)和蛋白(C)表达的抑制情况；D-F：CMV-RVG-siR E+T质粒分子转染U87MG细胞并检测siRNA的表达水平(D)，以及其对EGFR、TNC基因mRNA(E)和蛋白(F)表达的抑制情况。*表示p<0.05，**表示p<0.01，***表示p<0.005。
图3显示了CMV-siR E质粒表达的siRNA在各个组织的分布情况。注射质粒1、3、6、9、12、24、48小时后，处死小鼠，取小鼠组织：A：肝组织中检测siRNA表达元件和成熟siRNA水平；B：在肺、肾、肾、脾、脑、心脏、胰腺、肌肉、CD4 +T细胞等组织中分别检测siRNA水平；C：在上述时间点小鼠血浆中检测siRNA表达量，及其在血浆外泌体中的含量。
图4显示了CMV-siR E质粒分子对LLC原位植瘤肺癌小鼠模型的治疗效果及生 存情况统计。将LLC原位植瘤肺癌小鼠模型平均分组，每两天注射一次PBS、对照质粒(CMV-scrR)、吉非替尼或CMV-siR E质粒，为期2周，治疗前后分别通过CT扫描检测小鼠肿瘤大小，并统计生存情况。A：代表性CT扫描3D成像结果；B：小鼠肿瘤体积变化情况；C：生存统计。其中，*表示p<0.05，**表示p<0.01，***表示p<0.005。
图5显示了CMV-siR K质粒分子对KrasG12D；p53fl/fl转基因肺癌小鼠模型的治疗效果及生存情况统计。将KrasG12D；p53fl/fl转基因肺癌小鼠模型平均分组，每两天注射一次PBS、对照质粒(CMV-scrR)或CMV-siR K质粒，为期2周，治疗前后分别通过CT扫描检测小鼠肿瘤大小，并统计生存情况。A：代表性CT扫描3D成像结果；B：小鼠肿瘤体积变化情况；C：生存统计。其中，*表示p<0.05，**表示p<0.01，***表示p<0.005。
图6显示了CMV-RVG-siR E+T质粒分子对原位胶质母细胞瘤小鼠模型的治疗效果及生存情况统计。将胶质母细胞瘤原位植瘤小鼠模型平均分组，每两天注射一次PBS、对照质粒(CMV-scrR)、或CMV-RVG-siR E+T质粒，通过检测siRNA和蛋白质水平，验证了CMV-RVG-siR E+T质粒能够有效将siRNA递送至脑部，并抑制EGFR和TNC基因的表达。对小鼠模型进行为期2周的治疗，通过活体成像技术检测小鼠肿瘤大小变化情况，并统计生存情况。A：代表性活体成像扫描结果；B：小鼠肿瘤体积变化情况；C：生存统计。其中，*表示p<0.05，**表示p<0.01，***表示p<0.005。
图7显示了质粒给药的安全性检测。A-F：质粒给药对小鼠血清中谷丙转氨酶、谷草转氨酶、总胆红素、尿素、碱性磷酸酶和肌酐等生化指标的影响；G：质粒给药对小鼠组织结构的影响。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种siRNA组合物或载体，其含有(a)降低第一靶基因的表达的第一siRNA分子；(b)任选的，靶向肽元件的编码序列；和(c)任选的，降低第二靶基因的表达的第二siRNA分子；其中所述第一靶基因选自下组：EGFR、KRAS、或其组合，所述第二靶基因选自下组：EGFR、TNC、或其组合。并且所述siRNA组合物可降低两个或更多个基因的表达。本发明还意外的发现，本发明的siRNA组合物或载体可直接注射入体内，直接形成外泌体，用于治疗癌症。在此基础上，本发明人完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
如本文使用的，术语“宿主”、“受试者”、“所需对象”指任何哺乳动物或非哺乳动物。哺乳动物包括但不限于人类、脊椎动物诸如啮齿类、非人类灵长类，如牛、马、狗、猫、猪、绵羊、山羊、骆驼、大鼠、小鼠、野兔和家兔。
第一siRNA分子
在本发明中，第一siRNA分子指能够降低第一靶基因(如EGFR、KRAS)的表达的siRNA分子。
在一优选实施方式中，第一siRNA的序列如SEQ ID NO.:1或2所示。
SEQ ID NO.1:UGUGGCUUCUCUUAACUCCU(EGFR siRNA)；
SEQ ID NO.2:GCAAAUACACAAAGAAAGCCC(KRAS siRNA)。
第二siRAN分子
在本发明中，第二siRNA分子指能够降低第二靶基因(如EGFR、TNC)的表达的siRNA分子。
在一优选实施方式中，第二siRNA的序列如SEQ ID NO.:3所示。
SEQ ID NO.3:CACACAAGCCAUCUACACAUG(TNC siRNA)。
siRNA组合物
在本发明中，提供了一种siRNA组合物，包括：
降低第一靶基因的表达的第一siRNA分子；
任选的，靶向肽元件的编码序列；和
任选的，降低第二靶基因的表达的第二siRNA分子；
其中所述第一靶基因选自下组：EGFR、KRAS、或其组合。
在一优选实施方式中，所述第二靶基因选自下组：EGFR、TNC、或其组合。
在一优选实施方式中，所述第一靶基因和第二靶基因不同。
在本发明，本发明的siRNA组合物可降低两个或更多个基因的表达。并且，本发明的siRNA组合物可直接注射入体内，在体内直接形成外泌体，治疗癌症。
载体
本发明还提供一种载体，它含有本发明所述的siRNA组合物。所述的表达载体通常还含有启动子、复制起点和/或标记基因等。本领域的技术人员熟知的方法能用于构建本发明所需的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如卡拉霉素、庆大霉素、潮霉素、氨苄青霉素抗性。
在本发明中，代表性的启动子包括(但并不限于)：CMV启动子、U6、T7启动子或其组合。
靶向肽元件
在本发明中，靶向肽元件选自下组包括但并不限于，RVG-、LAMP2B。在一优选实施方式中，本发明的靶向肽元件包括狂犬病毒糖蛋白。狂犬病毒糖蛋白(rabies virus glycoprotein,RVG)是一种嗜神经性的蛋白质，能够与神经细胞表达的乙酰胆碱受体相结合。狂犬病毒为弹状病毒科狂犬病毒属，具有囊膜的单股负链RNA病毒。该病毒主要编码糖蛋白G，G蛋白以三聚体的形式锚定于病毒囊膜表面，并能够与细胞表面的受体结合，介导膜融合使病毒侵入细胞。同时，G蛋白是狂犬病毒主要的抗原蛋白，刺激机体产生中和抗体。RVG肽特异性结合神经元细胞所表达的胆碱体，RVG靶点在细胞膜外表达，引导外泌体通过血脑屏障，运输到神经细胞。
在一优选实施方式中，本发明的靶向肽元件为RVG-LAMP2b，即RVG与LAMP2B组成的融合蛋白。
治疗方法
本发明还提供了一种治疗癌症的方法，即，将安全有效量的本发明的siRNA组合物或载体或药物制剂施用于所需对象，从而治疗癌症。
本发明的主要优点包括：
(1)本发明首次将上述合成生物学设计理念与机体自身的miRNA分泌和循环机制相结合，开创性的地以哺乳动物自身的组织器官(主要是肝脏)作为天然生物细胞底盘，直接利用人工设计的质粒系统进行体内治疗。
(2)本发明首次建立了一种体内siRNA靶向递送方法，其代表性特征是利用可替换的合成生物学元件，构建成具有完整功能的基因环路。其中生物学元件包含核心元件和可替换元件2部分：核心元件由启动子元件和第一siRNA表达元件组成；可替换元件包括靶向元件和第二siRNA的表达元件。siRNA表达元件能够在体内有效表达一个或多个siRNA并自动组装进入外泌体中；靶向元件能够表达具有靶向功能的肽段，该肽段能够自动表达在外泌体膜表面，使外泌体具有靶向特定细胞或组织的能力；启动元件能够同时启动上述靶向肽段和siRNA表达。
(3)本发明将具有不同功能的元件构建到了质粒载体骨架上，单独将启动子元件和siRNA表达元件串联时，能够在体内表达外泌体包裹的siRNA；将启动子元件与多个siRNA表达元件串联时，能表达包裹有多种siRNA的外泌体；将启动子元件、靶向元件、siRNA表达元件串联时，能表达包裹有siRNA且具有靶向特定组织或细胞能力的外泌体。
(4)本发明通过尾静脉注射表达siRNA的质粒，siRNA可以在多个组织如肝、肺、肾、脾、胃等组织中检测到，但其前体分子仅能在肝组织中检测到，提示质粒分子可能在肝细胞中进行表达，并将siRNA分泌到其他组织中；检测血浆和血浆外泌体中的siRNA含量发现，血浆中的siRNA分子几乎全都集中于血浆外泌体中。将带有启动元件、RVG靶向元件、第一siRNA表达元件、第二siRNA表达元件的质粒经尾静脉注射导入体内后，siRNA能够穿过血脑屏障到达脑组织，抑制2种不同基因的表达。
(5)本发明设计的质粒分子可以在体内进行加工表达，产生siRNA，再以 外泌体的形式将siRNA分子分泌到其他组织器官。并且实现了靶向递送使其穿过血脑屏障到达脑组织发挥功能。利用该系统递送抑制EGFR、KRAS基因的siRNA，分别在肺癌原位植瘤和转基因动物肿瘤模型中得到了良好的治疗效果；通过联合靶向递送抑制TNC和EGFR基因的siRNA，穿过血脑屏障到达脑组织，在胶质母细胞瘤原位植瘤模型中取得了良好的治疗效果。
(6)本发明实现siRNA自体生产和运输的技术方法很大程度上解决了目前siRNA生产成本高、易降解的问题，是一种低成本、高效率的siRNA药物生产递送方式。同时本发明证明了基于该基因治疗模式的安全性。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
通用方法
(1)质粒构建：
1.双酶切实验
以BamH Ι(New England Biolab，货号：#R0136)，Xho Ι(Biolabs，货号：#R0146)双酶切实验为例：
反应体系：
NEBuffer 3.1 5.0μL
BamH Ι 1.0μL
Xho Ι 1.0μL
载体DNA 1.0μg
ddH 20 to 50μL
Total volume 50μL
反应程序：
37度，孵育60分钟
跑胶(1％琼脂糖)
胶回收，将骨架暂存于-20度冰箱。
2.退火
将2对合成好的寡聚单链DNA用ddH2O溶解成100μM，互补单链各取5μl两两混合，按表二给出体系进行退火。将2份oligo混合物在95度加热5分钟，然后放置室温20分钟，形成双链DNA。
oligo DNA退火体系
100μM top strand oligo 5μL
100μM bottom strand oligo 5μL
10×oligo annealing buffer 2μL
ddH2O 8μL
Total volume 20μL
3.连接
将退火的双链DNA继续稀释成10nM浓度，按表三给出体系在室温连接30分钟。
T4酶连接体系：
5×ligation buffer 4μL
载体 2μL
ds oligo(10nM) 4μL
T4 DNA ligase(1U/μL) 1μL
ddH2O 9μL
Total volume 20μL
4.转化
取10μL连接产物转化100μL感受态细胞DH5α，冰浴30分钟，42度热激90-120秒，冰浴5min。
涂LB平板(含50μg/ml壮观霉素)后，37℃孵育过夜。加入无抗性LB培养基，37度摇菌培养1小时。
取500μL，涂板(壮观)，37度培养16小时。
5.测序验证
每个转化平板分别挑取3个克隆，摇菌抽提质粒后进行测序，以验证重组克隆中插入片段序列是否与设计的寡聚单链DNA序列一致。
(2)细胞转染：
1.将细胞接种于培养平板中(按照实验目的选择合适规格)，并养至大约50％-80％细胞密度。
2.按Lipofectamine 2000转染说明书，将Lipofectamine 2000用OPTI-MEM稀释，并吹打混匀，静置备用(A液)。
3.同样参考转染说明书，吸取适量质粒用OPTI-MEM稀释为B液，备用。
4.将A、B液混合，吹打10-15次，静置20min。并将待转染的细胞培液换为OPTI-MEM。
5.在混合好的AB混合液均匀滴加入细胞中，轻轻摇匀。
6.转染6h后将培养基替换为2％胎牛血清的培养基，36h后收集细胞用于后续实验分析。
(3)RNA提取：
1.按每10 7个细胞或10mg组织中加入1mL的比例加Trizol(通风橱操作)，剧烈震荡充分混匀，室温静置10分钟。
2.加入Trizol体积1/5的三氯甲烷(通风橱操作)，剧烈震荡充分混匀，室温静置5分钟后，12000g离心20分钟。
3.小心吸取上清，避免碰触蛋白层，并加入上清2倍体积的异丙醇(预冷)，-20度静置至少1h。
4. 12000g，4度离心20分钟，加入Trizol等体积DEPC水配置的75％乙醇清洗。
5. 12000g，4度离心15分钟，完全弃去上清，室温晾干不超过10min。
6.用25μL DEPC水溶解。
(4)除内毒素质粒大提：
以6L菌液为例：
1.摇菌：每个2L锥形瓶装1L LB，6瓶共摇菌液6L。摇菌时间不超过16h
2.菌液装入3个离心瓶中，中心对称配平(国产瓶不超过1/2，进口瓶不超过2/3)，5000rpm，10min离心，倒掉上清收集菌体。
3.向每个离心瓶中加入75mL Solution 1，剧烈震荡至看不到块状物质。
4. 3个离心瓶每瓶分别加入150mL Solution 2，出现絮状粘稠物质，轻 柔摇动，不要太剧烈，裂解过程不超过10min。
5. 3个离心瓶每瓶分别加入112.5mL预冷的Solution 3，充分轻摇至沉淀散开，此时可见白色沉淀。
6.配平后5000rpm，20min，4度离心。用试剂盒中的CSI过滤器将上清滤入国产离心瓶中。
7.把进口瓶洗净、晾干，将国产瓶中滤液转移至进口瓶。
8. 3个离心瓶每瓶分别加入210mL异丙醇，颠倒约20下充分混匀。-20度沉淀1h以上。
9.将上面所得溶液5000rpm，20min，4度离心，倒掉上清。
10.向一个瓶子中加入60mL P1，剧烈混匀后，分别量取30mL加入到另外两个离心瓶中，即得到2个分别装有30mL P1的离心瓶，剧烈摇晃使沉淀溶解。
11. 37度静置10min.2个离心瓶每瓶分别加入30mL P2，温和颠倒数次，静置7-9min。
12. 2个离心瓶每瓶分别加入30mL P2，温和颠倒数次至溶液出现白色分散絮状沉淀，静置7-9min。
13. 5000rpm，10min，4度离心。
14.用试剂盒中的CSI过滤器将上清滤入1个离心瓶中。
15.加入19mL红色的去内毒素溶液ER，颠倒混匀。
16.加入60mL异丙醇，充分混匀，-20度沉淀1h以上。
17.柱平衡：取6个吸附柱，每个加入2.5mL BL，8000rpm，2min离心，倒掉废液。(角转子，圆底，用平衡液处理过的吸附柱最好立即使用)。
18.过柱：6个吸附柱每个吸附柱分别倒入10mL液体，8000rpm，2min离心，倒掉废液，至全部过滤完。
19. 6个吸附柱每个吸附柱分别倒入10mL缓冲液ED，8000rpm，2min离心，倒掉废液。
20. 6个吸附柱每个吸附柱分别倒入10mL漂洗液PW(提前加入无水乙醇)，8000rpm，2min离心，倒掉废液。
21.重复20。
22.每个吸附柱分别加入2mLddH2O，静置5min，7000rpm，2min离心。将液体重新倒回吸附柱中再离一次。
23.将液体混匀，测浓度，-20度保存。
(5)LLC原位肺癌模型：
为了产生原位肺癌模型，我们通过尾静脉静脉注射5×10 6LLC细胞到裸鼠中。30天后，使用非侵入性Micro-CT扫描监测小鼠以确保在肺中成功地形成肿瘤。然后，将荷瘤小鼠随机分为4组：每2天静脉注射PBS或5mg/kg CMV-scr R或CMV-siR E基因环路，1组5mg/kg吉非替尼灌胃，共治疗7次。治疗过程为期2周。
由于需要在特定时间点处死小鼠，用于取组织进行分子生物学分析，因此对于植瘤成功的小鼠进行随机分组，并用于评估存活时间和肿瘤进展。用于存活分析的小鼠，在治疗后一直监测小鼠而不进行任何进一步处理。对于肿瘤进展分析，仅使用Micro-CT分析在2周治疗期结束后仍存活的小鼠。在Micro-CT扫描后，处死小鼠，取肺组织，并使用组织病理学染色和免疫组织化学方法进行分析，进而采用。
(6)KRASLSL-G12D；p53fl/f1转基因肺癌模型：
1.将6周龄的KRASLSL-G12D；p53fl/f1小鼠用适量的5％水合氯醛麻醉。
2.按每只小鼠5×106PFU的用量吸取表达Cre的腺病毒Adeno-Cre，每只小鼠50μL体积用PBS稀释后备用。
3.在小鼠颈部外皮褪毛，沿着颈部腹面中轴纵切小口，暴露主气管。
4.用弯头镊子固定气道位置，并引导动脉监测针经口腔插入气管后，注射器推入腺病毒稀释液。
5.缝合外皮，并用红霉素软膏处理伤口，以防感染。
利用该方法，可以将Adeno-Cre准确、定向输送至小鼠肺部，而不会滞留在口腔和呼吸道。在吸入后不同时间(30,40和50天)用micro-CT监测确保肿瘤形成。在Adeno-Cre施用50天后，将小鼠随机分成两组，并通过尾静脉用5mg/kg CMV-scrR或CMV-siRK治疗2周(7次注射)。然后，监测小鼠以确定存活时间或评估肿瘤生长。
(7)小动物Micro-CT监测肺部肿瘤进展情况：
本文采用小动物Micro-CT分析的方式评估肺部肿瘤生长，因为即使没有任何造影剂，Micro-CT图像也清楚地区分肺肿瘤与周围组织，并且重建的3-D肺图像能更直观地反应出肿瘤在肺组织中的实际位置。使用Bruker公司 SkyScan 1176型Micro-CT分析仪进行Micro-CT扫描，该分析仪以35μM的分辨率扫描180°区域，旋转步长为0.800。该系统包括两个金属陶瓷管，配有固定的0.5毫米铝过滤器和两个1280×1024像素的数字X射线摄像机。在50kV和500μA下获得X射线图像。在仰卧位置扫描小鼠。
根据制造商(Bruker公司)的使用说明，使用N-Recon程序对微CT数据进行批量分类，处理和重建。随后使用DataViewer对重建的数据进行成像，辨别并鉴定出肿瘤位置后，进一步使用CTan程序计算肿瘤体积，利用CTVol程序完成全肺重构。
(8)外泌体的分离：
从小鼠收集静脉血样并置于血浆分离管中。在室温下使用800×g离心10分钟分离血浆，并在室温下以10,000×g离心15分钟除去细胞碎片。回收上清血浆，并使用Total Exosome Isolation试剂盒根据制造商的说明分离外泌体。
实施例1 不同可替换元件的验证以及完整质粒的干扰效率检测
分别构建了针对EGFR、TNC基因的质粒分子，将启动子元件与siRNA表达元件串联，构建了CMV-siR E并分别连入骨架载体(图1)，将质粒分子转染小鼠肺癌细胞系LLC中，36小时后利用qRT-PCR和Western blotting实验检测细胞中EGFR基因的mRNA(图2A-B)和蛋白质(图2C)表达水平。将启动子元件、靶向元件与针对EGFR、TNC基因的2种siRNA表达元件串联，构建了CMV-RVG-siR E+T(图1)，将质粒分子转染胶质母细胞瘤细胞系U87MG，36小时后利用qPCR和western实验检测细胞中EGFR、TNC基因的mRNA(图2D-E)和蛋白质(图2F)表达水平。结果表明，人工构建的siRNA表达质粒分子能够在细胞系中有效抑制各自基因的表达。
实施例2 质粒表达的siRNA在肝脏表达以及体内分布情况
将表达siRNA的质粒按照10mg/kg的剂量对正常小鼠进行尾静脉注射；分别在1、3、6、9、12、24、48小时后，处死小鼠，取小鼠的肝、肺、肾、脾、脑、心脏、胰腺、肌肉、CD4 +T细胞等组织分别检测siRNA水平。结果表明，在小鼠的肝脏组织中有大量siRNA的分布(图3A)，表达siRNA的元件仅能在肝脏中检测到。在血浆中能检测到siRNA(图3C)，且主要以微囊泡包裹的形式存在。而在肺、肾、脾、胰腺和CD4 +T细胞中均能检测到大量的siRNA表达 (图3B)，在其他组织中的表达量较低或无信号。
实施例3 质粒分子对肺部肿瘤模型的治疗效果
为了进一步确认质粒在体内的治疗效果，利用LLC肺癌原位植瘤小鼠模型作为实验对象，确认CMV-siR E质粒对肺部肿瘤的治疗效果。我们将原位植瘤成功的小鼠随机分为4组，分别按照10mg/kg的剂量注射PBS、对照质粒、CMV-siR E质粒以及灌胃吉非替尼药物。每两天给药一次，共治疗两周，治疗前后分别利用CT成像的方式检测肺部肿瘤变化情况(图4A)，并统计小鼠的生存情况。结果表明，在治疗前后，注射CMV-siR E质粒组的小鼠肺部肿瘤体积明显减小，部分小鼠中完全消失，而其他三组小鼠肿瘤均显著增大(图4B)。同时注射CMV-siR E质粒组小鼠生存期得到显著延长(图4C)。
进而，研究了CMV-siR K质粒对KrasG12D；p53fl/fl转基因肺癌小鼠模型的治疗效果，验证siRNA表达元件的可替换性。我们利用CT成像验证该转基因小鼠造模成功后，将小鼠随机分为3组，分别按照10mg/kg的剂量注射PBS、对照质粒(CMV-scrR)和CMV-siR K质粒。每两天给药一次，共治疗两周，治疗前后分别利用CT成像的方式检测肺部肿瘤变化情况(图5A)，并统计小鼠的生存情况。结果表明，在治疗前后，注射CMV-siR K质粒组的小鼠肺部肿瘤进展得到明显缓解，在部分小鼠中完全消失，而其他2组小鼠肿瘤均显著增大(图5B)。同时注射CMV-siR K质粒组小鼠生存期得到显著延长(图5C)。
实施例4 带有靶向肽元件的双siRNA质粒分子对胶质母细胞瘤小鼠模型的治疗效果
由于仅有启动元件和siRNA表达元件的质粒无法将siRNA有效递送至脑部，将表达狂犬病病毒肽段RVG的元件整合到质粒载体中，同时，为了达到更佳的抑制效果，将抑制EGFR基因和TNC基因的siRNA表达元件共同整合至质粒载体中，构建了CMV-RVG-siR E+T质粒分子，通过检测siRNA在脑组织中的表达情况(图6A-B)，证明了这样的设计能够有效将siRNA递送至脑组织。然后，利用小鼠胶质母细胞瘤原位模型，验证了该质粒的治疗效果。将小鼠随机分为3组，分别按照10mg/kg的剂量注射PBS、对照质粒(CMV-scrR)和CMV-RVG-siR E+T质粒，每两天给药一次，共治疗两周，通过活体成像跟踪检测肿瘤体积(图6C)。结果证明注射CMV-RVG-siR E+T质粒能够有效抑制质母细胞瘤，实验组小鼠肿瘤体积 显著减小，生存时间得到了明显的延长(图6D-E)。
实施例5 体内安全性检测
为了检测该治疗手段的安全性，我们又检测了对照组小鼠和注射质粒分子的实验组小鼠血清中谷丙转氨酶(图7A)、谷草转氨酶(图7B)、总胆红素(图7C)、尿素(图7D)、碱性磷酸酶(图7E)和肌酐(图7F)等生化指标的水平。结果显示，注射质粒分子的实验组小鼠上述指标与对照组没有明显差异，对肝、肺、肾、脾的切片表明尾静脉注射质粒不会造成组织损伤，是一种比较安全的给药方式(图7G)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种siRNA组合物，其特征在于，包括：
    降低第一靶基因的表达的第一siRNA分子；
    任选的，靶向肽元件的编码序列；和
    任选的，降低第二靶基因的表达的第二siRNA分子；
    其中所述第一靶基因选自下组：EGFR、KRAS、或其组合，并且所述siRNA组合物降低两个或更多个基因的表达。
  2. 如权利要求1所述的siRNA组合物，其特征在于，所述第二靶基因选自下组：EGFR、TNC、或其组合。
  3. 如权利要求1所述的siRNA组合物，其特征在于，所述第一靶基因和第二靶基因不同。
  4. 如权利要求1所述的siRNA组合物，其特征在于，所述第一siRNA分子具有如SEQ ID NO.:1或2所示的序列。
  5. 如权利要求1所述的siRNA组合物，其特征在于，所述第二siRNA分子具有如SEQ ID NO.:3所示的序列。
  6. 一种载体，其特征在于，包括：
    启动子元件；
    降低第一靶基因的表达的第一siRNA分子；
    任选的，靶向肽元件的编码序列；和
    任选的，降低第二靶基因的表达的第二siRNA分子；其中所述第一靶基因选自下组：EGFR、TNC、KRAS、或其组合，并且所述siRNA分子降低两个或更多个基因的表达。
  7. 如权利要求6所述的载体，其特征在于，所述载体具有5’－3’的式I所示的结构：
    Z0-Z1-Z2-Z3(I)
    其中，Z0为启动子元件；
    Z1为任选的靶向肽元件的编码序列；
    Z2为降低第一靶基因的表达的第一siRNA分子；
    Z3为任选的降低第二靶基因的表达的第二siRNA分子。
  8. 如权利要求6所述的载体，其特征在于，所述第二靶基因选自下组：EGFR、TNC、KRAS、或其组合。
  9. 一种权利要求1所述的siRNA组合物或权利要求6所述的载体的用途，其特征在于，用于制备治疗癌症的药物或制剂。
  10. 一种药物制剂，其特征在于，所述药物制剂含有：
    (a)权利要求6所述的载体；和
    (b)药学上可接受的载体。
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