CN101199859A - Inhibition lung cancer transfer and lung transfer tumor gene medicament - Google Patents
Inhibition lung cancer transfer and lung transfer tumor gene medicament Download PDFInfo
- Publication number
- CN101199859A CN101199859A CNA2007101339145A CN200710133914A CN101199859A CN 101199859 A CN101199859 A CN 101199859A CN A2007101339145 A CNA2007101339145 A CN A2007101339145A CN 200710133914 A CN200710133914 A CN 200710133914A CN 101199859 A CN101199859 A CN 101199859A
- Authority
- CN
- China
- Prior art keywords
- raav2
- lung
- tumor
- vas
- cct
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to materia medica, molecular medicine and disease prevention fields, in particular to a gene medicine based on recombinant adeno-associated virus and tumor angiogenesis inhibiting gene. The medicine can target at lung tissue and inhibit effectively cancer metastasis and the occurrence of pulmonary metastases.
Description
Technical field
The present invention relates to molecular medicine, molecular medicine and diseases prevention and treatment field, be specifically related to genomic medicine based on recombinant adeno-associated virus (rAAV) and tumor-blood-vessel growth suppressor gene, this medicine can targeting in lung tissue, suppress the transfer of pulmonary carcinoma and the generation of pulmonary metastases effectively.
Background technology
Primary bronchogenic carcinoma of lung (abbreviation pulmonary carcinoma) is a modal pulmonary primary malignant tumor, ranks first in the incidence rate of whole world tumor and the statistics of patient because of the tumor mortality rate, accounts for 18% of cancer patient sum.Lung cancer therapy medicine commonly used clinically at present has multiple, comprise taxine kind drug taxol (paclitaxel), topoisomerase II depressant teniposide (etoposide), platinum-like compounds carboplatin (carboplatin) and Docetaxel (docetaxel) etc. are used for the treatment of non-minicell pulmonary carcinoma (NSCLC), though certain curative effect is arranged, still barely satisfactory, most important weak point is, these chemotherapeutics still can not strictly be distinguished tumor tissues and normal structure, in use, the multiple toxic and side effects that often occurs together, the severe patient threat to life.
If tumor be attack to the person one sharp sword, neoplasm metastasis then can be rated as that deathblow.Discover, neoplasm metastasis is the reason of 90% tumour patient death, even in scientific and technical develop rapidly, the generation of tumor, treatment, diagnosis research have all had today of rapid progress, neoplasm metastasis still is the maximum stumbling-block that the cancer cure rate can't obviously promote, and pulmonary carcinoma is no exception.Tumor tissues discharges cancerous cell every day in blood, the cancerous cell that comes off is spread whole body with blood or lymph, in case condition maturity, the cancerous cell that is scattered can be as any histoorgan plantation of seed at health, and ramp gets up, and destroys organ function, finally causes patient death.And pulmonary is widely distributed owing to lymphoid tissue, and the blood flow operation is abundant, and the easier generation of lung carcinoma cell focus is at a distance shifted.Lung cancer metastasis can be through four approach: 1. direct extension: pulmonary carcinoma can be soaked into growth from the bronchus nidus along bronchial wall, directly to its nearside or distal end portion diffusion, can invade to the trachea carina plane, also can soak into growth to pulmonary parenchyma by empty overdraw tracheal wall, further can reach vertical diaphragm and pleura.The plantation diffusion of two-layer pleura can be invaded and thoracic wall and diaphragm, or to esophagus and pericardium.2. lymphatic metastasis: lymphatic metastasis just might take place in pulmonary carcinoma in early days.General small cell carcinoma is wanted Zao than the scale cancer transfer and is seen more.Lymphatic metastasis at first sees bronchopulmonary lymph nodes, hilar lymph node, the other lymph node of tracheal bronchus.Can there be the visible retroperitoneal lymph node of half to shift approximately to vertical phrenic lymph nodes (this is the remote pointer that shifts) pulmonary carcinoma postmortem case thus.3. bloody path shifts: pulmonary carcinoma shifts through bloody path, often causes that whole body extensively shifts and deadly dying, and general mortality rate is 92%.Particularly the bloody path transfer can take place in undifferentiated type pulmonary carcinoma such as small cell carcinoma, large cell carcinoma in early days, and scale cancer and adenocarcinoma are then later.4. implantation metastasis: when the tumor of organ spread to organ surface in the body cavity, oncocyte can come off and as sowing, be planted in the surface of each organ in body cavity and the body cavity, forms most metastatic tumors.This branch mode is called implantation metastasis or sowing, and pilosity is born in the carcinoma of organ in abdominal cavity or the thoracic cavity.Pulmonary carcinoma also is everlasting and is formed implantation metastasis widely in the thoracic cavity.It should be noted that operation also may cause the pulmonary carcinoma plantation to shift.
Malignant tumor late more can be transferred to pulmonary, can be that hematogenous spread, lymphatic metastasis or adjacent organs are directly invaded.Lungs are one of modal metastasis sites of malignant tumor, according to statistics, die among the patient of malignant tumor about 20% and lung takes place shift; Pulmonary metastases is mainly derived from breast carcinoma, renal carcinoma, colon cancer, rectal cancer, osteosarcoma, malignant melanoma, skin carcinoma, soft tissue sarcoma etc.; In case when shifting appearred in pulmonary, tumor had belonged to late period.The case that extensively shifts appears in the both sides lungs cannot surgical intervention, to single transfer tuberosity is only arranged, or though several metastasis arranged but all belong to the minority pulmonary metastases be limited in a lobe of the lung or the side lung, just consider operative treatment, so be a very thorny difficult problem to the treatment of pulmonary metastases.
In 1971, Folkman proposes tumor neogenetic blood vessels and generates hypothesis, think " tumor growth depends on angiogenesis; tumor-blood-vessel growth is because tumor secretion TAF (tumor angiogenesis factor) causes ", set up a series of models afterwards gradually this hypothesis has been verified, and on experiment basis, established the framework that whole tumor neogenetic blood vessels generates.Many important tumor angiogenesis factors and antineoplastic vascular somatomedin are found successively, cause at the drug research of various angiogenesis regulatory factors in the ascendant.
Kind of an anti-angiogenic medicaments enters clinical research surplus in the of worldwide about 70.But clinical research is the result show, and is single very limited to the curative effect of tumor with anti-angiogenic medicaments, also pronounced " death sentence " by FDA even the quilt that the Folkman research group is found is expressed the anti-angiogenesis Angiostatin and the Endostatin of very big hope.Research worker thinks that wherein the reason of most critical is to be difficult to reach active drug concentration in the tumor body, or can not keep active drug concentration for a long time.Gene therapy medicament can be realized the expression of destination protein in the intravital long-term stability of people, provides thinking for addressing the above problem.Domestic Li Lei etc. are carrier with recombinant adenovirus (rAd), have carried out inhibitory action research (Li Lei, Yuan Yaozong, Zhang Yongping, Qiao Minmin, the Lu Jian of Vasostatin gene therapy medicament to pancreatic cancer growth.The structure of Vasostatin gene recombinant adenovirus vector and to the inhibitory action of pancreatic cancer growth.Tumor, 2004,24 (6): 538-541).Chen Hong etc. are carrier with rAAV, carried out angiostatin (angiostatin) gene therapy medicament treatment rat brain glioma research (Chen Hong,
Sword is non-, appoints Radix Dichroae.The recombined glandulae correlation viral vectors mediated angiostatin gene therapy for rat glioma.Liaoning medical journal, 2005,19 (3): 113-114).So far, the carrier mediated Vasostatin of the still unmanned rAAV of employing, kallistatin and angiostatin carry out the treatment of pulmonary carcinoma and report, and more do not suppress the report of lung cancer metastasis and pulmonary metastases.
Can steady in a long-termly express because of the exogenous gene of rAAV mediation, do not have immunoreation or immunoreation extremely low, host range is extensive, becomes one of the most promising gene therapy vector.Aspect oncotherapy, carry genes such as p53, metalloprotease tissue inhibitive factor (TIMP1), tumor necrosin relative death inducing ligand (TRAIL), interferon (IFN), Endostatin and angiostatin with rAAV, shown that in zooperies such as colon cancer, pulmonary carcinoma and hepatocarcinoma the good restraining tumor takes place, tumor vessel forms and the tumor growth effect, improved antineoplastic immune function and the survival rate thereof of animal.
The inventor is a carrier with lung targeted characteristic rAAV in early-stage Study, has carried out multiple antineoplastic vascular somatomedin, as the animal experiment research of gene therapy medicaments such as Vasostatin, Angiostatin and Kallistatin to pulmonary carcinoma.Result of study shows, though above gene therapy medicament can suppress the growth of pulmonary carcinoma, but still can not realize effecting a radical cure the purpose of tumor.But amazing is that the said gene medicine can reduce the generation of lung cancer metastasis and pulmonary metastases greatly, provides hope for suppressing lung cancer metastasis and pulmonary metastases.
In addition, the research of antineoplastic vascular growth factor gene treatment, the pattern that all adopts intramuscular injection, intravenous injection etc. to be administered systemically, the antineoplastic vascular somatomedin of generation is low in target organ concentration, can not bring into play target organ tumor treatment purpose preferably.Angiogenesis is pathological processes such as tumor growth, also is physiological process such as repair in trauma, cardiovascular function, and the angiogenesis of non-targeting suppresses also can cause inevitably the generation of untoward reaction.
Summary of the invention
Purpose of the present invention is a therapeutic goal from solving still shiftless at present lung cancer metastasis and pulmonary metastases promptly, adopt the viral vector of lung targeted characteristic transfection and expression, at the local long-term expression antineoplastic vascular of lung growth factor gene, prevent and treat lung cancer metastasis and pulmonary metastases energetically, and, avoided the generation of untoward reaction the infecting of the virus-free carrier of other organ.
The invention discloses a kind of targeting gene therapy medicine, with the carrier mediated Vasostatin of rAAV, kallistatin and angiostatin are fine to treatment pulmonary carcinoma effect, in further studying, the inventor is surprised to find, when viral vector is selected rAAV2/1 for use, rAAV2/2 or rAAV2/5, promoter is selected cmv enhancer/chicken β-actin promoter for use, when CCT promoter or CC10 promoter, genomic medicine of the present invention is only at pulmonary's specifically expressing genes of interest, can not only effectively treat pulmonary carcinoma, and to suppressing the transfer of pulmonary carcinoma, and the pulmonary metastases that primary tumor causes had the good restraining effect, also help the generation of avoiding untoward reaction.
Targeting gene therapy medicine of the present invention, by the expression of viral vector mediation antineoplastic vascular growth factor gene, it is characterized in that: viral vector is selected from rAAV2/1, rAAV2/2 or rAAV2/5; The antineoplastic vascular growth factor gene is selected from Vasostatin, kallistatin or angiostatin; The promoter of gene expression frame is selected from cmv enhancer/chicken β-actin promoter, CCT promoter or CC10 promoter; The regulating transgene expression element is WPRE (woodchuck hepatitis viruspost-transcriptional regulatory element).Wherein preferred CCT promoter of the promoter of gene expression frame or CC10 promoter.
In the rAAV carrier that the present invention adopts, its gene expression frame contains promoter, transgenic, regulating transgene expression element, polyA tail successively, and the gene expression frame two ends are AAV2 ITR (inverted terminal repeat sequence).Genes of interest inserts in the above-mentioned expression cassette behind suitable enzyme action, obtains expression plasmid.Be equipped with recombinant virus through many plasmids of non-auxiliary virus legal system, just obtain gene therapy medicament of the present invention.
The rAAV of different serotypes, though its capsid protein has the homology of height, the nuance of aminoacid sequence has determined the difference of its cytotaxis (tropism).Cytotaxis's difference, each is variant to cause the type of AAV transfectional cell of different serotypes and efficiency of infection.The present invention is a gene expression plasmid with the rAAV2 plasmid, adopts the rAAV1-11 helper plasmid to pack respectively, obtains the rAAV virus of different serotypes.Adopt Lewis lung cancer cell (LLC) and people's pulmonary epithelial cells (HLEC) that the tropism of rAAV is screened, found that and select rAAV2/1 for use, or rAAV2/2, or rAAV2/5 is as lung targeting vector effect best (seeing embodiment 3).
Promoter plays a significant role to the power of gene expression, and has cell selective.The present invention adopts 293 cells, Lewis lung cancer cell (LLC) screens different promoteres with people's pulmonary epithelial cells (HLEC), determines to select for use cmv enhancer/chicken β-actin promoter, CCT promoter (CTP:phosphocholine cytidylyltransferase promoter), CC10 promoter (Clara cell 10-kd promoter) (seeing pharmacology test 7).
Pulmonary carcinoma primary tumor model mice is behind trachea perfusion medicine of the present invention, and medication group primary tumor dwindles to some extent than matched group, but there was no significant difference.Matched group is all seen tangible lung cancer metastasis at pleura, mediastina lymph node and both sides lung, but the lung cancer metastasis of medication group mice has obtained significant inhibition.
The preparation method of genomic medicine of the present invention adopts many plasmids of non-auxiliary virus method, promptly adopts calcium phosphate method with expression plasmid and helper plasmid cotransfection 293 cells, gathers in the crops recombinant virus after 60-72 hour, and is standby behind chromatography purification.
The mode that genomic medicine route of administration of the present invention preferably sucks with trachea perfusion or atomizing back.
Genomic medicine of the present invention can become various preparations with pharmaceutically acceptable preparing carriers, preferred spray or solution.
Genomic medicine disclosed by the invention can use separately, also can merge use.
Be the part pharmacology test of genomic medicine of the present invention below, wherein VAS is the vasostatin abbreviation, and KAL is the kallistatin abbreviation, and AS is the abbreviation of angiostatin, and hm-LLC is the abbreviation of high metastasis Lewis LungCarcinoma.
1.rAAV2/2-VAS to the inhibitory action of high transitivity LLC (hm-LLC) subcutaneous transplantation tumor to the lung transfer
Hm-LLC is high transitivity lung carcinoma cell, can form sporadic transfer lung cancer model behind the mouse hypodermic inoculation.
With rAAV2/2-VAS in-vitro transfection hm-LLC cell (MOI:5 * 10
4) after, subcutaneous vaccination (5 * 10
6Individual cell), adopt PBS and rAAV2/2-eGFP to be contrast to C57BL mouse.
Inoculate after 7 days, check the blood supply situation of respectively organizing the mouse subcutaneous transplanting tumor.The average supply vessels number of rAAV2/2-VAS group mice is 3.2 ± 0.8, significantly be less than PBS and rAAV2/2-eGFP matched group (7.8 ± 0.9 and 8.0 ± 0.8) (p＜0.001, n=5).Exist many to contain erythrocytic tract in tumor tissue section's (HE dyeing) demonstration matched group, and there is not this type of tract (see figure 1) in the rAAV2/2-VAS group.Illustrate that vasostatin has suppressed the vascularization of subcutaneous transplantation tumor.
E-cadherin is a class cell adhesion factor relevant with the tumor cell invasive, and its down-regulated expression shows that the metastasis tendency of tumor increases.Tumor tissue section's immunostaining shows (see figure 2), and the expression of rAAV2/2-VAS group E-cadherin obviously surpasses matched group, illustrates that its metastasis tendency is suppressed.
Inoculate after 21 days, rAAV2/2-VAS group subcutaneous transplantation tumor volume is 65% of a matched group.
The pulmonary of all rAAV2/2-eGFP control group mice, tangible transitivity pulmonary carcinoma tuberosity has all appearred, average tuberosity number is 20 ± 10, the tuberosity number of all control group mice is more than 10, and the average tuberosity number of pulmonary of rAAV2/2-VAS group mice is 9 ± 6 (p＜0.05, n=5), wherein the tuberosity number of 3 mices is less than 10.
Above presentation of results: rAAV2/2-VAS of the present invention is inhibited to tumor-blood-vessel growth, has limited growth of tumor.The more important thing is that this medicine can effectively reduce the metastasis tendency of tumor cell, reduce the generation of pulmonary metastases.
2. trachea perfusion rAAV2/1-CCT-KAL is to the inhibitory action of A549 pulmonary carcinoma primary tumor transfer
Because of the implant site of tumor cell has material impact to its growth, the development that pulmonary carcinoma primary tumor model more can anthropomorphic dummy's pulmonary carcinoma.The plastidogenetic pulmonary carcinoma primary tumor of mice left side lung injection A549 model, can observe cancer in situ growth, invade pleura and vertical phrenic lymph nodes and be transferred to the process of offside lung.
At BALB/c nude mice left side lung injection in 7 ages in week A549 cell (1 * 10
6) 1 week of back, through trachea perfusion rAAV2/1-CCT-KAL, dosage is 3 * 1011vp/ Mus.Matched group is PBS or rAAV2/1-eGFP.
After 50 days, all mices pulmonary carcinoma primary tumor of all having grown, the tumor volume no significant difference of experimental group and matched group.But the vertical phrenic lymph nodes weight of rAAV2/1-CC10-KAL group only be matched group 50% (p＜0.05, n=8).The right lung lymph node quantity of all mices of matched group (8/8) is all above 30, and the rAAV2/1-CCT-KAL group has 3 (3/8) mice right lung lymph node quantity to be less than 10.
Measure the microvessel density (see figure 3) of primary tumor by the CD34 immunostaining, the microvessel density that the result shows the rAAV2/1-CCT-KAL group only for matched group 0.63 (p＜0.05, n=8).
Above presentation of results: inhibited behind the trachea perfusion rAAV2/1-CCT-KAL to tumor-blood-vessel growth, reduced of the generation of pulmonary carcinoma primary tumor to the transfer of vertical diaphragm and offside lung.
3. trachea perfusion rAAV2/5-CCT-VAS is to the inhibitory action of subcutaneous transplantation tumor postoperative lung transfer
Hm-LLC cell (5 * 10
6) subcutaneous vaccination is to C57BL mouse, when treating that the tumor major diameter reaches the 0.5cm left and right sides (1 week back), the excision primary tumor causes tumor cell to shift to lung and quickens.
Postoperative mice on the same day is poured into rAAV2/5-CCT-VAS through trachea, and dosage is 2 * 10
11The vp/ Mus.Matched group is PBS or rAAV2/5-eGFP.
Postoperative 30 days, serious lung transfer phenomena takes place in all control group mice (n=4), hemothorax occurs, and the transfer tuberosity is arranged in the lung, and gross tumor volume is big, rich blood vessel, and bleeding is arranged.And the lung transfer phenomena does not take place in 1 mice (1/4) in the rAAV2/5-VAS group, and tuberosity only appears shifting in a small amount of lung in 2 mices (2/4), and average lung weightening finish reduces by 40% than matched group.
Every group has 6 mices to carry out the life span experiment, is treatment 10 weeks of back experimental period.The matched group mean survival time is 21 days, and the rAAV2/5-CCT-VAS group is 52 days, prolongs (see figure 4) more than 2 times.During 10 weeks mouse lung is carried out light microscopy checking, there is 1 mice not see neoplasm metastasis in the treatment group, but the histogenic immunity inspection finds have the micrometastasis of hiding to have (see figure 5) around this Mus lung blood vessel, illustrates that rAAV2/5-CCT-VAS is inhibited to the growth that transplanted tumor postoperative lung shifts.
4. trachea merging perfusion rAAV2/5-CCT-VAS and rAAV2/5-CCT-AS are to the inhibitory action of subcutaneous transplantation tumor postoperative lung transfer
Experimental technique is with 3, and the treatment group changes trachea into and merges perfusion rAAV2/5-CCT-VAS and rAAV2/5-CCT-AS, and dosage is 2 * 10
11The vp/ Mus.Matched group is PBS or rAAV2/5-eGFP.
Postoperative 30 days, the lung transfer phenomena does not take place in 1 mice (1/3) in the treatment group, and 2 mices (2/3) only occur shifting tuberosity in a small amount of lung, and average lung weightening finish reduces more than 40% than matched group.
The half life span surpasses 10 all (see figure 4)s in the treatment group, weight increase during 10 weeks, and behavior is normal.During 10 weeks mouse lung is carried out light microscopy checking, have 4 mices not see neoplasm metastasis in the treatment group.But the histogenic immunity inspection finds have the micrometastasis of hiding to have (see figure 5) around the lung blood vessel, illustrates that rAAV2/5-CCT-VAS and rAAV2/5-CCT-AS are inhibited to the growth that transplanted tumor postoperative lung shifts.
The 4th week and the 10th week are checked the expression (see figure 6) of angiostatin to the section of survival Mus lung tissue after the trachea medication.The 4th week and the 10th week after the trachea medication is in the expression of the visible angiostatin of pulmonary epithelial cells.
5. trachea perfusion rAAV2/1-CC10-VAS, rAAV2/1-CC10-AS or the two are share the inhibitory action that the lung primary tumor is shifted
C57B6 mice left side pulmonary parenchyma injection LLC cell (1 * 10
3), trachea perfusion therapy medicine or contrast after 3 days, dosage is 2 * 10
11The vp/ Mus.After 21 days, check pulmonary and mediastinal lymph nodes, estimate lung primary tumor and transfer case.
Table 1. is respectively organized lung primary tumor volume
| Group | Lung primary tumor volume (mm3) |
| PBS | 81.4±28.83 |
| rAAV2/1-eGFP | 73.39±16.60 |
| rAAV2/1-CC10-VAS | 60.3±28.0 |
| rAAV2/1-CC10-AS | 54.3±18.9 |
| RAAV2/1-CC10-VAS and rAAV2/1-CC10-AS | 57.3±14.5 |
The lung of treatment group as seen from Table 1 primary tumor volume is all less than matched group, but there was no significant difference.
Table 2. is respectively organized mediastinal lymph nodes weight
| Group | Mediastinal lymph nodes weight (mg) |
| PBS | 138.4±33.2 |
| rAAV2/1-eGFP | 162.8±42.2 |
| rAAV2/1-CC10-VAS | 84.8±43.5* |
| rAAV2/1-CC10-AS | 120.7±33.5 |
| RAAV2/1-CC10-VAS and rAAV2/1-CC10-AS | 59.6±27.7** |
*: p＜0.05; *: p＜0.01 is compared with PBS.
Mediastinal lymph nodes weight is the index that reflection lung primary tumor shifts, and the lung of treatment group as seen from Table 2 primary tumor shifts and obviously obtained inhibition.
6. the long-term specifically expressing of transgenic in lung behind the trachea perfusion rAAV2/5-CC10-KAL
Normal mouse trachea perfusion rAAV2/5-CC10-KAL, dosage is 2 * 10
11The vp/ Mus.After January, get the part mice and put to death, the RT-PCR method is checked the expression (Fig. 7) of Kallistatin in lung, the heart, liver, kidney, the small intestinal.As seen only there is the specifically expressing of Kallistatin in pulmonary, and other internal organs there is no expression.Genetically modified expression had lung specificity after trachea perfusion rAAV2/5-CC10-AS was described, had realized targeting.
Trachea perfusion rAAV2/5-CC10-KAL1,2 and March after, the RT-PCR method is checked the expression (Fig. 8) of Kallistatin in the lung.As seen the specifically expressing of the Kallistatin of pulmonary is sustainable more than 6 months.
7. different promoters is at the specifically expressing of HLEC pulmonary epithelial cells
With rAAV2/1-CC10-AS difference in-vitro transfection 293 cells, HLEC cell and LLC cell (MOI=10
4), the WesternBlot method is measured the expression power (Fig. 9) of angiostatin.
As can be seen from Figure 9, rAAV2/1-CC10-AS is better than 293 cells and LLC cell in the expression of HLEC cell.
With rAAV2/5-CCT-AS in-vitro transfection 293 cells, HLEC cell and LLC cell have also obtained similar result.
By above-mentioned pharmacological testing as seen, genomic medicine of the present invention not only has lung targeted characteristic, can treat pulmonary carcinoma, and what is more important genomic medicine of the present invention can suppress lung cancer metastasis and pulmonary metastases.
Description of drawings
Fig. 1. the tissue slice (HE dyeing) of mouse subcutaneous transplanting tumor behind hm-LLC7 days of rAAV2/5-VAS transfection.Last figure is the PBS matched group, and middle figure is the rAAV2/5-eGFP matched group, and figure below is a rAAV2/5-VAS treatment group.Amplification 200.Exist many to contain erythrocytic tract in the matched group (last figure and middle figure), and there is not this type of tract in rAAV2/2-VAS group (figure below).
Fig. 2. mouse subcutaneous transplanting is behind hm-LLC7 days of rAAV2/5-VAS transfection, and tumor tissue section carries out the E-cadherin immunostaining.Last figure is the PBS matched group, and middle figure is the rAAV2/5-eGFP matched group, and figure below is a rAAV2/5-VAS treatment group.Amplification 400.The expression of rAAV2/5-VAS treatment group (figure below) E-cadherin obviously increases.
Fig. 3. left side figure is rAAV2/1-eGFP control group A 549 pulmonary carcinoma primary tumor SABC section (CD34 dyeing, an amplification 100), and right figure is rAAV2/1-CCT-KAL treatment group A549 pulmonary carcinoma primary tumor SABC section (CD34 dyeing, an amplification 100).Treatment group (right figure) microvessel density is starkly lower than matched group (left figure).
Fig. 4. behind the subcutaneous primary tumor of excision hm-LLC, the survival rate of each group is (n=6) relatively.Each group is respectively rAAV2/5-CCT-VAS and rAAV2/5-CCT-AS treatment group (V+A, * *), rAAV2/5-CCT-VAS treatment group (V*), rAAV2/5-eGFP matched group (G), PBS matched group (P).(*p＜0.05，**p＜0.01)
Fig. 5. behind the subcutaneous primary tumor of excision hm-LLC, lung tissue section checks little neoplasm metastasis (arrow is the little neoplasm metastasis that exists around the pulmonary vein, amplification 200).Last figure is a rAAV2/5-CCT-VAS treatment group, and figure below is rAAV2/5-CCT-VAS and rAAV2/5-CCT-AS treatment group.
Fig. 6. the expression (brown colouring) of the section immunohistochemical analysis angiostatin of Mus lung tissue.Last left side figure be rAAV2/5-CCT-VAS and rAAV2/5-CCT-AS treatment group during 4 weeks, and last right side figure is rAAV2/5-CCT-VAS and rAAV2/5-CCT-AS treatment group 10 when all, and figure below is that rAAV2/5-eGFP matched group 4 is when all.Amplification 100.In the lung qi tube-surface epithelial cell of treatment group (last figure) tangible brown colouring is arranged, and matched group (figure below) does not have.
Fig. 7. last figure is that the trachea perfusion is after rAAV2/5-CC10-KAL30 days, the RT-PCR method is checked lung (Lung9,10), the heart (heart7,8), liver (liver5,6), kidney (kidney3,4), the expression of Kallistatin in the small intestinal (intestine1,2), contain the positive contrast of plasmid (PC) of Kallistatin cDNA.Figure below is for 18S rRNA being the methodology contrast.M is a molecular weight standard.Only in lung, detect the expression of Kallistatin.
Fig. 8. last figure is the trachea perfusion rAAV2/5-CC10-KAL1 month (1,2), and after February (3,4) and June (5,6), the RT-PCR method is checked the expression of Kallistatin in the lung, contains the positive contrast of plasmid (PC) of Kallistatin cDNA.Figure below is for 18S rRNA being the methodology contrast, the negative contrast of NC.M is a molecular weight standard.
Fig. 9 .293 cell, HLEC cell and LLC cell transfecting rAAV2/1-CC10-AS (MOI=10
4) after 60, the WesternBlot method is measured the expression of angiostatin.1 is the supernatant of rAAV2/1-CC10-AS rotaring redyeing 293 cell; 2 is the supernatant of rAAV2/1-CC10-AS transfection HLEC cell; 3 is the supernatant of rAAV2/1-CC10-AS transfection LLC cell; 4 is the supernatant of rAAV2/1-eGFP rotaring redyeing 293 cell; 5 is the supernatant of rAAV2/1-eGFP transfection HLEC cell; 6 is the supernatant of rAAV2/1-eGFP transfection LLC cell.
The specific embodiment
The preparation of rAAV2/2-eGFP
The composition of the expression cassette of eGFP expression plasmid is followed successively by: promoter is cmv enhancer/chicken β-actin promoter, eGFPcDNA, polyA, WPRE.The expression cassette two ends are AAV2 ITR.
Adopt many plasmids of calcium phosphate non-auxiliary virus legal system to be equipped with recombinant virus: with the eGFP expression plasmid, the AV helper plasmid, with rAAV2 helper plasmid cotransfection 293 cells, harvesting after 60-72 hour, with sodium deoxycholate (0.5%) and benzonase (30u/ml) cracking, centrifugal (3000g 30min) removes cell debris, caesium chloride density gradient centrifugation, promptly get Recombinant rAAV 2/2-eGFP, chromatography method purification.
The preparation of rAAV2/1-eGFP, rAAV2/3-eGFP, rAAV2/4-eGFP, rAAV2/5-eGFP, rAAV2/6-eGFP, rAAV2/7-eGFP, rAAV2/8-eGFP, rAAV2/9-eGFP, rAAV2/10-eGFP, rAAV2/11-eGFP
With reference to embodiment 1 step, the rAAV2 helper plasmid is changed to rAAV1, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11 respectively, all the other are operated with embodiment 1, obtain 11 kinds of different serotypes rAAV viruses.
The screening of lung tropism rAAV serotype
Adopt the Real-PCR method, the titre of the various serotype rAAV that mensuration embodiment 2 obtains.The primer is according to the WPRE sequential design, is respectively WPRE-F (5 '-TGGCGTGGTGTGCACTGT) and WPRE-R (5 '-GTTCCGCCGTGGCAATAG).
With LLC cell or HLEC cell inoculation to 24 orifice plate, overnight incubation.Add respectively and contain the various serotype rAAV viruses that embodiment 2 obtains (MOI 10
4) serum-free medium, hatch 5 hours after, replace with normal culture medium.After 72 hours, use fluorescence microscope, and carry out cell divide, and calculate transduction efficiency (n=3) with flow cytometer.
Table 3 different serotypes rAAV is to the transduction efficiency of LLC cell or HLEC cell
| RAAV serotype | LLC cell transduction efficient (%) | HLEC cell transduction efficient (%) |
| rAAV2/1 | 58 | 78 |
| rAAV2/2 | 53 | 50 |
| rAAV2/3 | 19 | 20 |
| rAAV2/4 | 34 | 29 |
| rAAV2/5 | 73 | 49 |
| rAAV2/6 | 45 | 32 |
| rAAV2/7 | 26 | 19 |
| rAAV2/8 | 23 | 18 |
| rAAV2/9 | 49 | 45 |
| rAAV2/10 | 38 | 28 |
| rAAV2/11 | 36 | 35 |
As seen various serotype rAAV are all different to the transduction efficiency of LLC cell or HLEC cell, but comparatively speaking, rAAV2/1, rAAV2/2 and rAAV2/5 are higher to the transduction efficiency of LLC cell or HLEC cell.
RAAV2/2-VAS, the preparation of rAAV2/1-VAS and rAAV2/5-VAS
People vasostatin full-length cDNA is increased by the people liver first chain cDNA by PCR.The exclusive primer of amplification vasostatin is Vas-F (5 '-AACTCGAGCCCGCCATGCTGCTATCC) and Vas-R (5 '-AAAAGCTTCTAGTTGTCTGGCCGCACAAT).Restriction enzyme site CTCGAG (Xho I) and AAGCTT (HindIII) are introduced into to make things convenient for sub-clone, have introduced termination codon CTA in primer Vas-R.The PCR condition is 94 ℃, 50 ℃ and 68 ℃ each 45 seconds, totally 36 circulations.The PCR product is through its correctness of sequence verification, and sub-clone obtains the rAAV2-VAS expression plasmid to the AAV-2 expression vector.The promoter of this expression plasmid is cmv enhancer/chicken β-actin promoter.For adding strongly expressed, after inserting gene, also introduced the WPRE element.
Adopt many plasmids of calcium phosphate non-auxiliary virus legal system to be equipped with recombinant virus: with this expression plasmid, the AV helper plasmid, with rAAV2 helper plasmid cotransfection 293 cells, harvesting after 60-72 hour, with sodium deoxycholate (0.5%) and benzonase (30u/ml) cracking, centrifugal (3000g 30min) removes cell debris, and caesium chloride density gradient centrifugation, affinitive layer purification promptly get Recombinant rAAV 2/2-VAS.
During the virus preparation, the rAAV2 helper plasmid is replaced with rAAV1 helper plasmid and rAAV5 helper plasmid respectively, all the other methods are the same, then obtain rAAV2/1-VAS and rAAV2/5-VAS virus.
The preparation of rAAV2/1-CCT-VAS, rAAV2/2-CCT-VAS, rAAV2/5-CCT-VAS
Cmv enhancer in embodiment 4 expression plasmids/chicken β-actin promoter is changed to the CCT promoter, and all the other steps are carried out according to embodiment 4 respectively.
The preparation of rAAV2/1-CC10-VAS, rAAV2/2-CC10-VAS, rAAV2/5-CC10-VAS
Cmv enhancer in embodiment 4 expression plasmids/chicken β-actin promoter is changed to the CC10 promoter, and all the other steps are carried out according to embodiment 4 respectively.
Embodiment 7
RAAV2/2-KAL, the preparation of rAAV2/1-KAL and rAAV2/5-KAL
Genes of interest vasostatin in embodiment 4 expression plasmids is changed to kallistatin, and all the other steps are carried out according to embodiment 4 respectively.
Embodiment 8
The preparation of rAAV2/1-CCT-KAL, rAAV2/2-CCT-KAL, rAAV2/5-CCT-KAL
Genes of interest vasostatin in embodiment 5 expression plasmids is changed to kallistatin, and all the other steps are carried out according to embodiment 5 respectively.
Embodiment 9
The preparation of rAAV2/1-CC10-KAL, rAAV2/2-CC10-KAL, rAAV2/5-CC10-KAL
Genes of interest vasostatin in embodiment 6 expression plasmids is changed to kallistatin, and all the other steps are carried out according to embodiment 6 respectively.
RAAV2/2-AS, the preparation of rAAV2/1-AS and rAAV2/5-AS
Genes of interest vasostatin in embodiment 4 expression plasmids is changed to angiostatin, and all the other steps are carried out according to embodiment 4 respectively.
Embodiment 11
The preparation of rAAV2/1-CCT-AS, rAAV2/2-CCT-AS, rAAV2/5-CCT-AS
Genes of interest vasostatin in embodiment 5 expression plasmids is changed to angiostatin, and all the other steps are carried out according to embodiment 5 respectively.
Embodiment 12
The preparation of rAAV2/1-CC10-AS, rAAV2/2-CC10-AS, rAAV2/5-CC10-AS
Genes of interest vasostatin in embodiment 6 expression plasmids is changed to angiostatin, and all the other steps are carried out according to embodiment 6 respectively.
Embodiment 13
The preparation of Recombinant rAAV 2/5-CCT-AS solution
Get sodium chloride 0.58g, sodium citrate 0.58g, citric acid 0.006g, 20% human albumin 1ml adds the about 80ml of water for injection, after the dissolving, adds the injection water to 100ml, and aseptic filtration must be diluted with buffer (pH6.5-7.5).
Get Recombinant rAAV 2/5-CCT-AS virus 5 * 10 that embodiment 11 methods make
14Vp, add dilution with buffer to 100ml, aseptic filtration, promptly.
Embodiment 14
The preparation of Recombinant rAAV 2/1-CCT-KAL solution
Get sodium chloride 0.58g, sodium citrate 0.58g, citric acid 0.006g, 20% human albumin 1ml adds the about 80ml of water for injection, after the dissolving, adds the injection water to 100ml, and aseptic filtration must be diluted with buffer (pH6.5-7.5).
Get Recombinant rAAV 2/1-CCT-KAL virus 5 * 10 that embodiment 8 methods make
14Vp, add dilution with buffer to 100ml, aseptic filtration, promptly.
Embodiment 15
The preparation of Recombinant rAAV 2/5-CCT-VAS solution
Get sodium chloride 0.58g, sodium citrate 0.58g, citric acid 0.006g, 20% human albumin 1ml adds the about 80ml of water for injection, after the dissolving, adds the injection water to 100ml, and aseptic filtration must be diluted with buffer (pH6.5-7.5).
Get Recombinant rAAV 2/5-CCT-VAS virus 5 * 10 that embodiment 5 methods make
14Vp, add dilution with buffer to 100ml, aseptic filtration, promptly.
Embodiment 16
The preparation of Recombinant rAAV 2/1-CC10-VAS solution
Get sodium chloride 0.58g, sodium citrate 0.58g, citric acid 0.006g, 20% human albumin 1ml adds the about 80ml of water for injection, after the dissolving, adds the injection water to 100ml, and aseptic filtration must be diluted with buffer (pH6.5-7.5).
Get Recombinant rAAV 2/1-CC10-VAS virus 5 * 10 that embodiment 6 methods make
14Vp, add dilution with buffer to 100ml, aseptic filtration, promptly.
Embodiment 17
The preparation of Recombinant rAAV 2/5-CC10-KAL spray
Get sodium chloride 0.58g, sodium citrate 0.58g, citric acid 0.006g, 20% human albumin 1ml adds the about 80ml of water for injection, after the dissolving, adds the injection water to 100ml, and aseptic filtration must be diluted with buffer (pH6.5-7.5).
Get Recombinant rAAV 2/5-CC10-KAL virus 5 * 10 that embodiment 9 methods make
14Vp, add dilution with buffer to 100ml, aseptic filtration divides to be filled in the spray container, promptly.
Embodiment 18
The preparation of Recombinant rAAV 2/1-CC10-AS solution
Get sodium chloride 0.58g, sodium citrate 0.58g, citric acid 0.006g, 20% human albumin 1ml adds the about 80ml of water for injection, after the dissolving, adds the injection water to 100ml, and aseptic filtration must be diluted with buffer (pH6.5-7.5).
Get Recombinant rAAV 2/1-CC10-AS virus 5 * 10 that embodiment 12 methods make
14Vp, add dilution with buffer to 100ml, aseptic filtration, promptly.
Claims (3)
1. targeting gene therapy medicine, by the expression of viral vector mediation antineoplastic vascular growth factor gene, it is characterized in that: viral vector is selected from rAAV2/1, rAAV2/2 or rAAV2/5; The antineoplastic vascular growth factor gene is selected from Vasostatin, kallistatin or angiostatin; The promoter of gene expression frame is selected from cmv enhancer/chicken β-actin promoter, CCT promoter or CC10 promoter; The regulating transgene expression element is WPRE.
2. the gene therapy medicament of claim 1 is used to prepare the purposes of the medicine for the treatment of lung cancer metastasis.
3. the gene therapy medicament of claim 1 is used to prepare the purposes of the medicine for the treatment of pulmonary metastases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101339145A CN101199859B (en) | 2007-10-15 | 2007-10-15 | Inhibition lung cancer transfer and lung transfer tumor gene medicament |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101339145A CN101199859B (en) | 2007-10-15 | 2007-10-15 | Inhibition lung cancer transfer and lung transfer tumor gene medicament |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010169434A Division CN101804210A (en) | 2007-10-15 | 2007-10-15 | Gene medicine for inhibiting lung cancer metastasis and pulmonary metastases |
| CN2010101707933A Division CN101822845B (en) | 2007-10-15 | 2007-10-15 | Genomic medicine for inhabiting lung cancer transfer and pulmonary metastatic tumor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101199859A true CN101199859A (en) | 2008-06-18 |
| CN101199859B CN101199859B (en) | 2011-10-05 |
Family
ID=39515343
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007101339145A Expired - Fee Related CN101199859B (en) | 2007-10-15 | 2007-10-15 | Inhibition lung cancer transfer and lung transfer tumor gene medicament |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101199859B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008725A (en) * | 2010-11-09 | 2011-04-13 | 北京大学 | Application of human UTP14a expression inhibitor to preparation of cell proliferation inhibitor |
| CN102286489A (en) * | 2011-06-27 | 2011-12-21 | 华侨大学分子药物学研究所 | Pilot-scale preparation of recombinant human vasostatin protein |
| CN104815338A (en) * | 2015-05-01 | 2015-08-05 | 许瑞安 | KAL (Kallistating) genetic recombination gland related virus vector carried gene therapy drug |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100431613C (en) * | 2005-12-31 | 2008-11-12 | 刁勇 | Two kinds of novel angiogenesis-inhibiting medicine and its uses in preventing and treating tumour |
-
2007
- 2007-10-15 CN CN2007101339145A patent/CN101199859B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008725A (en) * | 2010-11-09 | 2011-04-13 | 北京大学 | Application of human UTP14a expression inhibitor to preparation of cell proliferation inhibitor |
| CN102008725B (en) * | 2010-11-09 | 2012-06-06 | 北京大学 | Application of human UTP14a expression inhibitor in preparation of cell proliferation inhibitor |
| CN102286489A (en) * | 2011-06-27 | 2011-12-21 | 华侨大学分子药物学研究所 | Pilot-scale preparation of recombinant human vasostatin protein |
| CN104815338A (en) * | 2015-05-01 | 2015-08-05 | 许瑞安 | KAL (Kallistating) genetic recombination gland related virus vector carried gene therapy drug |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101199859B (en) | 2011-10-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1966082B (en) | Genetic medicine for preventing and treating cancer of colon and rectum, preparation process and use thereof | |
| JP2021097675A (en) | Aav vectors targeted to the central nervous system | |
| US7923436B2 (en) | Methods and compounds for controlled release of recombinant parvovirus vectors | |
| CA2710928C (en) | Parvovirus cancer therapy and combination with chemotherapy | |
| AU2001268149A1 (en) | Methods and compounds for controlled release of recombinant parvovirus vectors | |
| CN103623425A (en) | Medicament for inhibiting neovascularization diseases angiogenesis by using dual-target antagomir | |
| CN101199859B (en) | Inhibition lung cancer transfer and lung transfer tumor gene medicament | |
| RU2679442C1 (en) | Treatment of cancer by parvovirus in combination with bevacizumab | |
| CN101822845B (en) | Genomic medicine for inhabiting lung cancer transfer and pulmonary metastatic tumor | |
| CN109568299A (en) | Ambroxol purposes in preparing tumor chemotherapeutic drug Synergistic preparations | |
| KR20230170652A (en) | Local-regional perfusion of the kidney | |
| CN101804210A (en) | Gene medicine for inhibiting lung cancer metastasis and pulmonary metastases | |
| KR20230173088A (en) | Local-regional perfusion of the non-quiescent beating heart. | |
| CN106955292A (en) | A kind of pharmaceutical composition and purposes for treating the cancer of the esophagus | |
| CN104815338A (en) | KAL (Kallistating) genetic recombination gland related virus vector carried gene therapy drug | |
| CN1952160A (en) | Recombinant of intelligent adenovirus vector and khp53 gene and application thereof | |
| CN101683518B (en) | Application of FHL 1 in preparing medicament for treating tumour | |
| Ning et al. | Gene therapy with the angiogenesis inhibitor endostatin in an orthotopic lung cancer murine model | |
| CN107921075B (en) | For treating the parvovirus preparation of tumour | |
| US20050281785A1 (en) | Method of reducing side effects of cancer therapy using p53 recombinant adenovirus | |
| CN106047931B (en) | A kind of novel capsid protein gene modification adenovirus vector and application thereof | |
| Brentjens et al. | 401. Unexpected toxicity of cyclophosphamide followed by adoptively transferred CD19-targeted T cells in a patient with Bulky CLL | |
| CN101116747B (en) | Cytosine arabinoside and gland correlated virus composite preparations and uses thereof | |
| CN100355458C (en) | Use of artificial proliferative inhibiting gene-adenovirus expression carrier | |
| CN101138637A (en) | Antineoplastic medicament and adenovirus composite preparations and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111005 Termination date: 20141015 |
|
| EXPY | Termination of patent right or utility model |