CN116286821A - CRISPR/Cas13a system-based crRNA for specifically targeting IDH1R132H gene and application thereof - Google Patents

Abstract

The invention provides CRRNA based on CRISPR/Cas13a system specificity targeting IDH1R132H gene and application thereof, wherein the sequence of the CRRNA is shown as SEQ ID NO:14, the crRNA specifically targets the IDH1R132H gene. The CRISPR/Cas13a gene editing system-based targeting glioma key variant gene IDH1R132H realizes in-vivo molecular diagnosis and discovers that the gene has the effect of inhibiting intracranial tumor formation. In addition, the invention integrates the report RNA with the CRISPR/Cas13a system to realize high-sensitivity diagnosis.

Description

CRISPR/Cas13a system-based crRNA for specifically targeting IDH1R132H gene and application thereof

Technical Field

The invention belongs to the field of gene detection, and particularly relates to CRRNA based on CRISPR/Cas13a system specific targeting IDH1R132H gene and application thereof.

Background

An advantage of CRISPR/Cas-based biological detection is that RNA can be specifically recognized and nuclease activity initiated. The specificity of detecting target RNA is achieved by designing CRISPR RNA (crRNA) sequence to be complementary to the target RNA sequence. After recognition and binding of the target RNA to the crRNA, the single stranded RNA (ssRNA) cleavage capability of CRISPR/Cas13a is activated. The CRISPR/Cas13a can distinguish single base mismatch, which shows the remarkable induction precision of the system. Ultrasensitive and low cost RNA diagnostics are now highly desirable because immediate and accurate detection of RNA can be effective in monitoring and controlling outbreaks of infectious diseases as well as early detection of other pathological conditions, including cancer. After the Cas13a is correctly combined with the target RNA sequence, the nonspecific cleavage characteristic of the Cas13a is activated, so that a fluorescent reporter gene in a cleavage system is cleaved, and the conversion of the sequence information to be detected from a fluorescent signal is realized. Cas13 a-based diagnostic methods have been effectively applied to detect avian influenza a (H7N 9) virus, ebola virus, hepatitis B Virus (HBV), SARS-CoV-2 and miRNA. Therefore, the CRISPR/Cas13a system can be used as a diagnostic tool for rapidly detecting nucleic acid, can be used for early diagnosis of tumor-related diseases, has the advantages of low cost and easy development and use, is short in time consumption and high in sensitivity, and has great potential in detection of multiple target molecules.

Currently, IDH1/2 mutation is considered to be the earliest event in glioma onset, and is also one of the most important genetic changes in glioma biology. The development of a novel method for detecting glioma RNA with high sensitivity, accuracy and convenience is not only beneficial to glioma research, but also can be used as a glioma diagnostic tool.

Disclosure of Invention

In view of this, the present invention aims to overcome the defects in the prior art, and proposes a crRNA specifically targeting IDH1R132H gene based on CRISPR/Cas13a system and application thereof.

In order to achieve the above purpose, the technical scheme of the invention is realized as follows:

in a first aspect, the invention provides a crRNA specifically targeting glioma, the sequence of which is as set forth in SEQ id no:14, the crRNA specifically targets the IDH1R132H gene.

In a second aspect, the invention provides a CRISPR/Cas13a gene editing system comprising a crRNA specifically targeted to glioma as described above.

Preferably, the CRISPR-Cas13a gene editing system further comprises a Cas13a protein and a reporter RNA comprising a single stranded RNA comprising a plurality of consecutive bases U and a fluorescent group and a quenching group.

Preferably, the sequence of the reporter RNA is: 5'-FAM-UUUUUUUUUUUUUUUUUUUU-BHQ-3'.

In a third aspect, the invention provides an application of the CRISPR/Cas13a gene editing system in preparation of a reagent for diagnosing glioma.

In a fourth aspect, the invention provides an application of the CRISPR/Cas13a gene editing system in preparing a medicine for treating glioma.

Compared with the prior art, the invention has the following advantages:

the CRISPR/Cas13a gene editing system-based targeting glioma key variant gene IDH1R132H realizes in-vivo molecular diagnosis and discovers that the gene has the effect of inhibiting intracranial tumor formation. In addition, the invention integrates the reporter RNA with the CRISPR/Cas system to achieve high-sensitivity diagnosis.

Drawings

FIG. 1 is a graph of the Cas13a-crRNA14 trigger side effect of example 6 (FIG. 1A is the RNA integrity value measured on Agilent 2100; FIG. 1B is the 28S:18S ratio measured on Agilent 2100);

FIG. 2 is a graph showing the fluorescence change after transfection of different length reporter RNAs with the flow cytometer of example 8;

FIG. 3 shows the results of gel hysteresis test of cRGD-PP vector and plasmid DNA;

FIG. 4 is a graph showing the fluorescence and bioluminescence images of the cy5.5 of the nude mouse brain of example 11 (FIG. 4A is a graph showing the fluorescence and bioluminescence images of the cy5.5 of the living nude mouse brain containing the IDH1 mutation, FIG. 4B is a statistical result of the fluorescence and bioluminescence of the cy5.5 of the living nude mouse brain containing the IDH1 mutation, FIG. 4C is a graph showing the fluorescence and bioluminescence images of the cy5.5 of the isolated nude mouse brain containing the IDH1 mutation, FIG. 4D is a statistical result of the fluorescence and bioluminescence images of the cy5.5 of the isolated nude mouse brain containing the IDH1 mutation, FIG. 4E is a graph showing the fluorescence and bioluminescence images of the cy5.5 of the living nude mouse brain containing the FGFR3-TACC3 fusion mutation, FIG. 4F is a statistical result of the fluorescence and bioluminescence images of the cy5.5 of the isolated nude mouse brain containing the FGFR3-TACC3 fusion mutation, FIG. 4G is a statistical result of the fluorescence and bioluminescence images of the cy5.5 of the isolated nude mouse brain containing the FGFR3-TACC3 fusion mutation);

FIG. 5 is a graph of the results of intratumoral delivery inhibition of intracranial tumor formation by the CRISPR-Cas13a system of example 11 (FIG. 5A is representative bioluminescence images taken at days 7, 14 and 21 post-implantation of mice implanted with IDH1 intracranial tumor; FIG. 5B is representative quantified signal intensity taken at days 7, 14 and 21 post-implantation of mice implanted with IDH1 intracranial tumor; FIG. 5C is representative bioluminescence images taken at days 7, 14 and 21 post-implantation of mice implanted with FGFR3-TACC3 intracranial tumor; FIG. 5D is representative quantified signal intensity taken at days 7, 14 and 21 post-implantation of mice implanted with FGFR3-TACC3 intracranial tumor);

FIG. 6 is a graph of the survival of mice in example 11 (FIG. 6A is a graph of the survival of mice containing IDH1 mutations; FIG. 6B is a graph of the survival of mice containing FGFR3-TACC3 fusion mutations);

FIG. 7 is a graph showing the results of the immunohistochemical analysis of the sacrificed mice of example 11 (FIG. 7A is a representative image of H & E staining of intracranial tumor mice containing IDH1 mutation, FIG. 7B is a representative image of H & E staining of intracranial tumor mice containing FGFR3-TACC3 mutation, FIG. 7C is a representative image of immunohistochemical staining of intracranial tumor mice containing IDH1 mutation, FIG. 7D is a representative image of immunohistochemical staining of intracranial tumor mice containing FGFR3-TACC3 mutation, FIG. 7E is a statistical result of the immunohistochemical analysis of intracranial tumor mice containing IDH1 mutation, and FIG. 7F is a statistical result of the immunohistochemical analysis of intracranial tumor mice containing FGFR3-TACC3 mutation).

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the inventive concepts pertain. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to examples.

Example 1 design of crRNA

Following the design principle of the plagues, a crRNA library targeting specific fragments was designed, comprising 29 crrnas in total:

crRNA-1：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGACCUAUGAU GAUAGGUUUUACCCAUCC-3’(SEQ ID NO：1)；

crRNA-2：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGACCUAUGAUGAUAGGUUUUACCCAUC-3’(SEQ ID NO：2)；

crRNA-3：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUGACCUAUGAUGAUAGGUUUUACCCAU-3’(SEQ ID NO：3)；

crRNA-4：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGAUGACCUAUGAUGAUAGGUUUUACCCA-3’(SEQ ID NO：4)；

crRNA-5：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGAUGACCUAUGAUGAUAGGUUUUACCC-3’(SEQ ID NO：5)；

crRNA-6：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUGAUGACCUAUGAUGAUAGGUUUUACC-3’(SEQ ID NO：6)；

crRNA-7：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCAUGAUGACCUAUGAUGAUAGGUUUUAC-3’(SEQ ID NO：7)；

crRNA-8：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCAUGAUGACCUAUGAUGAUAGGUUUUA-3’(SEQ ID NO：8)；

crRNA-9：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAGCAUGAUGACCUAUGAUGAUAGGUUUU-3’(SEQ ID NO：9)；

crRNA-10：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAAGCAUGAUGACCUAUGAUGAUAGGUUU-3’(SEQ ID NO：10)；

crRNA-11：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUAAGCAUGAUGACCUAUGAUGAUAGGUU-3’(SEQ ID NO：11)；

crRNA-12：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUAAGCAUGAUGACCUAUGAUGAUAGGU-3’(SEQ ID NO：12)；

crRNA-13：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCAUAAGCAUGAUGACCUAUGAUGAUAGG-3’(SEQ ID NO：13)；

crRNA-14：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCAUAAGCAUGAUGACCUAUGAUGAUAG-3’(SEQ ID NO：14)；

crRNA-15：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCCAUAAGCAUGAUGACCUAUGAUGAUA-3’(SEQ ID NO：15)；

crRNA-16：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCCCAUAAGCAUGAUGACCUAUGAUGAU-3’(SEQ ID NO：16)；

crRNA-17：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUCCCCAUAAGCAUGAUGACCUAUGAUGA-3’(SEQ ID NO：17)；

crRNA-18：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUCCCCAUAAGCAUGAUGACCUAUGAUG-3’(SEQ ID NO：18)；

crRNA-19：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGAUCCCCAUAAGCAUGAUGACCUAUGAU-3’(SEQ ID NO：19)；

crRNA-20：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGAUCCCCAUAAGCAUGAUGACCUAUGA-3’(SEQ ID NO：20)；

crRNA-21：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUUGAUCCCCAUAAGCAUGAUGACCUAUG-3’(SEQ ID NO：21)；

crRNA-22：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACAUUGAUCCCC AUAAGCAUGAUGACCUAU-3’(SEQ ID NO：22)；

crRNA-23：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUAUUGAUCCC CAUAAGCAUGAUGACCUA-3’(SEQ ID NO：23)；

crRNA-24：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUAUUGAUCC CCAUAAGCAUGAUGACCU-3’(SEQ ID NO：24)；

crRNA-25：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUGUAUUGAU CCCCAUAAGCAUGAUGACC-3’(SEQ ID NO：25)；

crRNA-26：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUGUAUUGA UCCCCAUAAGCAUGAUGAC-3’(SEQ ID NO：26)；

crRNA-27：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUCUGUAUUG AUCCCCAUAAGCAUGAUGA-3’(SEQ ID NO：27)；

crRNA-28：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUCUGUAUUG AUCCCCAUAAGCAUGAUG-3’(SEQ ID NO：28)；

crRNA-29：

5’-GGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCUCUGUAUU GAUCCCCAUAAGCAUGAU-3’(SEQ ID NO：29)。

example 2 screening of optimal crRNA candidate sequences

The computer simulates the butt joint of crRNA and Cas13a protein and target RNA, calculates the binding energy of Cas13a-crRNA and Cas13a-crRNA-RNA complex, screens according to the binding energy, and the crRNA-14 with the smallest binding energy is regarded as the potential optimal crRNA candidate sequence screened by the computer because the smaller the binding energy is, the more stable the complex is.

Example 3 packaging of Cas13a lentiviruses

The Cas13a lentiviral expression vector was p GV341. And transferring a transfection system containing a plasmid expressing Cas13a, a packaging plasmid and the like into HEK293T cells by adopting a transient cotransfection method, so that the lentivirus is packaged in the cells. The packed lentiviral particles are secreted into the extracellular culture supernatant, and the supernatant is collected to obtain lentivirus.

EXAMPLE 4 Synthesis of crRNA

(1) DNA templates for crrnas were generated by PCR using T7 flanking primers. Primer extension was performed by heating at 95℃for 5 minutes to denature the reaction mixture, followed by quenching on ice for 10 minutes and incubation at 72℃for 30 minutes.

(2) T7 PCR products were transcribed in vitro using the T7sgRNA MICscriptTM kit (Biomics Biotech, jiangsu, china).

(3) The transcribed crRNA was purified using EzOmicsTM RNA Quick Clear kit (Biomics Biotech, jiangsu, china).

EXAMPLE 5 cell culture and transfection

Human glioma cells U87 cells and TBD0220 cells were cultured in DMEM and DMEM-F12 medium with 10% FBS, and 1% penicillin-streptomycin. Placed at 37℃and 5% CO ₂ Is replaced 1 time every 1 day. crRNA was transfected into cells with Lipofectamine R3000 (Invitrogen, california, usa).

Example 6 Agilent 2100 quality control verification side effects

The RNA extraction steps are as follows: (the whole course is as free of enzyme environment as possible)

(1) The cell culture medium was aspirated and discarded, 1 ml of Trizol was added to a 10 cm dish and lysed at room temperature for 5 minutes.

(2) 200ul of chloroform was added, mixed upside down, left at room temperature for 5 minutes and centrifuged at 12,000rpm for 20 minutes at 4 ℃.

(3) 200ul of isopropanol was added to the upper aqueous phase, and the mixture was left at room temperature for 20 minutes, at 12,000rpm, and centrifuged at 4℃for 20 minutes.

(4) The supernatant was aspirated, 75% ethanol was added, and the mixture was centrifuged at 12000rpm for 5 minutes at 4 ℃.

(5) The supernatant was aspirated, purged with absolute ethanol, discarded, air dried and dissolved in 10-20ul of DEPC water.

(7) 1ul of sample was taken and the read concentration values were measured with a nanodrop 2000.

The extracted RNA is detected on the Agilent 2100 quality control platform. The detection results are shown in fig. 1, and it can be seen from fig. 1 that Cas13a-crRNA14 of the present invention can trigger side effects and cause extensive RNA degradation.

Example 7 design of reporter RNA

The reporter RNA was synthesized in vitro by Ai Bosai biotechnology limited (Shanghai, china). The sequence is as follows:

5nt：FAM-UUUUU-BHQ；

10nt：FAM-UUUUUUUUUU-BHQ(SEQ ID NO：30)；

15nt：FAM-UUUUUUUUUUUUUUU-BHQ；(SEQ ID NO：31)

20nt：FAM-UUUUUUUUUUUUUUUUUUUU-BHQ；(SEQ ID NO：32)

example 8 flow cytometry detection of fluorescence changes after transfection of different Length reporter RNAs

(1) Cells were digested with pancreatin and resuspended with PBS.

(2) After the PBS was aspirated, the cells were resuspended in 70% ethanol and the single cell suspension was prepared by multiple pipetting and fixed at room temperature for 15min.

(3) Centrifugation at 2000rpm at room temperature for 5min and PBS washing 3 times.

(4) PI dye was diluted with PBS and cells were resuspended by addition of dye.

(5) And (5) detecting on the machine.

The detection results are shown in FIG. 2, and it can be seen from FIG. 2 that the optimal reporter RNA is 20nt in length by flow cytometry screening.

EXAMPLE 9Cas13a, crRNA plasmid amplification

(1) Transformation experiment: mu.l of the experimental plasmid to be amplified was aspirated and then placed in a main tube, one of which was about 100. Mu.l. After careful mixing with a pipette, the mixture was placed on ice for 15 minutes. After the ice bath was completed, the ice bath was placed at 42℃for thermal shock. The time is severely limited to 90 seconds.

(2) According to the experimental formula, the components of the solid culture medium are weighed out by an analytical balance, put into a flask, sterilized in an autoclave, when the temperature of the autoclave slowly reaches about 40 ℃, ampicillin with the concentration of 100 mug/ml is taken and put into a sterile culture dish, 100 mug of conversion products are added after solidification, a Z-shaped sample of a sterile glass rod is smeared back and forth for several times, the cover of the culture dish is covered, sealed by a preservative film, vertically fixed on a flat plate in a 37-DEG incubator for 1 hour, and then the culture dish is inverted and placed into the incubator for overnight culture.

(3) White colonies on solid medium were picked, added to ampicillin-containing medium, and the inoculating loop was moved to allow the colonies to completely mix with the liquid medium, sealed with sterile gauze, placed in an incubator at 37℃and cultured overnight with shaking.

(4) Plasmid extraction: a) 1.5 ml of the bacterial liquid was aspirated by a pipette, placed in an EP tube, and centrifuged at 12000rpm at 4℃for 30s. The supernatant was discarded and the pellet in the EP tube was completely dispersed with a shaker. b) Mu.l of solution I reagent (pre-chilled on ice) was added, mixed thoroughly by vortexing, and allowed to stand at room temperature for 5 minutes. c) 200ul of Solution II reagent was added (note: the reagents were prepared as-is), the centrifuge tube was shaken up and down several times and left at room temperature for 5 minutes. d) 150 μl of solution III reagent (the reagent must be pre-chilled in advance) was added to the kit, inverted and mixed for about 3-5 standing times of 5 minutes. e) Centrifuge 12000rpm at 4℃for 5min. Aspirate supernatant into another centrifuge tube. Equal amounts of phenol, chloroform and isoamyl alcohol (25:24:1) were added, inverted and mixed 5 times, and centrifuged at 12000rpm at 4℃for 3 minutes. f) The supernatant was aspirated into another centrifuge tube, 2.5 volumes of 95% glacial ethanol were added, and after thorough mixing, left at room temperature for 5 minutes. g) Centrifuge 12000rpm at 4℃for 20 min. The supernatant was aspirated, 1 ml of 70% ethanol was added, and the mixture was centrifuged at 12℃for 10 minutes at 1000rpm. h) The supernatant was discarded, the tube was placed at 37℃for 30 minutes, and the precipitate was allowed to dry naturally. An appropriate amount of TE was taken to dissolve the precipitate therein. Finally, 0.5mg of RNase was added from the kit and incubated at 37℃for 15 minutes to digest RNA in the centrifuge tube. Finally, the RNA concentration was measured by a Nanodrop2000 instrument.

Example 10 construction of Cas13a-FAM RNA fluorescence reporting System and gel hysteresis test

Plasmid DNA (pCas 13a and pCrRNA 14) and 20nt length of reporter RNA (0.05 mg/mL and 0.5mg/mL, respectively) obtained by the screening of example 8 were diluted in transfection buffer and mixed by vortexing at different weight ratios to construct Cas13a-FAM RNA fluorescence reporter system. The novel cation transfer vector cRGD-PEI-PBLG (cRGD-PP) was used as the delivery vehicle for the CRISPR/Cas13a system and after incubation for 10 min at room temperature, the 20 μl volume of the mixture was analyzed by 0.6% agarose gel electrophoresis (80 v,1 hour). As a result, as shown in FIG. 3, it was found from FIG. 3 that cRGD-PP was able to completely complex with DNA at a [ vector ]/[ DNA ] (wt/wt) ratio of 1.

cRGD-PP was wrapped around Cas13a, a 20nt length of the reporter RNA screened in example 8, and plasmids of crRNA14 for intratumoral delivery.

EXAMPLE 11 in vivo detection and evaluation of anti-tumor efficacy

Xenograft models of brain GBM patients were established in BALB/c nude mice. Cells of GBM patients were obtained with informed consent of the patients when they underwent surgery in a affiliated hospital at the university of hebrew. Tumor samples of the patient xenograft (PDX) model were collected, cultured with IDH1mut and F3-T3 overexpressing constructs, and then injected into animals. On day 7, GBM mice were intravenously injected with relevant formulations and prepared for bioluminescence imaging, as shown in fig. 4, it can be seen that mice injected with crRNA14 by tail vein exhibited a significant fluorescent signal compared to control group injected with crRNA14 in glioma mice model containing IDH1 mutation. The results obtained by analyzing the bioluminescence image of the isolated nude mice are consistent with the results obtained by analyzing the bioluminescence image of the living nude mice. This also gave consistent results in glioma mouse models containing FGFR3-TACC3 fusion mutations, confirming that the fluorescence reporting system can achieve in vivo molecular detection. Another group of animals followed brain tumor progression on days 7, 14 and 21 and analyzed by Kaplan-Meier survival curve, the results are shown in fig. 5 and 6, and it can be seen that the CRISPR-Cas13a gene editing system of the present invention can significantly inhibit tumor growth and prolong survival rate of treated mice. On day 21, after euthanasia of the mice, brain tumors were harvested and hematoxylin and eosin staining and immunohistochemical analysis were performed, and the results are shown in fig. 7, which shows that the CRISPR-Cas13a gene editing system of the present invention can significantly inhibit the growth of intracranial tumors caused by IDH1/2 gene mutation.

The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1. A crRNA that specifically targets glioma, characterized in that: the sequence is as shown in SEQ ID NO:14, the crRNA specifically targets the IDH1R132H gene.

2. A CRISPR/Cas13a gene editing system, characterized by: comprising the crRNA specifically targeting glioma.

3. The CRISPR/Cas13a gene editing system according to claim 2, characterized in that: the CRISPR/Cas13a gene editing system further comprises a Cas13a protein and a reporter RNA comprising a single stranded RNA of a plurality of consecutive bases U and a fluorescent group and a quenching group.

4. The CRISPR/Cas13a gene editing system according to claim 3, wherein: the sequence of the report RNA is as follows: 5'-FAM-UUUUUUUUUUUUUUU UUUUU-BHQ-3'.

5. Use of the CRISPR/Cas13a gene editing system of any one of claims 2-4 for the preparation of a reagent for diagnosing glioma.

6. Use of the CRISPR/Cas13a gene editing system of any one of claims 2-4 in the preparation of a medicament for treating glioma.

Images