DECORIN GENE DELIVERY SYSTEM AND CANCER TREATMENT
20170080106 ยท 2017-03-23
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2799/022
CHEMISTRY; METALLURGY
A61P17/02
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
A61K48/005
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
C12N2710/10033
CHEMISTRY; METALLURGY
C07K14/70578
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
International classification
A61K48/00
HUMAN NECESSITIES
C12N7/00
CHEMISTRY; METALLURGY
C07K14/705
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a novel gene delivery system and recombinant adenovirus comprising the decorin-encoding sequence to enhance transduction efficiency of transgenes, a pharmaceutical anti-tumor composition comprising the recombinant adenovirus, a pharmaceutical composition having improved tissue penetration potency and a pharmaceutical composition for treating a disease or disorder associated with accumulation of excess extracellular matrix.
Claims
1-16. (canceled)
17. A method for enhancing transduction efficiency of a recombinant adenovirus expression vector into a tumor cell in a solid tumor, the method comprising: (a) preparing the recombinant adenovirus expression vector, the recombinant adenovirus expression vector comprising a first nucleotide sequence of interest operably linked to a first promoter and a polyadenylation sequence (promoter-nucleotide sequence of interest-poly A sequence) and a second nucleotide sequence encoding decorin operably linked to a second promoter and a polyadenylation sequence (promoter-decorin-encoding nucleotide sequence-poly A sequence), wherein the promoter-nucleotide sequence of interest-poly A sequence and the promoter-decorin-encoding nucleotide sequence-poly A sequence are inserted into a deleted E3 region and E1 region, respectively, of the adenovirus genome sequence; and (b) infecting the tumor cell with the prepared recombinant adenovirus expression vector by administering to a subject having the solid tumor the prepared recombinant adenovirus expression vector via intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal route, or intratumoral injection, wherein expression of decorin in the tumor cell infected with the prepared recombinant adenovirus expression vector enhances transduction efficiency of the prepared recombinant adenovirus expression vector which has not yet infected the tumor cell by binding of the expressed decorin to type-I collagen fibril in an extracellular matrix of connective tissue surrounding the tumor cell.
18. The method according to claim 17, wherein the recombinant adenovirus expression vector is replication incompetent.
19. The method according to claim 17, wherein the recombinant adenovirus expression vector further comprises a nucleotide sequence of interest operably linked to a promoter and a polyadenylation sequence (promoter-nucleotide sequence of interest-poly A sequence).
20. The method according to claim 17, wherein the promoter-nucleotide sequence of interest-poly A sequence is inserted into the deleted E1 region or E3 region of the adenovirus genome sequence.
21. The method according to claim 20, wherein the promoter-nucleotide sequence of interest-poly A sequence is inserted into the deleted E1 region and the promoter-decorin-encoding nucleotide sequence-poly A sequence is inserted into the deleted E3 region.
22. The method according to claim 20, wherein the promoter-nucleotide sequence of interest-poly A sequence is inserted into the deleted E3 region and the promoter-decorin-encoding nucleotide sequence-poly A sequence is inserted into the deleted E1 region.
23. A method for enhancing transduction efficiency of a recombinant adenovirus expression vector into a tumor cell in a solid tumor, the method comprising: (a) preparing the recombinant adenovirus expression vector, the recombinant adenovirus expression vector comprising a nucleotide sequence encoding decorin operably linked to a promoter and a polyadenylation sequence (promoter-decorin-encoding nucleotide sequence-poly A sequence), wherein the promoter-decorin-encoding nucleotide sequence-poly A sequence is inserted into a deleted E1 region or E3 region of the adenovirus genome sequence; and (b) infecting the tumor cell with the prepared recombinant adenovirus expression vector by administering to a subject having the solid tumor the prepared recombinant adenovirus expression vector via intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal route, or intratumoral injection, wherein expression of decorin in the tumor cell infected with the prepared recombinant adenovirus expression vector enhances transduction efficiency of the prepared recombinant adenovirus expression vector which has not yet infected the tumor cell.
24. The method according to claim 23, wherein the expression of decorin enhances transduction efficiency of the prepared recombinant adenovirus expression vector by binding of the expressed decorin to type-I collagen fibril in an extracellular matrix of connective tissue surrounding the tumor cell.
25. The method according to claim 23, wherein the prepared recombinant adenovirus expression vector comprises an inactivated E1B 19 gene, an inactivated E1B 55 gene, or an inactivated E1B 19 and E1B 55 genes.
26. The method according to claim 23, wherein the prepared recombinant adenovirus expression vector comprises an active E1A gene.
27. A method for treating a cancer of a subject having a solid tumor, the method comprising: administering to the subject a recombinant adenovirus expression vector via intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal route, or intratumoral injection, wherein the recombinant adenovirus expression vector comprises a first nucleotide sequence encoding an antitumor protein operably linked to a first promoter and a polyadenylation sequence (promoter-antitumor protein-encoding nucleotide sequence-poly A sequence) and a second nucleotide sequence encoding decorin operably linked to a second promoter and a polyadenylation sequence (promoter-decorin-encoding nucleotide sequence-poly A sequence); wherein expression of decorin in cells of the solid tumor infected with the recombinant adenovirus expression vector enhances transduction efficiency of the recombinant adenovirus expression vector which has not yet infected the tumor cells.
28. The method according to claim 27, wherein the promoter-antitumor protein-encoding nucleotide sequence-poly A sequence is inserted into the deleted E1 region or E3 region of the adenovirus genome sequence.
29. The method according to claim 27, wherein the promoter-decorin-encoding nucleotide sequence-poly A sequence is inserted into the deleted E1 region or E3 region of the adenovirus genome sequence.
30. The method according to claim 27, wherein the promoter-antitumor protein-encoding nucleotide sequence-poly A sequence is inserted into the deleted E1 region and the promoter-decorin-encoding nucleotide sequence-poly A sequence is inserted into the deleted E3 region.
31. The method according to claim 27, wherein the promoter-antitumor protein-encoding nucleotide sequence-poly A sequence is inserted into the deleted E3 region and the promoter-decorin-encoding nucleotide sequence-poly A sequence is inserted into the deleted E1 region.
32. The method according to claim 27, wherein the recombinant adenovirus expression vector comprises an inactivated E1B 19 gene, an inactivated E1B 55 gene, or an inactivated E1B 19 and E1B 55 genes.
33. The method according to claim 27, wherein the recombinant adenovirus expression vector comprises an active E1A gene.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0115] The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims.
Examples
Materials and Methods
[0116] 1. Cell Lines and Cell Culture
[0117] Cell lines for experiments were tumor cell lines such as human brain cancer cell lines (U343, U87MG), cervical cancer cell line (C33A), liver cancer cell line (Hep3B), lung cancer cell line (A549) and mouse melanoma (B16BL6), human normal cell lines (CBHEL, MRC5, IMR90 and W138) and 293 cell line carrying the early gene of adenovirus, E1 region. All cell lines except B16BL6 cell line were available from the ATCC (American Type Culture Collection; Manassas, Va., USA), and B16BL6 mouse melanoma cell line was gifted from Dr. Y. S. Park's research group at the Yonsei University of Korea.
[0118] All cell lines except B16BL6 cell line were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL), penicillin and streptomycin and maintained at 37 C. under 5% CO.sub.2 atmosphere. B16BL6 cell line was cultured in RPMI medium (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL), penicillin and streptomycin and maintained at 37 C. under 5% CO.sub.2 atmosphere.
[0119] 2. Experimental Animals
[0120] In vivo anti-tumor experiments were conducted using 6- to 8-week-old male nude mice (BALB/c-nu) and C57BL/6 mice purchased from Charles River Korea (Seoul, Korea). Mice were maintained under controlled lighting cycle (12-hr light:12-hr dark), temperature (222 C.) and humidity (55-60%), and had free access to radiation-sterilized solid feeds (Orient, Seoul, Korea) and water.
[0121] 3. Generation and Titration of Recombinant Adenoviruses Expressing Decorin
[0122] <1> Generation of Replication-Incompetent Adenoviruses
[0123] We generated EVE3-gene deleted replication-incompetent adenoviruses expressing the decorin gene and the reporter gene lac Z. The pdl-LacZ viral vector was prepared by inserting the lac Z gene as a reporter into the deleted E1 region in the vmdl324Bst vector (gifted from Dr. Verca, University of Fribourgh, Switzerland; Heider, H. et al., Biotechniques, 28(2):260-265, 268-270(2000)). For preparing this vector, the pcDNA-hygro-LacZ plasmid (Invitrogen, Carlsbad, Calif., USA) was digested with HindIII and NaeI to isolate the CMV promoter, lacZ gene and polA and the isolated three sequences were inserted into the E1 adenoviral shuttle vector, pE1sp1A to prepare pE1sp1A/CMV-LacZ shuttle vector. The prepared pE1sp1A/CMV-LacZ shuttle vector was digested with XmnI and cotransformed with vmdl324Bst adenovirus linearized by BstBI into E. coli BJ5183 (Dr. Verca. University of Fribourgh, Switzerland) to induce homologous recombination, obtaining pdl-LacZ adenovirus.
[0124] For constructing decorin-expressing adenoviruses, we constructed the pcDNA3.1-DCNG (containing the wild type decorin cDNA) by inserting the decorin gene (DCNG, D. G. Seidler, University Hospital of Munster, Germany) into the expression vetor pcDNA3.1. Then, the decorin mutant DCNQ having weaker binding affinity to collagen was prepared by inducing a point mutation of E180 amino acid to E180Q in the sixth leucine-rich repeat of the wild type decorin core protein, followed by inserting the mutant gene into the pcDNA3.1 vector to give the expression vector pcDNA3.1-DCNQ. In addition, the decorin mutant DCNK lack of binding affinity to collagen was prepared by inducing a point mutation of E180 amino acid to E180K of the wild type decorin core protein, followed by inserting the mutant gene into the pcDNA3.1 vector to give the expression vector pcDNA3.1-DCNK. Each of the vectors prepared above was digested with EcoRI and XbaI to give a 1 kb DNA fragment, which was inserted into the pCA14 vector (Microbix, Ontario, Canada) to generate pCA14-DCNG, pCA14-DCNQ and pCA14-DCNK vectors.
[0125] Each of the pCA14-DCNG, pCA14-DCNQ and pCA14-DCNK vectors thus prepared was digested with BgIII to obtain the CMV-DCN-polA expression cassette expressing the decorin gene under the control of the CMV promoter, after which the cassette was inserted into the adenoviral E3 shuttle vector, pSP72E3, to obtain the adenoviral E3 shuttle vectors, pSP72E3-DCNG, pSP72E3-DCNQ and pSP72E3-DCNK.
[0126] The adenoviral E3 shuttle vectors thus prepared were linearized with PvuI or XmnI and cotransformed into E. coli BJ5183 together with the adenoviral total vector, pdl-LacZ linearized with SpeI for homologous recombination, generating the adenoviral vectors dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK which express the lac Z gene and decorin simultaneously (
[0127] <2> Generation of Tumor-Specific Oncolytic Adenovirus
[0128] We generated tumor-specific oncolytic adenoviruses expressing the decorin gene. Specifically, each of dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK was linearized with PvuI or XmnI and cotransformed into E. coli BJ5183 together with the E1B 19 kDa/E1B 55 kDa-deleted pAdE1B19/55 adenovirus vector linearized with SpeI (KFCC 11288) for homologous recombination, generating the Ad-E1B-DCNG, Ad-E1B-DCNQ and Ad-E1B-DCNK adenovirus vectors, respectively (
[0129] All adenoviruses were propagated in 293 cells and their titration was performed according to limited dilution or plaque assay (Hitt, M. et. al., Construction and propagation of human adenovirus vectors. Cell biology: a laboratory handbook. New York: Academic Press Inc, 479-490(1994)), followed by concentration using CsCl gradient and purification.
[0130] 4. Examination of Decorin Expression Pattern
[0131] To examine the decorin expression pattern induced by recombinant adenoviruses of the present invention, tumor-specific oncolytic adenoviruses having the decorin gene (Ad-E1B-DCNG, Ad-E1B-DCNQ and Ad-E1B-DCNK) and Ad-E1B adenovirus as a control were infected to the human liver cancer Hep3B at 3 MOI. At 48 hr after infection, the medium used was recovered and subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Then, the proteins on the gel were electro-transferred to PVDF membrane, incubated with the primary anti-decorin antibody (D. G. Seidler, University Hospital of Munster, Germany) and anti-1-actin antibody (Sigma, St. Louis, Mo., USA), and then incubated with the HRP (horse radish peroxidase)-conjugated secondary antibody (sc-2004; Santa Cruz Biotech., Santa Cruz, Calif.), after which the expression patterns of decorin were revealed using the ECL detection kit (sc-2004; Santa Cruz Biotech).
[0132] 5. Comparable Evaluation of Transduction Efficiency
[0133] To evaluate the transduction efficiency of replication-incompetent adenoviruses expressing LacZ, various human tumor cell lines (U343, U87MG, C33A, Hep3B and A549) and human normal cell lines (CBHEL, IMR90 and W138) were plated onto 24-well plates and infected with dl-LacZ, dl-LacZ-DCNG, dl-LacZ-DCNQ or dl-LacZ-DCNK viruses at an MOI (multiplicity of infection) of 0.1-100. On day 2 after infection, the cell lines were incubated with X-Gal reagent (PBS containing 1 mg/ml, 5 mM K.sub.3Fe(CN).sub.6, 5 mM K.sub.4Fe(CN).sub.6 and 2 mM MgCl.sub.2) at 37 C. for 6 hr for X-gal staining to confirm the transduction efficiency of the LacZ gene induced by the expression of the decorin gene.
[0134] 6. Evaluation on Spreading and Penetration of Adenovirus in Tumor Spheroid
[0135] U343, U87MG, C33A and A549 xenografts were established subcutaneously by injecting cells into the abdomen of 6- to 8-week-old nude mice and once the tumors reached to 150-200 mm.sup.3 in volume, fresh tumor tissue was extracted at surgery, 1-2 mm fragments of the tumor tissue were dissected. These explants were plated individually on 0.75% agarose-coated plates and cultured in DMEM (Gibco BRL) supplemented with 5% FBS (Gibco BRL) and penicillin/streptomycin (Gibco BRL) at 37 C. under 5% CO.sub.2 atmosphere. Medium was renewed once every week. Prior to infection with adenoviruses, spheroids with diameter of 2 mm were transferred to 0.75% agarose-coated 48-well plates and 150 l of DMEM (containing 5% FBS) were added, after which viruses were added at 110.sup.6, 110.sup.7, or 110.sup.8 PFU (plaque-forming unit). 3-days later, the medium was aspirated and spheroids were fixed in a fixation solution for X-gal staining. The surface of X-gal stained spheroids was observed under a stereoscopic microscope (Olympus optical Co., LTD, Tokyo, Japan). For the observation on penetration of adenovirus into tumor spheroid, the X-gal stained tumor spheroids were embedded in O.C.T. compound (Sakura Finetec, Torrance, Calif.) and snap frozen. 10 m frozen section was then placed onto gelatin-coated slide glass. Moreover, the ratio of X-gal stained portion in tumor spheroid was given using the MetaMorph program (Meta imaging series, Version 6.1, Universal imaging corporation TM, Downingtown, Pa.).
[0136] 7. Evaluation on Spreading and Penetration of Adenoviruses In Vivo
[0137] U343, U87MG, C33A, and Hep3B xenografts were established subcutaneously by injecting cells into the abdomen of 6- to 8-week-old nude mice and once the tumors reached to 150-200 mm.sup.3 in volume, mice were randomized into two groups and dl-LacZ and dl-LacZ-DCNG adenovirus at 510.sup.7-110.sup.8 PFU was intratumorally injected five times into the tumors. For A549 xenograft model, A549 cell line was subcutaneously injected into mice and once the tumors reached to 150-200 mm.sup.3 in volume, each of dl-LacZ, dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK at 510.sup.8 PFU was intratumorally injected five times into the tumors. On day 3 after the last injection, animals were sacrificed and tumors were taken, after which they were fixed in 4% paraformaldehyde (PFA) at 4 C. for 4-8 hr and dehydrated in 30% sucrose solution for 12 hr. The dehydrated tumor tissues were embedded in O.C.T. compound and snap frozen, followed by performing X-gal staining described above.
[0138] 8. Analysis of Cytopathic Effect (CPE)
[0139] To evaluate the oncolytic activity of decorin-expressing adenoviruses, human tumor cell lines (U343, U87MG, C33A, Hep3B and A549) and human normal cell lines (CBHEL, MRC5, IMR90 and W138) were plated onto 24-well plates and then infected with Ad-E1, Ad-E1B, Ad-E1B-DCNG, Ad-E1B-DCNQ or Ad-E1B-DCNK adenovirus at MOIs 0.1-100. At the time that cells infected with any one of the viruses exhibited complete cell lysis at the low titer, the dead cells were washed out and cells on the plate were then stained with 0.5% crystal violet in 50% methanol.
[0140] 9. Plaque Development Assay
[0141] To observe the change of plaque size over decorin expression, 310.sup.5 Hep3B cells were placed to 6-well plates and infected with Ad-E1B, Ad-E1B-DCNG, Ad-E1B-DCNQ and Ad-E1B-DCNK adenovirus at 110.sup.4 MOI after one day of cell growth. After 4 hr of incubation, the infected cells were overlayed with agarose-DMEM mixture of 2DMEM (containing 10% FBS and penicillin/streptomycin) at 37 C. and 1.4% UltraPure agarose (Invitrogen, Carsbad, Calif.) at 42 C. and then incubated. Following about 4-16 days of incubation, the size of plaques formed on plates was observed, agarose overlay was removed by soaking with 1 ml of 10% trichoroacetic acid for 30 minutes and the remaining cells were stained with 0.5% crystal violet in 50% methanol. The number of plaques formed was counted.
[0142] 10. Flow Cytometry Analysis for Apoptosis Potential
[0143] To examine apoptosis induced by decorin, human tumor cell lines, U343, U87MG, C33A, Hep3B and A549 were introduced to 25T culture flasks and 24 hr later, were infected with each adenovirus at MOIs of 0.5-50. Cells were treated with 0.1-1 M CPT-11 (camptothecin-11) as a positive control and treated with PBS as a negative control. After 48 hr, 72 hr and 96 hr of infection, the infected cells were collected and fixed in 70% ethanol at 4 C. over 24 hr. Following the fixation, the cells were incubated at 4 C. with a mixture of PI (propidium iodide, 50 g/ml) and RNase (ribonuclease) for 15 min and the FACS analysis was then performed.
[0144] In addition, to examine early apoptosis induced by decorin, several human tumor cell lines were infected with each adenovirus as described above. The infected cells were collected and then processed for Annexin V/PI dual staining according to manufacturer's instruction in the ApoAlert V-FITC (fluorescein isothiocyanate) apoptosis detection kit (Clontech, Palo Alto, Calif.), followed by flow cytometric analysis.
[0145] 11. TUNEL Assay
[0146] U343 (510.sup.4), U87MG (510.sup.4), C33A (510.sup.5), Hep3B (410.sup.5) and A549 (510.sup.4) cells were plated onto a chamber slide and then infected with adenovirus at an MOI of 0.2-20. Following 24 hr and 48 hr of infection, medium was removed and TUNNEL (terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling) assay was carried out according to the manufacturer's instruction of ApopTag kit (Intergen, Purchase, N.Y.). For color development, cells were incubated with peroxidase-conjugated avidin and DAB (diaminobenzidine; DAKO, Carpinteria, Calif.). At the time that color of cells became brown, cells were counterstained with 0.5% methyl green for 10 min and observed under microscope in more than 4 selected fields. The ratio of stained cells to total cells was calculated.
[0147] 12. Anti-Tumor Effects and Survival Rates of Decorin-Expressing Adenovirus In Vivo
[0148] The effect of decorin-expressing adenoviruses on the growth of human tumor spheroid formed in nude mice was assessed. Tumors were implanted on the abdomen of 6- to 8-week-old nude mice (Charles River Japan Inc.) by subcutaneous injection of 110.sup.7 human cancer cell lines (U343, U87MG, C33A, Hep3B and A549) in 100 l of HBSS (Hanks' balanced salt solution, Gibco BRL). When tumors reached to 50-80 mm.sup.3 in volume, adenoviruses at 110.sup.8-510.sup.8 PFU were administered intratumorally three times every other day and the growth pattern and survival rate of tumors were observed. The volume of tumors was calculated with the major axis and minor axis measured using a caliper: tumor volume=(minor axis mm).sup.2(major axis mm)0.523.
[0149] 13. Observation of the Change of Tumor Characteristics Induced by the Administration of Decorin-Expressing Replication-Competent Adenoviruses
[0150] When U343, U87MG, C33A, Hep3B or A549 tumor formed in the abdomen of nude mice reached to about a range of 50-80 mm.sup.3, adenoviruses at 110.sup.8-510.sup.8 PFU were administered intratumorally three times. Following 3 days of injection, the tumor tissues were extracted and their paraffin blocks were prepared. The blocks were cut into 3-1 m slides and deparaffinized in xylene and then in graded alcohols (100%, 95%, 80% and 70%), followed by staining with hematoxylin and eosin. For the observation of distribution of collagen, a component of connective tissue, 3-m paraffin-embedded slides were stained using bouin, hematoxylin and biebrich's scarlet acid fuchsin. The staining reagents were purchased from DAKO ARK (Dako, Carpinteria, Calif.). In addition, the immunohistochemistry staining for the hexon region of adenoviruses was carried out. The slides were deparaffinized as described above and incubated with the primary anti-adenoviral hexon antibody (MAB 8052 chemicon, Temecula, Calif.) and then with the secondary goat anti-rat IgG-HRP (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). The color development was performed using DAB (DAKO, Carpinteria, Calif.).
[0151] To observe the occurrence of apoptosis in tumors, TUNNEL assay was carried out according to the manufacturer's instruction of ApopTag kit (Intergen, Purchase, N.Y.). For the color development, cells were incubated with peroxidase-conjugated avidin and then DAB (DAKO, Carpinteria, Calif.). At the time that color of cells became brown, cells were counterstained with 0.5% methyl green for 10 min and observed under microscope.
[0152] 14. Examination for Expression Pattern of MMP by Zymoaraphy
[0153] To observe the change of MMP activity, various human tumor cell lines (U343, U87MG, C33A, Hep3B and A549) were introduced to 75T culture flasks and 24-hr later, were infected with PBS, Ad-E1B or Ad-E1B-DCNG adenovirus at an MOI 1-100 and then, incubated for 48 hours. The cells were additionally incubated for 24 hr in refreshed DMEM without FBS, and the medium was collected and concentrated. The proteins present in the medium were quantified using the protein analysis kit (Bio-Rad, Hercules, Calif., USA) and their same amounts were electrophoresed on a gelatin-substrate gel. After the electrophoresis, the gel was subject to gelatinolysis at 37 C. for 18 hr, and stained with Coomassie brilliant blue to observe the expressions of MMP-2 and MMP-9. In addition, each experiment was independently conducted three times and the thickness of bands formed were compared by QuntityOne2.1 program (BIO-Rad Laboratories, Hercules, Calif.).
[0154] 15. Changes of Metastatic Potential Over Decorin Expression Using Spontaneous Metastasis Model
[0155] To assess changes of metastatic potential over decorin overexpression, B16BL6 cells (210.sup.5/mouse) were administered subcutaneously into the right hind foot pad of 6-8-week-old male C57BL/6 mice (Charles River Korea, Seoul, Korea) to form primary tumors. Once the primary tumor reached to 100-200 mm.sup.3 in volume, PBS, Ad-E1B or Ad-E1B-DCNG were injected directly into tumors three times every other day. On day 5 after the last injection, the primary tumors were surgically removed by amputating below knee under mild anesthesia. On day 20 following primary tumor removal, the weight of metastatic tumor lesions in the lungs of the mice was assessed.
[0156] 16. Evaluation on Transduction Efficiency and Tissue Penetration Potency of Decorin-Expressing Adenoviruses Using Tumor Tissues from Breast Cancer Patient
[0157] Tumor tissues and adjacent normal tissues from breast cancer patients were collected, cut into 1-2 mm sections and then plated onto 24-well plates, after which they were cultured for 4 hr in IMDM (Isocove's Modified Dulbecco's Medium) supplemented with 5% FBS, 10 M/L insulin and 1 M/L hydrocortisone. Each of dl-LacZ and dl-LacZ-DCNG adenoviruses at 110.sup.8 PFU were added into the plates containing breast tumor and normal tissues, and incubated at 37 C. in 5% CO.sub.2 incubator for 5 days. Following the incubation, the medium was removed from the plates, and breast tumor and normal tissues were fixed in a fixation solution and X-gal stained. The surface of X-gal stained tumor tissues was observed under a stereoscopic microscope (Olympus optical Co., LTD, Tokyo, Japan).
[0158] 17. Evaluation on Transduction Efficiency of Decorin-Expressing Adenovirus in Primary Keloid Cells
[0159] The primary keloid cell line at passage 2 obtained from keliod patients was plated onto 24-well plates and then infected with dl-LacZ or dl-LacZ-DCNG adenovirus at an MOI of 0.1-50, followed by incubating at 37 C. in 5% CO.sub.2 incubator. At 48 hr of viral infection, cells were X-gal stained to reveal the transduction efficiency of adenoviruses.
[0160] 18. Evaluation on Spreading and Penetration Potency Using Keloid Spheroid Model
[0161] The primary keloid cells (110.sup.5) at passage 2 from keliod patients were added into a 15 ml falcon tube and centrifuged at 500g for 5 min to obtain keloid spheroid, followed by culturing at 37 C. for 5 days. The keloid spheroid was transferred to 0.75% agarose-coated 48-well plate and 150 l of DMEM (containing 5% FBS) were added, after which dl-LacZ or dl-LacZ-DCNG adenoviruses at 110.sup.7 PFU was added to the medium. Following 3 days of viral infection, the keolid spheroid was fixed in a fixation solution and X-gal stained. The X-gal stained spheroids was observed under a stereoscopic microscope.
[0162] 19. Evaluation on Spreading and Penetration Potency to Tissues from Keloid Patients
[0163] The keloid tissues were extracted from keliod patients and dissected to 1-2 mm sections. The sections were cultured in 0.75% agarose-coated incubator in DMEM containing 5% FBS and penicillin/streptomycin. The medium used was refreshed once or twice every week and the keloid tissues were cultured for more than one week. Keloid tissues in a diameter of 2 mm were transferred to 0.75% agarose-coated 48-well plate and 150 l of DMEM (containing 5% FBS) were added, followed by the infection with 110.sup.8 PFU dl-LacZ or dl-LacZ-DCNG adenoviruses. Following 3 days of viral infection, the keolid tissue was fixed in a fixation solution and X-gal stained. The surface of X-gal stained tissues was observed under a stereoscopic microscope. To evaluate the spreading and penetration potency of adenoviruses into tissues, the X-gal stained keloid tissues were embedded in O.C.T. compound and snap frozen. 10 m frozen section was then placed onto gelatin-coated slide glass for microscopic observation.
Results
[0164] 1. Construction of Decorin-Expressing Adenoviruses and Expression Pattern of Decorin
[0165] To visually evaluate the alteration of penetration efficiency into tissues depending on decorin expression, replication-incompetent dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK adenoviruses expressing LacZ as a reporter were constructed. Furthermore, tumor-specific oncolytic Ad-E1B-DCNG, Ad-E1B-DCNQ and Ad-E1B-DCNK adenoviruses were constructed to enhance the transduction efficiency of replication-competent adenovirus into tissues (
[0166] For assessing the decorin expression pattern of adenoviruses constructed, Hep3B was infected with tumor-specific oncolytic adenoviruses Ad-E1B, Ad-E1B-DCNG, Ad-E1B-DCNQ and Ad-E1B-DCNK at 3 MOIs and medium was recovered for Western blotting. Cells infected with Ad-E1B as a negative control for tumor-specific oncolytic adenovirus were revealed not to express decorin, whereas all those infected with dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK showed expression of decorin (
[0167] 2. Comparable Evaluation of Transduction Efficiency
[0168] To evaluate the transduction efficiency of replication-incompetent adenoviruses, various human tumor cell lines (U343, U87MG, C33A, Hep3B and A549) and human normal cell lines (CBHEL, IMR90 and W138) were infected with each of dl-LacZ, dl-LacZ-DCNG, dl-LacZ-DCNQ and dl-LacZ-DCNK viruses at an MOI 0.1-100 and then, we observed the expression pattern of LacZ gene by performing X-gal staining after 48 hr of infection. The expression of LacZ in all tumor cell lines infected with dl-LacZ-DCNG was highly increased compared to those infected with dl-LacZ, demonstrating that the expression of decorin dramatically improves the transduction efficiency of adenovirus. Interestingly, a high titer of replication-incompetent adenoviruses showed to induce cell death. In particular, the infection with dl-LacZ-DCNG virus at MOI of no less than 10 resulted in complete cell death in C33A cells, suggesting that overexpression of decorin can successfully induce cell death (
[0169] 3. Evaluation on Spreading and Penetration Potency of Adenovirus to In Vitro Tumor Tissue Using Tumor Spheroids
[0170] To evaluate the transduction efficiency and tissue penetration potency of recombinant adenoviruses to tumor spheroids, various human tumor cell lines were subcutaneously injected into nude mice and once the tumors reached to 150-200 mm.sup.3 in volume, fresh tumor tissues was extracted. The tumor tissues extracted were dissected into 1-2 mm sections and infected with adenoviruses at 110.sup.6, 110.sup.7, or 110.sup.8 PFU. X-gal staining was carried out after 48 hr of infection. Compared to the treatment with dl-LacZ, dl-LacZ-DCNK and dl-LacZ-DCNQ at 110.sup.6 PFU, the same dose of dl-LacZ-DCNG showed stronger X-gal staining on the surface of tumor spheroid. The treatment with adenoviruses of no less than 110.sup.7 PFU led to darker X-gal staining on the overall surface of tumor spheroid (
[0171] 4. Evaluation on the Transduction Efficiency of d-LacZ-DCNG Adenovirus in Tumor Mass In Vivo
[0172] In order to investigate whether the enhanced transduction efficiency and viral spread of dl-LacZ-DCNG seen in tumor spheroids in vitro would lead to an increase in gene delivery to tumor mass in vivo, tumor xenograft models were used. Each of dl-LacZ and dl-LacZ-DCNG adenoviruses at 110.sup.8-510.sup.8 PFU was intratumorally injected into the tumor mass of U343, U87MG, C33A, Hep3B, and A549 formed in the abdomen of nude mice. Three days later, tumors were taken and sectioned for X-gal staining. While dl-LacZ exhibited the low level of LacZ expression and the stained region was restricted to the virus injection site, dl-LacZ-DCNG showed much higher LacZ expression and the stained region was found to be widely spread to other regions than the virus injection site (
[0173] Furthermore, dl-GFP and dl-GFP-DCNG adenoviruses expressing GFP (green fluorescence protein) were intratumorally injected into C33A tumor mass formed in the abdomen of nude mice at 510.sup.8 PFU. Three days later, tumors were taken and frozen sectioned for observation under fluorescent microscope. In tumor mass injected with dl-GFP, GFP was limitedly expressed along the needle track formed by viral infection; however, GFP was strongly and widely expressed in tumor mass injected with dl-GFP-DCN (
[0174] 5. Assessment on Tumor Cell Killing Effect of Decorin-Expressing Oncolytic Adenovirus
[0175] To reveal that the increase in penetration and spreading of decorin-expressing adenovirus contributes to enhanced tumor cell killing effect of tumor-specific oncolytic adenoviruses, a CPE assay was carried out. Each of human tumor cell lines (U343. U87MG, C33A, Hep3B and A549) and human normal cell lines (CBHEL, MRC5, IMR90 and W138) was infected with dl-LacZ (negative control), Ad-E1B, Ad-E1B-DCNG, Ad-E1B-DCNQ and Ad-E1B-DCNK adenoviruses at MOIs of 0.1-100 and the tumor cell killing effect was analyzed. As shown in
[0176] 6. Plaque Formation of Decorin-Expressing Oncolytic Adenovirus
[0177] To visualize the effect of decorin expression on the cytopathic ability and viral spread into surrounding cells, plaque formation in a solid medium containing agarose was compared. Hep3B cells were infected with Ad-E1B, Ad-E1B-DCNG, Ad-E1B-DCNQ and Ad-E1B-DCNK adenoviruses and plaque formation was then analyzed. As shown in
[0178] 7. Apoptosis Induced by Decorin-Expressing Adenovirus
[0179] The replication incompetent adenovirus, dl-LacZ-DCNG at high titer was revealed to induce the death of cells that were detached from the bottom of culture plates as shown in Result 2. Therefore, we examined whether decorin expression is responsible for cytotoxic effect. First, to determine whether decorin induces apoptosis, flow cytometric assay was carried out after PI staining for analyzing an increase rate of subG.sub.1 cell population containing randomly fragmented DNAs due to apoptosis. Various human tumor cell lines were infected with Ad-E1B, Ad-E1B-DCNG, Ad-E1B-DCNQ and Ad-E1B-DCNK adenoviruses and harvested after 48-96 hr post-infection for measuring an increase in subG.sub.1 cell population. CPT was used as a positive control for the induction of apoptosis. A549 cells infected with Ad-E1B showed about 3.11% of subG.sub.1 cell population and those infected with Ad-E1B-DCNG showed about 26.52% of subG.sub.1 cell population. Such increased subG.sub.1 cell population by adenoviral infection was also observed in other cell lines (U343, U87MG, C33A and Hep3B) (
[0180] A representative of human tumor cell lines was infected with replication-incompetent adenoviruses, dl-LacZ or dl-LacZ-DCNG adenovirus and harvested for measuring the increase in subG.sub.1 cell population. As results observed in oncolytic adenoviruses described above, dl-LacZ-DCNG gave rise to larger subG.sub.1 cell population than dl-LacZ (
TABLE-US-00001 TABLE 1 subG1 cell population (%) Tumor cell lines PBS CPT Ad-E1B Ad-EIB-DCNG U343 0.89 59.86 0.98 44.33 U87MG 2.00 14.78 8.00 15.93 C33A 2.37 26.44 2.44 19.56 Hep3B 1.37 5.99 7.92 13.91 A549 0.84 17.96 3.11 26.52
TABLE-US-00002 TABLE 2 Tumor subG1 cell population (%) cell lines PBS CPT dl-LacZ dl-LacZ-DCNG U343 1.68 29.02 2.96 22.30 U87MG 3.93 12.51 8.52 15.68 C33A 2.96 68.20 5.75 46.63 Hep3B 3.00 16.60 4.22 9.92 A549 6.54 21.59 11.73 26.94
[0181] Further, to accurately examine the effect of decorin expression on cell killing potency, the progress of apoptosis induced by oncolytic adenoviruses was assessed by Annexin V-FITC and PI dual staining. Annexin V-FITC is used to detect the translocation of phosphatidylserin (PS) to the external membrane leaflet as an early marker for apoptosis, and PI is used to identify necrosis by binding to nuclear chromatin as a late marker for apoptosis. Therefore, Annexin V-FITC.sup./PI.sup., Annexin V-FITC.sup.+/PI.sup. and PI.sup.+ represent healthy, apoptotic and necrotic cells, respectively. Of the CPT-treated U343 cells, 32.15% (Annexin V-FITC.sup.+/PI.sup.) of the cells were apoptotic, while the cells infected with Ad-E1B and Ad-E1B-DCNG showed 24.15% and 44.85% apoptotic rate, respectively, indicating that Ad-E1B-DCNG adenovirus induces enhanced apoptosis rate compared to Ad-E1B (
[0182] To examine the apoptotic rate induced by replication-incompetent decorin-expressing adenoviruses, various cells including U343, U87MG, C33A, Hep3B and A549 were treated with PBS, CPT, dl-LacZ or dl-LacZ-DCNG and the progress of apoptosis was then assessed by Annexin V-FITC and PI dual staining. As shown in
[0183] TUNNEL assay was performed for identifying DNA fragmentation as a characteristic of early apoptosis. It was shown in
TABLE-US-00003 TABLE 3 Tumor Proportion of apoptotic cells (%) cell line PBS CPT Ad-E1B Ad-E1B-DCNG U343 10.5 5.83 53.5 7.45 32.5 12.5 69.7 5.40 U87MG 2.5 1.11 83.0 29.29 16.5 5.21 77.0 17.98 C33A 5.65 3.29 60.1 25.91 45.2 7.61 79.8 20.51 Hep3B 1.65 0.61 71.2 15.73 38.5 2.65 69.7 15.64 A549 3.5 0.83 37.5 5.35 34.8 11.3 75.21 1.22 Tumor Proportion of apoptotic cells (%) cell line PBS CPT dl-LacZ dl-LacZ-DCNG U343 1.54 2.98 74.67 12.38 15.07 7.43 61.82 15.20 U87MG 1.94 1.58 40.00 13.17 15.00 2.44 44.44 7.24 C33A 3.61 2.45 70.51 15.90 18.56 9.99 54.74 13.29 Hep3B 3.33 1.56 75.86 3.11 23.58 9.23 45.28 7.34 A549 3.00 2.21 69.80 8.38 7.84 2.42 52.08 13.66
[0184] A representative of human tumor cell lines was infected with replication-incompetent adenovirus (dl-LacZ or dl-LacZ-DCNG) or tumor-specific oncolytic adenovirus (Ad-E1B or Ad-E1B-DCNG), and harvested 48-96 hr after infection for TUNNEL analysis. As results obtained from oncolytic adenoviruses, the decorin-expressing dl-LacZ-DCNG and Ad-E1B-DCNG adenoviruses exhibited much higher apoptosis rate than the dl-LacZ adenovirus (
[0185] 8. Evaluation on Anti-Tumor Effect of Decorin-Expressing Oncolytic Adenovirus In Vivo
[0186] To investigate in vivo anti-tumor effect of decorin-expressing Ad-E1B-DCNG, tumors xenografts formed in nude mice were infected three times every other day with Ad-E1B or Ad-E1B-DCNG at 110.sup.8-510.sup.8 PFU and the growth pattern of tumors was observed. For human brain tumor U87MG, the negative control PBS resulted in the considerable growth of tumor to 1089.22 mm.sup.3, whereas Ad-E1B and Ad-E1B-DCNG led to the significant suppression of tumor growth to 115.70 mm.sup.3 and 11.87 mm.sup.3, respectively (
[0187] After 25 days post-treatment, all of 9 mice treated with PBS were dead (
[0188] The survival rate of tumor-bearing mice was examined for the decorin-expressing adenovirus treatment. For C33A tumor bearing mice, 80 days after the beginning of the treatment, 100% of the animals treated with Ad-E1B-DCNG) were still viable, whereas only 50% of Ad-E1B-treated mice were viable (death of mice; tumor volume >2000 mm for C33A) in the same time period (
[0189] 9. Change of Tumor Characteristics Induced by Decorin-Expressing Replication-Competent Adenovirus
[0190] Various human tumor cell lines (U343, U87MG, C33A, Hep3B and A549) formed in the abdomen of nude mice was infected three times with Ad-E1B and Ad-E1B-DCNG. Following 3 days of injection, the tumor tissues were extracted and stained with hematoxylin and eosin for histological characterization. As shown in
[0191] Summarizing, it could be recognized that Ad-E1B-DCNG adenovirus replicates actively in the viral injection site and spreads widely, contributing to the induction of apoptosis and necrosis.
[0192] 10. Investigation of Collagen Distribution in Tumor Mass Using Masson's Trichrome Staining
[0193] Human brain tumor cell line U343 formed in nude mice was injected three times with Ad-E1B or Ad-E1B-DCNG. Following 3 days of injection, the tumor tissues were extracted and stained with Masson's trichrome to analyze the distribution of collagen (stained blue color), major component of extracellular matrix, trichrome stain). U343 tumor mass treated with Ad-E1B was frequently observed to be stained in blue color within its inner portion; however, that treated with Ad-E1B-DCNG showed no blue staining within its inner portion. Instead, collagen in the form of capsule was observed in normal tissues surrounding tumors (
[0194] For U87MG xenograft model, the tumor mass injected with Ad-E1B-DCNG showed little or no blue-stained region due to very low level of collagen as that injected with Ad-E1B (
[0195] 11. Examination for Expression Pattern of MMP by Zymography
[0196] To examine whether the decrease in extracellular matrix is induced by MMP, the activities of MMP-2 and MMP-9 were verified by zymography (
[0197] 12. Inhibition of Tumor Metastasis by Decorin-Expressing Oncolytic Adenovirus
[0198] It is generally known that decorin reacts with extracellular matrix components to promote the expressions of MMP-1 and MMP-2, thereby degrading extracellular matrix. The change in metastatic potential over decorin expression was examined using spontaneous metastasis model. B16BL6 cells (210.sup.5/mouse) were administered subcutaneously into the right hind foot pad of C57BL/6 mice and, once the tumor volume reached to a volume of 100-200 mm.sup.3, PBS, Ad-E1B or Ad-E1B-DCNG was injected directly into the tumor three times every other day. On day 5 after last injection, the primary tumors were surgically removed by amputating below knee under mild anesthesia. On day 20 following primary tumor removal, the weight of metastatic tumor lesions in the lungs of the mice was assessed. As shown in
[0199] 13. Evaluation of Transduction Efficiency and Tissue Penetration Potency on Decorin-Expressing Adenovirus Using Tumor Tissues from Breast Cancer Patients
[0200] To examine whether the enhanced transduction efficiency and tissue penetration potency of decorin-expressing adenoviruses as verified in Examples described above is also exhibited in primary human tumor tissues, tumor tissues and adjacent normal tissues from breast cancer patients were collected, cut into 1-2 mm diameter sections and cultured on 0.75% agarose-coated plates, followed by infecting with dl-LacZ or dl-LacZ-DCNG at 110.sup.8 PFU. On day 5 after viral injection, X-gal staining was performed. The surface of tumor tissues injected with dl-LacZ-DCNG showed darker blue color compared to that injected with dl-LacZ (
[0201] Breast tumor spheroids (<1 cm.sup.3) from breast cancer patients were plated into 12-well plates containing 5% IMDM supplemented with insulin (10 mol) and hydrocortisone (1 mol) and infected with dl-GFP or dl-GFP-DCNG at 110.sup.7 PFU. On day 5 after viral infection, the tumor spheroids were observed under fluorescence microscope. In tumor spheroids injected with dl-GFP, GFP was limitedly expressed on the surface of tumor spheroids; however, GFP was strongly and widely expressed in most of tissues in tumor spheroids injected with dl-GFP-DCN (
[0202] 14. Evaluation on Transduction Efficiency of Decorin-Expressing Adenovirus on Primary Keloid Cell
[0203] Keloid is one of disorders caused by the extensive formation of extracellular matrix. To assess the therapeutic efficacy of decorin-expressing adenoviruses on keloid, the primary keloid cell line from keliod patients was infected with dl-LacZ or dl-LacZ-DCNG adenovirus at an MOI of 0.1-50 and then subject to X-gal staining. It was observed that dl-LacZ-DCNG induced much stronger LacZ expression than dl-LacZ, demonstrating that the decorin expression is responsible for the significant increase in the transduction efficiency into keloid cells (
[0204] 15. Evaluation on Transduction Efficiency Using Keloid Spheroid Model
[0205] To verify the improved transduction efficiency of the decorin-expressing adenovirus into keloid tissues, keloid cell spheroids prepared using primary keloid cells at passage 2 from keloid patients were infected with dl-LacZ or dl-LacZ-DCNG adenovirus at 110.sup.7 PFU and subject to X-gal staining for microscopic observation. The surface of keloid cell spheroids was more intensively stained for dl-LacZ-DCNG than dl-LacZ (
[0206] 16. Evaluation on Spreading and Penetration Potency to Tissues from Keloid Patients
[0207] To examine whether the enhanced transduction efficiency of dl-LacZ-DCNG revealed by using keloid cell spheroids is also reproducible in keloid tissues from patients, keloid tissues from patients were infected with dl-LacZ or dl-LacZ-DCNG at 110.sup.8 PFU and subject to X-gal staining for microscopic observation. While dl-LacZ-treated keloid tissues showed weak LacZ expression, dl-LacZ-DCNG-treated keloid tissues were intensively X-gal stained (
[0208] Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.