Patent application title: DEFECTIVE SINDBIS VIRAL VECTORS
Inventors:
Alicia Hurtado (New York, NY, US)
Daniel Meruelo (Scarborough, NY, US)
Assignees:
New York University
IPC8 Class: AC07H2100FI
USPC Class:
536 231
Class name: Nitrogen containing n-glycosides, polymers thereof, metal derivatives (e.g., nucleic acids, oligonucleotides, etc.) dna or rna fragments or modified forms thereof (e.g., genes, etc.)
Publication date: 2008-12-04
Patent application number: 20080300394
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Patent application title: DEFECTIVE SINDBIS VIRAL VECTORS
Inventors:
Alicia Hurtado
Daniel Meruelo
Agents:
DARBY & DARBY P.C.
Assignees:
New York University
Origin: NEW YORK, NY US
IPC8 Class: AC07H2100FI
USPC Class:
536 231
Abstract:
Disclosed herein are new defective Sindbis viral vectors made from wild
type Ar-339 Sindbis virus, with differences in replicase and envelope
proteins between JT vectors and consensus Sindbis virus sequences, and
also between JT and Ar-339 vectors. Also disclosed are plasmids used for
the production of the vectors, methods for producing the vectors, methods
for treating mammals suffering from tumors and pharmaceutical
formulations for use in the treatment methods.Claims:
1. A purified, isolated nucleic acid comprising the nucleotide sequence as
set forth in nucleotides 1-4831 of SEQ ID NO.: 36.
2. The nucleic acid of claim 1, further comprising a bacteriophage promoter.
3. The nucleic acid of claim 2, further comprising a bacterial antibiotic resisitance gene.
4. The nucleic acid of claim 3 wherein said promoter is the T7 promoter.
5. The nucleic acid of claim 3 wherein said promoter is the SP6 promoter.
6. A purified, isolated nucleic acid comprising the nucleotide sequence as set forth in nucleotides 1-8048 as set forth in SEQ ID NO.: 39.
7. The nucleic acid of claim 6, further comprising a bacteriophage promoter.
8. The nucleic acid of claim 7, further comprising a bacterial antibiotic resistance gene.
9. The nucleic acid of claim 7 wherein said promoter is the T7 promoter.
10. The nucleic acid of claim 7 wherein said promoter is the SP6 promoter.
11. A purified, isolated nucleic acid comprising a nucleic acid consisting of nucleotides 1-4831 as set forth in SEQ ID NO.: 36.
12. A purified, isolated nucleic acid comprising a nucleotide sequence consisting of nucleotides 1-4831 as set forth in SEQ ID NO.: 36 and a bacteriophage promoter.
13. A purified, isolated nucleic acid comprising a nucleotide sequence consisting of nucleotides 1-4831 as set forth in SEQ ID NO.: 36, a bacteriophage promoter and a bacterial antibiotic resistance gene.
14. A purified, isolated nucleic acid comprising a nucleotide sequence consisting of nucleotides 1-8048 as set forth in SEQ ID NO.: 39.
15. A purified, isolated nucleic acid comprising a nucleotide sequence consisting of nucleotides 1-8048 as set forth in SEQ ID NO.: 39 and a bacteriophage promoter.
16. A purified, isolated nucleic acid comprising a nucleotide sequence consisting of nucleotides 1-8048 as set forth in SEQ ID NO.: 39, a bacteriophage promoter and a bacterial antibiotic resistance gene.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation of patent application Ser. No. 11/876,522, filed Oct. 22, 2007, which is a divisional of patent application Ser. No. 11/392,926, filed Mar. 28, 2006, which claims priority under 35 U.S.C. § 119(e) from Provisional Patent Application Ser. Nos. 60/666,432 and 60/755,428 filed Mar. 29, 2005 and Dec. 30, 2005, respectively, which are incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003]The present invention is directed to defective Sindbis viral vectors, plasmids used to produce such vectors, pharmaceutical formulations containing the vectors, and methods for their production and use to treat mammals suffering from tumors.
BACKGROUND OF THE INVENTION
[0004]Sindbis virus, a member of the alphavirus genus in the Togaviridae family, is a single-stranded, enveloped, positive-sense RNA virus (Strauss & Strauss, 1994). In nature, it is transmitted via mosquito bites to mammals. Thus, as Sindbis virus has evolved as a blood-borne vector, this hematogenous delivery property enables Sindbis vectors to reach tumor cells throughout the circulation (Tseng et al 2004a,b).
[0005]PCT/US02/09432 published as WO 02/076468 entitled TUMOR THERAPY WITH ALPHAVIRUS-BASED AND HIGH AFFINITY LAMININ RECEPTOR-TARGETED VECTORS discloses a method for treating solid tumors in mammals using Alphavirus vectors. The method comprised administering to a mammal harboring a tumor an amount of an Alphavirus vector effective to treat the tumor. The vector was said to have a preferential affinity for high affinity laminin receptors (HALR). Tumor cells were said to express greater levels of HALR compared to normal cells of the same lineage. The anti-tumor effect was said to be due to the fact that Sindbis virus infection induced apoptosis in infected cells.
[0006]PCT/US 2004/026671 for A METHOD FOR DETECTING CANCER CELLS AND MONITORING CANCER THERAPY discloses the use of Sindbis viral vectors to identify cancer cells in the body of a mammal and monitor anti-cancer therapy.
[0007]With the aim of broadening the knowledge of the way Sindbis vectors work for cancer gene therapy, two different kinds of Sindbis vectors, SP6-H/SP6-R, derived from wild type Ar-339, and JT-BB/JT-Rep derived from an Ar-339 laboratory adapted strain, Toto 1101 have been studied. Sindbis virus Ar-339 was first isolated in August 1952, from a pool of mosquitoes (Culex pipiens and C. univittatus) trapped in the Sindbis health district in Egypt (Hurlbut 1953; Taylor and Hurlbut 1953; Frothingham 1955; Taylor et al. 1955). Toto 1101 was made out of the heat resistant (HR) strain initially derived from AR-339 (Burge and Pfefferkorn, 1966). The first studies done with JT vectors in animal models showed good targeting of tumor cells and significant reduction of metastatic implant size (Tseng et al. 2002). Further studies of these vectors in tumor-induced SCID mice were done using the new imaging technique of IVIS®, that allows in vivo detection of viral vector and tumor cells in the same animal. In tumor-induced SCID mice there was a good correlation between vectors and tumor cells (Tseng et al. 2004b). Although these positive results in vector targeting and in vivo growth reduction of tumors and mouse survival, which are very promising for gene therapy, survival of all mice in these tumor models has not yet been achieved.
[0008]Therefore, what is need in the art are improved Sindbis viral vectors for use as anti-tumor agents.
BRIEF SUMMARY OF THE INVENTION
[0009]Disclosed herein are new vectors made from wild type Ar-339 Sindbis virus, with differences in replicase and envelope proteins between JT vectors and consensus Sindbis virus sequences, and also between JT and Ar-339 vectors. The chimeras combining both strains were produced and studied in tumor-induced SCID mice by the IVIS® imaging technique. Surprisingly JT envelope proteins targeted tumors more effectively than Ar-339 while Ar-339 replicase showed increased efficiency in tumor reduction. To analyze which residues would be responsible for tumor targeting, mutants of Ar-339 E2 envelope protein were made and tested by IVIS® imaging in ES-2 induced and tumor free mouse models. The change of only one amino acid from Glu to Lys at position 70 of Ar-339 E2, suppressed the ability to target metastatic tumor implants in mice. Double E2 mutant Mut-2, with K70 and V251 did not revert the targeting. Only when the whole sequence of JT E2 was substituted in the Ar-339 helper was the ability of targeting metastatic tumor implants recovered, though with less intensity. Thus, residue 70 in the outer leaf of the E2 protein is essential for tumor specific targeting of Sindbis vectors.
[0010]In one aspect, the present invention provides a purified, isolated nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 37 (SP6-H).
[0011]In another aspect, the present invention provides a purified, isolated nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO. 38 (SP6-HK 70).
[0012]In another aspect, the present invention provides a purified, isolated nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO. 40 (SP6-HK70-V251).
[0013]In a further aspect, the present invention provides a purified, isolated nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 39 (SP6-H-13-K70-E181-V251).
[0014]In yet a further aspect, the present invention provides a method for producing defective Sindbis viral vectors comprising the steps of [0015](a) providing a linearized replicon plasmid comprising the nucleotide sequence as set forth in SEQ ID NO: 36 and a linearized Helper plasmid selected from SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 39; [0016](b) transcribing said replicon plasmid and one Helper plasmid to produce RNA; [0017](c) collecting the RNA transcribed in step (b) and transfecting cells with said RNA; [0018](d) incubating said transfected cells for a time and at a temperature effective for producing defective Sindbis viral vectors; and [0019](e) collecting said defective Sindbis viral vectors from the medium of said transfected cells.
[0020]These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present specification, claims and figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021]FIG. 1 Sindbis virus Ar-339 cDNA cloning.
[0022]Ar-339 11703 nt genomic RNA, is illustrated schematically in grey line, viral subgenomic promoter (PSG) nt 7334 to 7646, is represented as grey solid box. The viral genome was cloned in 6 PCR overlapping fragments. The viral replicase (grey dotted line) and the subgenomic promoter were cloned in 4 PCR fragments: CDNA-1, CDNA-2, CDNA-3A and CDNA-3B. The Sindbis subgenomic promoter and structural proteins sequence (gray dashed line) were cloned in 2 PCR overlapping fragments CDNA-4 and CDNA-5. The position of restriction enzyme sites in the PCR fragments that were used for further cloning strategy are indicated.
[0023]FIG. 2. Sindbis replicon and helper plasmids.
[0024]The Sindbis virus Ar-339 genome was split in two to generate both replicon and helper plasmids. Viral sequences are represented in grey. Virus nucleotide numbers are indicated and follow the Strauss et al. 1984 sequence.
[0025]FIG. 3. Summary of vector constructions.
[0026]The first step generated the plasmid T7-pUC-PolyA#114 that contains the bacterial segment (ampicillin resistance, pMB1 replication origin), bacteriophage promoter T7, the two multicloning sites (MCS1 and MCS2) and the 3' end of the virus sequence (grey dashed line). To generate the replicon, restriction enzyme (RE) digested and gel purified DNA fragments from CDNA-1, CDNA-2 and CDNA-3 were cloned sequentially into T7-pUC-PolyA#114. To generate the helper, first the MfeI/BamHI fragment from the CDNA-1 plasmid was cloned into T7-pUC-PolyA#114, and bands from CDNA-4 and CDNA-5 were cloned into this plasmid. The sequences of the primers used to clone the Ar-339 cDNA fragments are shown in Table I (Appendix A).
[0027]FIGS. 4(A and B). Suppression of disease progression by Ar-339 and JT chimeric vectors. A) ES-2/Fluc cells (1.5×106) were i.p. inoculated into SCID mice on day 0. The next day (day 1), mice were imaged using the IVIS® Imaging System using D-luciferin as the substrate and were split into five groups of five mice each: control, which received no vector treatment, vector A (JT-BB/SP6-RhRLuc), vector B (SP6-H/JT-RephRluc), vector C(SP6-H/SP6-RhRluc) and vector D (JT-BB/JT-RephRluc). On day 5 the groups received daily i.p. treatments of corresponding Sindbis vectors and were IVIS® imaged on days 1, 5, 13 and 19. All vector treatments suppressed the tumor growth on the mesentery and diaphragm and reduced the signals on the omentum compared with control mice. B) Quantitative analysis of the whole-body total photon counts of control and Sindbis-treated mice. Error bars represent the SEM.
[0028]FIG. 5. Survival curves of mice treated with Ar-339 and JT vectors. Survival curve of mice described in FIG. 4. Vector A was the most efficient in prolonging the survival of mice bearing ES-2/Fluc tumors.
[0029]FIGS. 6 (A and B). Colocalization in peritoneal cavity of vector C. A) Vector C (SP6-H/SP6-RhRluc) infection colocalized with the metastasized ES-2/Fluc tumors in the peritoneal cavity as determined by the IVIS® system. SCID mice were i.p. inoculated with 1.5×106 ES-2/Fluc cells. Five days later, while the disease was still microscopic, inoculated mice received a single i.p. treatment of Vector C and were imaged the next day. The first IVIS® imaging was done by i.p. injection of Rluc substrate, coelenterazine, followed by a 5-minute acquiring interval (left panel). Thirty minutes after the coelenterazine injection, when the short-lived Rluc signals faded away, Fluc substrate, D-luciferin, was i.p. injected to determine the ES-2/Fluc tumor locations (right panel). B) Correlation analysis of vector C shows a high correspondence between tumor cells and vector infection in the peritoneal cavity.
[0030]FIGS. 7(A and B). Background infection of Ar-339 and JT chimeric vectors. A) Five tumor-free mice per group were i.p. injected on day 0 with one dose of vector A (JT-BB/SP6-RFluc), vector B (SP6-H/JT-RepFluc) or vector C(SP6-H/SP6-RFluc) and next day (day 1) IVIS® imaged for vector luciferase signal). The peritoneum was removed for imaging of the peritoneal cavity and the organs were harvested and imaged for the representative mice (rows 3 to 5). All vectors showed infection in fat tissue, and in vector B and C groups some mice showed a low background signal on ribs but not in organs B) Some mice per group received a second i.p. injection of the vectors of day 2, and were IVIS® imaged in: the peritoneal cavities (second row) and organs (bottom rows) on day 3. Very low signals were observed in fat tissue for vectors B and C.
[0031]FIGS. (8A and B). Tumor targeting of Ar-339 and JT chimeric vectors. A) SCID female mice were injected i.p. on day 0 with 2×106 ES-2 cells/mouse. On day 4 five mice per group were i.p. injected with one doses of vector A (JT-BB/SP6-RFluc), vector B (SP6-H/JT-RepFluc) or vector C(SP6-H/SP6-RFluc) and next day (day 5) IVIS® imaged for vector luciferase signals. The peritoneum was removed for imaging of the peritoneal cavity and the organs were harvested and imaged. All vectors targeted tumor implants. Tumors on the peritoneum, pancreas-omentum and bowel are circled. B) Some mice per group received a second i.p. injection of the vectors of day 6, and the peritoneal cavities (second row) and organs (bottom rows) were IVIS® imaged on day 7. One mouse per group (#28, 33, 38) was not injected to serve as a luciferase background control. Vectors B and C showed decreased bioluminescence signals in tumors compared with the first injection (A).
[0032]FIG. 9A-9E. SP6-H Ar-339 E2 mutants. Amino acids changed in Ar-339 E2 mutants Mut-1 (9C), Mut-2 (9D) and Mut-4 (9E). Sequences and residues from the JT-BB plasmid are shown in 9A, those corresponding to Ar-339 are represented in 9B.
[0033]FIGS. 10(A and B). Background infection of Ar-339 E2 mutants. A) Five tumor-free mice per group were i.p. injected on day 0 with one dose of vector A (JT-BB/SP6-RFluc), vector C(SP6-H/SP6-RFluc), Mut-1 (SP6-H-K70/SP6-RFluc), Mut-2(SP6-H-K70-V251/SP6-RFluc) and Mut-4(SP6-H-13-K70-E181-V251/SP6-RFluc). The next day, mice were IVIS® imaged for vector luciferase signals. The peritoneum was removed for imaging of the peritoneal cavity of representative mice and the organs were harvested and also imaged. The E2 mutant vectors did not show background infection of fat tissue as observed with vectors A and C. E2 mutant organ arrays were also IVIS® imaged at Bin 10 resolution to increase the detection limit (bottom row), arrows point to regions with signals. B) Some mice per group received a second i.p. injection of the vectors of day 2, organs were harvested and IVIS® imaged. Mice circled (#17, 24, 27, 32, and 39) were not injected to serve as luciferase background controls. Only using high sensitivity Bin 10 resolution, low bioluminescence signals (indicated with arrows) were detected in mice 21, 22 and 38 of vector groups A, C and Mut-4 respectively.
[0034]FIGS. 11(A and B). Tumor targeting of Ar-339 E2 mutants. A) SCID female mice were i.p. injected on day 0 with 2×106 ES-2 cells/mouse. On day 4, five mice per group were i.p. injected with one dose of vector A (JT-BB/SP6-RFluc), vector C(SP6-H/SP6-RFluc), Mut-1(SP6-H-K70/SP6-RFluc), Mut-2(SP6-H-K70-V251/SP6-RFluc) or Mut-4(SP6-H-13-K70-E181-V251/SP6-RFluc), and the next day (day 5) IVIS® imaged for vector luciferase signals. The peritoneum was removed for imaging of the peritoneal cavity and the organs were harvested and imaged. Vectors A and C efficiently targeted tumors, Mut-4 showed low bioluminescence signals, Mut-1 and Mut-2 did not show luminescence. For the three mutants, IVIS® images at Bin 10 resolution were taken of full mice and organ arrays. Only at this high sensitivity did some mice of Mut-1 and Mut-2 groups show very low residual signals in metastatic implants (lower panels), arrows point to regions with signals. B) Some mice per group received a second i.p. injection of the vectors on day 6, and peritoneal cavities and organs were IVIS® imaged on day 7. One mouse per group (#80, 85, 89, 94 and 99) was not injected to serve as luciferase background controls. For the three E2 mutants, high sensitivity Bin 10 images were also taken. Vectors showed equivalent infection pattern as for first injection.
[0035]FIGS. 12 (A and B) Colocalization in peritoneal cavity of vector Mut-4. A) Vector Mut-4 infection colocalized with the metastasized ES-2/Fluc tumors in the peritoneal cavity as determined by the IVIS® Imaging System. SCID mice were i.p. inoculated with 1.5×106 ES-2/Fluc cells. Five days later, while the disease was still microscopic, inoculated mice received a single i.p. treatment of vector Mut-4 and were imaged the next day. The first IVIS® imaging was done by i.p. injection of Rluc substrate, coelenterazine, followed by a 5-minute acquiring interval (left panel). Thirty minutes after the coelenterazine injection, when the short-lived Rluc signals faded away, Fluc substrate, D-luciferin, was i.p. injected to determine the ES-2/Fluc tumor locations (right panel). B) Correlation analysis of vector Mut-4 shows a high correspondence between tumor cells and vector infection in the peritoneal cavity.
[0036]FIG. 13. (A-C) Suppression of disease progression by Ar-339 and Mut-4 chimeric vectors. A) ES-2/Fluc cells (1.5×106) were i.p. inoculated into SCID mice on day 0. The next day (day 1), mice were imaged using the IVIS® Imaging System with D-luciferin as substrate and were split into four groups of five mice each: control which received no vector treatment, vector D, vector C and vector Mut-4. The groups received daily i.p. treatments of corresponding Sindbis vectors (106 TU) and were IVIS® imaged on days 1, 5, 13 and 19 after the start of treatment. All vector treatments suppressed the tumor growth on the mesentery and diaphragm and reduced the signals on the omentum compared with control mice. Image scale Min 8×103 Max 105 counts/pixel. B) Quantitative analysis of the whole-body total photon counts of control and Sindbis-treated mice. Error bars represent the SEM. C) Survival curve of mice.
DETAILED DESCRIPTION OF THE INVENTION
[0037]The term Aabout@ or Aapproximately@ means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, Aabout@ can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, Aabout@ can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.
[0038]In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989 (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F. M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994.
[0039]Amino acid residues in proteins are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Trytophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
[0040]As used herein, the term Atumor@ refers to a malignant tissue comprising transformed cells that grow uncontrollably. Tumors include leukemias, lymphomas, myelomas, plasmacytomas, and the like; and solid tumors. Examples of solid tumors that can be treated according to the invention include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, neuroglioma, and retinoblastoma.
[0041]The phrase Apharmaceutically acceptable@, as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term Apharmaceutically acceptable@ means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
[0042]The term Atherapeutically effective@ applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a mammal in need thereof. As used herein with respect to viral vectors of the invention, the term Atherapeutically effective amount/dose@ refers to the amount/dose of a vector or pharmaceutical composition containing the vector that is sufficient to produce an effective anti-tumor response upon administration to a mammal.
[0043]Described below are preferred replication defective Sindbis viral vectors for use in the present invention termed vector C, Mut-1, Mut-2 and Mut-4 and the plasmids used to produce them. Mut-1, Mut-2 and Mut-4 contain mutations in the E2 envelope protein and are alternatively referred to herein as "E2 Mutants".
[0044]The E2 mutants of the present invention were produced using unique helper and replicase plasmids. The present invention provides four novel helper plasmids (SP6-H (SEQ ID NO:37) SP6-H-K70 (SEQ ID NO:38), SP6-H-K70-V251 (SEQ ID NO:40) and SP6-H-13-K70-E181-V251 (SEQ ID NO:39)) and one replicon plasmid (SP6-R (SEQ ID NO:36)). Helper plasmid SP6-H, which does not contain any amino acid changes but has a different nucleotide sequence, is set forth in SEQ ID NO:37. The 4 helper plasmids are used to produce Vector C, Mut-1, -2 and -4 vectors, respectively, when produced using the novel replicon plasmid.
[0045]In order to produce viral vectors in this system, two plasmids are used, the replicon and the helper. The replicon contains the viral replicase, the viral packaging signal, nt 945 to nt 1075, (Weiss B et al. 1994; Frolova et al 1997); the viral subgenomic promoter, multicloning site 1 (MCS1) to allow for the insertion and expression of the gene of interest, and the 3' end of the virus (nt 11394 to 11703) to allow viral (-) strand RNA synthesis. A second multicloning site (MCS2) allows for the linearization of the plasmid for in vitro transcription. The helper plasmid contains the first 425 nt of the virus, followed by the 3' end of the virus from nt 7334 to nt 11703 which includes the subgenomic promoter, the capsid and the viral envelope proteins (E3, E2, 6K and E1) and the 3' end (nt 11394 to 11703).
[0046]Both plasmids share the following viral sequences: the first 425 nt and the 309 nt of the 3' end and the sub genomic promoter.
[0047]Both plasmids have several non-viral elements in common, the replication origin (rep pMB1) and the Ampicillin resistance gene from the pUC cloning plasmid; the promoter for in vitro transcription (T7 or SP6) and the MCS2. In the construction process a plasmid containing the pUC sequences, SP6 or T7 promoter, the multicloning sites, and the 3' viral end, which are common to both vectors, was first generated. The specific viral sequences were then cloned into this plasmid (FIG. 3).
[0048]In order to produce the viral vectors of the present invention, one pair of plasmids are linearized using restriction enzymes such as PacI, NotI, or XhoI, transcribed in vitro, the RNAs collected and electroporated into cells. For in vitro transcription, a promoter is inserted before the Sindbis viral sequences. Preferably, the promoter is a bacteriophage promoter, for us with its respective RNA polymerase such as SP6 or T7 (Ambion Austin, Tex.).
[0049]Cells for use in the present invention include BHK-21 cells (available from the American Type Culture Collection, ATCC, Manassas, Va. as CRL 6281), ES-2 cells, (ATTC, CRL 1978), ES-2/Fluc cells that were derived from the ES-2 line by transfection of a plasmid, pIRES2-Luc/EGFP and the MOSEC cell line (clone ID8). The transcribed RNAs (i.e., one helper and one replicon plasmid) are electroporated into the cells at a concentration ranging between about 0.75 mg/mL and about 1.25 mg/mL. The ratio of viral RNA to cell concentration ranges between about 30-50 μg RNA per 6×106 cells. Electroporation is performed using equipment commercially available from e.g., Bio Rad (Hercules, Calif.)
[0050]The transfected cells are fed medium containing 5% fetal bovine serum (FBS) and incubated at 37° C. for about 12 hours. The medium is then discarded, replaced with 9 ml of Opti-MEM I medium (GIBCO-BRL Invitrogen, San Diego Calif.) and incubated at about 37° C. for about 24 hours. Then, supernatents are collected and centrifugated at 2,400 rpm (=1,500 g) for 15 min to eliminate cell debris. Clear supernatants, containing the viral vector, were collected, aliquoted and stored at -80° C.
[0051]The viral vectors can be used as anti-tumor agents as described in International Application No. PCT/US02/09432 published as WO 02/076468. Although the Mut-1 vectors do not bind to the high affinity laminin receptors (HALR), they are useful as controls and to monitor non-viral effects of the vectors. Since Mut-1 vectors do not enter cells, the contribution of host factors in the anti-cancer response can be studied.
[0052]The plasmids of the present invention can be used to transfer of cells and create packaging cell lines for the continuous production of defective Sindbi viral vectors as described in copending Ser. No. 10/983,432 and in Paper Example 1 below.
[0053]The amount of viral vectors produced may be determined as described below in the Examples. Briefly, clear supernatants are serial diluted in Opti-MEM I medium and 300 μL of each vector dilution are added to a 35 mm well in 12-well plates, containing 2×105 cells. After incubation for 1 hour at room temperature, the cells are washed with PBS (Phosphate buffered saline) and incubated with 2 mL of αMEM at 37° C. for about 24 hours. Media is removed, cells are washed with PBS and cell lysates are prepared and assayed for the different reporter activity: β-galactosidase, firefly luciferase or Renilla luciferase. Vector titers were estimated as the highest dilution having detectable reporter activity. Detection of reporter activities is described in Example 2.
[0054]Viral vectors can be produced by linearizing helper and replicon plasmids after the polyA sequence, followed by separately performed in vitro transcription reactions. Usually, 1.6 μg of plasmid yields 15-25 μg of mRNA/reaction. Then 30-50 μg of both RNAs are co-electroporated into 6×106 BHK-21 cells, which are then incubated in 10 ml of αMEM containing 5% FBS at 37° C. for about 12 h. Then, the medium is replaced with 9 ml of Opti-MEM I medium (GIBCO-BRL Invitrogen, San Diego Calif.) supplemented with 0.7 μM CaCl2, cells are incubated at 37° C. for about 24 h, supernatants collected and centrifuged at =1,500 g for 15 min to eliminate cell debris. The procedure can be scaled up using the following electroporation ratios: 5-8 μg helper and replicon mRNA's per 106 BHK-21 and 9 mL Ca-Opti-MEM media per reaction.
[0055]The viral vectors of the present inventions have the following properties which are summarized in Strauss and Strauss 1994. Briefly, as Alphaviruses replicate exclusively in the cytoplasm, there is no possibility of adventitious splicing. Because they are replication incompetent and packaging defective, the vectors are incapable of spread by reinfection. The vector replicates to high copy number inside the cell, and large quantities of mRNA are produced, leading to production of large amounts of the protein of interest
[0056]Viral vectors obtained as described herein can be formulated in a pharmaceutical composition for administration to a patient. As used herein, a Apharmaceutical composition@ includes the active agent, i.e., the viral vector, and a pharmaceutically acceptable carrier, excipient, or diluent. The phrase Apharmaceutically acceptable@ refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term Apharmaceutically acceptable@ means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term Acarrier@ refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
[0057]For human therapy, the viral vectors of the present invention may be prepared in accordance with good manufacturing process (GMP) standards, as set by the Food & Drug Administration (FDA). Quality assurance (QA) and quality control (QC) standards will include testing for replication competent virus (if the virus vector is replication defective), activity (colony forming units [CFU] per number of viral particles, tested by induction of apoptosis or cytopathic effect (CPE), or by expression of a marker gene such as β-galactosidase), toxicity, and other standard measures.
[0058]In order to treat the tumors, the pharmaceutical composition is administered by any route that will permit homing of the vector to the tumor cells. Preferably, administration is parenteral, including, but not limited to, intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. As disclosed herein, viral vectors can be also administered to the tumor-bearing animal via intranasal or oral route (see Hardy, In: The Arbiviruses: Epidemiology and Ecology, Ch. 4, pp. 87-126). Importantly, however, in contrast to other viral vectors in gene therapy, administration of the Sindbis vectors of the invention need not be locally to the tumor. Indeed, one of the advantages of this invention is the high specificity and affinity of the vector for cancer cells, even micrometastases that cannot be resected or located by standard techniques (e.g., CAT scanning, MRI scanning, etc.).
[0059]In the therapeutic treatments of the invention, a therapeutically effective amount of the vector is administered to a patient. As used herein, the term Atherapeutically effective amount@ means an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. Specifically, a therapeutically effective amount will cause one or more of the following: apoptosis of tumor cells; necrosis of tumor cells; elimination or prevention of tumor metastases; reduction in the rate of tumor growth; reduction in tumor size or tumor shrinkage; scab formation over cutaneous tumor; elimination of the tumor; remission of the cancer; an increase in the time for reappearance of the cancer; and increased time of survival of the patient. The frequency and dosage of the vector therapy can be titrated by the ordinary physician using standard dose-to-response techniques, but generally will be in the range of from about 106 to about 1012 viral vector particles per dose administered daily, weekly, biweekly, or monthly at least twice and preferably at least three times.
[0060]In order to explore if wild type Sindbis would improve the vector systems, four vectors were generated combining the helper and replicase segments of both strains (JT-BB/SP6-R; SP6-H/JT-Rep; SP6-H/SP6-R; JT-BB/JT-Rep) and tumor size reduction and survival in tumor-induced SCID mice was studied. Unexpectedly, it was found that those vectors carrying Ar-339 helper (SP6-H) were less efficient in targeting tumors than the JT vectors and that those carrying Ar-339 replicase (JT-BB/SP6-R and SP6-H/SP6-R) were more efficient in tumor reduction. To study the surprising phenotype of the helper, both viral sequences were analyzed and compared. One amino acid difference was found in the capsid protein (Pro 67 (JT-BB) to Gln (Ar-339)); three amino acid changes in envelope protein E1 (Ala72 (JT-BB) to Val (Ar-339), Gly75 (JT-BB) to Asp (Ar-339) and Ser237 (JT-BB) to Ala (Ar-339)) and four changes in envelope protein E2 (Ile3(JT-BB) to Thr(AR-339); Lys70(JT-BB) to Glu(Ar-339); Glu181(JT-BB) to Lys(Ar-339) and Val251(JT-BB) to Ala(Ar-339)). To determine which amino acids were critical for the vector properties, different chimeras from both vectors were generated containing mixed sequences from both strains. The analysis in animal models showed that the Ar-339 E2 envelope protein sequence was primarily responsible for tumor metastases targeting although the optimum amino acid pattern was not defined. To address this question, vectors were generated containing sequences with mixed combinations of the four amino acids in E2, for JT and Ar-339, and tested by IVIS® imaging in ES-2 induced and tumor-free mouse models. Surprisingly, the change of only one amino acid from Glu to Lys at position 70 of Ar-339 E2, suppressed the ability to target metastatic tumor implants in mice and also showed no fat tissue background. This result was unexpected, considering that this mutant (Mut-1) repeatedly showed titers equivalent to the Ar-339 vector when measured in ES-2 cultured cells. Double mutant Mut-2, with K70 and V251 did not revert the targeting. Only when the whole sequence of JT E2 was substituted in the Ar-339 helper was the ability to target metastatic tumor implants recovered, a though with less intensity. Thus, residue 70 in the outer leaf of the E2 protein is essential for tumor specific targeting of vectors.
[0061]Described herein are amino acids of Sindbis viral vectors involved in specific infection of metastatic tumor implants in the mouse peritoneal cavity. Sequence analysis of cloned Ar-339 and JT viral vectors has been an important tool for the discovery of these amino acids.
[0062]The sequence analysis comparison with the published Strauss sequence, revealed 2 or 3 amino acid changes in viral replicase for JT and Ar-339 respectively. The Ar-339 nsP1 Ile 441 is a reverse mutation to an alphavirus conserved residue; this amino acid may also be related to viral adaptation to BHK-21 tissue culture. Ar-339 replicase is more efficient in suppression of disease progression than JT, the change from polar Cys in JT to aliphatic residue, Ile 441, may play a role in enhancing replicase activity. Interestingly, both JT and Ar-339 vectors have the same 2 amino acid changes in nsP2 protein versus the Strauss sequence. JT and Ar-339 nsP2 residues are more conserved among the Sindbis-like Alphavirus group.
[0063]Comparing the Strauss sequence with the region coding for the structural part of the JT and Ar-339 vectors, the changed amino acids were found mainly in the viral spike, although in different residues, suggesting a different evolutionary lineage of both strains. E1 D75 and A237 of Ar-339 are highly conserved in Sindbis-like alphaviruses; all viruses in this group carry D75. There is no virus in the group that has serine at 237, which only occurs in the JT vector. These data suggest that E1 G75 and S237 of JT vector may more likely be point mutations that arose in this laboratory strain. Further studies with E1 mutants should reveal the implication of these E1 residues in the specific tumor targeting of the viral vector. Most of the sequence variabilities have been found at the E2 envelope protein in the leaf-like domain at the viral spike; these residues are also poorly conserved in the Sindbis-like alphavirus group. The role of these E2 spike mutations in mouse tumor models in vivo was examined.
[0064]The fact that the Mut-1 vector shows the same titer as the Ar-339 vector in ES-2 cells, but does not efficiently infect ES-2 metastatic tumor implants in mice, represents a powerful tool for the study and the improvement of Sindbis vectors for gene therapy. One cause of the loss of tumor targeting in vivo could be a reduced stability of the vector in mice. Alignment of protein sequences among 17 different viruses of the Sindbis-like alphavirus group showed that ten out of the 17 members have a gap in Sindbis E2 residues 68-71, including Semliki Forest virus (SFV) which has a comparable structure to Sindbis virus. The viral spike is composed of three E1-E2 heterodimers that lean against each other. There is a gap between the base of neighboring E1-E2 heterodimers which would allow E2 to move out of the center of the spike during fusion (Zhang et al. 2002). In addition, previous studies with stable deletion mutants in the E2 receptor binding domain, also show equivalent in vitro titers but drastic reduction of infectivity in live Aedes aegypti mosquitoes (Myles et al. 2003). Without wishing to be bound by theory, it is believed that these data suggest that there is structural flexibility in this area of the spike, therefore, alterations in residue 70 shouldn't be critical for Mut-1 vector stability in vivo. The loss of vector infectivity would be more likely to occur via a decrease in cell binding affinity, especially in vivo, where the environmental conditions for vector infection are more restrictive.
[0065]In Sindbis and Semliki Forest virus (SFV), the residues involved in host cell fusion and binding to cellular receptors are located in the viral spike. It has been described that only one change from the small non-polar Valine, at position 75, to the acidic Aspartic acid in E1 SFV spike subunit, modifies the cell-cell fusion properties of the virus (Levy-Mintz and Kielian, 1991). Amino acids in these E1 and E2 spike domains are, thus, important in spike configuration and virus infectivity.
[0066]The vector Ar-339, having hydrophobic (V72), and acidic (D75) residues in E1 and a glutamic acid (E70) in E2 protein, is able to efficiently target tumor cells in vivo. In Mut-1, in which E2 residue 70 is changed to lysine, there is a change in polarity and charge of the amino acid that would change the conformation of the spike and so the cell binding properties of the vector. This hypothesis is supported by the difference in cell tropism observed in vitro between Mut-1 and Ar-339. Only the recovery of the full sequence of JT-BB E2 in Mut-4 results in higher titers in BHK-21 cells. If these differences were observed in vitro, where the conditions for cell binding are optimized, it is possible that in vivo factors involved in cell-vector interactions might be able to prevent vector adherence to the viral receptor. Ar-339 and Mut-1 vectors have the same titer in ES-2 cells in vitro, but in mouse metastatic implants, where ES-2 cells are in a different environments and could have receptor variations, small affinity differences between both vectors are revealed.
[0067]The present invention is described further below in examples which are intended to describe the invention without limiting the scope thereof.
[0068]In the examples below, the following materials and methods were used.
EXAMPLE 1
Sindbis cDNA Cloning
Common Techniques
[0069]Virus Propagation
[0070]Sindbis virus strain Ar-339 (Original) was obtained from ATCC (Manassas, Va., Item #VR-68) and propagated on a secondary chicken embryo fibroblast cell line, CEF. Cells were cultured in EMEM media (BioWhittaker, cat#12-684) supplemented with 10% Fetal bovine sera, NaHCO3 1.5 g/l; I-Glutamine, 292 mg/l and Penicillin/Streptomycin, 100 U/ml. Two T-75 flasks with 80% confluent CEF cell monolayers, were infected with 2.5×107 pfu and 5×107 pfu, respectively, of Sindbis virus Ar-339, diluted in 1 ml of Opti-Mem I media (GIBCO-BRL Invitrogen, San Diego Calif.; cat#31985-070) supplemented with 0.7 μM CaCl2. Virus-infected cells were incubated at 37° C. for 1 h, 10 ml of EMEM media/flask added, and cells were incubated overnight at 37° C. Supernatants containing the Sindbis "innocula" were harvested and stored at -80° C. until used in further infections. CEF cells were collected for total RNA extraction.
[0071]RNA Extraction
[0072]5 T-75 flasks with 90% confluent CEF monolayers were incubated 1 h at 37° C. with 1:100; 1:20; 1:10; and 1:5 dilutions (in Ca-OPTI-MEM media) of previously obtained "Sindbis innocula". Ten ml of EMEM media/flask was then added and incubated overnight at 37° C. After collecting the supernatant, 2 ml of Trizol (Invitrogen, San Diego, Calif.; cat #15596-018) per flask was added to the infected cells, the extract collected and stored at -80° C. Total RNA from infected cells was prepared following the manufacturer's (Trizol) protocol. Briefly, 1 ml of Trizol cell extract was vortexed for 15 seconds, 200 μL of chloroform added, vortexed, spun at 15,000 g for 10 min at room temperature (rt), the aqueous upper phase transferred to a clean tube, 750 μL of isopropanol added, incubated at rt for 15 min, spun again, the supernatant removed and the pellet washed with ethanol (70% in DEPC water). The pellet was air dried at rt for 5-10 min, and resuspended in 50 μL of DEPC water.
[0073]Sindbis c-DNA Cloning. Sindbis virus RNA was cloned in 6 overlapping fragments (CDNA-1, CDNA-2, CDNA-3A, CDNA-3-B, CDNA-4 and CDNA-5) into sequencing plasmid pCR4Blunt-TOPO (Invitrogen, San Diego Calif.; cat#45-0245). The position of the fragments is shown in FIG. 1 and the sequences of the primers used are shown in Table I (Appendix A). The primers were designed to take advantage of the unique restriction sites of the virus. In primer SV-C3R RE XbaI was introduced to allow CDNA-3 cloning. For each fragment the RT and the PCR reactions were performed with the same pair of primers. The cloning procedure was the same for all of the fragments except for the conditions of the PCR cycles.
[0074]Reverse transcriptase (RT) reactions were performed with: 5 μg from total RNA from infected cells, reaction buffer (1×); forward and reverse specific primers (2.5 μM/each); dNTPs (1 mM); DTT (5 mM) and 15 u of ThermoScript® RNase H-Reverse Transcriptase (Invitrogen, cat#12236-014), in a final volume of 20 μL. RT was diluted 1:1 in distilled water prior to use in PCR.
[0075]The Pfx-PCR reactions were performed with 1 μL of RT reaction, reaction buffer (1×), MgSO4(1 mM), dNTPs (0.3 mM/each), forward and reverse primers (0.3 μM/each) and 1 U Platinum Pfx DNA Polymerase (Invitrogen, cat#11708-013) in final volume of 25 μl. RT and PCR reactions were performed in an Eppendorf "Mastercycler Gradient" Thermal Cycler. Taq-PCR reactions contained reaction buffer (1×); dNTP's (200 μM), forward and reverse primers (0.5 μM/each) and 1 u Taq-DNA-polymerase (Fisher Scientific, Pittsburgh, Pa., cat#FB 600025) in a final volume of 20 μl. In both cases PCR products were analyzed by electrophoresis in 1% agarose gels and DNA bands cut out and purified with a QIAEXII gel extraction kit (Qiagen, Valencia Calif.; cat# 20021)
[0076]All enzymes were purchased from New England Biolabs (NEB, Beverly, Mass.). After digestions with restriction enzymes (RE), extracted DNAs were CIP dephosphorylated (1 h at 37° C.) and/or phosphorylated with T4-Polynucleotide-Kinase (1 h, 37° C.), enzymes were inactivated (70° C., 10 min or 65° C. 20 min) and reactions were run in 1% agarose gels. Bands were cut out, extracted, and quantified by gel electrophoresis by comparison with bands of known DNA concentration. Ligations were carried out with T4-DNA-Ligase (NEB) (16° C. 14 h) or with the Quick ligation kit (NEB) (5 min at room temperature (rt)). Transformations were performed using RapidTransm® TAM1 extra competent E. coli (Active Motif, Carlsbad, Calif.; cat#11099). After transforming E. coli with the ligations, the first screening of positive bacteria was done by Taq-PCR of E. coli colonies. Positive plasmids were checked by restriction enzymes. Sequencing was done at the Skirball Institute of Biomolecular Medicine, NYU.
[0077]CDNA-1 plasmid. RT reaction was performed with primers CDNA-1F/CDNA1-R at 55° C. 1 h. Two Pfx-PCR were performed: 94° C. 5 min; 35 cycles [94° C. 30 s; 66° C., 30 s (band D) or [67° C., 30 s; 72° C., 2.5 min and 72° C., 2.5 min](band E). PCR products were run in 1% agarose gels and both 2.2 Kb bands, D and E, were isolated and cloned separately into the pCR4Blunt-TOPO plasmid. Positive colonies were screened using the same PCR conditions with 65° C. as the annealing temperature and Taq-DNA-Polymerase. Two plasmids were selected (one from each PCR band) and the PCR band D in plasmid CDNA-1_Topo#64 was completely sequenced. The sequence was compared to the Ar-339 sequence published by Strauss et al., Virology 133, 1984. The differences found were compared with the sequences of PCR band E. Identical differences were found in bands D and E to confirm that they came from the virus and not from PCR mutations.
[0078]CDNA-2 plasmid. The Reverse transcriptase reaction was performed with primers CDNA-2F/CDNA-2R for 1 h at 55° C. Three PCR in gradient 94° C., 5 min; 35 cycles of [94° C., 30 s; 64.7° C., 30 s (band 1); or 66° C., 30 s (band 2); or 67° C., 30 s (band 3); 72° C., 2.5 min] and 72° C., 2.5 min. After cloning the 2 kb bands separately in pCR4Blunt-TOPO, PCR screening of colonies was done at an annealing temperature of 64.5° C. Band 2 in plasmid CDNA-2#213 was fully sequenced, and any mutations found were checked by comparison with the sequences of PCR bands 1 and 3.
[0079]CDNA-3A plasmid. Three RT reactions were performed in gradient with primers CDNA-3F/SV-6932R at 53.2° C., 55.5° C. and 60.8° C. respectively for 1 h 30 min. Three Pfx PCR were performed with 1 μL of each RT, respectively: 94° C., 5 min; 35 cycles of [94° C., 30 s; 53° C., 30 s; 72° C., 3 min] and 72° C. 3 min. The 2.68 kbp bands T, U and V were cloned separately and band V in plasmid C3A_Topo#735 was fully sequenced. No changes were found compared with the published Ar-339 sequences (Strauss et al. 1984).
[0080]CDNA-3B plasmid. RT reaction was performed with primers SV-6882F/SV-C3R at 60.8° C. for 1 h 30 min. Three Pfx-PCR in gradient were performed with 94° C., 5 min; 35 cycles of [94° C., 30 s; 55.3° C., 30 s (band J); or 56° C., 30 s (band K); or 58.3° C. 30 s (band L); 72° C., 1 min] and 72° C., 2 min. The three 774 bp bands were cloned separately and band L, in plasmid C3B-Topo#334, was fully sequenced. The mutations found were confirmed by sequencing bands J and K.
[0081]CDNA-3 (CDNA-3A+3B) plasmid. HpaI RE is not unique in the Sindbis sequence, so in order to clone CDNA3A and CDNA3B fragments into the new reporter vector, it was necessary to generate first a Topo plasmid containing both sequences, and then clone fragment CDNA-3(CDNA-3A+3B) in the AvrII/XbaI site. Plasmid C3B-Topo#334 was HpaI and SacI digested and the 774 bp viral band was isolated from the agarose gel and ligated (T4-DNA-Ligase 16° C. 14 h) to HpaI/SacI digested C3A_Topo#735 plasmid. Transformants were screened by PCR with Taq-Polymerase, and primers CDNA-3F, SV-C3R at: 94° C., 10 min; 35 cycles of [94° C. 30 s; 55° C., 30 s; 72° C., 3 min] and 72° C. 2 min. The positive plasmid was named C3A+B_Topo#810, and the junctions were sequenced.
[0082]CDNA-4 plasmid. The RT reaction was performed with primers CDNA-4F/CDNA4-R at 52° C. for 1 h 30 min. Three PCRs were performed in gradient with 94° C., 5 min; 35 cycles of [94° C., 45 s; 49.7° C., 30 s (band M); or 55° C., 30 s (band N); or 57.2° C., 30 s (band O); 72° C. 2.5 min] and 72° C. 2.5 min. The 2 kbp bands were isolated and cloned separately, band N in plasmid CDNA-4_Topo#422 was fully sequenced. The mutations found were confirmed by sequencing bands M and O.
[0083]CDNA-5 plasmid. RT reactions performed with primers CDNA-5F/CDNA5-R at 49.7° C. for 1 h 30 min. Three PCR were made in gradient with 94° C., 5 min; 35 cycles of [94° C., 30 s; 46.8° C., 30 s (band Q); or 48.6° C., 30 s (band R); or 50.5° C., 30 s (band S); 72° C. 2 min] and 72° C., 2 min. The 2.35 kbp bands were cloned separately and band S in plasmid CDNA-5_Topo#525 was fully sequenced. The mutations found were confirmed by sequencing bands Q and R. Vectors construction.
[0084]Polylinker cloning in pUC plasmid. In summary, the SP6 and T7 Polylinker bands were constructed out of primers in two fragments, a 5' end containing the SP6 promoter (primer pairs Poly1-SP6(+)/Poly-2(-)) or T7 promoter (primers Poly1-T7(+)/Poly-2(-)) and a common 3' end (primers Poly-3(+)/Poly-4(-)) the sequences of the primers are given in Table II (Appendix A). 5' and 3' bands were digested and ligated to generate the polylinker and then were ligated to the AflIII/AatII pUC band containing the E. coli replication origin and Ampicillin resistance gene to generate T7-pUC or SP6-pUC plasmids.
[0085]T7-pUC plasmid. Two separate reactions were performed with primers pairs Poly1-T7(+)/Poly-2(-) or Poly-3(+)/Poly-4(-). For both reactions, conditions were: primers (5 μg each), buffer (1×), dNTPs (1.5 mM), BSA (50 μg/ml). Mixes were boiled for 5 min and cooled down to room temperature for primer annealing, 3 units/reaction of T4-DNA-Polymerase was then added to a final volume of 20 μl, and incubated at 37° C., for 30 min for chain extension. 0.5 μL of each reaction was used as a template in two PCR reactions: one with template Poly1-T7(+)/Poly-2(-) and primers PCR-Poly1-F/PCR-Poly2-R and other with template Poly-3(+)/Poly-4(-) and primers PCR-Poly-3F/PCR-Poly-4R. For both PCRs, the conditions were: reaction buffer (1×), dNTP's (200 μM), forward and reverse primers (0.5 μM/each) and 1 unit of Taq-DNA-polymerase in a final volume of 20 μl. For each primary reaction, 3 secondary tubes were prepared and PCR reactions were performed in gradient for annealing temperature: 94° C., 5 min; 35 cycles of [94° C., 30 s; 40° C., 30 s (tube 1); or 45.4° C., 30 s (tube 2); or 50.7° C., 30 s (tube 3); 72° C., 30 s] and 72° C. 1 min. Bands were stronger for the 40° C. annealing temperature. The 145 bp bands were then isolated and digested with enzymes: AflIII and XbaI for PolyT7/Poly2-R and, AatII and XbaI for Poly3(+)/Poly 4(-). After inactivating the enzymes at 65° C. 20 min, the ligation of equimolar amounts of both bands was carried out at 25° C. for 1 h with T4-DNA-ligase. The 1811 nt AatII/AflIII band of the pUC plasmid was added, and incubated at 16° C. overnight. E. coli Tam1 competent cells were transformed with the ligations and positively selected colonies screened by double digestion with AatII and AflIII. The positive plasmid was named T7-pUC#32 and was checked by sequencing.
[0086]SP6-pUC plasmid. The reaction mix: Poly1-SP6(+) and Poly-2(-) primers (5 μg each), buffer (1×), dNTPs (1.5 mM), BSA(50 μg/ml), was boiled for 5 min and cooled down to rt for primer annealing. Then 3 units/reaction of T4-DNA-Polymerase was added to a final volume of 20 μl, and incubated at 37° C., 30 min. 0.5 μl/Rxn was used as a template in a gradient PCR reaction with PCR-Poly1-F/PCR-Poly2-R primers (0.5 μM/each) reaction buffer (1×), dNTP's (200 μM), and 1 unit of Taq-DNA-polymerase in a final volume of 20 μl. PCR conditions: 94° C., 5 min; 35 cycles of [94° C., 30 s; 40° C., 30 s (tube 4); or 45.4° C., 30 s (tube 5); or 50.7° C., 30 s (tube 6); 72° C., 30 s] and 72° C. 1 min. Bands were isolated in agarose gels, pooled together and purified. The DNA obtained was digested with enzymes AflIII and XbaI for 2 h at 37° C. After inactivating the enzymes at 65° C., 20 min, the digested DNA band was ligated to the AatII and XbaI digested Poly-3(+)/Poly-4(-) band at 25° C. for 1 h with T4-DNA-ligase. Five μL of the ligation was used as the template for a second gradient PCR reaction carried out with primers PCR-Poly1-F and PCR-Poly4-R, (same concentrations as previous PCR) and cycles: 94° C. 2 min, 18 cycles of [94° C., 30 s; 40° C., 30 s (tube 4); or 43.1° C., 30 s (tube 5); or 45.4° C., 30 s (tube 6); 72° C. 30 s] and 72° C. 1 min. Bands were isolated in an agarose gel, pooled together, purified and AflIII/AatII digested at 37° C. for 16 h. After enzyme inactivation (65° C., 20 min), the AflIII/AatII SP6 band was ligated to the 1811 nt AatII/AflIII band of the pUC plasmid with Quick T4-DNA-ligase (New England Biolabs) at rt for 5 min and transformed into E. coli Tam1 cells. Screening of colonies was performed as for T7 polylinker. The plasmid was named SP6-pUC#51.
[0087]T7-pUC-PolyA#114 plasmid. The Sindbis 3' end from nt 11392 to 11694 was obtained by PCR on plasmid CDNA-5_Topo #525 with primers: PolyA-F (5' CCCCAATGATCCGACCA 3'') (SEQ ID NO:1) and PolyA-R (5' AAAACAAATTTTGTTGATTAATAAAAG 3') (SEQ ID NO:2). PCR conditions: reaction buffer (1×), MgSO4(1 mM), dNTPs (0.3 mM each), primers (0.3 μM each) and 1 unit of Platinum Pfx DNA Polymerase in a final volume of 25 μl. Three PCR reactions were performed in gradient for annealing temperature: 94° C., 5 min; 35 cycles of [94° C., 45 s; 53.2° C., 30 s (tube 1); or 55.5° C., 30 s (tube 2); or 60.8° C., 30 s (tube 3); 72° C. 45 s] and 72° C. 1 min. The 53.2° C. band was stronger and its DNA was isolated from the gel, phosphorylated with 10 units of T4-Polynucleotide-Kinase (Biolabs) at 37° C., 30 min. After inactivation at 70° C. 10 min, the DNA was ligated to plasmid T7-pUC#32 (previously digested with HpaI and dephosphorylated) using T4-DNA-ligase at 16° C. for 14 h. Screening of recombinants was performed by PCR of colonies using the same primers, Taq-Polymerase and cycles: 94° C., 5 min; 35 cycles of [94° C., 45 s; 53° C., 30 s; 72° C., 45 s] and 72° C. 1 min. The orientation of the insert was analyzed with restriction enzymes AflIII/AseI. The positive plasmid was confirmed by sequencing, and named T7-pUC-PolyA#114. From this vector the final vector T7-ARep#68 and SP6-pUC-PolyA#914 was generated to construct the final vector SP6-Arep#68.
[0088]Replicon vector constructions. T7-ARep plasmid: Viral cDNA fragments CDNA-1, CDNA-2, and CDNA-3 were cloned in T7-pUC-PolyA#114 to generate a new reporter vector T7-ARep#68. Plasmid CDNA-1_Topo#64 was digested with MfeI and BglII RE and the 2247 bp viral band isolated and ligated, with the quick ligase kit, to T7-pUC-PolyA#114, MfeI, BglII digested and CIP dephosphorylated TAM1 transformant bacteria were screened by PCR with Taq-Polymerase and primers CDNA-1F and CDNA-1R at: 94° C. 10 min, 35 cycles of [94° C., 30 s; 65° C., 30 s; 72° C. 2.5 min] and 72° C. 2 min. The positive plasmid was named T7-pUC-PolyA-C1#11 and was AvrII/BglII digested and CIP dephosphorylated and ligated, with Quick ligase, to the CDNA-2 1950 bp viral band, obtained after AvrII/BglII digestion of plasmid CDNA-2_Topo#213. Screening of positive colonies was made by PCR with Taq polymerase and primers CDNA-2F and CDNA-2R at: 94° C., 10 min; 35 cycles of [94° C., 30 s; 64° C., 30 s; 72° C. 2.5 m] and 72° C. for 3 min. The plasmid was named T7-pUC-PolyA-C1-C2#35. C3A+B_Topo#810 was digested with XbaI/AvrII and BsshII, the 3350 nt viral C3 band separated in an agarose gel, isolated and ligated (Quick Ligase Kit) to XbaI/AvrII digested T7-pUC-PolyA-C1-C2#35 plasmid. Transformants were analyzed by XbaI/AvrII digestion. The new reporter vector was named T7-ARep#68, and was fully sequenced.
[0089]SP6-ARep plasmid. The reporter vector under the SP6 promoter was cloned in three steps. First, the SP6 promoter was cloned into T7-pUC-PolyA#114, then CDNA-1 was inserted and in the last step, the CDNA-2+CDNA-3 band from T7-ARep#68 was cloned.
[0090]SP6-pUC#51 was SphI and AflIII digested and the 154 nt band isolated and ligated (T4-DNA-ligase) to the T7-pUC-PolyA#114 SphI/AflIII/CIP band. Plasmids were screened by MboII digestion and checked by sequencing. The positive plasmid was named SP6-pUC-PolyA#902, and was digested with MfeI and BglII, CIP dephosphorylated and ligated (quick ligase) to the MfeI/BglII CDNA-1 band. Colonies were analyzed by PCR with CDNA-1F/CDNA-1R primers at: 94° C. 10 min, 35 cycles of [94° C., 30 s; 65° C., 30 s; 72° C., 2.5 min] and 72° C. 2 min. The positive plasmid was also analyzed by MfeI/BglII digestion and named SP6-pUC-PolyA-C1#306. Plasmid T7-ARep#68 was BglII/XbaI digested, and a 5.6 kb CDNA-2+CDNA-3 band was isolated from an agarose gel and ligated to BglII/XbaI/CIP digested SP6-pUC-PolyA-C1#306 plasmid. Screening was done by BglII and BglII/XbaI digestions. The new vector was named SP6-Arep#701.
[0091]Helper vector constructions. 77-AH#17 plasmid. In a first step Sindbis virus nts 1 to 425 were cloned into T7-pUC-PolyA#114. In a second step, both CDNA-4 and CDNA-5 viral fragments were cloned to generate a new helper vector. Sindbis nt 1 to 425 were amplified by PCR using as a template 54 ng of CDNA-1_Topo#64, primers (0.5 μM/each) SIN1-19F (5' ATTGACGGCGTAGTACACA 3') (SEQ ID NO:3) and H-BamR (5' GTATCAAGTAGGATCCGGAG 3') (SEQ ID NO:4) which adds a BamHI RE to allow CDNA-4 fragment cloning, reaction buffer (1×), dNTP's (200 μM), and 1 unit of Taq-DNA-polymerase in final volume of 20 μl. Two PCR reactions were performed in gradient for the following annealing temperature: 94° C. 5 min, 28 cycles of [94° C., 30 s; 45.3° C., 30 s (tube 1); or 46.4° C., 30 s (tube 2); 72° C., 30 s] and 72° C. 1 min. Bands were analyzed in agarose 1.3% gels, extracted, pooled together and digested with MfeI/BamHI. Plasmid T7-pUC-PolyA#114 was MfeI/BamHI digested and CIP dephosphorylated, the band isolated from an agarose gel and ligated to the 350 bp MfeI/BamHI PCR band. Colonies were screened by Taq-PCR with primers SIN1-19F/H-BamR at: 94° C., 10 min; 45 cycles of [94° C., 30 s; 45° C., 30 s; 72° C. 30 s] and 72° C. 1 min. A positive plasmid was checked by sequencing and named T7-pUC-PolyA-5'#604. This plasmid was digested with BamHI and NsiI and CIP dephosphorylated to ligate with viral inserts.
[0092]The ligation of CDNA-4 and CDNA-5 was through RE BclI. This enzyme is dam methylation dependent, so to demethylate the DNA, plasmids harboring CDNA-4_Topo#422 and CDNA-5_Topo#525 were transformed into dam.sup.-/dcm.sup.-1 E. coli strain GM2163 (New England Biolabs). CDNA-4_Topo#422(dam-) was BamHI and BclI digested and plasmid CDNA-5_Topo#525 was digested with BclI and NsiI. In both cases, the enzymes were inactivated at 70° C. for 15 min. The ligation was performed with T4-DNA-Ligase (16° C. for 14 h) with equimolar amounts of the three bands: T7-pUC-PolyA-5'#604 (BamHI/NsiI/CIP), CDNA-4_Topo#422(dam.sup.-) (BamHI/BclI) and CDNA-5_Topo#525(dam.sup.-) (BclI/NsiI). Colonies were screened by BamHI and NsiI digestion. The positive plasmid was named T7-AH#17 and was fully sequenced.
[0093]SP6-AH plasmid. Plasmid SP6-pUC-PolyA#902 was digested with MfeI and NsiI, CIP treated and the 2.4 kb band was ligated (T4-DNA-ligase) to the 4.5 kb T7-AH#17 MfeI/NsiI band. Colonies were screened by Taq PCR with primers CDNA-5F and CDNA-5R at: 94° C., 10 min; 25 cycles of [94° C., 30 s; 53° C., 30 s; 72° C. 3 min]. Positive plasmids were checked by NsiI, and NsiI/MfeI digestions and sequenced. The resulting plasmid was named SP6-AH#318
[0094]T7-R AND SP6-R plasmids. The four plasmids (SP6-AH#318, SP6-ARep#701, T7-AH#17 and T7-ARep#68) were fully sequenced, and in all four, a deletion of one T at the 3' end of the virus before the polyA, nt 11686 was found. In order to have the same sequence as the virus Ar-339, the deletion was fixed.
[0095]The Ar-339 sequence was placed first in plasmid T7-pUC-polyA#114. The new 3' end was obtained by PCR with primers: PolyA-F (5' CCCCAATGATCCGACCA 3'') (SEQ ID NO:5) and END-R (5' AAAACAAAATTTTGTTGATTAATAAAAG 3') (SEQ ID NO:6) and cloned into T7-pUC#32, as described previously for T7-pUC-polyA#114 cloning. The new plasmid T7-pUC-3end#9 was sequenced and used to generate new helpers and reporters.
[0096]To generate the reporter vectors, the T7-pUC-3end#9 plasmid was digested with XbaI and XhoI and the 423 bp band was cloned into the SP6-Arep#701 XbaI/XhoI 9504 bp band and into the T7-ARep#68 XbaI/XhoI 9504 bp band to generate new reporters SP6-R#406 and T7-R#202, respectively.
[0097]T7-H AND SP6-H plasmids. Plasmid T7-pUC-3end#9 was digested with NsiI and XhoI and the 311 bp band was cloned into the SP6-AH#38 NsiI/XhoI 6399 bp band and into the T7-AH#17 NsiI/XhoI 6399 bp band, to generate, respectively, the new helpers SP6-H#432 and T7-H#226. The new reporters and helper plasmids were fully sequenced.
[0098]SP6-HE2 mutants. Mutants were made on SP6-H plasmid following the kit QuickChange IIx site-directed mutagenesis (Stratagene, La Jolla, Calif.). Briefly, 10 ng of SP6-H#432 were incubated with complementary primers: E2-13-F/E2-13-R or E2-K70-F/E2-K70-R or E2-E181-F/E2-E181-R or E2-V251-F/E2-V251-R, reaction buffer, dNTPs and 2.5 units of pfuUltra HF DNA polymerase. PCR reactions were 95° C. 1 min, 18 cycles of: 95° C. 50 s, 60° C. 50 s, 68° C. 7 min, and final elongation of 68° C. 7 min.
[0099]After the PCR reactions 10 U of restriction enzyme DpnI was added and reaction incubated 37° C. 1 hour to digest methylated parental DNA. XL-10-Gold competent E. coli cells were transformed and the colonies analyzed by restriction enzyme digestion (RE). Mutations were verified by sequencing. Multiple mutants were made following the same protocol using previous mutants as template for PCR. The sequence of primers used and nt changes are shown in Table III (Appendix A). Plasmids made: SP6-H-13#5; SP6-H-K70#59; SP6-H-13-K70#66; SP6-H-13-K70-E181#3; SP6-H-K70-V251# SP6-H-13-K70-E181-V251. In order to make sure that there were not additional mutations due to the PCR technique, the 1270 bp BssHII/BanII fragment of each clone was subcloned in the sequenced plasmid SP6-H.
[0100]Reporter gene cloning in replicon vectors. The LacZ gene was cloned in the PmlI/XbaI site of vectors SP6-ARep#701, SP6-R#406, T7-ARep#68 and T7-R#202 to generate SP6-ARepLacZ, SP6-RLacZ, T7-ARepLacZ and T7-RLacZ, respectively.
[0101]The firefly luciferase gene was excised at the NheI/XbaI sites from pGL3 plasmid (Promega Co, Madison Wis.) and cloned in the XbaI site of vectors: SP6-ARep#701, SP6-R#406, T7-ARep#68 and T7-R#202 to generate SP6-ARepFluc, SP6-RFluc, T7-ARepFluc and T7-RFluc, respectively.
[0102]The Renilla luciferase gene from plasmid phRL-CMV (Promega Co, Madison Wis.) was cloned in the XbaI site of the replicons, to generate plasmids: SP6-RhRluc and T7-RhRluc.
EXAMPLE 2
[0103]Cells. BHK-21, and ES-2 cells were obtained from the American Type Culture Collection (ATCC). BHK-21 cells were maintained in αMEM (JRH Bioscience) with 5% FBS. ES-2 cells were derived from a patient with clear cell carcinoma, a type of ovarian cancer that has a poor prognosis and is resistant to several chemotherapeutic agents including cisplatin. ES-2 cells were cultured in McCoy's 5A medium (Mediatech) with 5% FBS. All basal media were supplemented with 100 μg/ml of penicillin-streptomycin and 0.5 μg/ml of amphotericin B (both from Mediatech). ES-2/Fluc cells were derived from the ES-2 line by transfection of a plasmid, pIRES2-Luc/EGFP, that expresses a bicistronic mRNA transcript containing both firefly luciferase and EGFP genes. To construct the pIRES2-Luc/EGFP plasmid, a DNA fragment containing the luciferase gene was obtained from pGL-3 basic plasmid (Promega) and then subcloned into the multicloning sites of the pIRES2-EGFP plasmid (BD Biosciences Clontech).
[0104]The mouse ovarian MOSEC cell line (clone ID8) was a generous gift from Dr. Katherine F. Roby (University of Kansas Medical Center, Kansas City) and was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4% FBS and 1× insulin-transferrin-selenium (Mediatech, Inc).
[0105]In vitro transcription and vector preparation. The plasmids carrying the Sindbis replicon or JT-BB helper RNAs were linearized with PacI, NotI or XhoI, before in vitro transcription using the mMESSAGE mMACHINE RNA transcription kit (T7 and SP6 version; Ambion). Both helper and replicon RNA transcripts (20 μl each) were then electroporated into BHK-21 cells and incubated in 10 ml of αMEM containing 5% FBS at 37° C. for 12 h. The medium was replaced with 9 ml of Opti-MEM I medium (GIBCO-BRL, Invitrogen San Diego Calif.) supplemented with 0.7 μM CaCl2. After 24 h, the culture medium was collected and stored at -80° C.
[0106]Vector titering. The titers of Sindbis vectors were assayed in BHK-21, ES-2, ES-2/Fluc or MOSEC cells. Serial dilutions (300 μL each) of vector were added to 2×105 BHK-21 cells in 12-well plates. After incubation for 1 hour at room temperature, the cells were washed with PBS and incubated with 2 mL of αMEM at 37° C. for 24 hours.
[0107]LacZ expression was determined by two methods: staining and counting blue cells/well or reading absorbance. For the first, cells were fixed in PBS containing 0.5% glutaraldehyde at room temperature for 20 minutes, washed three times with PBS, and then stained with PBS containing 1 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside; (Fisher Scientific, Pittsburgh, Pa.), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mM MgSO4 at 37° C. for 3 hours. After staining with the X-Gal solution, cells that expressed LacZ were blue. Blue-stained cells were counted and vector titers were estimated by determining the number of transducing units (TU) per mL of aliquot. With the second method, cells were lysed with 200 μL of M-PER lysis buffer (Pierce Biotechnology, Rockford, Ill.). 50 μL of the cell lysates were added into 50 μL of All-in-One-Galactosidase Assay Reagent (Pierce Biotechnology) and incubated at room temperature for 5 minutes before reading at 405 nm. Vector titers were estimated as the last dilution having detectable absorbance.
[0108]Firefly luciferase activity in cell lysates was determined by aspirating the culture medium from the wells and adding 200 μL per well of culture medium and 200 μL per well of Steady-Glo® reagent (Promega Corp., Madison, Wis.). Plates were incubated with gentle rocking for 5 minutes until the cells detached from the plates. The cell lysates were then transferred to 12×47 mm cuvettes (BD Pharmingen, San Diego, Calif.), and the luciferase activity of each lysate was determined by taking a 30-second reading with a LUMI-ONE portable luminometer (Bioscan, Inc., Washington, D.C.).
[0109]Renilla luciferase activity was determined by following the protocol in "Renilla Luciferase assay system" (Promega Corp., Madison, Wis.). Briefly, cells were washed with PBS and 250 μl/well of lysis buffer was added. 20 μL of substrate were added to 100 μL of extract and the luciferase activity of each lysate was determined by taking a 30-second reading with a LUMI-ONE portable luminometer (Bioscan, Inc., Washington, D.C.).
[0110]Animal models. All animal experiments were done in accordance with NIH and institutional guidelines. To determine the therapeutic effects of Sindbis virus vectors, SCID mice (female, 6-8 week old; Taconic, Germantown, N.Y.) were i.p. injected with 1.5×106 ES-2/Fluc cells/mouse on day 0 and imaged with the IVIS® system the next day (day 1) to confirm the presence of tumor cells. Then on day 4, 5 mice/vector received i.p. treatment with vectors carrying the Renilla luciferase: A(JT-BB/SP6-RhRluc), B (SP6-H/JT-RephRluc), C(SP6-H/SP6-RhRluc), D (JT-BB/JT-RephRluc). All vectors had the same titer in ES-2/Fluc cells, and mice were i.p. injected daily with ˜106 TU in 0.5 ml Opti-MEM I/mouse. Control mice (n=5) received no Sindbis vector treatment. Disease progression was later determined by IVIS® imaging on days 1, 5, 9 and 13.
[0111]Survival curves were compared with log rank test. All the P values presented in this study are two-tailed.
[0112]For colocalization experiments, two SCID mice/vector were i.p. inoculated with 2×106 ES-2/Fluc cells on day 0 and received one i.p. treatment of vector C (˜106 TU in 0.5 mL of OptiMEM I) on day 5. The next day (day 6), mice were i.p. injected with 0.3 mL of 0.2 mg/mL coelenterazine (Biotium, Inc., Hayward, Calif.) followed by IVIS® imaging for Renilla luciferase activity. 30 minutes later, the same mice were i.p. injected with 0.3 mL of 15 mg/mL D-luciferin (Biotium, Inc) and a second IVIS® imaging for Firefly luciferase activity was performed.
[0113]Cell tropism experiments, were done in two groups of SCID mice with 5 mice/vector: one without tumor induction and the second one in mice with induced tumors.
[0114]The tumor free animals were i.p. injected with Fluc vectors on day 0, imaged by IVIS® on day 1, received a second injection of vectors on day 2 and on day 3 were IVIS® imaged again.
[0115]For the second group, SCID female mice were injected i.p. on day 0 with 2×106 ES-2 cells/mouse and on day 4 mice were injected with vectors carrying the luciferase reporter genes. After the first whole-body IVIS® imaging on day 1, the peritoneum was removed for another IVIS® imaging of the peritoneal cavity. The remaining mice of the group, except one for background control, had a second i.p. injection of vectors on day 6 and imaged again on day 7.
[0116]In vivo bioluminescence detection with the IVIS® Imaging System. A cryogenically cooled IVIS® Imaging System Series 100 (Xenogen) was used with Living Image acquisition and analysis software (Version 2.11, Xenogen Corp. Alameda, Calif.) to detect the bioluminescence signals in mice. For firefly luciferase detection, each mouse was injected i.p. with 0.3 ml of 15 mg/ml beetle luciferin (potassium salt; Promega Corp., Madison, Wis.) in PBS. After 5 min, mice were anesthetized with 0.3 ml of avertin (1.25% of 2,2,2-tribromoethanol in 5% tert-amyl alcohol) or isofluran-mixed oxygen. The imaging system first took a photographic image in the chamber under dim illumination; this was followed by luminescent image acquisition. The overlay of the pseudocolor images represents the spatial distribution of photon counts produced by active luciferase. An integration time of 1 min was used for luminescent image acquisition for all mouse tumor models. Living Image software (Wave Metrics Inc., Lake Oswego, Oreg.) was used to integrate the total bioluminescence signals (in terms of proton counts) obtained from mice. The in vitro detection limit of the IVIS® Imaging System is 1,000 ES-2/Fluc cells.
[0117]Ar-339 Sequence Analysis
[0118]Ar-339 virus was amplified in chicken embryo fibroblasts and cloned into sequencing plasmids as six separate overlapping fragments (FIG. 1). CDNA-3B and CDNA-4 overlap the 312 bp fragment (nt 7334-7646) that contains the viral subgenomic promoter. In order to avoid mutations due to RT or PCR reactions, for each plasmid three different RT reactions were performed and each one served as template for one PCR reaction. The three PCR amplified bands of each fragment were cloned separately, sequenced and compared to verify the virus sequence. The Ar-339 sequence obtained was compared to the Sindbis published sequence (Strauss E G, Rice C M and Strauss J H 1984) and to the sequence of Sindbis vectors that we used previously in our laboratory, JT-BB and JT-Rep. (Tseng et al 2004a,b). The results are shown in Table IV (Appendix A). The differences in sequence between the Strauss sequence and JT vectors are described in Table V (Appendix A). Functional changes between JT and AR-339 plasmids are summarized in Table VI (Appendix A).
[0119]In the viral replicase, comparing the Strauss map with Ar-339, three point mutations in nsp1 were found: nt 353 a silent mutation; nt 1380 and 1381. Both change amino acid 441 from Cys to Ile in Ar339. In the Sindbis-like virus supergroup, the methyltransferase nsp1 has four conserved motifs I (a.a 31 to 51), II (a.a 87 to 86), III (168 to 220) and IV(243 to 260) (Rozanov et al. 1992). Cys 441 to Ile is not in the carboxyterminal domain required for enzymatic activity (K468 to L512)(Wang et al. 1996). Nsp2 has three mutations compared to the Strauss sequence, one silent at nt 3698 (A to G) and two (nt 2992 and 3579) that change amino acids 438 (Pro toLeu) and 634 (Lys to Glu) respectively. Both amino acids are outside the active helicase and protease domains of nsp2 (Rikkonen et al. 1994). Sindbis virus with Pro at 438, as described in the Strauss sequence, has lethal effects on virus replication (Rice et al. 1987). In nsp4 there was a silent mutation at nt 7337 (T to C).
[0120]Regarding the structural proteins, the Ar-339 capsid protein had two mutations compared with the Strauss sequence, one silent at nt 8345 (C to A) and one at nt 7846 that changed Pro 67 to Gln. In the consensus Strauss sequence and JT vectors a Proline occurs at position 67. This residue is conserved in different isolates of virus in Australia, and for MK6962, a New Guinea isolate, a Tyr (T) is present at this site (Sammels et al. 1999). This change is in the 11 to 74 amino acid region that doesn't bind to Sindbis RNA (Geigenmuller-Gnirke et al. 1993) and is not in E2 or capsid proteins interaction domains residues 36-39, 108 to 111, 172, 180 to 183, 201, 231-234, 240 or 254 (Katherine E. Owen and Richard J. Kuhn, 1996; H. Lee and D. T. Brown 1994).
[0121]There were also two silent mutations in E1 at nt 10392 (T to C) and 10469 (T to A) and two differences in the Ar-339 with the Strauss map were found at positions Ala 72 to Val in Ar-339 and 237 (Ser to Ala), which are both located in domain II. Residues of this domain are involved in E1-E1 interaction in the virus spike (Zhang W et al. 2002).
[0122]Most of the coding changes were found in the envelope protein E2, in which the antigenic sites and the binding receptor domain of the virus have been described. Comparing the Strauss sequence with Ar-339, five amino acid changes were found located in the external leaf-like domain of the E2 protein, which extends from the amino terminus to residue 218 (Zhang W et al. 2002). Changes are in amino acids: 3 (Ile to Thr); 23 (Val to Ala); 70 (Lys to Glu) and also two of mutations, 172 (Arg to Gly) and 181 (Glu to Lys), occur in the putative binding receptor domain (amino acids 170 to 220). No changes were found in the endodomain that interacts with the capsid protein (from 391 to 483) or with the E2-E1 interaction region.
[0123]An analysis of the amino acid changes between the JT plasmids and the Ar-339 sequence revealed only one mutation in the replicase, Cys 441 to Ile of nsp1. In the structural proteins there were a total of eight differences, only one in the capsid protein, Pro 67 to Gln; and seven in the E1 and E2 envelope proteins. Three mutations in E1: Ala 72 to Val; Gly 75 to Asp and Ser 237 to Ala. Most of the differences were found in the E2 protein, three in the leaf-Like domain; Ile 3 to Thr; Lys70 to Glu and Glu 181 to Lys and one in the ectodomain, Val 251 to Ala. V251 is important for virus maturation in CEF (Li M L et al. 1999).
[0124]Vector Constructions
[0125]To construct the Ar-339 vectors, the Sindbis genome was split into two plasmids: the replicon and the helper (FIG. 2). This vector system is designed to electroporate in vitro transcribed viral RNA into the susceptible cell line to produce replicative defective Sindbis virus, called viral vector, that contains, as a genome, the replicase RNA and lacks the structural genes. For in vitro transcription, a bacteriophage promoter is required before the viral sequence.
[0126]In order to compare the yield of viral vectors in this system, two pairs of vectors were produced, one pair with replicon and helper under the control of the SP6 promoter (SP6-H and SP6-R), and the other pair under the control of the T7 promoter (T7-H and T7-R).
[0127]The replicon contains the viral replicase, with the packaging signal, nt 945 to nt 1075, (Weiss B et al. 1994; Frolova et al 1997); the viral subgenomic promoter, multicloning site 1 (MCS1) to allow for the insertion and expression of the gene of interest, and the 3' end of the virus (nt 11394 to 11703) to allow viral (-) strand RNA synthesis. A second multicloning site (MCS2) allows the linearization of the plasmid for in vitro transcription.
[0128]The helper plasmid contains the first 425 nt of the virus, followed by the 3' end of the virus from nt 7334 to nt 11703 which includes the subgenomic promoter, the capsid and the viral envelope proteins (E3, E2, 6K and E1) and the 3' end (nt 11394 to 11703).
[0129]Both plasmids share the following viral sequences: the first 425 nt, the 309 nt of the 3' end and the sub genomic promoter.
[0130]Both plasmids have several non-viral elements in common, the replication origin (rep pMB1) and the Ampicillin resistance gene from the pUC cloning plasmid; the promoter for in vitro transcription (T7 or SP6) and the MCS2. In the construction process a plasmid containing the pUC sequences, SP6 or T7 promoter, the multicloning sites, and the 3'viral end, which are common to both vectors, was first generated. The specific viral sequences were then cloned into this plasmid (FIG. 3).
[0131]SP6 and T7 Promoters
[0132]For in vitro transcription systems, the RNA yield using SP6 or T7 RNA polymerase for long RNA transcripts could differ. To study if the promoter would make a difference in the titer of the viral vectors production, BHK-21 cells were electroporated with two sets of in vitro transcribed RNAs: SP6-AH and SP6-ARepLacZ, to generate the SP6-LacZ viral vector, and with T7-AH and T7-ARepLacZ for the T7-LacZ vector. The comparison of both vector's titers in BHK-21 cell, in repeated experiments, gave equivalent titers ≈106 transducing units (TU)/mL. In terms of infective particles production, both promoters work with the same efficiency in this system.
[0133]Sequencing of the four plasmids (SP6-AH, SP6-ARep, T7-AH and T7-ARep) revealed a deletion of one T at the 3' end of the virus before the poly A (SV nt 11686). To study the effect of this deletion on viral vectors, four new plasmids without the deletion were constructed (SP6-H, SP6-R, T7-H and T7-R) and titers of viral vectors from both sets of plasmids were compared. No significant difference was observed indicating that the deletion of Ti 1686 is not critical for vector replication. As these four sets of vectors showed the same in vitro titer, in order to standardize results the experiments with mice were performed using SP6-H and SP6-R plasmids to synthesize viral vectors.
EXAMPLE 3
Biological Properties
Cell Tropism
[0134]Most of the amino acid differences found between JT and Ar-339 vectors were in the envelope proteins. One of them was related to virus adaptation to BHK-21, E2 Lys 70 to Glu (McNight K et al, 1996) and two of them were located in the receptor-binding domain of the E2 protein. To analyze if the amino acid changes had any effect on the viral vector's infectivity, JT, Ar-339 and chimeric viral vectors were produced and titered in three cell lines: BHK-21 (baby hamster kidney), ES-2 and Mosec human and mouse ovarian cancer cell lines respectively. The results are shown in Table VII (Appendix A). Vectors that carry JT-BB helper (JT-BB/SP6-ARepLacZ and JT-BB/JT-RepLacZ) had titers two logarithms higher in BHK-21 than in the other two cell lines; when the helper was SP6-H, the difference observed was only one log. The infectivity of the vectors in vitro was similar in both ovarian cancer cell lines, ES-2 and Mosec. This difference was observed in repeated experiments.
[0135]Disease Progression
[0136]In order to compare the ability in targeting and suppression of disease progression by Sindbis vectors Ar-339 and JT, JT and Ar-339 chimeric vectors were produced and tested in the ES-2/Fluc mouse metastatic ovarian cancer model described previously (Tseng et al. 2004b). Five female SCID mice per vector group were injected i.p. with 1.5×106 ES-2/Fluc cells (day 0) and IVIS® imaged the next day to verify the presence of ES-2/Fluc cells. Cells were left to grow for four days before daily treatment with vectors was started. There were five mouse groups, one of which did not receive vector treatment, whereas the remaining 4 had vectors: A(JT-BB/SP6-RhRluc), B (SP6-H/JT-RephRluc), C(SP6-H/SP6-RhRluc) and D (JT-BB/JT-RephRluc). As these strains showed different cell tropism in BHK-21 cells, vectors were titered in the same cell line used to induce the tumor ES-2/Fluc, and titers for all vectors were standardized at 106 TU/mL. Total whole body photon counts were determined by IVIS® imaging on days 1, 5, 13, and 19 to determine disease progression of ES-2/Fluc metastases (FIG. 4). Survival curves were also compared (FIG. 5).
[0137]Vector A, carrying the Ar-339 replicase (SP6-RhRluc) and JT structural proteins was more efficient in reducing tumor progression and gave better survival of the animals. The vectors carrying the same structural proteins, vector B versus C and A versus D were compared. In both cases there was more tumor reduction with Ar-339 replicase (SP6-RhRluc). Regarding the structural part, when the vectors carrying the same replicase were analyzed, in one case SP6-H seemed to be more effective (Vectors D versus B), but in the case of A versus C, where the photon count difference is larger, JT-BB is more efficient in tumor targeting. The small differences between vectors B, C and D correlates with similar animal survival data, although both structural proteins and replicase function in the efficiency of the vectors in vivo, these data suggest that an improvement in the targeting of Ar-339 replicase (SP6-R) to tumor cells would lead to more efficient gene therapy vectors.
Co-Localization
[0138]To establish the degree and specificity of Ar-339 Sindbis infection of tumor cells, IVIS® imaging studies were performed that measured independent bioluminescent signals from tumor cells and vectors. The ES-2/Fluc cells expressed the firefly luciferase gene, that uses D-luciferin as substrate, and the vectors carried a different luciferase gene cloned from soft coral Renilla renifomis (Rluc) that uses coelenterazine to generate bioluminescence. The two luciferases are highly substrate specific and do not cross-react (Bhaumik S and Gambhir SS. 2002). Each anesthetized mouse was first treated with coelenterazine the image was collected (FIG. 6A, left panel), then treated with D-luciferin for a second IVIS® imaging, this time of ES-2/Fluc cells (FIG. 6A right panel). The bioluminescence signals generated in the same animal from Sindbis/Rluc and ES-2/Fluc, were quantitated using Living Image software. The images of Rluc and Fluc signals were grided (12 8, 96 boxed regions), and corresponding regions were analyzed for statistical correlation (FIG. 6B). A highly significant correlation was established (P<0.0001) indicating that a single i.p. delivery of Ar-339 Sindbis vector lead to the efficient infection of the metastasized tumor cells throughout the peritoneal cavity. In several mice an additional infection outside the peritoneal cavity was observed.
[0139]Ar-339 Targeting
[0140]To analyze tissues or organs targeted by the Ar-339 strain, new chimeric vectors were made with firefly luciferase (Fluc) as the reporter gene, since its stronger bioluminescent signal allows the study of vectors in animal organs. Each vector was tested in two groups of 5 SCID female mice: tumor-free and 5 day ES-2 metastasis induced mice. To assess which part of the C vector was responsible for the chest bioluminiscence, three Fluc chimeric vectors were made: A(JT-BB/SP6-RFluc), B(SP6-H/JT-RepFluc) and C (JT-BB/JT-RepFluc). As previously, vectors were titered in ES-2 cells. Tumor free mice received one dose of vector (104 TU/mL) at day 0 and were IVIS® imaged next day (FIG. 7A). All three groups showed a low background signal in fat tissue. Two out of five vector B mice and one of five mice in vector C group showed some additional bioluminescent signal in the chest, as previously observed in the colocalization experiment. To investigate if vectors were infecting organs in these mice, intraperitoneal cavity and harvested organs were also IVIS® imaged. The chest signal observed corresponded to connective tissue in the ribs, while organs had no background signal. To study if repeated doses of these vectors could lead to accumulative infection in tumor-free mice, a second dose was i.p. injected on day 2 and the image repeated on day 3. The results (FIG. 7B) showed low background signal in fat tissue for vectors B and C and no signal at all for vector A, indicating that the background is transient and shouldn't affect the target effectiveness of Ar-339 vectors in repeated treatment.
[0141]Previous studies of JT/Fluc vector in 5 day ES-2 tumor induced mice, showed that the vector specifically targets metastasized ES-2 cells after one injection and also in a second dose two days later (Tseng et al. 2004). To study if the difference between sequences could affect the specificity of Ar-339 vector, these 3 chimeric vectors were tested in the same model. Mice injected with 2×106 ES-2 cells on day 0, received one i.p. dose of vectors on day 5 and were IVIS® imaged on day 6. The peritoneal cavity and organs of two mice per group were imaged. As is shown in FIG. 8A for all vectors, bioluminescence correlated with ES-2 metastatic implants. At day 7 two mice per group received a second i.p. dose of vector and one mouse was not injected to serve as a luciferase background signal control (FIG. 8B). Vector A showed a similar signal compared with previous doses, but vectors carrying Ar-339 structural proteins, B and C, showed decreased bioluminescence signals in tumors compared with the first injection. The difference in reinfection suggests that amino acid changes in structural proteins could play an important role in targeting metastases by repetitive treatment with vector.
[0142]In order to determine which mutations were critical for the vector properties, a chimeric vector was generated, QE2, that contains E2 from JT-BB and the remaining structural proteins from Ar-339. When we compared with Ar-339 vector in the same IVIS® animal model as in the previous experiment, in tumor free animals, a low background in fat tissue with the first dose was observed and no signal in a second dose. In ES-2 5 day induced tumor mice, vector QE2 targeted tumor and was able to re-infect animals, though the bioluminescent signal was not as strong as for Ar-339 vector (data not shown). This indicates that Ar-339 sequence in the E2 envelope protein was primarily responsible for the targeting pattern, though the optimal amino acid pattern was still not clear. To address this question site directed mutagenesis was performed on the Ar-339 E2 envelope protein.
[0143]E2 Mutants
[0144]The E2 envelope has been described as the protein that is primarily responsible for cell tropism and infectivity of Sindbis virus. More specifically, Lys 70 is implicated in BHK-21 specificity (McNight K et al, 1996) and also residues 69 to 72 have been related to targeting vertebrate cells (Ohno K et al. 1997; Dubuisson J and Rice CM, 1993). As the Ar-339 strain has a Glu in this position, 3 point mutants were generated that contain Lys at position 70 (FIG. 9). In Mut-1 only Ar-339 Glu 70 was changed to Lys. Amino acid 251 of E2 is highly conserved between different Sindbis isolates (Sammels L M et al. 1999) and mutation to Valine at this position has been related with host range phenotype (Li M L et al. 1999). To explore the influence of this amino acid in vector targeting, Mut-2 was produced that has Lys70 and also Ar-339 Ala 251 mutated to Val. Residues at positions 3 and 181 are located in the external leaf-like domain of the E2 protein and 181 is in the receptor binding domain. To assess if the combination of these 4 amino acid changes in the E2 is responsible for the difference in infection of Ar-339 vectors, mutant Mut-4 was produced with Lys 70, and Val 251 plus changes in Ar-339 Thr 3 to Ile and Ar-339 Lys 181 to Glu (FIG. 9). The mutants were tested in tumor-free and ES-2 5 day induced tumor mice as previously described for vectors A, B and C. To assure that the difference in signals were due to the E2 mutations, all five vectors tested carried the same replicase SP6-RFluc: vector A (JT-BB/SP6-RFluc), C(SP6-H/SP6-RFluc), Mut-1 (SP6-H-K70/SP6-RFluc) Mut-2 (SP6-H-K70-V251/SP6-RFluc) and Mut-4 (SP6-H-I3-K70-E 181-V251/SP6-RFluc).
[0145]Vectors were titered in BHK-21 cells and in ovarian cell lines ES-2 and Mosec (Table VIII (Appendix A)). Mutants showed a greater difference between A and C when titered in BHK-21 than in ovarian cell lines.
[0146]In tumor-free mice vectors A and C gave background in some of the animals. Only one out of the five vector C mice showed a low bioluminescent signal in the ribs (FIG. 10A). In second doses of these vectors the background was even less noticeable (FIG. 10B). Mut-1 and Mut-2 did not produce background bioluminescent signal in either dose. Mut-4 showed barely detectable signal in fat tissue, much less intense than vectors A and C.
[0147]In ES-2 tumor induced mice (FIG. 11A) mice #79 in A and #88 in C vector groups did not show bioluminescence; these mice did not develop ES-2 metastatic implants in the peritoneal cavity (these mice are not shown in the Figure).
[0148]This result showed a dramatic decrease in the infectivity of mutants Mut-1 and Mut-2 and an important reduction in Mut-4 after the first and second dose (FIG. 11). The change of only one amino acid in Ar-339 E2 at position 70 makes vector C lose the specificity in targeting ES-2 tumor metastases in vivo. The double mutant with Valine at position 251, Mut-2, doesn't revert the vector tropism. Mut-4 combines vector A E2 with Ar-339 E1, E3 and capsid sequences. The fact that Mut-4 could not revert to full infectivity indicates that the interaction between E2 and E1 in the vector spike could also play an important role in vector targeting.
EXAMPLE 4
Suppression of Disease Progression by Ar-339, MUT-4 and JT Vectors
[0149]In previous mouse experiments Vector C showed background infections in some of the animals treated. Although this effect was transient and vector C was efficient in suppression of disease progression, it would be preferable for gene therapy to use a viral vector that does not cause background tissue infections. In tumor targeting experiments of SP6-HE2 mutants, vector Mut-4 showed tumor targeting although with less intensity than vector C (FIG. 11) and did not show background infection (compare FIGS. 10A and 10B). With the aim of improving this vector system, the ability of Mut-4 in tumor reduction was studied and vector-tumor colocalization analyzed, using the same model described for vectors A, B, C and D as in previous experiments.
[0150]Materials and Methods
[0151]Animal models. All animal experiments were done in accordance with NIH and institutional guidelines. To determine the therapeutic effects of Sindbis virus vectors, SCID mice (female, 6-8 week old; Taconic, Germantown, N.Y.) were i.p. injected with 1.5×106 ES-2/Fluc cells/mouse on day 0 and imaged with the IVIS® system the next day (day 1) to confirm the presence of tumor cells. Then on day 4, 10 mice/vector received i.p. treatment with vectors carrying the Renilla luciferase: Mut-4 (SP6-HI3K70E181V251/SP6-RhRluc), C(SP6-H/SP6-RhRluc) and D (JT-BB/JT-RephRluc). All vectors had the same titer in ES-2/Fluc cells, and mice were i.p. injected daily with ˜106 TU in 0.5 ml Opti-MEM I/mouse. Control mice (n=5) received no Sindbis vector treatment. Disease progression was later determined by IVIS® imaging on days 1, 5, 9 and 13.
[0152]Survival curves were compared with log rank test. All the P values presented in this study are two-tailed.
[0153]For colocalization experiments, two SCID mice/vector were i.p. inoculated with 1.5×106 ES-2/Fluc cells on day 0 and received one i.p. treatment of vector Mut-4 (˜106 TU in 0.5 mL of OptiMEM I) on day 5. The next day (day 6), mice were i.p. injected with 0.3 mL of 0.2 mg/mL coelenterazine (Biotium, Inc., Hayward, Calif.) followed by IVIS® imaging for Renilla luciferase activity. 30 minutes later, the same mice were i.p. injected with 0.3 mL of 15 mg/mL D-luciferin (Biotium, Inc) and a second IVIS® imaging for Firefly luciferase activity was performed.
[0154]Results
[0155]Colocalization. To measure the degree and specificity of Mut-4 Sindbis infection of tumor cells, IVIS® imaging studies were performed that measured independent bioluminescent signals from tumor cells and vector Mut-4 (SP6-HI3K70E181V251/SP6-RhRluc). The ES-2/Fluc cells expressed the Firefly luciferase gene, that uses D-luciferin as a substrate, and the vector carried a different luciferase gene cloned from soft coral Renilla renifomis (hRluc) that uses coelenterazine to generate bioluminescence. The two luciferases are highly substrate specific and do not cross-react (Bhaumik, S, and Gambhir, S. S. 2002). Each anesthetized mouse was first treated with coelenterazine and the image was collected (FIG. 12A, left panel), then treated with D-luciferin for sequential IVIS® imaging of ES-2/Fluc cells (FIG. 12A, right panel). The bioluminescence signals generated in the same animal from vector and ES-2/Fluc were quantitated using Living Image software. The images of Rluc and Fluc signals were grided (12×8, 96 boxed regions), and the corresponding regions were analyzed for statistical correlation (FIG. 12B). A highly significant correlation was established (P<0.0001) indicating that a single i.p. delivery of Mut-4 Sindbis vector leads to very efficient infection of the metastasized tumor cells throughout the peritoneal cavity.
[0156]Disease progression. In order to compare the ability of Sindbis vectors Ar-339 and Mut-4 in targeting and suppression of disease, vectors were constructed and tested in the ES-2/Fluc mouse metastatic ovarian cancer model described previously. Ten female SCID mice per vector group were injected i.p. with 1.5×106 ES-2/Fluc cells (day 0) and IVIS® imaged the next day to verify the presence of ES-2/Fluc cells in the mice. Cells were left to grow for four days before daily treatment with vectors was started. There were four groups of animals, one of which did not receive vector treatment, the remaining 4 were injected daily with 106 TU/ml doses of vectors carrying Renilla luciferase reporter gene: C(SP6-H/SP6-RhRluc), Mut-4 (SP6-HI3K70E181V251/SP6-RhRluc). and D (JT-BB/JT-RephRluc).
[0157]Total whole body photon counts were determined by IVIS® imaging on days 1, 5, 13, and 19 to determine disease progression of ES-2/Fluc metastases. Survival curves were also compared (FIG. 13). Mice treated with vectors C and Mut-4 showed similar photo count reduction and survival proportions. These data suggest that vector Mut-4 (SP6-HI3K70E181V251/SP6-RhRluc) has similar in vivo efficiency in tumor reduction as vector C(SP6-H/SP6-RhRluc) and so can also be used in gene therapy. Both vectors, C and Mut-4, showed significantly improved tumor reduction and mouse survival compared to vector D.
PAPER EXAMPLE 1
Production of C6/36 Packaging Cell Line with Rederived Ar-339 Plasmids
[0158]Plasmids SP6-H and SP6-R can be used to engineer insect plasmids to generate a mosquito C6/36-derived packaging cell line producing Ar-339 Sindbis vectors. Three plasmids with the Opal2 mosquito promoter (described in Theilmann et al., J. Virology, 1995, 69(12):7775-7781) are required to constitutively express viral sequences in mosquito cells. The replicon plasmid (pIZ-Ar339-R) will have the replicase, subgenomic promoter and gene of interest and the other two will contain the split helper sequences, one with capsid protein only (PIB-Ar339-C) and the second one with E1, E2, E3 and K6 envelope proteins (pIZ-Ar339-H). In a first step the C6/36 cell line (available from the American Type Culture Collection, ATCC, Manassas, Va. as ATCC CRL 1660) will be transfected with replicon plasmid (pIZ-Ar339-R) and clones will be selected. In a second step the previous clones containing the replicon will be transfected with the capsid plasmid (PIB-Ar339-C) and replicon and capsid positive clones selected. In the last step, the 2nd helper plasmid (pIZ-Ar339-H) will be transfected into the previously isolated clones to generate the packaging cell line that express all three plasmids.
[0159]1.--Cloning of SP6-R Replicase into PIZ/V5-HIS Plasmid
[0160]In order to clone the Ar-339 replicase in PIZ/V5-His (Invitrogen, San Diego, Calif.) it is necessary to introduce a SacI RE site before the SP6 promoter.
[0161]1.1--PCR Reactions will be performed on SP6-R#406 plasmid using primers SacI_SP6F/cDNA-1R, and then the 2340 bp band will be cloned in pcr4blunt_topo vector (Invitrogen, San Diego, Calif.). The new plasmids TOPO-Rep1
[0162]Primer sequences:
TABLE-US-00001 SacI_SP6F GGCTAGAGCTCATTTAGGTGACA (SacI) cDNA-1R GTAACAAGATCTCGTGCCGTGACA
[0163]1.2.--The SP6-R#406 plasmid will be digested with BglII/NotI and cloned the 5776 bp band into BglII/NotI TOPO-Rep1 to make new plasmid TOPO-Ar339-R
[0164]1.3.--The 8085 bp SacI/NotI TOPO-Ar339-R band will be cloned into SacI/NotI PIZ/V5-His to generate pIZ-Ar339-R.
[0165]2.--Cloning of Ar-339 Helper P
[0166]In order to minimize the presence of recombinant replicative competent virus, the helper genome will be split into two plasmids: PIB-Ar339-C and pIZ-Ar339-H.
[0167]2.1 Construction of pIB-Ar339-C
[0168]The Ar-339 Capsid DNA sequence will be cloned into pIB-V5-His (Invitrogen, San Diego, Calif.) at the BamHI/SpeI site of the vector. PCR will be performed on plasmid SP6-H#432 using primers C1-F and C1-R to obtain the Ar-339 capsid 1133 bp DNA band.
TABLE-US-00002 C1-F GGA TCT CCG GAT CCC CTG AAA AGG (BamHI) C1-R GTG ACC AGT GGA CTA GTG GAC CAC TCT TC (SpeI)
[0169]The band will be digested and cloned into the pIB-V5-His vector at the BamHI/SpeI site to make the new plasmid pIB-Ar-339-C.
[0170]2.2 Construction of pIZ-Ar339-H
[0171]The first step is to clone the 5' end of Ar-339 (from nt 425 to 692) into pIZ/V5-His. PCR reactions will be performed on SP6#432 plasmid with primers 416B-F and 676NB-R. Restriction sites will be included in this no coding sequence to allow further cloning of helper sequence.
TABLE-US-00003 416B-F GGA TCT CCG GAT CCC CTG AAA AGG CTG T (BamHI) 676NB-R GAT GAA AGG ATC CTC GCG AAC TAT TTA GGA CCA CCG (BamHI/NruI)
[0172]The 296 bp band will be digested with BamHI and cloned in the BamHI site of pIZ/V5-His to make pIZ-5END plasmid.
[0173]In a second step SP6-H#432 plasmid will be digested with NruI and XhoI and the 3435 bp band will be cloned into the NruI/XhoI site of the pIZ-5END plasmid.
REFERENCES
[0174]Bhaumik S, Gambhir S S. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci USA 2002; 99:377-82. [0175]Burge B W, Pfefferkorn E R. "Complementation between temperature-sensitive mutants of Sindbis virus". Virology. 1966 October; 30(2):214-23. [0176]Dubuisson J, Rice C M. "Sindbis virus attachment: isolation and characterization of mutants with impaired binding to vertebrate cells." J. Virol. 1993 June; 67(6):3363-74. [0177]Frolova E, Frolov I, Schlesinger S. "Packaging signals in alphaviruses." J. Virol. 1997 January; 71(1):248-58. [0178]Frothingham (1955). "Tissue culture applied to the study of Sindbis virus." Am. J. Trop. Med. Hyg. 4: 863-871. [0179]Geigenmuller-Gnirke U, Nitschko H, Schlesinger S. "Deletion analysis of the capsid protein of Sindbis virus: identification of the RNA binding region." J. Virol. 1993 March; 67(3): 1620-6. [0180]Hurlbut, H. S. (1953). 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"ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2." J. Virol. 1994 September; 68(9):5804-10. [0190]Rozanov M N, Koonin E V, Gorbalenya A E. "Conservation of the putative methyltransferase domain: a hallmark of the `Sindbis-like` supergroup of positive-strand RNA viruses." J Gen Virol. 1992 August; 73 (Pt 8):2129-34. [0191]Sammels L M, Lindsay M D, Poidinger M, Coelen R J, Mackenzie J S. "Geographic distribution and evolution of Sindbis virus in Australia." J Gen Virol. 1999 March; 80 (Pt 3):739-48. [0192]Strauss E G, Rice C M, Strauss J H. "Complete nucleotide sequence of the genomic RNA of Sindbis virus." Virology. 1984 February; 133(1):92-110. [0193]Strauss, J. H. and Strauss, E. G. "The alphaviruses: gene expression, replication, and evolution" Microbiol Rev. 1994 September; 58(3): 491-562. [0194]Taylor, R M and H S Hurlbut (1953). "Isolation of coxsackie-like viruses from mosquitoes." J. Egypt. Med. Assoc. 36: 489-494. [0195]Taylor, R M, H S Hurlbut, T H Work, J R Kingsbury and T E Frothingham (1955). "Sindbis virus: A newly recognized arthropod-transmitted virus." Am. J. Trop. Med. Hyg. 4: 844-846. [0196]Tseng J C, Levin B, Hirano T, Yee H, Pampeno C, Meruelo D. In vivo antitumor activity of sindbis viral vectors. J Natl Cancer Inst (Bethesda) 2002;94: 1790-802. [0197]Tseng J C, Levin B, Hurtado A, et al. Systemic tumor targeting and killing by Sindbis viral vectors. Nat Biotechnol 2004a; 22:70-7. [0198]Tseng J C, Hurtado A, Yee H, Levin B, Boivin C, Benet M, Blank S V, Pellicer A, Meruelo D. "Using sindbis viral vectors for specific detection and suppression of advanced ovarian cancer in animal models." Cancer Res. 2004b Sep. 15; 64(18):6684-92. [0199]Wang H L, O'Rear J, Stollar V. "Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity." Virology. 1996 Mar. 15; 217(2):527-31. [0200]Weiss B, Geigenmuller-Gnirke U, Schlesinger S. "Interactions between Sindbis virus RNAs and a 68 amino acid derivative of the viral capsid protein further defines the capsid binding site." Nucleic Acids Res. 1994 Mar. 11; 22(5):780-6. [0201]Zhang W, Mukhopadhyay S, Pletnev S V, Baker T S, Kuhn R J, Rossmann M G "Placement of the structural proteins in Sindbis virus." J. Virol. 2002 November; 76(22): 11645-58.
[0202]The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
[0203]It is further to be understood that all values are approximate, and are provided for description.
[0204]Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.
APPENDIX A
TABLE-US-00004 [0205]TABLE I Primers used for Sindbis cDNA cloning. cDNA PRIMER SEQUENCE (5'→3') SV NT bp cDNA- ATTGACGGCGTAGTACAC 1- cDNA-1 1F (SEQ ID NO: 7) 20 2276 bp cDNA- GTAACAAGATCTCGTGCCGTGACA 2299- 2276 1R (Bgl II) 2276 bp (SEQ ID NO: 8) cDNA- GGCACGAGATCTTGTTACCAGC 2281- cDNA-2 2F (Bgl II) 2303 (SEQ ID NO: 9) cDNA- CTTTCTTTCCTAGGCACACAGTCATTCTT 4265- 2012 2R (Avr II) 4293 bp (SEQ ID NO: 10) cDNA- GACTGTGTGCCTAGGAAAGAAAGTG 4271- cDNA- 3F (Avr II) 4295 3A (SEQ ID NO: 11) SV- CACACCCAGGTCCTCCAAGATC 6932- 2682 6932R (SEQ ID NO: 12) 6953 bp SV- GCATCATTCGACAAAAGCCAAG 6882- cDNA- 6882F (SEQ ID NO: 13) 6903 3B SV-C3R CTCTTCTAGAGGTGGTGGTGTTGTAGTATT 7626- 774 (XbaI) 7656 bp (SEQ ID NO: 14) cDNA- GGATCCCCTGAAAAGGCTGTTTAAG 7334- cDNA-4 4F (BamHI) 7359 (SEQ ID NO: 15) cDNA- TCATGTCTGATCAAGTCCGGTGA 9370- 2014 4R (BclI) 9348 bp (SEQ ID NO: 16) cDNA- GGACTTGATCAGACATGACGACCA 9353- CDNA-5 5F (BclI) 9376 (SEQ ID NO: 17) cDNA- TTTTTGAAATGTTAAAAACAAAATTTTGTTG 11678- 2350 5R (SEQ ID NO: 18) 11703 bp The restriction endonuclease recognition sites are underlined. Sindbis virus nucleotide numbers follow Strauss et al (1984) sequence (Accession# NC_001547.1)
TABLE-US-00005 TABLE II Primers used to generate vector polylinkers. PRIMER (RE) SEQUENCE 5'→3' Poly1- CCCACATGTGGGAGGCTAGAGTACTTAATACGACTCACTATAGGATTGACGGCGTAGTACACACTAT- TGAATCAAACAGCCGACC T7(+) (SEQ ID NO: 19) (AflIII) Poly1- CCCACATGTGGGAGGCTAGAGTACATTTAGGTGACACTATAGAAATTGACGGCGTAGTACACACTAT- TGAATCAAACAGCCGACC SP6(+) (SEQ ID NO: 20) (AflIII) Poly- GGCGCGCCTCTAGACTAGCCTAGGTATGGAAGATCTTCCGCGGATCCGCCTAGTGCAATTGGTCGGCT- GTTTGATTCAAT 2(-) (SEQ ID NO: 21) (XbaI) Poly- AGGCTAGTCTAGAGGCGCGCCGATCTCACGTGAGCATGCGTTTAAACTGGGCCCAATGTTAACATTTC- AAAAAAAAAAAAAAAAA 3(+) (SEQ ID NO: 22) XbaI) Poly- GGTGATGACGTCCTCGAGGCGGCCGCTTAATTAATTTAAATTTTTTTTTTTTTTTTTTTTTTTTTTTT- TTTTTTTTTTGAAATGT 4(-) (SEQ ID NO: 23) (AatII) PCRPoly- ATATATATCCCACATGT 1F (SEQ ID NO: 24) (AflIII) PCRPoly- GCGCGCCTCTAGA 2R (SEQ ID NO: 25) PCRPoly- AGGCTAGTCTAGAGGC 3F (SEQ ID NO: 26) PCRPoly- GGTGATGACGTCCT 4R (SEQ ID NO: 27) (AatII) Restriction endonuclease recognition sites (RE) are underlined. Overlapping sequences are shown in bold.
TABLE-US-00006 TABLE III PRIMERS USED IN SITE-DIRECTED MUTAGENESIS OF E2 GENE. PRIMER SEQUENCE 5'→3' RE E2-I3- GCAAAAGAAGCGTCATCGATGACTTTACCCTGACCAGC Cla F (SEQ ID NO: 28) I E2-I3- GCTGGTCAGGGTAAAGTCATCGATGACGCTTCTTTTGC Cla R (SEQ ID NO: 29) I E2- CTACATGTCGCTTAAGCAGGATCACACCGTTAAAG Afl K70-F (SEQ ID NO: 30) II E2- CTTTAACGGTGTGATCCTGCTTAAGCGACATGTAG Afl K70-R (SEQ ID NO: 31) II E2- CGGGCCCGCACGCTTATACATCCTACCTGGAAGAATCATC Sma E181-F (SEQ ID NO: 32) I E2- GATGATTCTTCCAGGTAGGATGTATAAGCGTGCGGGCCCG Sma E181-R (SEQ ID NO: 33) I E2- GACTTGATCCGACATGACGACCACACGGTCCAAGGG Mme V251-F (SEQ ID NO: 34) I E2- CCCTTGGACCGTGTGGTCGTCATGTCGGATCAAGTC Mme V251-R (SEQ ID NO: 35) I Nucleotides changed are underlined and the new restriction sites generated are indicated (RE)
TABLE-US-00007 TABLE IV Nucleotide differences between Ar-339, JT vectors and Sindbis virus Strauss sequence (Strauss et al. 1984) Nucleotide numbers follow the Strauss et al sequence. (Accession# NC_001547.1
TABLE-US-00008 TABLE V Nucleotide differences between JT vectors and Strauss sequence Strauss et al 1984 sequence (Accessio# NC_001547.1)
TABLE-US-00009 TABLE VI Summary of amino acid differences between JT and Ar-339 vectors
TABLE-US-00010 TABLE VII TITER OF CHIMERIC VIRAL VECTORS. BHK-21 cells ES-2/Fluc cells Mosec cells VIRAL VECTOR (TU/mL) (TU/mL) (TU/mL) JT-BB/SP6-ARepLacZ 3 × 106 3 × 104 3 × 104 SP6-AH/JT-RepLacZ 3 × 104 3 × 103 3 × 103 SP6-AH/SP6-ARepLacZ 3 × 104 3 × 103 3 × 103 JT-BB/JT-RepLacZ 3 × 106 3 × 104 3 × 104 Vectors were titered in BHK-21, ES-2/Fluc and Mosec cell lines. TU transducing units
TABLE-US-00011 TABLE VIII TITERS OF E2 MUTANT VECTORS. BHK-21 ES-2/Fluc Mosec VIRAL VECTOR (TU/mL) (TU/mL) (TU/mL) A (JT-BB/SP6-RFluc) 107 105 105 C (SP6-H/SP6-RFluc) 105 103 104 Mut-1 (SP6H-K70/SP6-RFluc) 104 103 105 Mut-2 (SP6H-K70-V251/SP6-RFluc) 105 104 102 Mut-4 (SP6H-I3-K70-E181-V251/SP6-RFluc) 106 104 105 Vectors were titered in BKH-21, ES-2/Fluc and Mosec cell lines. TU transducing units
Sequence CWU
1
40117DNAartificialprimer 1ccccaatgat ccgacca
17227DNAartificialprimer 2aaaacaaatt ttgttgatta
ataaaag 27319DNAartificialprimer
3attgacggcg tagtacaca
19420DNAartificialprimer 4gtatcaagta ggatccggag
20517DNAartificialprimer 5ccccaatgat ccgacca
17628DNAartificialprimer
6aaaacaaaat tttgttgatt aataaaag
28718DNAartificialprimer 7attgacggcg tagtacac
18824DNAartificialprimer 8gtaacaagat ctcgtgccgt
gaca 24922DNAartificialprimer
9ggcacgagat cttgttacca gc
221029DNAartificialprimer 10ctttctttcc taggcacaca gtcattctt
291125DNAartificialprimer 11gactgtgtgc ctaggaaaga
aagtg 251222DNAartificialprimer
12cacacccagg tcctccaaga tc
221322DNAartificialprimer 13gcatcattcg acaaaagcca ag
221430DNAartificialprimer 14ctcttctaga ggtggtggtg
ttgtagtatt 301525DNAartificialprimer
15ggatcccctg aaaaggctgt ttaag
251623DNAartificialprimer 16tcatgtctga tcaagtccgg tga
231724DNAartificialprimer 17ggacttgatc agacatgacg
acca 241831DNAartificialprimer
18tttttgaaat gttaaaaaca aaattttgtt g
311985DNAartificialprimer 19cccacatgtg ggaggctaga gtacttaata cgactcacta
taggattgac ggcgtagtac 60acactattga atcaaacagc cgacc
852085DNAartificialprimer 20cccacatgtg ggaggctaga
gtacatttag gtgacactat agaaattgac ggcgtagtac 60acactattga atcaaacagc
cgacc 852180DNAartificialprimer
21ggcgcgcctc tagactagcc taggtatgga agatcttccg cggatccgcc tagtgcaatt
60ggtcggctgt ttgattcaat
802285DNAartificialprimer 22aggctagtct agaggcgcgc cgatctcacg tgagcatgcg
tttaaactgg gcccaatgtt 60aacatttcaa aaaaaaaaaa aaaaa
852385DNAartificialprimer 23ggtgatgacg tcctcgaggc
ggccgcttaa ttaatttaaa tttttttttt tttttttttt 60tttttttttt ttttttttga
aatgt 852417DNAartificialprimer
24atatatatcc cacatgt
172513DNAartificialprimer 25gcgcgcctct aga
132616DNAartificialprimer 26aggctagtct agaggc
162714DNAartificialprimer
27ggtgatgacg tcct
142838DNAartificialprimer 28gcaaaagaag cgtcatcgat gactttaccc tgaccagc
382938DNAartificialprimer 29gctggtcagg gtaaagtcat
cgatgacgct tcttttgc 383035DNAartificialprimer
30ctacatgtcg cttaagcagg atcacaccgt taaag
353135DNAartificialprimer 31ctttaacggt gtgatcctgc ttaagcgaca tgtag
353240DNAartificialprimer 32cgggcccgca cgcttataca
tcctacctgg aagaatcatc 403340DNAartificialprimer
33gatgattctt ccaggtagga tgtataagcg tgcgggcccg
403436DNAartificialprimer 34gacttgatcc gacatgacga ccacacggtc caaggg
363536DNAartificialprimer 35cccttggacc gtgtggtcgt
catgtcggat caagtc 36369928DNASindbis virus
36attgacggcg tagtacacac tattgaatca aacagccgac caattgcact accatcacaa
60tggagaagcc agtagtaaac gtagacgtag acccccagag tccgtttgtc gtgcaactgc
120aaaaaagctt cccgcaattt gaggtagtag cacagcaggt cactccaaat gaccatgcta
180atgccagagc attttcgcat ctggccagta aactaatcga gctggaggtt cctaccacag
240cgacgatctt ggacataggc agcgcaccgg ctcgtagaat gttttccgag caccagtatc
300attgtgtctg ccccatgcgt agtccagaag acccggaccg catgatgaaa tatgccagta
360aactggcgga aaaagcgtgc aagattacaa acaagaactt gcatgagaag attaaggatc
420tccggaccgt acttgatacg ccggatgctg aaacaccatc gctctgcttt cacaacgatg
480ttacctgcaa catgcgtgcc gaatattccg tcatgcagga cgtgtatatc aacgctcccg
540gaactatcta tcatcaggct atgaaaggcg tgcggaccct gtactggatt ggcttcgaca
600ccacccagtt catgttctcg gctatggcag gttcgtaccc tgcgtacaac accaactggg
660ccgacgagaa agtccttgaa gcgcgtaaca tcggactttg cagcacaaag ctgagtgaag
720gtaggacagg aaaattgtcg ataatgagga agaaggagtt gaagcccggg tcgcgggttt
780atttctccgt aggatcgaca ctttatccag aacacagagc cagcttgcag agctggcatc
840ttccatcggt gttccacttg aatggaaagc agtcgtacac ttgccgctgt gatacagtgg
900tgagttgcga aggctacgta gtgaagaaaa tcaccatcag tcccgggatc acgggagaaa
960ccgtgggata cgcggttaca cacaatagcg agggcttctt gctatgcaaa gttactgaca
1020cagtaaaagg agaacgggta tcgttccctg tgtgcacgta catcccggcc accatatgcg
1080atcagatgac tggtataatg gccacggata tatcacctga cgatgcacaa aaacttctgg
1140ttgggctcaa ccagcgaatt gtcattaacg gtaggactaa caggaacacc aacaccatgc
1200aaaattacct tctgccgatc atagcacaag ggttcagcaa atgggctaag gagcgcaagg
1260atgatcttga taacgagaaa atgctgggta ctagagaacg caagcttacg tatggctgct
1320tgtgggcgtt tcgcactaag aaagtacatt cgttttatcg cccacctgga acgcagacca
1380tcgtaaaagt cccagcctct tttagcgctt ttcccatgtc gtccgtatgg acgacctctt
1440tgcccatgtc gctgaggcag aaattgaaac tggcattgca accaaagaag gaggaaaaac
1500tgctgcaggt ctcggaggaa ttagtcatgg aggccaaggc tgcttttgag gatgctcagg
1560aggaagccag agcggagaag ctccgagaag cacttccacc attagtggca gacaaaggca
1620tcgaggcagc cgcagaagtt gtctgcgaag tggaggggct ccaggcggac atcggagcag
1680cattagttga aaccccgcgc ggtcacgtaa ggataatacc tcaagcaaat gaccgtatga
1740tcggacagta tatcgttgtc tcgccaaact ctgtgctgaa gaatgccaaa ctcgcaccag
1800cgcacccgct agcagatcag gttaagatca taacacactc cggaagatca ggaaggtacg
1860cggtcgaacc atacgacgct aaagtactga tgccagcagg aggtgccgta ccatggccag
1920aattcctagc actgagtgag agcgccacgt tagtgtacaa cgaaagagag tttgtgaacc
1980gcaaactata ccacattgcc atgcatggcc ccgccaagaa tacagaagag gagcagtaca
2040aggttacaaa ggcagagctt gcagaaacag agtacgtgtt tgacgtggac aagaagcgtt
2100gcgttaagaa ggaagaagcc tcaggtctgg tcctctcggg agaactgacc aaccctccct
2160atcatgagct agctctggag ggactgaaga cccgacctgc ggtcccgtac aaggtcgaaa
2220caataggagt gataggcaca ccggggtcgg gcaagtcagc tattatcaag tcaactgtca
2280cggcacgaga tcttgttacc agcggaaaga aagaaaattg tcgcgaaatt gaggccgacg
2340tgctaagact gaggggtatg cagattacgt cgaagacagt agattcggtt atgctcaacg
2400gatgccacaa agccgtagaa gtgctgtacg ttgacgaagc gttcgcgtgc cacgcaggag
2460cactacttgc cttgattgct atcgtcaggc cccgcaagaa ggtagtacta tgcggagacc
2520ccatgcaatg cggattcttc aacatgatgc aactaaaggt acatttcaat caccctgaaa
2580aagacatatg caccaagaca ttctacaagt atatctcccg gcgttgcaca cagccagtta
2640cagctattgt atcgacactg cattacgatg gaaagatgaa aaccacgaac ccgtgcaaga
2700agaacattga aatcgatatt acaggggcca caaagccgaa gccaggggat atcatcctga
2760catgtttccg cgggtgggtt aagcaattgc aaatcgacta tcccggacat gaagtaatga
2820cagccgcggc ctcacaaggg ctaaccagaa aaggagtgta tgccgtccgg caaaaagtca
2880atgaaaaccc actgtacgcg atcacatcag agcatgtgaa cgtgttgctc acccgcactg
2940aggacaggct agtgtggaaa accttgcagg gcgacccatg gattaagcag ctcactaaca
3000tacctaaagg aaactttcag gctactatag aggactggga agctgaacac aagggaataa
3060ttgctgcaat aaacagcccc actccccgtg ccaatccgtt cagctgcaag accaacgttt
3120gctgggcgaa agcattggaa ccgatactag ccacggccgg tatcgtactt accggttgcc
3180agtggagcga actgttccca cagtttgcgg atgacaaacc acattcggcc atttacgcct
3240tagacgtaat ttgcattaag tttttcggca tggacttgac aagcggactg ttttctaaac
3300agagcatccc actaacgtac catcccgccg attcagcgag gccggtagct cattgggaca
3360acagcccagg aacccgcaag tatgggtacg atcacgccat tgccgccgaa ctctcccgta
3420gatttccggt gttccagcta gctgggaagg gcacacaact tgatttgcag acggggagaa
3480ccagagttat ctctgcacag cataacctgg tcccggtgaa ccgcaatctt cctcacgcct
3540tagtccccga gtacaaggag aagcaacccg gcccggtcga aaaattcttg aaccagttca
3600aacaccactc agtacttgtg gtatcagagg aaaaaattga agctccccgt aagagaatcg
3660aatggatcgc cccgattggc atagccggtg cagataagaa ctacaacctg gctttcgggt
3720ttccgccgca ggcacggtac gacctggtgt tcatcaacat tggaactaaa tacagaaacc
3780accactttca gcagtgcgaa gaccatgcgg cgaccttaaa aaccctttcg cgttcggccc
3840tgaattgcct taacccagga ggcaccctcg tggtgaagtc ctatggctac gccgaccgca
3900acagtgagga cgtagtcacc gctcttgcca gaaagtttgt cagggtgtct gcagcgagac
3960cagattgtgt ctcaagcaat acagaaatgt acctgatttt ccgacaacta gacaacagcc
4020gtacacggca attcaccccg caccatctga attgcgtgat ttcgtccgtg tatgagggta
4080caagagatgg agttggagcc gcgccgtcat accgcaccaa aagggagaat attgctgact
4140gtcaagagga agcagttgtc aacgcagcca atccgctggg tagaccaggc gaaggagtct
4200gccgtgccat ctataaacgt tggccgacca gttttaccga ttcagccacg gagacaggca
4260ccgcaagaat gactgtgtgc ctaggaaaga aagtgatcca cgcggtcggc cctgatttcc
4320ggaagcaccc agaagcagaa gccttgaaat tgctacaaaa cgcctaccat gcagtggcag
4380acttagtaaa tgaacataac atcaagtctg tcgccattcc actgctatct acaggcattt
4440acgcagccgg aaaagaccgc cttgaagtat cacttaactg cttgacaacc gcgctagaca
4500gaactgacgc ggacgtaacc atctattgcc tggataagaa gtggaaggaa agaatcgacg
4560cggcactcca acttaaggag tctgtaacag agctgaagga tgaagatatg gagatcgacg
4620atgagttagt atggatccat ccagacagtt gcttgaaggg aagaaaggga ttcagtacta
4680caaaaggaaa attgtattcg tacttcgaag gcaccaaatt ccatcaagca gcaaaagaca
4740tggcggagat aaaggtcctg ttccctaatg accaggaaag taatgaacaa ctgtgtgcct
4800acatattggg tgagaccatg gaagcaatcc gcgaaaagtg cccggtcgac cataacccgt
4860cgtctagccc gcccaaaacg ttgccgtgcc tttgcatgta tgccatgacg ccagaaaggg
4920tccacagact tagaagcaat aacgtcaaag aagttacagt atgctcctcc accccccttc
4980ctaagcacaa aattaagaat gttcagaagg ttcagtgcac gaaagtagtc ctgtttaatc
5040cgcacactcc cgcattcgtt cccgcccgta agtacataga agtgccagaa cagcctaccg
5100ctcctcctgc acaggccgag gaggcccccg aagttgtagc gacaccgtca ccatctacag
5160ctgataacac ctcgcttgat gtcacagaca tctcactgga tatggatgac agtagcgaag
5220gctcactttt ttcgagcttt agcggatcgg acaactctat tactagtatg gacagttggt
5280cgtcaggacc tagttcacta gagatagtag accgaaggca ggtggtggtg gctgacgttc
5340atgccgtcca agagcctgcc cctattccac cgccaaggct aaagaagatg gcccgcctgg
5400cagcggcaag aaaagagccc actccaccgg caagcaatag ctctgagtcc ctccacctct
5460cttttggtgg ggtatccatg tccctcggat caattttcga cggagagacg gcccgccagg
5520cagcggtaca acccctggca acaggcccca cggatgtgcc tatgtctttc ggatcgtttt
5580ccgacggaga gattgatgag ctgagccgca gagtaactga gtccgaaccc gtcctgtttg
5640gatcatttga accgggcgaa gtgaactcaa ttatatcgtc ccgatcagcc gtatcttttc
5700cactacgcaa gcagagacgt agacgcagga gcaggaggac tgaatactga ctaaccgggg
5760taggtgggta catattttcg acggacacag gccctgggca cttgcaaaag aagtccgttc
5820tgcagaacca gcttacagaa ccgaccttgg agcgcaatgt cctggaaaga attcatgccc
5880cggtgctcga cacgtcgaaa gaggaacaac tcaaactcag gtaccagatg atgcccaccg
5940aagccaacaa aagtaggtac cagtctcgta aagtagaaaa tcagaaagcc ataaccactg
6000agcgactact gtcaggacta cgactgtata actctgccac agatcagcca gaatgctata
6060agatcaccta tccgaaacca ttgtactcca gtagcgtacc ggcgaactac tccgatccac
6120agttcgctgt agctgtctgt aacaactatc tgcatgagaa ctatccgaca gtagcatctt
6180atcagattac tgacgagtac gatgcttact tggatatggt agacgggaca gtcgcctgcc
6240tggatactgc aaccttctgc cccgctaagc ttagaagtta cccgaaaaaa catgagtata
6300gagccccgaa tatccgcagt gcggttccat cagcgatgca gaacacgcta caaaatgtgc
6360tcattgccgc aactaaaaga aattgcaacg tcacgcagat gcgtgaactg ccaacactgg
6420actcagcgac attcaatgtc gaatgctttc gaaaatatgc atgtaatgac gagtattggg
6480aggagttcgc tcggaagcca attaggatta ccactgagtt tgtcaccgca tatgtagcta
6540gactgaaagg ccctaaggcc gccgcactat ttgcaaagac gtataatttg gtcccattgc
6600aagaagtgcc tatggataga ttcgtcatgg acatgaaaag agacgtgaaa gttacaccag
6660gcacgaaaca cacagaagaa agaccgaaag tacaagtgat acaagccgca gaacccctgg
6720cgactgctta cttatgcggg attcaccggg aattagtgcg taggcttacg gccgtcttgc
6780ttccaaacat tcacacgctt tttgacatgt cggcggagga ttttgatgca atcatagcag
6840aacacttcaa gcaaggcgac ccggtactgg agacggatat cgcatcattc gacaaaagcc
6900aagacgacgc tatggcgtta accggtctga tgatcttgga ggacctgggt gtggatcaac
6960cactactcga cttgatcgag tgcgcctttg gagaaatatc atccacccat ctacctacgg
7020gtactcgttt taaattcggg gcgatgatga aatccggaat gttcctcaca ctttttgtca
7080acacagtttt gaatgtcgtt atcgccagca gagtactaga agagcggctt aaaacgtcca
7140gatgtgcagc gttcattggc gacgacaaca tcatacatgg agtagtatct gacaaagaaa
7200tggctgagag gtgcgccacc tggctcaaca tggaggttaa gatcatcgac gcagtcatcg
7260gtgagagacc accttacttc tgcggcggat ttatcttgca agattcggtt acttccacag
7320cgtgccgcgt ggcggacccc ctgaaaaggc tgtttaagtt gggtaaaccg ctcccagccg
7380acgacgagca agacgaagac agaagacgcg ctctgctaga tgaaacaaag gcgtggttta
7440gagtaggtat aacaggcact ttagcagtgg ccgtgacgac ccggtatgag gtagacaata
7500ttacacctgt cctactggca ttgagaactt ttgcccagag caaaagagca ttccaagcca
7560tcagagggga aataaagcat ctctacggtg gtcctaaata gtcagcatag tacatttcat
7620ctgactaata ctacaacacc accacctcta gaggcgcgcc gatctcacgt gagcatgcgt
7680ttaaactggg cccaatgttc cccaatgatc cgaccagcaa aactcgatgt acttccgagg
7740aactgatgtg cataatgcat caggctggta cattagatcc ccgcttaccg cgggcaatat
7800agcaacacta aaaactcgat gtacttccga ggaagcgcag tgcataatgc tgcgcagtgt
7860tgccacataa ccactatatt aaccatttat ctagcggacg ccaaaaactc aatgtatttc
7920tgaggaagcg tggtgcataa tgccacgcag cgtctgcata acttttatta tttcttttat
7980taatcaacaa aattttgttt ttaacatttc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
8040aaaaaaaatt taaattaatt aagcggccgc ctcgaggacg tcaggtggca cttttcgggg
8100aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
8160catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
8220tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
8280tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
8340ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
8400ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
8460cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
8520ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
8580tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
8640gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
8700ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
8760aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
8820acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
8880tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
8940cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
9000gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
9060taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
9120tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
9180cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
9240ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
9300accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
9360cttcagcaga gcgcagatac caaatactgg tcttctagtg tagccgtagt taggccacca
9420cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
9480tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
9540taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
9600gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
9660agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
9720ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
9780acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
9840caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtgggaggc
9900tagagtacat ttaggtgaca ctatagaa
9928376711DNASindbis virus 37attgacggcg tagtacacac tattgaatca aacagccgac
caattgcact accatcacaa 60tggagaagcc agtagtaaac gtagacgtag acccccagag
tccgtttgtc gtgcaactgc 120aaaaaagctt cccgcaattt gaggtagtag cacagcaggt
cactccaaat gaccatgcta 180atgccagagc attttcgcat ctggccagta aactaatcga
gctggaggtt cctaccacag 240cgacgatctt ggacataggc agcgcaccgg ctcgtagaat
gttttccgag caccagtatc 300attgtgtctg ccccatgcgt agtccagaag acccggaccg
catgatgaaa tatgccagta 360aactggcgga aaaagcgtgc aagattacaa acaagaactt
gcatgagaag attaaggatc 420tccggatccc ctgaaaaggc tgtttaagtt gggtaaaccg
ctcccagccg acgacgagca 480agacgaagac agaagacgcg ctctgctaga tgaaacaaag
gcgtggttta gagtaggtat 540aacaggcact ttagcagtgg ccgtgacgac ccggtatgag
gtagacaata ttacacctgt 600cctactggca ttgagaactt ttgcccagag caaaagagca
ttccaagcca tcagagggga 660aataaagcat ctctacggtg gtcctaaata gtcagcatag
tacatttcat ctgactaata 720ctacaacacc accaccatga atagaggatt ctttaacatg
ctcggccgcc gccccttccc 780ggcccccact gccatgtgga ggccgcggag aaggaggcag
gcggccccga tgcctgcccg 840caacgggctg gcttctcaaa tccagcaact gaccacagcc
gtcagtgccc tagtcattgg 900acaggcaact agacctcaac ccccacgtcc acgccagcca
ccgcgccaga agaagcaggc 960gcccaagcaa ccaccgaagc cgaagaaacc aaaaacgcag
gagaagaaga agaagcaacc 1020tgcaaaaccc aaacccggaa agagacagcg catggcactt
aagttggagg ccgacagatt 1080gttcgacgtc aagaacgagg acggagatgt catcgggcac
gcactggcca tggaaggaaa 1140ggtaatgaaa cctctgcacg tgaaaggaac catcgaccac
cctgtgctat caaagctcaa 1200atttaccaag tcgtcagcat acgacatgga gttcgcacag
ttgccagtca acatgagaag 1260tgaggcattc acctacacca gtgaacaccc cgaaggattc
tataactggc accacggagc 1320ggtgcagtat agtggaggta gatttaccat ccctcgcgga
gtaggaggca gaggagacag 1380cggtcgtccg atcatggata actccggtcg ggttgtcgcg
atagtcctcg gtggagctga 1440tgaaggaaca cgaactgccc tttcggtcgt cacctggaat
agtaaaggga agacaattaa 1500gacgaccccg gaagggacag aagagtggtc cgcagcacca
ctggtcacgg caatgtgttt 1560gctcggaaat gtgagcttcc catgcgaccg cccgcccaca
tgctataccc gcgaaccttc 1620cagagccctc gacatccttg aagagaacgt gaaccatgag
gcctacgata ccctgctcaa 1680tgccatattg cggtgcggat cgtctggcag aagcaaaaga
agcgtcactg acgactttac 1740cctgaccagc ccctacttgg gcacatgctc gtactgccac
catactgaac cgtgcttcag 1800ccctgttaag atcgagcagg tctgggacga agcggacgat
aacaccatac gcatacagac 1860ttccgcccag tttggatacg accaaagcgg agcagcaagc
gcaaacaagt accgctacat 1920gtcgcttgag caggatcaca ccgttaaaga aggcaccatg
gatgacatca agattagcac 1980ctcaggaccg tgtagaaggc ttagctacaa aggatacttt
ctcctcgcaa aatgccctcc 2040aggggacagc gtaacggtta gcatagtgag tagcaactca
gcaacgtcat gtacactggc 2100ccgcaagata aaaccaaaat tcgtgggacg ggaaaaatat
gatctacctc ccgttcacgg 2160taaaaaaatt ccttgcacag tgtacgaccg tctgaaagaa
acaactgcag gctacatcac 2220tatgcacagg ccgggaccgc acgcttatac atcctacctg
aaagaatcat cagggaaagt 2280ttacgcaaag ccgccatctg ggaagaacat tacgtatgag
tgcaagtgcg gcgactacaa 2340gaccggaacc gtttcgaccc gcaccgaaat cactggttgc
accgccatca agcagtgcgt 2400cgcctataag agcgaccaaa cgaagtgggt cttcaactca
ccggacttga tcagacatga 2460cgaccacacg gcccaaggga aattgcattt gcctttcaag
ttgatcccga gtacctgcat 2520ggtccctgtt gcccacgcgc cgaatgtaat acatggcttt
aaacacatca gcctccaatt 2580agatacagac cacttgacat tgctcaccac caggagacta
ggggcaaacc cggaaccaac 2640cactgaatgg atcgtcggaa agacggtcag aaacttcacc
gtcgaccgag atggcctgga 2700atacatatgg ggaaatcatg agccagtgag ggtctatgcc
caagagtcag caccaggaga 2760ccctcacgga tggccacacg aaatagtaca gcattactac
catcgccatc ctgtgtacac 2820catcttagcc gtcgcatcag ctaccgtggc gatgatgatt
ggcgtaactg ttgcagtgtt 2880atgtgcctgt aaagcgcgcc gtgagtgcct gacgccatac
gccctggccc caaacgccgt 2940aatcccaact tcgctggcac tcttgtgctg cgttaggtcg
gccaatgctg aaacgttcac 3000cgagaccatg agttacttgt ggtcgaacag tcagccgttc
ttctgggtcc agttgtgcat 3060acctttggcc gctttcatcg ttctaatgcg ctgctgctcc
tgctgcctgc cttttttagt 3120ggttgccggc gcctacctgg cgaaggtaga cgcctacgaa
catgcgacca ctgttccaaa 3180tgtgccacag ataccgtata aggcacttgt tgaaagggca
gggtatgccc cgctcaattt 3240ggagatcact gtcatgtcct cggaggtttt gccttccacc
aaccaagagt acattacctg 3300caaattcacc actgtggtcc cctccccaaa aatcaaatgc
tgcggctcct tggaatgtca 3360gccggccgtt catgcagact atacctgcaa ggtcttcgga
ggggtctacc cctttatgtg 3420gggaggagcg caatgttttt gcgacagtga gaacagccag
atgagtgagg cgtacgtcga 3480actgtcagca gattgcgcgt ctgaccacgc gcaggcgatt
aaggtgcaca ctgccgcgat 3540gaaagtagga ctgcgtatag tgtacgggaa cactaccagt
ttcctagatg tgtacgtgaa 3600cggagtcaca ccaggaacgt ctaaagactt gaaagtcata
gctggaccaa tttcagcatc 3660gtttacgcca ttcgatcata aggtcgttat ccatcgcggc
ctggtgtaca actatgactt 3720cccggaatat ggagcgatga aaccaggagc gtttggagac
attcaagcta cctccttgac 3780tagcaaggat ctcatcgcca gcacagacat taggctactc
aagccttccg ccaagaacgt 3840gcatgtcccg tacacgcagg ccgcatcagg atttgagatg
tggaaaaaca actcaggccg 3900cccactgcag gaaaccgcac ctttcgggtg taagattgca
gtaaatccgc tccgagcggt 3960ggactgttca tacgggaaca ttcccatttc tattgacatc
ccgaacgctg cctttatcag 4020gacatcagat gcaccactgg tctcaacagt caaatgtgaa
gtcagtgagt gcacttattc 4080agcagacttc ggcgggatgg ccaccctgca gtatgtatcc
gaccgcgaag gtcaatgccc 4140cgtacattcg cattcgagca cagcaactct ccaagagtcg
acagtacatg tcctggagaa 4200aggagcggtg acagtacact ttagcaccgc gagtccacag
gcgaacttta tcgtatcgct 4260gtgtgggaag aagacaacat gcaatgcaga atgtaaacca
ccagctgacc atatcgtgag 4320caccccgcac aaaaatgacc aagaatttca agccgccatc
tcaaaaacat catggagttg 4380gctgtttgcc cttttcggcg gcgcctcgtc gctattaatt
ataggactta tgatttttgc 4440ttgcagcatg atgctgacta gcacacgaag atgaccgcta
cgccccaatg atccgaccag 4500caaaactcga tgtacttccg aggaactgat gtgcataatg
catcaggctg gtacattaga 4560tccccgctta ccgcgggcaa tatagcaaca ctaaaaactc
gatgtacttc cgaggaagcg 4620cagtgcataa tgctgcgcag tgttgccaca taaccactat
attaaccatt tatctagcgg 4680acgccaaaaa ctcaatgtat ttctgaggaa gcgtggtgca
taatgccacg cagcgtctgc 4740ataactttta ttatttcttt tattaatcaa caaaattttg
tttttaacat ttcaaaaaaa 4800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa atttaaatta
attaagcggc cgcctcgagg 4860acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
ctatttgttt atttttctaa 4920atacattcaa atatgtatcc gctcatgaga caataaccct
gataaatgct tcaataatat 4980tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
cccttattcc cttttttgcg 5040gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
tgaaagtaaa agatgctgaa 5100gatcagttgg gtgcacgagt gggttacatc gaactggatc
tcaacagcgg taagatcctt 5160gagagttttc gccccgaaga acgttttcca atgatgagca
cttttaaagt tctgctatgt 5220ggcgcggtat tatcccgtat tgacgccggg caagagcaac
tcggtcgccg catacactat 5280tctcagaatg acttggttga gtactcacca gtcacagaaa
agcatcttac ggatggcatg 5340acagtaagag aattatgcag tgctgccata accatgagtg
ataacactgc ggccaactta 5400cttctgacaa cgatcggagg accgaaggag ctaaccgctt
ttttgcacaa catgggggat 5460catgtaactc gccttgatcg ttgggaaccg gagctgaatg
aagccatacc aaacgacgag 5520cgtgacacca cgatgcctgt agcaatggca acaacgttgc
gcaaactatt aactggcgaa 5580ctacttactc tagcttcccg gcaacaatta atagactgga
tggaggcgga taaagttgca 5640ggaccacttc tgcgctcggc ccttccggct ggctggttta
ttgctgataa atctggagcc 5700ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
cagatggtaa gccctcccgt 5760atcgtagtta tctacacgac ggggagtcag gcaactatgg
atgaacgaaa tagacagatc 5820gctgagatag gtgcctcact gattaagcat tggtaactgt
cagaccaagt ttactcatat 5880atactttaga ttgatttaaa acttcatttt taatttaaaa
ggatctaggt gaagatcctt 5940tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
cgttccactg agcgtcagac 6000cccgtagaaa agatcaaagg atcttcttga gatccttttt
ttctgcgcgt aatctgctgc 6060ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
tgccggatca agagctacca 6120actctttttc cgaaggtaac tggcttcagc agagcgcaga
taccaaatac tgtccttcta 6180gtgtagccgt agttaggcca ccacttcaag aactctgtag
caccgcctac atacctcgct 6240ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
agtcgtgtct taccgggttg 6300gactcaagac gatagttacc ggataaggcg cagcggtcgg
gctgaacggg gggttcgtgc 6360acacagccca gcttggagcg aacgacctac accgaactga
gatacctaca gcgtgagcta 6420tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
ggtatccggt aagcggcagg 6480gtcggaacag gagagcgcac gagggagctt ccagggggaa
acgcctggta tctttatagt 6540cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
tgtgatgctc gtcagggggg 6600cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
ggttcctggc cttttgctgg 6660ccttttgctc acatgtggga ggctagagta catttaggtg
acactataga a 6711386711DNASindbis virus 38attgacggcg
tagtacacac tattgaatca aacagccgac caattgcact accatcacaa 60tggagaagcc
agtagtaaac gtagacgtag acccccagag tccgtttgtc gtgcaactgc 120aaaaaagctt
cccgcaattt gaggtagtag cacagcaggt cactccaaat gaccatgcta 180atgccagagc
attttcgcat ctggccagta aactaatcga gctggaggtt cctaccacag 240cgacgatctt
ggacataggc agcgcaccgg ctcgtagaat gttttccgag caccagtatc 300attgtgtctg
ccccatgcgt agtccagaag acccggaccg catgatgaaa tatgccagta 360aactggcgga
aaaagcgtgc aagattacaa acaagaactt gcatgagaag attaaggatc 420tccggatccc
ctgaaaaggc tgtttaagtt gggtaaaccg ctcccagccg acgacgagca 480agacgaagac
agaagacgcg ctctgctaga tgaaacaaag gcgtggttta gagtaggtat 540aacaggcact
ttagcagtgg ccgtgacgac ccggtatgag gtagacaata ttacacctgt 600cctactggca
ttgagaactt ttgcccagag caaaagagca ttccaagcca tcagagggga 660aataaagcat
ctctacggtg gtcctaaata gtcagcatag tacatttcat ctgactaata 720ctacaacacc
accaccatga atagaggatt ctttaacatg ctcggccgcc gccccttccc 780ggcccccact
gccatgtgga ggccgcggag aaggaggcag gcggccccga tgcctgcccg 840caacgggctg
gcttctcaaa tccagcaact gaccacagcc gtcagtgccc tagtcattgg 900acaggcaact
agacctcaac ccccacgtcc acgccagcca ccgcgccaga agaagcaggc 960gcccaagcaa
ccaccgaagc cgaagaaacc aaaaacgcag gagaagaaga agaagcaacc 1020tgcaaaaccc
aaacccggaa agagacagcg catggcactt aagttggagg ccgacagatt 1080gttcgacgtc
aagaacgagg acggagatgt catcgggcac gcactggcca tggaaggaaa 1140ggtaatgaaa
cctctgcacg tgaaaggaac catcgaccac cctgtgctat caaagctcaa 1200atttaccaag
tcgtcagcat acgacatgga gttcgcacag ttgccagtca acatgagaag 1260tgaggcattc
acctacacca gtgaacaccc cgaaggattc tataactggc accacggagc 1320ggtgcagtat
agtggaggta gatttaccat ccctcgcgga gtaggaggca gaggagacag 1380cggtcgtccg
atcatggata actccggtcg ggttgtcgcg atagtcctcg gtggagctga 1440tgaaggaaca
cgaactgccc tttcggtcgt cacctggaat agtaaaggga agacaattaa 1500gacgaccccg
gaagggacag aagagtggtc cgcagcacca ctggtcacgg caatgtgttt 1560gctcggaaat
gtgagcttcc catgcgaccg cccgcccaca tgctataccc gcgaaccttc 1620cagagccctc
gacatccttg aagagaacgt gaaccatgag gcctacgata ccctgctcaa 1680tgccatattg
cggtgcggat cgtctggcag aagcaaaaga agcgtcactg acgactttac 1740cctgaccagc
ccctacttgg gcacatgctc gtactgccac catactgaac cgtgcttcag 1800ccctgttaag
atcgagcagg tctgggacga agcggacgat aacaccatac gcatacagac 1860ttccgcccag
tttggatacg accaaagcgg agcagcaagc gcaaacaagt accgctacat 1920gtcgcttaag
caggatcaca ccgttaaaga aggcaccatg gatgacatca agattagcac 1980ctcaggaccg
tgtagaaggc ttagctacaa aggatacttt ctcctcgcaa aatgccctcc 2040aggggacagc
gtaacggtta gcatagtgag tagcaactca gcaacgtcat gtacactggc 2100ccgcaagata
aaaccaaaat tcgtgggacg ggaaaaatat gatctacctc ccgttcacgg 2160taaaaaaatt
ccttgcacag tgtacgaccg tctgaaagaa acaactgcag gctacatcac 2220tatgcacagg
ccgggaccgc acgcttatac atcctacctg aaagaatcat cagggaaagt 2280ttacgcaaag
ccgccatctg ggaagaacat tacgtatgag tgcaagtgcg gcgactacaa 2340gaccggaacc
gtttcgaccc gcaccgaaat cactggttgc accgccatca agcagtgcgt 2400cgcctataag
agcgaccaaa cgaagtgggt cttcaactca ccggacttga tcagacatga 2460cgaccacacg
gcccaaggga aattgcattt gcctttcaag ttgatcccga gtacctgcat 2520ggtccctgtt
gcccacgcgc cgaatgtaat acatggcttt aaacacatca gcctccaatt 2580agatacagac
cacttgacat tgctcaccac caggagacta ggggcaaacc cggaaccaac 2640cactgaatgg
atcgtcggaa agacggtcag aaacttcacc gtcgaccgag atggcctgga 2700atacatatgg
ggaaatcatg agccagtgag ggtctatgcc caagagtcag caccaggaga 2760ccctcacgga
tggccacacg aaatagtaca gcattactac catcgccatc ctgtgtacac 2820catcttagcc
gtcgcatcag ctaccgtggc gatgatgatt ggcgtaactg ttgcagtgtt 2880atgtgcctgt
aaagcgcgcc gtgagtgcct gacgccatac gccctggccc caaacgccgt 2940aatcccaact
tcgctggcac tcttgtgctg cgttaggtcg gccaatgctg aaacgttcac 3000cgagaccatg
agttacttgt ggtcgaacag tcagccgttc ttctgggtcc agttgtgcat 3060acctttggcc
gctttcatcg ttctaatgcg ctgctgctcc tgctgcctgc cttttttagt 3120ggttgccggc
gcctacctgg cgaaggtaga cgcctacgaa catgcgacca ctgttccaaa 3180tgtgccacag
ataccgtata aggcacttgt tgaaagggca gggtatgccc cgctcaattt 3240ggagatcact
gtcatgtcct cggaggtttt gccttccacc aaccaagagt acattacctg 3300caaattcacc
actgtggtcc cctccccaaa aatcaaatgc tgcggctcct tggaatgtca 3360gccggccgtt
catgcagact atacctgcaa ggtcttcgga ggggtctacc cctttatgtg 3420gggaggagcg
caatgttttt gcgacagtga gaacagccag atgagtgagg cgtacgtcga 3480actgtcagca
gattgcgcgt ctgaccacgc gcaggcgatt aaggtgcaca ctgccgcgat 3540gaaagtagga
ctgcgtatag tgtacgggaa cactaccagt ttcctagatg tgtacgtgaa 3600cggagtcaca
ccaggaacgt ctaaagactt gaaagtcata gctggaccaa tttcagcatc 3660gtttacgcca
ttcgatcata aggtcgttat ccatcgcggc ctggtgtaca actatgactt 3720cccggaatat
ggagcgatga aaccaggagc gtttggagac attcaagcta cctccttgac 3780tagcaaggat
ctcatcgcca gcacagacat taggctactc aagccttccg ccaagaacgt 3840gcatgtcccg
tacacgcagg ccgcatcagg atttgagatg tggaaaaaca actcaggccg 3900cccactgcag
gaaaccgcac ctttcgggtg taagattgca gtaaatccgc tccgagcggt 3960ggactgttca
tacgggaaca ttcccatttc tattgacatc ccgaacgctg cctttatcag 4020gacatcagat
gcaccactgg tctcaacagt caaatgtgaa gtcagtgagt gcacttattc 4080agcagacttc
ggcgggatgg ccaccctgca gtatgtatcc gaccgcgaag gtcaatgccc 4140cgtacattcg
cattcgagca cagcaactct ccaagagtcg acagtacatg tcctggagaa 4200aggagcggtg
acagtacact ttagcaccgc gagtccacag gcgaacttta tcgtatcgct 4260gtgtgggaag
aagacaacat gcaatgcaga atgtaaacca ccagctgacc atatcgtgag 4320caccccgcac
aaaaatgacc aagaatttca agccgccatc tcaaaaacat catggagttg 4380gctgtttgcc
cttttcggcg gcgcctcgtc gctattaatt ataggactta tgatttttgc 4440ttgcagcatg
atgctgacta gcacacgaag atgaccgcta cgccccaatg atccgaccag 4500caaaactcga
tgtacttccg aggaactgat gtgcataatg catcaggctg gtacattaga 4560tccccgctta
ccgcgggcaa tatagcaaca ctaaaaactc gatgtacttc cgaggaagcg 4620cagtgcataa
tgctgcgcag tgttgccaca taaccactat attaaccatt tatctagcgg 4680acgccaaaaa
ctcaatgtat ttctgaggaa gcgtggtgca taatgccacg cagcgtctgc 4740ataactttta
ttatttcttt tattaatcaa caaaattttg tttttaacat ttcaaaaaaa 4800aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa atttaaatta attaagcggc cgcctcgagg 4860acgtcaggtg
gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 4920atacattcaa
atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 4980tgaaaaagga
agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 5040gcattttgcc
ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 5100gatcagttgg
gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 5160gagagttttc
gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 5220ggcgcggtat
tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 5280tctcagaatg
acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 5340acagtaagag
aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 5400cttctgacaa
cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 5460catgtaactc
gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 5520cgtgacacca
cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 5580ctacttactc
tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 5640ggaccacttc
tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 5700ggtgagcgtg
ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 5760atcgtagtta
tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 5820gctgagatag
gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 5880atactttaga
ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 5940tttgataatc
tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 6000cccgtagaaa
agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 6060ttgcaaacaa
aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 6120actctttttc
cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 6180gtgtagccgt
agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 6240ctgctaatcc
tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 6300gactcaagac
gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 6360acacagccca
gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 6420tgagaaagcg
ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 6480gtcggaacag
gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 6540cctgtcgggt
ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 6600cggagcctat
ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 6660ccttttgctc
acatgtggga ggctagagta catttaggtg acactataga a
6711396711DNASindbis virus 39attgacggcg tagtacacac tattgaatca aacagccgac
caattgcact accatcacaa 60tggagaagcc agtagtaaac gtagacgtag acccccagag
tccgtttgtc gtgcaactgc 120aaaaaagctt cccgcaattt gaggtagtag cacagcaggt
cactccaaat gaccatgcta 180atgccagagc attttcgcat ctggccagta aactaatcga
gctggaggtt cctaccacag 240cgacgatctt ggacataggc agcgcaccgg ctcgtagaat
gttttccgag caccagtatc 300attgtgtctg ccccatgcgt agtccagaag acccggaccg
catgatgaaa tatgccagta 360aactggcgga aaaagcgtgc aagattacaa acaagaactt
gcatgagaag attaaggatc 420tccggatccc ctgaaaaggc tgtttaagtt gggtaaaccg
ctcccagccg acgacgagca 480agacgaagac agaagacgcg ctctgctaga tgaaacaaag
gcgtggttta gagtaggtat 540aacaggcact ttagcagtgg ccgtgacgac ccggtatgag
gtagacaata ttacacctgt 600cctactggca ttgagaactt ttgcccagag caaaagagca
ttccaagcca tcagagggga 660aataaagcat ctctacggtg gtcctaaata gtcagcatag
tacatttcat ctgactaata 720ctacaacacc accaccatga atagaggatt ctttaacatg
ctcggccgcc gccccttccc 780ggcccccact gccatgtgga ggccgcggag aaggaggcag
gcggccccga tgcctgcccg 840caacgggctg gcttctcaaa tccagcaact gaccacagcc
gtcagtgccc tagtcattgg 900acaggcaact agacctcaac ccccacgtcc acgccagcca
ccgcgccaga agaagcaggc 960gcccaagcaa ccaccgaagc cgaagaaacc aaaaacgcag
gagaagaaga agaagcaacc 1020tgcaaaaccc aaacccggaa agagacagcg catggcactt
aagttggagg ccgacagatt 1080gttcgacgtc aagaacgagg acggagatgt catcgggcac
gcactggcca tggaaggaaa 1140ggtaatgaaa cctctgcacg tgaaaggaac catcgaccac
cctgtgctat caaagctcaa 1200atttaccaag tcgtcagcat acgacatgga gttcgcacag
ttgccagtca acatgagaag 1260tgaggcattc acctacacca gtgaacaccc cgaaggattc
tataactggc accacggagc 1320ggtgcagtat agtggaggta gatttaccat ccctcgcgga
gtaggaggca gaggagacag 1380cggtcgtccg atcatggata actccggtcg ggttgtcgcg
atagtcctcg gtggagctga 1440tgaaggaaca cgaactgccc tttcggtcgt cacctggaat
agtaaaggga agacaattaa 1500gacgaccccg gaagggacag aagagtggtc cgcagcacca
ctggtcacgg caatgtgttt 1560gctcggaaat gtgagcttcc catgcgaccg cccgcccaca
tgctataccc gcgaaccttc 1620cagagccctc gacatccttg aagagaacgt gaaccatgag
gcctacgata ccctgctcaa 1680tgccatattg cggtgcggat cgtctggcag aagcaaaaga
agcgtcatcg atgactttac 1740cctgaccagc ccctacttgg gcacatgctc gtactgccac
catactgaac cgtgcttcag 1800ccctgttaag atcgagcagg tctgggacga agcggacgat
aacaccatac gcatacagac 1860ttccgcccag tttggatacg accaaagcgg agcagcaagc
gcaaacaagt accgctacat 1920gtcgcttaag caggatcaca ccgttaaaga aggcaccatg
gatgacatca agattagcac 1980ctcaggaccg tgtagaaggc ttagctacaa aggatacttt
ctcctcgcaa aatgccctcc 2040aggggacagc gtaacggtta gcatagtgag tagcaactca
gcaacgtcat gtacactggc 2100ccgcaagata aaaccaaaat tcgtgggacg ggaaaaatat
gatctacctc ccgttcacgg 2160taaaaaaatt ccttgcacag tgtacgaccg tctgaaagaa
acaactgcag gctacatcac 2220tatgcacagg ccgggcccgc acgcttatac atcctacctg
gaagaatcat cagggaaagt 2280ttacgcaaag ccgccatctg ggaagaacat tacgtatgag
tgcaagtgcg gcgactacaa 2340gaccggaacc gtttcgaccc gcaccgaaat cactggttgc
accgccatca agcagtgcgt 2400cgcctataag agcgaccaaa cgaagtgggt cttcaactca
ccggacttga tccgacatga 2460cgaccacacg gtccaaggga aattgcattt gcctttcaag
ttgatcccga gtacctgcat 2520ggtccctgtt gcccacgcgc cgaatgtaat acatggcttt
aaacacatca gcctccaatt 2580agatacagac cacttgacat tgctcaccac caggagacta
ggggcaaacc cggaaccaac 2640cactgaatgg atcgtcggaa agacggtcag aaacttcacc
gtcgaccgag atggcctgga 2700atacatatgg ggaaatcatg agccagtgag ggtctatgcc
caagagtcag caccaggaga 2760ccctcacgga tggccacacg aaatagtaca gcattactac
catcgccatc ctgtgtacac 2820catcttagcc gtcgcatcag ctaccgtggc gatgatgatt
ggcgtaactg ttgcagtgtt 2880atgtgcctgt aaagcgcgcc gtgagtgcct gacgccatac
gccctggccc caaacgccgt 2940aatcccaact tcgctggcac tcttgtgctg cgttaggtcg
gccaatgctg aaacgttcac 3000cgagaccatg agttacttgt ggtcgaacag tcagccgttc
ttctgggtcc agttgtgcat 3060acctttggcc gctttcatcg ttctaatgcg ctgctgctcc
tgctgcctgc cttttttagt 3120ggttgccggc gcctacctgg cgaaggtaga cgcctacgaa
catgcgacca ctgttccaaa 3180tgtgccacag ataccgtata aggcacttgt tgaaagggca
gggtatgccc cgctcaattt 3240ggagatcact gtcatgtcct cggaggtttt gccttccacc
aaccaagagt acattacctg 3300caaattcacc actgtggtcc cctccccaaa aatcaaatgc
tgcggctcct tggaatgtca 3360gccggccgtt catgcagact atacctgcaa ggtcttcgga
ggggtctacc cctttatgtg 3420gggaggagcg caatgttttt gcgacagtga gaacagccag
atgagtgagg cgtacgtcga 3480actgtcagca gattgcgcgt ctgaccacgc gcaggcgatt
aaggtgcaca ctgccgcgat 3540gaaagtagga ctgcgtatag tgtacgggaa cactaccagt
ttcctagatg tgtacgtgaa 3600cggagtcaca ccaggaacgt ctaaagactt gaaagtcata
gctggaccaa tttcagcatc 3660gtttacgcca ttcgatcata aggtcgttat ccatcgcggc
ctggtgtaca actatgactt 3720cccggaatat ggagcgatga aaccaggagc gtttggagac
attcaagcta cctccttgac 3780tagcaaggat ctcatcgcca gcacagacat taggctactc
aagccttccg ccaagaacgt 3840gcatgtcccg tacacgcagg ccgcatcagg atttgagatg
tggaaaaaca actcaggccg 3900cccactgcag gaaaccgcac ctttcgggtg taagattgca
gtaaatccgc tccgagcggt 3960ggactgttca tacgggaaca ttcccatttc tattgacatc
ccgaacgctg cctttatcag 4020gacatcagat gcaccactgg tctcaacagt caaatgtgaa
gtcagtgagt gcacttattc 4080agcagacttc ggcgggatgg ccaccctgca gtatgtatcc
gaccgcgaag gtcaatgccc 4140cgtacattcg cattcgagca cagcaactct ccaagagtcg
acagtacatg tcctggagaa 4200aggagcggtg acagtacact ttagcaccgc gagtccacag
gcgaacttta tcgtatcgct 4260gtgtgggaag aagacaacat gcaatgcaga atgtaaacca
ccagctgacc atatcgtgag 4320caccccgcac aaaaatgacc aagaatttca agccgccatc
tcaaaaacat catggagttg 4380gctgtttgcc cttttcggcg gcgcctcgtc gctattaatt
ataggactta tgatttttgc 4440ttgcagcatg atgctgacta gcacacgaag atgaccgcta
cgccccaatg atccgaccag 4500caaaactcga tgtacttccg aggaactgat gtgcataatg
catcaggctg gtacattaga 4560tccccgctta ccgcgggcaa tatagcaaca ctaaaaactc
gatgtacttc cgaggaagcg 4620cagtgcataa tgctgcgcag tgttgccaca taaccactat
attaaccatt tatctagcgg 4680acgccaaaaa ctcaatgtat ttctgaggaa gcgtggtgca
taatgccacg cagcgtctgc 4740ataactttta ttatttcttt tattaatcaa caaaattttg
tttttaacat ttcaaaaaaa 4800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa atttaaatta
attaagcggc cgcctcgagg 4860acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
ctatttgttt atttttctaa 4920atacattcaa atatgtatcc gctcatgaga caataaccct
gataaatgct tcaataatat 4980tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
cccttattcc cttttttgcg 5040gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
tgaaagtaaa agatgctgaa 5100gatcagttgg gtgcacgagt gggttacatc gaactggatc
tcaacagcgg taagatcctt 5160gagagttttc gccccgaaga acgttttcca atgatgagca
cttttaaagt tctgctatgt 5220ggcgcggtat tatcccgtat tgacgccggg caagagcaac
tcggtcgccg catacactat 5280tctcagaatg acttggttga gtactcacca gtcacagaaa
agcatcttac ggatggcatg 5340acagtaagag aattatgcag tgctgccata accatgagtg
ataacactgc ggccaactta 5400cttctgacaa cgatcggagg accgaaggag ctaaccgctt
ttttgcacaa catgggggat 5460catgtaactc gccttgatcg ttgggaaccg gagctgaatg
aagccatacc aaacgacgag 5520cgtgacacca cgatgcctgt agcaatggca acaacgttgc
gcaaactatt aactggcgaa 5580ctacttactc tagcttcccg gcaacaatta atagactgga
tggaggcgga taaagttgca 5640ggaccacttc tgcgctcggc ccttccggct ggctggttta
ttgctgataa atctggagcc 5700ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
cagatggtaa gccctcccgt 5760atcgtagtta tctacacgac ggggagtcag gcaactatgg
atgaacgaaa tagacagatc 5820gctgagatag gtgcctcact gattaagcat tggtaactgt
cagaccaagt ttactcatat 5880atactttaga ttgatttaaa acttcatttt taatttaaaa
ggatctaggt gaagatcctt 5940tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
cgttccactg agcgtcagac 6000cccgtagaaa agatcaaagg atcttcttga gatccttttt
ttctgcgcgt aatctgctgc 6060ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
tgccggatca agagctacca 6120actctttttc cgaaggtaac tggcttcagc agagcgcaga
taccaaatac tgtccttcta 6180gtgtagccgt agttaggcca ccacttcaag aactctgtag
caccgcctac atacctcgct 6240ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
agtcgtgtct taccgggttg 6300gactcaagac gatagttacc ggataaggcg cagcggtcgg
gctgaacggg gggttcgtgc 6360acacagccca gcttggagcg aacgacctac accgaactga
gatacctaca gcgtgagcta 6420tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
ggtatccggt aagcggcagg 6480gtcggaacag gagagcgcac gagggagctt ccagggggaa
acgcctggta tctttatagt 6540cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
tgtgatgctc gtcagggggg 6600cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
ggttcctggc cttttgctgg 6660ccttttgctc acatgtggga ggctagagta catttaggtg
acactataga a 6711406711DNASindbis virus 40attgacggcg
tagtacacac tattgaatca aacagccgac caattgcact accatcacaa 60tggagaagcc
agtagtaaac gtagacgtag acccccagag tccgtttgtc gtgcaactgc 120aaaaaagctt
cccgcaattt gaggtagtag cacagcaggt cactccaaat gaccatgcta 180atgccagagc
attttcgcat ctggccagta aactaatcga gctggaggtt cctaccacag 240cgacgatctt
ggacataggc agcgcaccgg ctcgtagaat gttttccgag caccagtatc 300attgtgtctg
ccccatgcgt agtccagaag acccggaccg catgatgaaa tatgccagta 360aactggcgga
aaaagcgtgc aagattacaa acaagaactt gcatgagaag attaaggatc 420tccggatccc
ctgaaaaggc tgtttaagtt gggtaaaccg ctcccagccg acgacgagca 480agacgaagac
agaagacgcg ctctgctaga tgaaacaaag gcgtggttta gagtaggtat 540aacaggcact
ttagcagtgg ccgtgacgac ccggtatgag gtagacaata ttacacctgt 600cctactggca
ttgagaactt ttgcccagag caaaagagca ttccaagcca tcagagggga 660aataaagcat
ctctacggtg gtcctaaata gtcagcatag tacatttcat ctgactaata 720ctacaacacc
accaccatga atagaggatt ctttaacatg ctcggccgcc gccccttccc 780ggcccccact
gccatgtgga ggccgcggag aaggaggcag gcggccccga tgcctgcccg 840caacgggctg
gcttctcaaa tccagcaact gaccacagcc gtcagtgccc tagtcattgg 900acaggcaact
agacctcaac ccccacgtcc acgccagcca ccgcgccaga agaagcaggc 960gcccaagcaa
ccaccgaagc cgaagaaacc aaaaacgcag gagaagaaga agaagcaacc 1020tgcaaaaccc
aaacccggaa agagacagcg catggcactt aagttggagg ccgacagatt 1080gttcgacgtc
aagaacgagg acggagatgt catcgggcac gcactggcca tggaaggaaa 1140ggtaatgaaa
cctctgcacg tgaaaggaac catcgaccac cctgtgctat caaagctcaa 1200atttaccaag
tcgtcagcat acgacatgga gttcgcacag ttgccagtca acatgagaag 1260tgaggcattc
acctacacca gtgaacaccc cgaaggattc tataactggc accacggagc 1320ggtgcagtat
agtggaggta gatttaccat ccctcgcgga gtaggaggca gaggagacag 1380cggtcgtccg
atcatggata actccggtcg ggttgtcgcg atagtcctcg gtggagctga 1440tgaaggaaca
cgaactgccc tttcggtcgt cacctggaat agtaaaggga agacaattaa 1500gacgaccccg
gaagggacag aagagtggtc cgcagcacca ctggtcacgg caatgtgttt 1560gctcggaaat
gtgagcttcc catgcgaccg cccgcccaca tgctataccc gcgaaccttc 1620cagagccctc
gacatccttg aagagaacgt gaaccatgag gcctacgata ccctgctcaa 1680tgccatattg
cggtgcggat cgtctggcag aagcaaaaga agcgtcactg acgactttac 1740cctgaccagc
ccctacttgg gcacatgctc gtactgccac catactgaac cgtgcttcag 1800ccctgttaag
atcgagcagg tctgggacga agcggacgat aacaccatac gcatacagac 1860ttccgcccag
tttggatacg accaaagcgg agcagcaagc gcaaacaagt accgctacat 1920gtcgcttaag
caggatcaca ccgttaaaga aggcaccatg gatgacatca agattagcac 1980ctcaggaccg
tgtagaaggc ttagctacaa aggatacttt ctcctcgcaa aatgccctcc 2040aggggacagc
gtaacggtta gcatagtgag tagcaactca gcaacgtcat gtacactggc 2100ccgcaagata
aaaccaaaat tcgtgggacg ggaaaaatat gatctacctc ccgttcacgg 2160taaaaaaatt
ccttgcacag tgtacgaccg tctgaaagaa acaactgcag gctacatcac 2220tatgcacagg
ccgggaccgc acgcttatac atcctacctg aaagaatcat cagggaaagt 2280ttacgcaaag
ccgccatctg ggaagaacat tacgtatgag tgcaagtgcg gcgactacaa 2340gaccggaacc
gtttcgaccc gcaccgaaat cactggttgc accgccatca agcagtgcgt 2400cgcctataag
agcgaccaaa cgaagtgggt cttcaactca ccggacttga tccgacatga 2460cgaccacacg
gtccaaggga aattgcattt gcctttcaag ttgatcccga gtacctgcat 2520ggtccctgtt
gcccacgcgc cgaatgtaat acatggcttt aaacacatca gcctccaatt 2580agatacagac
cacttgacat tgctcaccac caggagacta ggggcaaacc cggaaccaac 2640cactgaatgg
atcgtcggaa agacggtcag aaacttcacc gtcgaccgag atggcctgga 2700atacatatgg
ggaaatcatg agccagtgag ggtctatgcc caagagtcag caccaggaga 2760ccctcacgga
tggccacacg aaatagtaca gcattactac catcgccatc ctgtgtacac 2820catcttagcc
gtcgcatcag ctaccgtggc gatgatgatt ggcgtaactg ttgcagtgtt 2880atgtgcctgt
aaagcgcgcc gtgagtgcct gacgccatac gccctggccc caaacgccgt 2940aatcccaact
tcgctggcac tcttgtgctg cgttaggtcg gccaatgctg aaacgttcac 3000cgagaccatg
agttacttgt ggtcgaacag tcagccgttc ttctgggtcc agttgtgcat 3060acctttggcc
gctttcatcg ttctaatgcg ctgctgctcc tgctgcctgc cttttttagt 3120ggttgccggc
gcctacctgg cgaaggtaga cgcctacgaa catgcgacca ctgttccaaa 3180tgtgccacag
ataccgtata aggcacttgt tgaaagggca gggtatgccc cgctcaattt 3240ggagatcact
gtcatgtcct cggaggtttt gccttccacc aaccaagagt acattacctg 3300caaattcacc
actgtggtcc cctccccaaa aatcaaatgc tgcggctcct tggaatgtca 3360gccggccgtt
catgcagact atacctgcaa ggtcttcgga ggggtctacc cctttatgtg 3420gggaggagcg
caatgttttt gcgacagtga gaacagccag atgagtgagg cgtacgtcga 3480actgtcagca
gattgcgcgt ctgaccacgc gcaggcgatt aaggtgcaca ctgccgcgat 3540gaaagtagga
ctgcgtatag tgtacgggaa cactaccagt ttcctagatg tgtacgtgaa 3600cggagtcaca
ccaggaacgt ctaaagactt gaaagtcata gctggaccaa tttcagcatc 3660gtttacgcca
ttcgatcata aggtcgttat ccatcgcggc ctggtgtaca actatgactt 3720cccggaatat
ggagcgatga aaccaggagc gtttggagac attcaagcta cctccttgac 3780tagcaaggat
ctcatcgcca gcacagacat taggctactc aagccttccg ccaagaacgt 3840gcatgtcccg
tacacgcagg ccgcatcagg atttgagatg tggaaaaaca actcaggccg 3900cccactgcag
gaaaccgcac ctttcgggtg taagattgca gtaaatccgc tccgagcggt 3960ggactgttca
tacgggaaca ttcccatttc tattgacatc ccgaacgctg cctttatcag 4020gacatcagat
gcaccactgg tctcaacagt caaatgtgaa gtcagtgagt gcacttattc 4080agcagacttc
ggcgggatgg ccaccctgca gtatgtatcc gaccgcgaag gtcaatgccc 4140cgtacattcg
cattcgagca cagcaactct ccaagagtcg acagtacatg tcctggagaa 4200aggagcggtg
acagtacact ttagcaccgc gagtccacag gcgaacttta tcgtatcgct 4260gtgtgggaag
aagacaacat gcaatgcaga atgtaaacca ccagctgacc atatcgtgag 4320caccccgcac
aaaaatgacc aagaatttca agccgccatc tcaaaaacat catggagttg 4380gctgtttgcc
cttttcggcg gcgcctcgtc gctattaatt ataggactta tgatttttgc 4440ttgcagcatg
atgctgacta gcacacgaag atgaccgcta cgccccaatg atccgaccag 4500caaaactcga
tgtacttccg aggaactgat gtgcataatg catcaggctg gtacattaga 4560tccccgctta
ccgcgggcaa tatagcaaca ctaaaaactc gatgtacttc cgaggaagcg 4620cagtgcataa
tgctgcgcag tgttgccaca taaccactat attaaccatt tatctagcgg 4680acgccaaaaa
ctcaatgtat ttctgaggaa gcgtggtgca taatgccacg cagcgtctgc 4740ataactttta
ttatttcttt tattaatcaa caaaattttg tttttaacat ttcaaaaaaa 4800aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa atttaaatta attaagcggc cgcctcgagg 4860acgtcaggtg
gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 4920atacattcaa
atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 4980tgaaaaagga
agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 5040gcattttgcc
ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 5100gatcagttgg
gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 5160gagagttttc
gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 5220ggcgcggtat
tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 5280tctcagaatg
acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 5340acagtaagag
aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 5400cttctgacaa
cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 5460catgtaactc
gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 5520cgtgacacca
cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 5580ctacttactc
tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 5640ggaccacttc
tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 5700ggtgagcgtg
ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 5760atcgtagtta
tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 5820gctgagatag
gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 5880atactttaga
ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 5940tttgataatc
tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 6000cccgtagaaa
agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 6060ttgcaaacaa
aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 6120actctttttc
cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 6180gtgtagccgt
agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 6240ctgctaatcc
tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 6300gactcaagac
gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 6360acacagccca
gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 6420tgagaaagcg
ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 6480gtcggaacag
gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 6540cctgtcgggt
ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 6600cggagcctat
ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 6660ccttttgctc
acatgtggga ggctagagta catttaggtg acactataga a 6711
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