CN103055325A - Specific gene-virus therapeutic drug for colorectal cancer - Google Patents

Abstract

The invention discloses a specific gene-virus therapy drug for colorectal cancer. Using adenovirus, the natural promoter of the early gene E1A is replaced by the colorectal cancer-specific promoter CEA to form an oncolytic adenovirus, which carries the colorectal cancer-specific anti-cancer gene ST13. If it is not strong enough to basically eliminate all cancers, the following genes can be added to the same vector to share with it, such as TRAIL, IL-24, MnSOD, CD, Smac, GM-CSF, IFN, IL-12, p53, RNAi , microRNA, Caspase 3 and Bax, or linking two genes with various schemes. The invention is a drug for colorectal cancer-specific gene-virus therapy, which has a high therapeutic effect targeting colorectal cancer and basically has no effect on normal cells.

Description

Specific gene-viral therapy for colorectal cancer

technical field

The invention belongs to the field of Cancer Targeting Gene-Viro-Therapy (CTGVT), in particular to Cancer Targeting Gene-Viro-Therapy Specific for Colorectal Cancer. CTGVT-CRC), and more specifically, a colorectal cancer-specific gene-viral therapy.

Background technique

From 1999 to 2001, Liu Xinyuan created a cancer treatment strategy called Cancer Targeting Gene-Viro-Therapy (CTGVT), which is to insert anti-cancer genes into oncolytic virus , OV), so CTGVT strategy is OV-(gene) strategy or Gene Armed Oncolytic Virus Therapy (GAOVT) strategy, the latter is also OV-(gene), CTGVT (GAOVT) combines the advantages of gene therapy and oncolytic virus therapy Combined, because the oncolytic virus itself has an anti-cancer effect, it may specifically replicate hundreds of times in cancer cells, and the anti-cancer gene inserted in it can also replicate hundreds of times, so its anti-cancer effect is greatly improved. It is much better than the corresponding single gene therapy, and also much better than the corresponding single oncolytic virus therapy, so it has become an international hot topic now. Although gene therapy was rated as one of the top ten scientific achievements in the world by Science in 2009, it is the result of genetic diseases with single gene deficiency. For complex diseases such as cancer with multiple gene mutations, the resistance of gene therapy The anti-cancer effect of CTGVT (GAOVT) is far inferior to that of CTGVT (GAOVT). The gene therapy is Ad-(gene), and the carrier Ad used in it has no cancer targeting and no replication ability in cancer cells (that is, non-replicating Ad). Its anticancer effect is far inferior to that of CTGVT.

As shown in Figure 5, the adenovirus has early genes E1, E2, E3, E4 and late genes L1, L2, L3, L4, L5. Among them, E1 can be divided into E1A and E1B. The most important one is E1A, followed by E1B. The natural promoter of E1A is replaced by a colorectal cancer-specific promoter, so the adenovirus can only infect and replicate in colorectal cancer cells.

Oncolytic virus (OV) refers to a virus that is specifically expressed and infected and replicated in tumors. It has cancer cell targeting and can replicate, but it is not specific for colorectal cancer. Any virus (Adenovirus (adenovirus), Simplex Herpes Virus (HSV-1) (herpes virus), Poxvirus (vaccinia virus)) can be formed into OV after modification. The gene carried by the oncolytic virus is OV-gene, but Ad-gene is gene therapy (Ad has no targeting and no ability to replicate).

Contents of the invention

In the construction and application of CTGVT, that is, OV-(gene), OV (Oncolytic Virus) mainly determines its targeting, which can be constructed by various methods to make it produce different targeting. This application only involves specific The field of targeted colorectal cancer aims to provide a colorectal cancer-specific gene-viral therapy drug, CTGVT-CRC and its application.

Gene mainly determines the lethality, and the killing effect of light OV is limited, so it is necessary to add a killing gene to enhance its anticancer effect to form OV-(gene), that is, CTGVT (GAOVT), to form a strong anticancer drug, which is also effective against CTGVT. More improvements are needed, such as the combination of two CTGVTs, which has a stronger anticancer effect and can often basically eliminate all transplanted tumor genes. In addition, if cancer stem cells are specifically targeted, there is the possibility of eradicating cancer and eradicating it completely.

The present invention specifically adopts the following technical solutions:

In the present invention, the natural promoter of E1A itself is replaced by the colorectal cancer-specific promoter CEA, so that the adenovirus becomes an oncolytic adenovirus specific for colorectal cancer, which can be abbreviated as (CRC)·OncoAd, which is a The OV vector targeting colorectal cancer is the fundamental requirement of this patent. In addition, the expression of E1B can also be modified. If 55KD is deleted, it will be E1B (Δ55). Δ means deletion, which is called ZD55. Written as D55; E1B has two genes, namely 19KD and 55KD genes, if these two genes are double-deleted, then it is ΔE1B. The natural promoter of Ad·E1B can also be replaced by HIF (hypoxia-inducible factor) to form Ad·HIF·E1B. ).

As for genes, for colorectal cancer, or add colorectal cancer-specific tumor suppressor genes, such as ST13, to form Ad·CEA·E1A·D55-(ST13), if the anti-cancer ability is not strong enough, it can also be added in the same An anti-cancer gene such as TRAIL with strong anti-cancer effect but no specificity is added to the carrier to form Ad·CEA·E1A·D55-(TRAIL) and combined with the above-mentioned CTGVT-CRC with ST13, a better anti-cancer effect can be obtained. cancer effect. In addition to TRAIL, IL-24, MnSOD, Smac, GM-CSF, IFN, IL-12, p53, RNAi, microRNA, Caspase 3, Bax, etc. can also be used. When using two genes, one of them must be colorectal Specific anti-cancer genes, two genes can be constructed by the following method: A. use two recombinants, each with an anti-cancer gene; B. use two gene expression cassettes to express in one recombinant; C. two Genes are connected with linkers. Such as F·2A or IETD four amino acids.

In the first aspect of the present invention, there is provided a colorectal cancer-specific gene-viral therapy drug, that is, CTGVT-CRC, which is characterized in that the drug inserts a colorectal cancer-specific anti-cancer gene into the colorectal produced from cancer-specific oncolytic viruses.

The oncolytic virus is an oncolytic adenovirus, the early gene E1 is divided into E1A and E1B, and the promoter of E1A is replaced by a colorectal cancer-specific promoter.

According to the present invention, the colorectal cancer-specific promoter is the colorectal cancer-specific promoter CEA or other colorectal cancer-specific promoters.

According to the present invention, firstly, the specific anti-cancer gene of colorectal cancer, such as ST13, can also increase other anti-cancer genes with good anti-cancer effect, such as TRAIL, IL-24, MnSOD, CD, Smac, GM-CSF, IFN , IL-12, p53, RNAi, microRNA, Caspase 3, or Bax, etc., use two genes, but there must be a colorectal-specific gene such as ST13.

If it is two anti-cancer genes, they can be used in combination with each other in different ways, and the ways are:

A. Use two recombinants;

B. Expression cassettes with two genes in one recombinant; or

C. The two genes are connected with a linker and added to a vector (that is, one expression). The linker can be four amino acids of F·2A or IETD.

According to the present invention, the E1B can be transformed as follows:

(1), the natural promoter of E1B is replaced by HIF;

(2) Delete 55KD in E1B;

(3) Two genes 19KD and 55KD in E1B were deleted (ΔE1B).

The invention is a drug for colorectal cancer-specific gene-virus therapy, which has a high targeted anti-colorectal cancer therapeutic effect and basically has no effect on normal cells.

Description of drawings

Figure 1A shows the schematic diagram of the construction principle of Ad·(ST13)·CEA·E1A(Δ24).

Figure 1B shows the specific construction process of Ad·(ST13)·CEA·E1A(Δ24).

Figure 1C is the identification of Ad·(ST13)·CEA·E1A(Δ24), by PCR, using primers at both ends of the CEA promoter to obtain 424bp.

Figure 1D is the identification of Ad·(ST13)·CEA·E1A(Δ24), by PCR, using primers at both ends of ST13 to obtain 1110bp.

Figure 2A shows the construction principle of CTGVT-CRC with double genes.

Figure 2B shows the specific construction process of CTGVT-CRC with double genes.

Figure 3A shows the dose-dependent in vitro anticancer effect of Ad·(ST13)·CEA·E1A(Δ24).

Figure 3B shows the time-dependent in vitro anticancer effect of Ad·(ST13)·CEA·E1A(Δ24).

Figure 4A shows the anticancer effects of various substances, and Figure 4B shows the survival rate of mice in the cancer model.

Figure 5 is a map of the adenovirus genome.

specific implementation plan

CTGVT-CRC is a colorectal cancer-specific oncolytic adenovirus (CRC)-OncoAd carrying a colorectal cancer-specific anti-oncogene (CRC)-gene, namely (CRC)·OncoAd——(CRC)-gene.

(CRC)·OncoAd is constructed by replacing the natural promoter of Ad·E1A with the colorectal cancer-specific promoter CEA (Carcinoma Embryonic Antigen), that is, Ad·CEA·E1A, in which E1A can also be modified. In this patent E1A Ad 923-946 bases are deleted by 24bp, targeting Rb-deleted tumors. As for Ad’s E1B, any modification (ie ∽E1B) or no modification is acceptable. ∽E1B can target cancer cells, but has no specificity Sex, Ad·CEA(Δ24)·E1B. The genes carried by Ad·CEA·E1A·E1B are colorectal cancer-specific anti-cancer genes (such as ST13), etc. The overall result is Ad·(ST13)·CEA·E1A(Δ24)·E1B, and the genes are in brackets Indicates the expression box of this gene (Δ24 means deletion of 24bp, non-gene, so the brackets are not the expression box). If the anti-cancer is not ideal, common anti-cancer genes with strong anti-cancer effects can be used, such as TRAIL, which constitutes Ad·CEA·E1A·E1B-(TRAIL), which is shared with the above-mentioned CTGVT-CRC with ST13. Also called CTGVT-CRC. In addition, in addition to TRAIL, other genes such as MnSOD and IL-24 can also be used. In addition, the two genes can also be joined by a linker (TRAIL-Linker-ST13) as a gene added to (CRC) · OncoAd, then become Ad · CEA · E1A · ∽E1B (TRAIL-Linker-ST13). Figure 2 uses TRAIL to enhance its killing effect, Ad·CEA·E1A·E1B(Δ55)-(TRAIL-IETD-ST13).

The present invention will be described in further detail below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed.

1. Construction example of CTGVT-CRC

Example 1:

Figure 1A: Schematic diagram of the construction principle of p Ad·(ST13)·CEA·E1A(Δ24)

Delete 24bp (deleted from 923-946bp) in the E1A of the adenovirus, and replace its natural promoter with the CEA promoter for regulation, and add a ST13 expression cassette (ST13) before CEA.

Figure 1B: The specific steps and molecular structure of Ad·(ST13)·CEA·E1A(Δ24) construction.

(1) Insert the CEA promoter into the plasmid pAd·E1A(Δ24) double-digested with XhoI and SnaB I to construct plasmid pAd·CEA·E1A(Δ24);

(2) Digest pCA13-ST13 (commercially available) with EcoRV and HindIII, cut off the ST13 gene, and connect it to the pMD18-T Simple-HCMV-MCS-polyA (SV40) vector cut with the same enzyme to form pMD18- T Simple-HCMV-ST13-polyA(SV40) (Note: pMD18-T Simple has commercial products).

(3) Digest pMD 18-T Simple-HCMV-ST13-polyA(SV40) with SalI to obtain the "HCMV-ST13-polyA(SV40)" expression cassette; insert it into pAd·CEA·E1A(Δ24) with XhoI Among the cleavage sites, constituting pAd ST13 CEA E1A (Δ24) (pMD18-T Simple is commercially available), since SalI and XhoI are homologous enzymes, the two can be ligated, but the cleavage site disappears after ligation.

(4) The purified pAd·(ST13)·CEA·E1A(Δ24) plasmid and the adenovirus large plasmid pBHGE3 were recombined in HEK293 to construct Ad·(ST13)·CEA·E1A(Δ24).

(5) After the successful construction of the recombinant virus, the viral DNA was extracted and used after identification and construction.

Example 2: Construction of Ad·CEA·E1A·E1B(Δ55)-(TRAIL-IETD-ST13) (Figure 2)

Figure 2A: Schematic diagram of the construction principle of Ad·CEA·E1A·E1B(Δ55)-(TRAIL-IETD-ST13)

Figure 2B: The specific construction process of Ad·CEA·E1A·E1B(Δ55)-(TRAIL-IETD-ST13)

(1) pZD55 was cut with XhoI-SnaB1, and CEA with the same restriction site was loaded into it to obtain pZD55·CEA promoter (9234bp);

(2) The 9234bp plasmid was cut with XhoI-MfeI and joined with pSW (6568bp) digested with the same enzyme to obtain pSW·CEA·E1A·E1B(Δ55) (9033bp) (pSW was cut with XhoI-MfeI by the pShuttle of the existing commercial product Open, made by adding E1A·E1B);

(3) Cut pCA13·TRAIL-IETD-ST13 with BglII and load it into a 9033bp plasmid to obtain pSW·CEA·TRAIL-IETD-ST13, namely pAd·CEA·E1A·E1B(Δ55KD)-(TRAIL-IETD-ST13 )(11582bp) (Δ in the first bracket means deletion of 55KDa gene, non-expression box, the last bracket is expression box);

(4) After recombining the purified pSW·CEA·E1A·E1B(Δ55KD)-(TRAIL-IETD-ST13) with the large adenovirus plasmid pBHGE3 in Bj5183 bacteria, they were then transferred to HEK293 cells for packaging, and finally Ad· CEA·E1A·E1B (Δ55KD)-(TRAIL-IETD-ST13) virus.

(5) After the successful construction of the recombinant virus, the viral DNA was extracted and used after identification and construction.

2. Application examples of CTGVT-CRC

Example 3: In vitro (invitro) anticancer effect of Ad·(ST13)·CEA·E1A (Δ24) (Figure 3A and Figure 3B)

Figure 3A: Dose-dependent anticancer effect in vitro

Infect ONYX-015, Ad·(EGFP)·CEA·E1A(Δ24) and Ad·(ST13)·CEA·E1A(Δ24) with the following different doses of virus (MO1 0.1, 1.0, 5.0), respectively.

Four days later, it was detected by MTT method, and the result was the mean ± SD of three detections.

The results can be seen from Figure 3A, anticancer drugs have a greater killing effect on different colorectal cancer cells SW620, HT116, HT29 (Figure 3A), while all drugs have no killing effect on normal cell groups QSG7701, WI38 (Figure 3A ), the killing effect on Hela is less than that of colorectal cancer.

Figure 3B: Time-dependent in vitro anticancer effect

Inoculate cancer cells and normal cells on a 24-well plate (the number of cells is 5-10×10 ³ ), when the cells grow to near saturation (eg 80% saturation) (saturation means that the cells cover the entire well). For different cells: including normal control cells QSG7701, WI38 and non-CRC control cells Hela (cervical cancer), colorectal cancer cells SW620, HT116, HT29, after being infected with 10 MOI of different viruses for 24, 48, 72, and 96 hours, respectively.

The results can be seen from Figure 3B, anticancer drugs have greater killing effect on different colorectal cancer cells SW620, HT116, HT29 (Figure 3B), while all drugs have no killing effect on normal cell groups QSG7701, WI38 (Figure 3B ), the killing effect on Hela is less than that of colorectal cancer.

Example 4: In vivo (in vivo) anticancer effect of Ad·(ST13)·CEA·E1A(Δ24) (Figure 4)

4-week-old female nude mice (nude mice) were selected and purchased from Shanghai Experimental Animal Center. All experimental operations were in accordance with the guidelines of the National Institutes of Health for the protection and use of experimental animals. 3×10 ⁶ SW620 colorectal cancer cells were used In 150μl DMEM culture, subcutaneously inoculate the dorsal side of each mouse, wait for the tumor to grow to 80-150mm ³ , divide the mice into 4 groups randomly, PBS and other drug groups, such as ONYX-015, Ad·(AGFP) ·CEA·E1A·D55, Ad·(ST13)·CEA·E1A(Δ24) treatment group, intratumoral injection treatment once a day, 0.5×108 pfu each time for 4 consecutive times, the total dose of each group was 2×10 ⁹ pfu, Then the length and width of the tumor were measured weekly with a caliper ruler, and the size (V) of the tumor was calculated according to the following formula, V=1/2 length×width ² .

The results are shown in Figure 4A and Figure 4B, Figure 4A and Figure 4B show the in vivo (in vivo) anticancer ability of Ad·(ST13)·CEA·E1A (Δ24), wherein Figure 4A shows the anticancer effect of various substances , the abscissa is time, and the ordinate is the size of the tumor volume. The smaller the tumor size, the stronger the anticancer effect, and the order of anticancer strength is: Ad·(ST13)·CEA·D55-(ST13)＞Ad·(EGFP)·CEA·D55>ONYX-015>>PBS; Fig. 4B is the survival rate of the mice. In the Ad·(ST13)·CEA·E1A(Δ24) group, a single nude mouse did not die, but in the PBS group, almost all mice died quickly, leaving only one mouse alive.

Claims (6)

1. a colorectal cancer-specific gene-viral therapy drug, i.e. CTGVT-CRC (Colorectal Cancer), is characterized in that, the medicine is that the colorectal cancer-specific anticancer gene is inserted into the colorectal cancer-specific lysate produced from tumor viruses. 2 . The colorectal cancer-specific gene-viral therapy drug according to claim 1 , wherein the colorectal cancer-specific promoter can be CEA or other colorectal cancer-specific promoters. 3. The colorectal cancer-specific gene-viral therapy drug according to claim 1, characterized in that the anti-cancer gene is colorectal cancer-specific ST13. 4. The specific gene-viral therapy drug for colorectal cancer as claimed in claim 3, if the anti-cancer ability is not strong enough, other following anti-cancer genes can be added to the same carrier to share with it: Such as TRAIL, IL-24, MnSOD, CD, Smac, GM-CSF, IFN, IL-12, p53, RNAi, microRNA, Caspase 3, or Bax. 5. The specific gene-viral therapy drug for colorectal cancer as claimed in claim 4, when using two anti-cancer genes, it can be used in combination in different ways, and its mode is: A. Use two recombinants; B. Expression cassettes with two genes in one recombinant; or C. The two genes are joined together with a linker and added to a vector. The linker can be four amino acids of F·2A or IETD. 6. The specific gene-viral therapy drug for colorectal cancer as claimed in claim 1, wherein E1B is modified as follows: (1), the natural promoter of E1B is replaced by HIF; (2) Delete 55KD in E1B; (3) Deletion of two genes 19KD and 55KD in E1B at the same time.

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