CN104815338A - KAL (Kallistating) genetic recombination gland related virus vector carried gene therapy drug - Google Patents
KAL (Kallistating) genetic recombination gland related virus vector carried gene therapy drug Download PDFInfo
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- CN104815338A CN104815338A CN201510219195.3A CN201510219195A CN104815338A CN 104815338 A CN104815338 A CN 104815338A CN 201510219195 A CN201510219195 A CN 201510219195A CN 104815338 A CN104815338 A CN 104815338A
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Abstract
The invention relates to a KAL (Kallistating) genetic recombination gland related virus vector carried gene therapy drug rAAV9-KAL. The treatment can be performed on the portability lung cancer through the gene drug and the gene drug can also be applied to the in-situ lung cancer, the liver cancer, the colon cancer, the breast cancer and other tumors according to experimental research results; the gene drug is suitable for an rAAV9 type vector and also suitable for other serotype vectors rAAV1-rAAV8 and rAAV10-rAAV12; the gene drug is suitable for a single-chain rAAV vector and also suitable for a double-chain rAAV vector.
Description
Technical field:
The present invention relates to a kind of gene therapy medicament utilizing recombined glandulae correlation viral vectors.
Background technology:
At present in China, pulmonary carcinoma is the patients with lung cancer of one of malignant tumor of harm people ' s health, the treatment of standardization Radiotherapy chemotherapy, and mean survival time (MST) is 28.7 months, is the number one killer threatening patients ' lives! Along with the development of biotechnology, genomic medicine becomes the new treatment means of one that people pay close attention to gradually.Said genomic medicine, refers to and utilizes gene sequence data, obtains new drug candidates through bioinformatic analysis, high flux gene expression, high flux functional screening and the exploitation of inside and outside pharmacodynamic study.Kallistatin (KAL) is a kind of serpin, the albumen that energy specificity is combined with blood plasma type kallikrein and is not combined with tectotype kallikrein, be present in Various Tissues and body fluid, be human endogenous's property angiostatin, there is the effects such as antiinflammatory, antioxidation, resisting hypertension, antitumor.At present, multiple labs active Immunotherapy of Cancer Induced of KAL, the tumor related to comprises breast carcinoma, colon cancer, hepatocarcinoma, gastric cancer and pulmonary carcinoma etc.Although have been reported title, utilize kallistatin albumen or the plasmid containing kallistatin gene for the treatment of lung tumors, but, exist the half-life shorter, need the problems such as high dose in a short time continues medication puzzled, therefore in the urgent need to find a kind of pulmonary carcinoma targeting, can the carrier of long-term express, for carrying kallistatin gene, play the effect for the treatment of pulmonary carcinoma better.
In order to solve the problem better, this laboratory selected have extensive host range, can mediate foreign gene long-term expression, security performance good, the mankind do not have pathogenic, can transfection division or non-division mammalian cell and have 9 type recombined glandulae correlation viral vectors (rAAV9) [Michelfelder of pulmonary's targeting, S. etc., PLoS One, 2011,6 (8): e23101; Zincarelli, C. etc., Mol Ther, 2009,16 (6): 1073-1080; Bell, C.L. etc., J Clin Invest, 2011,121 (6): 2427-2435; Walia, J.S.MolTher.2015,2 (3): 414-422].CDNA rAAV9 carrier being carried kallistatin of the present invention, lumbar injection is in people's pulmonary carcinoma mice with tumor body, and being negative control with PBS, take cisplatin as positive control, observes 49 days, finds the propagation of the obvious Tumor suppression of rAAV9-KAL energy.Treatment pulmonary carcinoma genomic medicine rAAV9-KAL provided by the invention, there is not yet relevant report both domestic and external so far.
Summary of the invention:
Lung cancer therapy genomic medicine (rAAV9-KAL) provided by the invention, comprises the following steps:
(1) gene recombination plasmid is the cDNA gene containing kallistatin, gene expression frame two ends are inverted terminal repeat sequence (the inverted ter min al repeat of AAV2 serotype, ITR), bovine growth hormone poly A element (bovine growth hormone polyadenylation signal, BGH poly A), wherein also containing ampicillin selectors AMP, woodchuck hepatitis post-transcriptional control element WPRE (Woodchuck hepatitisPost-transcriptional Regulatory Element) enhancer, CAG chicken β-actin promoter promoter.Vector plasmid is the cap gene of the rep gene of serotypes A AV2, AAV9.
(2) three plasmid calcium phosphate transfection methods produce rAAV9-KAL, adopt CHT chromatograph associating ANX chromatograph to carry out purification.
(3) set up pulmonary carcinoma mice with tumor model, adopt lumbar injection form to carry out administration, observed and recorded tumor size weekly, by gross tumor volume after 7 weeks, weight and kallistatin protein expression situation, evaluate the antitumous effect of rAAV9-KAL.
Wherein: described lung cancer therapy rAAV9-KAL dosage, is selected from 1 × 10
2vG/mL is to 1 × 10
16any one of VG/mL; Route of administration is suitable for lumbar injection, intravenous injection, intratumor injection, intramuscular injection, subcutaneous injection, oral etc.
RAAV genomic medicine of the present invention, not only can treat Lewis lung carcinoma, also may be used for original position pulmonary carcinoma.
RAAV genomic medicine of the present invention, may be used for the tumors such as hepatocarcinoma, colon cancer, breast carcinoma.
RAAV genomic medicine of the present invention, is not only applicable to rAAV9 type carrier, is applicable to other serotype vectors rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12 etc. yet.
RAAV genomic medicine of the present invention, is not only applicable to strand rAAV carrier, is applicable to double-strand rAAV carrier yet.
Accompanying drawing illustrates:
Fig. 1. the extracorporeal anti-tumor function of different serotypes rAAV-KAL
Fig. 2. the kallistatin expressing quantity of different way of administration
Fig. 3. different serotypes rAAV-KAL is to subcutaneous transplantation function of tumor
Fig. 4. single double-strand rAAV-KAL antitumor action
Fig. 5. the antitumor action of various dose rAAV9-KAL
Fig. 6 .KAL albumen is in the expression and distribution situation of each organ
Fig. 7. the immunohistochemical staining of lung and tumor tissues KAL
Fig. 8. organize HE to dye
Fig. 9. mice serum biochemical analysis
embodiment:
Following examples are only and help those skilled in the art to understand the present invention better, but do not limit the present invention in any way.
" embodiment 1 " antitumor activity in vitro
Trypsinization degree of converging is the cell of 60%-70%, and every hole adds 100 μ L 5 × 10
4individual/mL cell in 96 orifice plates, simultaneously with the blank cultures of 100 μ L as blank; After 12h, cell attachment, adds rAAV1-KAL, rAAV2-KAL, rAAV5-KAL, rAAV6-KAL, rAAV8-KAL and rAAV9-KAL according to MOI=10000 and infects, measure after 48h; Every hole adds the CCK-8 solution of 10ul; Absorbance is measured at 450n m continue to hatch 1h in cell culture incubator after.According to cell proliferation rate (%)=(1-test group OD450/ matched group OD450) × 100%, calculate Cytostatic to tumor cell rate.From data analysis, several rAAV carrier all can effective transfection H446 cell, and wherein 2,5,9 type inhibitory action better (see Figure 1A).
The vivoexpression test of " embodiment 2 " KAL albumen
Trypsinization degree of converging is the cell of 60%-70%, and every hole adds 100 μ L 1 × 10
5individual/mL cell in 96 orifice plates, simultaneously with the blank cultures of 100 μ L as blank; After 12h, cell attachment, add rAAV1-KAL, rAAV2-KAL, rAAV5-KAL, rAAV6-KAL, rAAV8-KAL and rAAV9-KAL according to MOI=10000 to infect, 3 multiple holes, cultivate 72h, collecting cell culture supernatant, adopts ELISA method, measures the KAL protein expression situation in cell conditioned medium liquid.All serotype vectors are energy transfection H446 cell all, but take temperature from KAL protein expression, the effect of rAAV2 and rAAV9 is best (see Figure 1B).
" embodiment 3 " vivo medicine-feeding approach is tested
The cleaning grade Km mice in 7-8 week, after adaptability grows 7 days, be divided into 5 groups at random, often organize 3, be respectively: normal group (Normal), intravenous injection group (intravenous injection, I.V.), lumbar injection group (intraperitoneal injection, I.P.), intramuscular injection group (intramuscular injection, I.M.) and subcutaneous injection group (Intracutaneous injection, I.C.).According to 5 × 10
11vG/ administration, every only 100 μ L, respectively after the same day, 1d, 3d, tail venous blood sampling weekly, 7 weeks posterior orbits are got blood prostration mortar and are put to death, and measure KAL protein expression situation in serum.Data result shows, different way of administration, and the expression of KAL albumen is different, the expressing quantity of 10-18d compares, I.V.>I.P.>I.M> I.H. (Fig. 2 A).Wherein, the carrier protein expression efficiency of intraperitoneal injection is about 80% of intravenously administrable, and protein expression trend is consistent (Fig. 2 B).
The anti-tumor in vivo test of " embodiment 4 " people's Pulmonary carcinoma nude mice Transplanted tumor model
(1) the H446 human lung carcinoma cell cultivated, according to 2 × 10
6individual/cell number is only inoculated in nude mice dorsal sc, and after 7-10 days, gross tumor volume is bred to 50mm
3time, become matched group (PBS), test group (rAAV), administration group at random, often organize 6, observe after 49 days, eye socket gets blood, and dislocation is put to death.Measure diameter of tumor weekly, calculate gross tumor volume (tumor volume, TV): TV=1/2 × a × b
2, wherein a, b represent length, width and height respectively.Relative tumour volume (relative tumor volume, RTV) is calculated, RTV=V according to measurement result
t/ V
0.Wherein V
0for (d during point cage administration
0) measuring gained gross tumor volume, Vt is gross tumor volume when measuring each time.Antitumor activity evaluation index is Relative tumor rate of increase T/C (%).T/C%=TRTV*100%/CRTV (TRTV: treatment group RTV; CRTV: negative control group RTV).The standard of curative effect evaluation: T/C%>60% is invalid; T/C%≤60%, and be effective through statistical procedures P<0.05.
(2) inhibitory action of people's Pulmonary carcinoma nude mice transplantation tumor of different serotypes administered vehicle
Be vaccinated with the transplanted tumor mice of H446 cell, inoculate after 7-10 days, gross tumor volume is bred to 50mm
3time, random point 4 groups, often organize 6, be divided into negative control group (PBS), ssrAAV2-KAL, ssrAAV5-KAL, ssrAAV9-KAL test group, intraperitoneal injection 100 μ L, test group administration titre is 5 × 10
12vG/mL, observe after 49 days, eye socket gets blood, dislocation is put to death, peel off tumor, measure gross tumor volume, calculate the relative appreciation rate of tumor, check through T, one factor analysis of variance, P<0.05, has statistical significance, the Relative tumor rate of increase 35% of rAAV9 group, has significant difference (see Fig. 3) compared with negative control group.
(3) inhibition of single double-strand rAAV9-KAL lung carcinoma subcutaneous transplantation tumor compares
Be vaccinated with the transplanted tumor mice of H446 cell, inoculate after 7-10 days, gross tumor volume is bred to 50mm
3time, random point 4 groups, often organize 6, be divided into PBS negative control group (NC), the positive group (PC) of cisplatin, ssrAAV9-KAL test group (SS) and double-strand dsrAAV9-KAL test group (DS), intraperitoneal injection 100 μ L, cisplatin administration concentration (0.2mg/mL), test group administration concentration is 5 × 10
12vG/mL, observes 49d, measures and calculate gross tumor volume, calculates the relative appreciation rate of tumor, and through T inspection, one factor analysis of variance, P<0.05, has statistical significance (see Fig. 4).Compare with positive group with feminine gender group, double-strand or strand have significant difference, but, do not have significant difference therebetween.
(4) the rAAV9-KAL antineoplastic action research of various dose
Be vaccinated with the transplanted tumor mice of H446 cell, inoculate after 7-10 days, when gross tumor volume is bred to 50mm3, random point 5 groups, often organize 6, be divided into PBS negative control group (NC), the positive group (PC) of cisplatin administration, the high, medium and low dosetest group of ssrAAV9-KAL (sH, sM, sL), intraperitoneal injection 100 μ L, cisplatin administration concentration (0.2mg/mL), test group administration concentration is respectively 5 × 10
12, 1 × 10
12, 2 × 10
11vG/mL, observes 49d, measures gross tumor volume, according to V=1/2 × a × b2, (a and b represents long and wide respectively) calculates gross tumor volume, according to formula T/C%=TRTV*100%/CRTV, calculate the relative appreciation rate of tumor, the rate of increase (see Fig. 5) of statistics tumor.P<0.05, has statistical significance.The high dose group rate of increase is 36.7%, and the rate of increase of middle dosage group is 48.5%, and the rate of increase of low dose group is 69.8%.
(5) KAL albumen is in the distribution of tumor tissues and pulmonary and expression
The paraffin section de-waxing rehydration of pulmonary and tumor tissues, after antigen retrieval, enzyme-deactivating, adopts kallistatin primary antibodie to hatch, then adds ELIAS secondary antibody and hatch, and after DAB colour developing, haematoxylin is redyed, mounting, basis of microscopic observation.In pulmonary and tumor tissue section, the KAL protein expression of yellowish-brown is expressed obviously in rAAV9-KAL administration group, and feminine gender is organized and be showed no dyeing (see Fig. 6) in positive group.
(6) the expression in vivo test of KAL albumen
By the nude mice of acquisition or mouse tissue, cut into tiny fragment, the ratio adding 100-200 μ L according to every 20mg tissue adds lysate (containing 1mM PMSF); Use glass homogenizer homogenate, until fully cracking.The centrifugal 3-5min of 10000-14000g, gets supernatant, can carry out follow-up test operation.Result shows, and the expression of KAL albumen is the highest in liver, is secondly kidney, the heart, harmonization of the stomach intestinal (see Fig. 7).
" embodiment 4 " acute toxicity test
The cleaning grade Km mice in 7-8 week, male and female half and half, after adaptability grows 7 days, are divided into 4 groups at random, and often organize 10 (male and female half and half), normal group and test group, test group lumbar injection rAAV9-KAL carrier 100 μ L, concentration is respectively 3.0 × 10
12vG/, 1.0 × 10
12vG/, 3.3 × 10
11vG/ only, observes 14d, often organizes and all death state does not occur.Often organize the skin of mice, mucosa, hair color, eyes, breathing, circulation, autonomous and central nervous system's behavior expression be showed no exception.Eye socket gets blood, and dislocation is put to death, the taking-up of each several part tissue, and paraffin section, HE dyes observation.The tissue such as lung, the heart, liver, kidney is showed no exception (see Fig. 8).Serum is used for biochemical indicator and detects, the hepatic and renal function of blood serum bio-chemical analysis mice, and at total protein (TP), total albumin (ALB), does not have significant difference between each group; Blood glucose and total bilirubin (TBIL) all have rising in rAAV administration group, compare, have significant difference with normal group; Aspartate transaminase (AST), alanine aminotransferase (ALT) are showed no obvious rising, and the liver function of visible rAAV carrier to mice does not have a significant effect.In rAAV administered vehicle group, carbamide (BUN) in serum and creatinine (CREA) measurement result raise, be singly there is no significant difference (see Fig. 9) compared with matched group, illustrate that the renal function of rAAV carrier on mice does not affect.
Claims (7)
1. the genomic medicine of a pulmonary carcinoma targeting, it is characterized in that, described genomic medicine is the 9 type recombined glandulae correlation viral vectors rAAV9-KAL of the cDNA carrying kallistatin, gene recombination plasmid is the cDNA gene containing kallistatin, gene expression frame two ends are inverted terminal repeat sequence (the inverted ter min alrepeat of AAV2 serotype, ITR), bovine growth hormone poly A element (bovine growthhormone polyadenylationsignal, BGH poly A), wherein also containing ampicillin selectors AMP, woodchuck hepatitis post-transcriptional control element (Woodchuck hepatitis Post-transcriptional Regulatory Element) enhancer WPRE, CAG chicken β-actin promoter promoter vector plasmid is the rep gene of serotypes A AV2, the cap gene of AAV9.
2. the application in lung cancer therapy medicine prepared by genomic medicine according to claim 1.
3. application according to claim 2, be on pulmonary carcinoma mice with tumor model, adopt the administration of lumbar injection mode, observed and recorded tumor size weekly, by gross tumor volume, weight and kallistatin protein expression situation after 7 weeks, evaluate the antitumous effect of rAAV9-KAL.
4. the application described in claim 2,3, described lung cancer therapy rAAV9-KAL dosage, is selected from 1 × 10
2vG/mL is to 1 × 10
16any one of VG/mL.
5. the application described in claim 2,3, its genomic medicine not only can treat Lewis lung carcinoma, also may be used for treatment original position pulmonary carcinoma.
6. genomic medicine according to claim 1, is not only applicable to rAAV9 type carrier, is applicable to other serotype vectors rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12 etc. yet.
7. genomic medicine according to claim 1 can be not only the rAAV carrier of strand, is also applicable to the rAAV carrier of double-strand.
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| CN112852879A (en) * | 2021-01-15 | 2021-05-28 | 华侨大学 | HBV PreS1 polypeptide modified human hepatocyte specific targeting rAAV2 vector and preparation method and application thereof |
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