Fast and Accurate Three-Plasmid Oncolytic Adenovirus Recombinant Packaging System AD5MIXPLUS and Application Thereof

Abstract

A fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system Ad5MixPlus and an application thereof are provided. The system is composed of three adenovirus recombinant plasmids. The core technology of the system is that two sets of different site recombination sequences are skillfully loaded on a first 5-type adenovirus right arm backbone plasmid large vector, then two small shuttle plasmids respectively provide a right arm-modified Hexon/E3/Fiber sequence and an E1a expression cassette controlled by a left arm tumor-specific promoter, and the difficulties and obstacles to the modification of the adenovirus backbone large vector are overcome. After two rounds of site-specific recombination, the ideal oncolytic adenovirus is packaged accurately and quickly.

Claims

1. A fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system, characterized in that the said fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system involving the following 3 adenovirus recombinant plasmids: a) adenovirus right arm backbone plasmid: the said adenovirus right arm backbone plasmid is loaded with two sets of recombinant sequences at different sites, one set of attL/attR in the Fiber/Hexon/E3 region, and the other set of Cre/loxP in the E1 region; the E3 region is also inserted the ccdB lethal gene of DB3.1 E. coli strain and competent cells; b) adenovirus right arm shuttle plasmid: the said adenovirus right arm shuttle plasmid contains the reconstructed chimeric Hexon sequence and chimeric Fiber sequence; the E3 region is preset with multiple cloning sites for exogenous gene insertion; Hexon/E3/Fiber sequence contains attL1/attL2 recombination sites at both ends; c) adenovirus left arm shuttle plasmid: the said adenovirus left arm shuttle plasmid is inserted tumor-specific promoter-controlled adenovirus early replication gene and loxP recombination site at its multiple cloning sites; wherein, the first round of attL/attR site-specific recombination is performed between the adenovirus right arm shuttle plasmid and the adenovirus right arm backbone plasmid based on attL1/attL2 at both ends of the Hexon/E3/Fiber sequence, resulting that the sequence between attL1/attL2 in the adenovirus right arm shuttle plasmid replaces the sequence between attR1/attR2 in the adenovirus right arm backbone plasmid; the second round of Cre/loxP site-specific recombination is performed between the adenovirus left arm shuttle plasmid and the adenovirus right arm backbone plasmid, resulting that the E1a expression cassette controlled by the tumor-specific promoter in the adenovirus left arm shuttle plasmid is inserted into the E1 region of the adenovirus right arm backbone plasmid; the required oncolytic adenovirus is packaged after the above two rounds of site-specific recombination.

2. The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of claim 1, wherein the chimeric Hexon sequence is a chimeric sequence formed by Hexon of Ad5 and Hexon or its mutant sequence of Ad48, Ad9, Ad37, Ad43 or any other serotype adenovirus.

3. The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of claim 1, wherein the sequence of the said chimeric Hexon is SEQ ID NO: 5.

4. The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of claim 1, wherein the chimeric Fiber sequence is a chimeric sequence formed by Fiber of Ad5 and Fiber or its mutant sequence of Ad11b, Ad3, Ad14, Ad16, Ad21, Ad35, Ad50, Ad55 or any other serotype adenovirus.

5. The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of claim 1, wherein the sequence of the said chimeric Fiber is SEQ ID NO: 6.

6. The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of claim 1, wherein the tumor-specific promoter is selected from: (a) promoter, enhancer and mutant sequence of carcinoembryonic antigen; (b) promoter, enhancer and mutant sequence of alpha fetoprotein; (c) promoters, enhancers and mutant sequences of receptor tyrosine kinases (including EGFR, Her-2, Her-3 and Her-4) of the human epidermal growth factor receptor family (EGFRs); (d) promoter, enhancer and mutant sequence of breast cancer related antigen DF3/MUC1; (e) promoter, enhancer and mutant sequence of vascular endothelial growth factor (VEGF) receptor KDR; (f) promoter, enhancer and mutant sequence of L-plastin; (g) promoters, enhancers and mutant sequences of members of the inhibitor of apoptosis protein family (IAP); (h) promoters, enhancers and mutant sequences of prostaglandin-specific antigens; (i) conserved sequences of hypoxia response elements regulated by hypoxia inducible factor-1 (HIF-1); (j) promoter, enhancer and mutant sequence of transcription factor E2F; (k) promoter, enhancer and mutant sequence of hTERT.

7. The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of claim 1, wherein the sequence of the tumor-specific promoter is SEQ ID NO: 7.

8. The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of claim 1, wherein the said adenovirus early replication gene is E1a or E1b, wherein E1a is wild or mutant of E1a, and the E1b is E1b-55 kDa, E1b-19 kDa or mutants thereof.

9. The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of claim 1, wherein the sequence of the said E1a expression cassette is SEQ ID NO: 8.

10. An application of the three-plasmid oncolytic adenovirus recombinant packaging system of claim 1 in the preparation of oncolytic adenovirus or antitumor drugs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 shows the structure of the adenovirus right arm backbone plasmid pAd5MixPlus.

[0040] FIG. 2 shows the structure of the adenovirus right arm shuttle plasmid pAdH548F511LR.

[0041] FIG. 3 shows the structure of the adenovirus left arm shuttle plasmid pAdSVPcreLoxP.

[0042] FIG. 4 shows the recombinant packaging process of oncolytic adenovirus AdSVPH548F511 based on the Ad5MixPlus system.

[0043] FIG. 5 shows the structure of the oncolytic adenovirus AdSVPH548F511.

[0044] FIG. 6 shows the replication activity of the oncolytic adenovirus AdSVPH548F511 detected by the TCID50 method.

[0045] FIG. 7 shows the killing activity of the oncolytic adenovirus AdSVPH548F511 on tumor cells. (A) MTT method, (B) RTCA method.

[0046] FIG. 8 shows the inhibitory effect of the oncolytic adenovirus AdSVPH548F511 on renal cell carcinoma xenograft.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0047] The specific embodiments of the present invention are described in detail with reference to drawings.

Example 1 Fast and Accurate Three-Plasmid Oncolytic Adenovirus Recombinant Packaging System Ad5MixPlus of the Present Invention

[0048] The fast and accurate three-plasmid oncolytic adenovirus recombinant packaging system of the present invention involves 3 adenovirus recombinant plasmids, adenovirus right arm backbone plasmid of Ad5MixPlus system, adenovirus right arm shuttle plasmid of Ad5MixPlus system, and adenovirus left arm shuttle plasmid of Ad5MixPlus system. The recombination and packaging process of these 3 adenovirus recombinant plasmids and Ad5MixPlus is as follows.

[0049] 1. Adenovirus Right Arm Backbone Plasmid of Ad5MixPlus System

[0050] The first adenovirus recombinant plasmid, adenovirus right arm backbone plasmid pAd5MixPlus, was constructed based on adenovirus type 5 and was loaded with two sets of recombinant sites, one attL/attR in Fiber/Hexon/E3 region and the other Cre/loxP in E1 region (FIG. 1). The ccdB lethal gene that causes the death of the DB3.1 E. coli strain and competent cells was inserted in the E3 region to screen successful recombinant clones. The principle is that the successful recombinant vector lost the ccdB lethal gene between attR1 and attR2 sites, and there was no ccdB lethal gene expression, so the competent bacteria survived and formed clones, while the unsuccessful recombinant vector expressed the original ccdB gene and resulted in bacterial death.

[0051] The full-length sequence of adenovirus right arm backbone plasmid pAd5MixPlus is SEQ ID NO: 1.

[0052] 2. Adenovirus Right Arm Shuttle Plasmid of Ad5MixPlus System

[0053] The second adenovirus recombinant plasmid, adenovirus right arm shuttle plasmid pAdH548F511LR, contains the modified Ad5H48 chimeric Hexon sequence and Ad5F11b chimeric Fiber sequence. Multiple cloning sites for exogenous genes insertion were preset in the E3 region. Hexon/E3/Fiber sequence contains attL1/attL2 recombination sites at both ends (FIG. 2).

[0054] The full-length sequence of adenovirus right arm shuttle plasmid pAdH548F511LR is SEQ ID NO: 2.

[0055] 3. Adenovirus Left Arm Shuttle Plasmid of Ad5MixPlus System

[0056] The third adenovirus recombinant plasmid, adenovirus left arm shuttle plasmid pAdSVPcreLoxP, contains an E1a expression cassette controlled by tumor-specific promoter Survivin and a loxP recombination site (FIG. 3).

[0057] 4. Recombinant Packaging Procedure for Ad5MixPlus System

[0058] Adenovirus right arm shuttle plasmid pAdH548F511LR was recombined with adenovirus right arm backbone plasmid pAd5MixPlus at attL1/attL2 located at both ends of the Hexon/E3/Fiber sequence in DB3.1 E. coli competent cells. After the first round of recombination mentioned above, the sequence between attR1/attR2 in pAd5MixPlus was replaced by the sequence between attL1/attL2 in pAdH548F511LR. Then, the adenovirus left arm shuttle plasmid pAdSVPcreLoxP was recombined with pAd5MixPlus in eukaryotic cells, which is the second round of recombination, leading to that the E1a expression cassette controlled by the tumor-specific promoter in pAdSVPcreLoxP was inserted into the E1 region of pAd5MixPlus. The ideal oncolytic adenovirus was packaged accurately and quickly after the above two rounds of site-specific recombination. The recombinant packaging procedure for Ad5MixPlus system was shown in FIG. 4. The structure of the oncolytic adenovirus AdSVPH548F511 was shown in FIG. 5.

[0059] The complete genome sequence of the oncolytic adenovirus AdSVPH548F511 of the present invention is: SEQ ID NO: 4.

[0060] The contents that need special explanation are as follows.

[0061] 1. Modification of Chimeric Hexon Sequence

[0062] The second adenovirus recombinant plasmid, adenovirus right arm shuttle plasmid pAdH548F511LR, contains the modified Ad5H48 chimeric Hexon sequence. Due to exposure to the surface of adenovirus, the hypervariable region (HVR) of Hexon is the key site for the difference of liver infection ability and immunogenicity between different serotypes of adenovirus. Using genetic engineering to modify the adenovirus vector, seven HVRs of Hexon on the surface of Ad5 are selectively chimeric with the corresponding regions of Hexon of rare serotype viruses, which is an effective method to help adenovirus evade pre-existing immunity and avoid liver adsorption.

[0063] The modified Ad5H48 chimeric Hexon of the present invention was prepared by replacing the corresponding sequence of Ad5 with the HVR of type 48 adenovirus of subgroup D. The population generally lacks neutralizing antibodies against Ad48, and Ad48 has weak liver affinity. Therefore, interception from neutralizing antibodies and uptake by the liver can be avoided and virus survivability is improved by replacing the corresponding part of Ad5 with the HVR of Ad48 to construct Ad5 and Ad48 chimeric Hexon adenovirus. The chimeric Hexon sequence described in this present invention also can be a chimeric sequence constructed by Hexon of Ad5 and Hexon of any other serotype adenovirus, such as Ad9, Ad37, Ad43, or a mutant sequence thereof.

[0064] The complete sequence of Ad5H48 chimeric Hexon is SEQ ID No: 5.

[0065] 2. Modification of Chimeric Fiber Sequence

[0066] The second adenovirus recombinant plasmid, adenovirus right arm shuttle plasmid pAdH548F511LR, contains the modified Ad5F11b chimeric Fiber sequence. The human adenovirus family has 51 known serotypes and is divided into 6 subgenus (A to F). CAR is the main recognition receptor of each subgenus adenovirus except B (Ad5 belongs to Subgenus C). Adenoviruses of subgenus B are further divided into subgroups B1 and B2. Ad11b, Ad14, and Ad35 are group B2 adenoviruses; Ad3, Ad16, Ad21, and Ad50 are group B1 adenoviruses. In recent years, subgenus B adenovirus derivatives have attracted much attention as attractive gene therapy vectors because they can infect target cells such as hematopoietic cells, hematopoietic stem cells, dendritic cells (DCs) and malignant tumor cells, which are often not easily infected by commonly used adenovirus vectors such as Ad5. Different from many adenoviruses that infect cells through CAR receptors, subgenus B adenoviruses use CD46 as recognition receptor. CD46 is a widely expressed complement regulatory protein that is present on the surface of almost all human cells. The chimeric Fiber of Ad5F11b is prepared by replacing the corresponding sequence of adenovirus type 5 Fiber with the Fiber knob of Ad11b, so that the chimeric virus has high infection characteristics to hematopoietic cells, stem cells and tumor cells. The chimeric Fiber sequence described in the invention also can be a chimeric sequence constructed by the Fiber of Ad5 and Fiber of any other serotype adenovirus, such as Ad3, Ad14, Ad16, Ad21, Ad35, Ad50, Ad55, or a mutant sequence thereof.

[0067] The complete sequence of Ad5F11b chimeric Fiber is SEQ ID No: 6.

[0068] 3. Tumor Specific Promoter

[0069] The third adenovirus recombinant plasmid, adenovirus left arm shuttle plasmid pAdSVPcreLoxP, contains an E1a expression cassette controlled by tumor-specific promoter Survivin inserted at multiple cloning sites of pAdSVPcreLoxP. Survivin promoter has attracted much attention due to its high specificity and wide tumor spectrum. Survivin is rarely expressed in normal tissues, but highly selectively expressed in malignant tumors. It is highly expressed in most tumors such as lung cancer, liver cancer, colon cancer, pancreatic cancer, prostate cancer and breast cancer, and is closely related to tumor recurrence and metastasis and poor prognosis of patients, making it a broad-spectrum molecular target for tumor gene therapy. The oncolytic adenovirus regulated by the Survivin promoter can target cancer cells, replicate in cancer cells, and lyse cancer cells, and at the same time mediate the high-efficiency expression of anti-tumor target genes. Therefore, oncolytic adenovirus regulated by Survivin promoter is expected to be used to obtain broad-spectrum and safe anti-cancer effect against most human tumors. In addition to being a Survivin promoter, the tumor specific promoter of the invention can also be any of the following: (a) promoter, enhancer and mutant sequence of carcinoembryonic antigen; (b) promoter, enhancer and mutant sequence of alpha fetoprotein; (c) promoters, enhancers and mutant sequences of receptor tyrosine kinases (including EGFR, Her-2, Her-3 and Her-4) of the human epidermal growth factor receptor family (EGFRs); (d) promoter, enhancer and mutant sequence of breast cancer related antigen DF3/MUC1; (e) promoter, enhancer and mutant sequence of vascular endothelial growth factor (VEGF) receptor KDR; (f) promoter, enhancer and mutant sequence of L-plastin; (g) promoters, enhancers and mutant sequences of members of the inhibitor of apoptosis protein family (IAP); (h) promoters, enhancers and mutant sequences of prostaglandin-specific antigens; (i) conserved sequences of hypoxia response elements regulated by hypoxia inducible factor-1 (HIF-1); (j) promoter, enhancer and mutant sequence of transcription factor E2F; (k) promoter, enhancer and mutant sequence of hTERT.

[0070] The nucleotide sequence of the tumor-specific promoter Survivin in this invention is SEQID NO: 7.

[0071] 4. Adenovirus Early Replication Gene E1a

[0072] The third adenovirus recombinant plasmid, adenovirus left arm shuttle plasmid pAdSVPcreLoxP, contains an E1a expression cassette controlled by tumor-specific promoter Survivin. Adenovirus early replication gene E1a is placed under the regulation of tumor-specific promoter to achieve the purpose of tumor-specific replication and oncolysis of the virus. The sequence of the early replication gene of adenovirus described in this invention can be a wild sequence of E1a, a mutant sequence of E1a, or E1b-55 kDa, E1b-19 kDa or their mutant sequences.

[0073] The nucleotide sequence of E1a in the E1a expression cassette controlled by the tumor-specific promoter in this invention is SEQ ID NO: 8.

Example 2 Specific Replication Activity of Oncolytic Adenovirus AdSVPH548F511

[0074] Hepatocellular carcinoma cells (HCCLM3, HepG2, Huh-7, MHCC97H, MHCC97L), normal hepatocytes (WRL-68) or normal fibroblasts (BJ) in logarithmic phase were collected and counted. The cells were seeded on 96-well plates at 1×10.sup.4 cells/well, changing to serum-free culture medium after cell adherence. The oncolytic adenovirus AdSVPH548F511 was added to the culture medium at MOI=1. After 2 h of viral infection, the medium was changed to 5% serum medium (This is the starting time of infection 0 h), and the cells were further cultured for 48 h and 96 h. The cells were collected at these three time points, and the virus titer was detected by TCID50 method. The results showed that AdSVPH548F511 had a very strong ability of specific replication in liver cancer cells, and the replication multiples were all above 10,000 times at 48 h, with the maximum was above 50,000 times. After 96 h, it reached 100,000 to 800,000 times. The replication ability of AdSVPH548F51 in normal cells WRL-68 or BJ was very low, and the highest replication multiple at 96 h was below 200 times (FIG. 6).

Example 3 Killing Activity of Oncolytic Adenovirus AdSVPH548F511 on Tumor Cells

[0075] Hepatocellular carcinoma cells (HepG2, MHCC97H), normal hepatocytes (L02) and normal fibroblasts (BJ) in logarithmic phase were collected and counted. The cells were seeded on 96-well plates at 1×10.sup.4 cells/well, changing to serum-free culture medium after cell adherence. The effect of AdSVPH548F511 on cell viability was detected by MTT assay. Cell Proliferation Kit I (MTT) was purchased from Roche Diagnostics GmbH. The viruses were added into culture wells with a gradient of MOIs, with 8 replicates per treatment. Then the cells were cultured in the incubator. After 2 h, the medium was replaced with serum medium for further culture, with the addition amount of 100 μl/well. After 48 hours, the culture medium was discarded, 0.1 mol/L PBS solution was added to the culture well with the dosage 100 μl/hole. Then MTT labeling reagent was added to the culture well to the final concentration of 0.5 mg/ml, and the culture plates were placed in incubator. After 4 h, 100 μl/well of Solubilization solution (10% SDS in 0.01 mol/L HCl) was added to the culture well, and the cells were cultured overnight in an incubator. The Model 550 Microplate Reader (BIO-RAD) was used to measure the light absorption value at 570 nm wavelength, and the corrected wavelength was 655 nm. The survival curve was drawn, and the IC50 value was calculated. The results showed that AdSVPH548F511 had strong killing activity against HepG2 and MHCC97H with IC50 values of 35.16 and 212.4, respectively, while AdSVPH548F511 had no significant effect on normal BJ cells with IC50 value of 20035. It can be seen that oncolytic adenovirus AdSVPH548F511 has the ability to specifically kill and destroy cancer cells (FIG. 7A).

[0076] At the same time, the killing activity of AdSVPH548F511 on cells was detected in real time, dynamically and quantitatively by Real Time Cellular Analysis (RTCA). E-Plate plate was added with culture medium and background impedance was measured. Hepatocellular carcinoma cells (HepG2) or normal liver cells (L02) in logarithmic growth phase were collected, counted and added to the E-Plate detection plate, and placed in the ultra-clean worktable at room temperature for 30 min. The virus was diluted in serum-free medium and added to E-Plate at MOI=5, 10 or 20. The E-Plate detection plate was placed on the detection platform pre-placed in the incubator for real-time dynamic cell proliferation detection, and the real-time dynamic cell growth curves were plotted. The results showed that the killing activity of AdSVPH548F511 on HepG2 cells increased with the increase of MOIs and time. However, the killing activity of AdSVPH548F511 on normal liver cell L02 was not high. L02 cells were inhibited to some extent only when given high-MOI virus infection (FIG. 7 B).

Example 4 Antitumor Animal Experiment of Oncolytic Adenovirus AdS VPH548F511

[0077] Eight healthy purebred male BALB/c nude mice aged 6-8 weeks were provided by the Shanghai Experimental Animal Center of the Chinese Academy of Sciences and kept in a clean animal laboratory. Renal carcinoma OSRC-2 cells in logarithmic growth phase were adjusted to 1×10.sup.7 cells/ml with PBS. Near-axillary skin on the abdominal side of nude mice was disinfected and subcutaneously injected with 100 μL cell suspension. Then the nude mice were raised under the condition of constant temperature, ventilation and sterility. Tumor growth was regularly observed daily and the presence of a rice-size tumor underneath the inoculation site was considered a success. Model animals were randomly divided into two groups, virus group (AdSVP) n=5, control group (PBS) n=3. Animals were numbered and vernier caliper was used to measure the tumor size. Then treatment was started. The virus AdSVPH548F511 with a concentration of 2×10.sup.8 pfu/100 μl was injected directly into the tumor at multiple sites, once every other day, 5 times in total. Control animals were injected with PBS instead of virus, 100 μl×5 times. The tumor size was measured regularly, and the tumor volume was calculated with the formula of ‘a×b.sup.2×0.5’ (a: maximum diameter, b: minimum diameter). The growth curve of transplanted tumor was plotted (FIG. 8). The results showed that AdSVPH548F511 could significantly inhibit the growth of renal cell carcinoma OSRC-2 xenograft.

[0078] The above are only the preferred embodiments of the present invention. It will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. Such changes and modifications are intended to be encompassed by the scope of the following claims.