EP1720911A1 - Proteine de fusion comprenant un polypeptide tatdmt - Google Patents

Proteine de fusion comprenant un polypeptide tatdmt

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Publication number
EP1720911A1
EP1720911A1 EP04703525A EP04703525A EP1720911A1 EP 1720911 A1 EP1720911 A1 EP 1720911A1 EP 04703525 A EP04703525 A EP 04703525A EP 04703525 A EP04703525 A EP 04703525A EP 1720911 A1 EP1720911 A1 EP 1720911A1
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Prior art keywords
tatdmt
cells
transcription
zinc
fusion protein
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Man-Wook Hur
Jung-Min 311-1003 Gyeonghyang Apt. KIM
Yeon-Soo Kim
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • HIN-1 Human immunodeficiency virus type 1
  • LTR long terminal repeat
  • the HIN-1 long terminal repeat (LTR) contains promoter elements responsible for the initiation of viral transcription. After synthesis, the full-length viral R ⁇ A is either transported directly into the cytoplasm and becomes translated into structural proteins or it is spliced into subgenomic R ⁇ As that encode the several viral regulatory proteins.
  • One of these regulatory proteins is the Tat protein, which is localized within the nucleus. Tat is essential for viral replication, and high level expression of HIN genome requires transcription by Tat. The control of viral gene transcription is of prime importance in the viral replication.
  • Control of transcription at the HIN-1 LTR promoter involves complex interactions among czs-acting elements, viral trans-activator, and cellular proteins.
  • the key players of transcription regulation are Spl, ⁇ F- ⁇ B, Tat, P-TEFb, and TAR sequence. Basal transcription is contributed by Spl and TFIID.
  • Tat drastically activates transcription either by increasing transcriptional initiation and/or elongation, and is essential for viral replication. Tat binds to the TAR sequence of the stalled short HIN-1 transcript, and interacts with Spl, ⁇ F- ⁇ B, TFIID and TFIIH . Tat also interacts with the cellular kinase (P-TEFb) and establishes the Tat-TAR-P-TEFb complex.
  • P-TEFb cellular kinase
  • zinc-fingers are highly variable in terms of D ⁇ A sequence they recognize.
  • Naturally occurring zinc-finger proteins show extremely diverse DNA-binding specificities, and recognize many different DNA sequences.
  • Fig. 1 shows that TatdMt fusions are targeted efficiently to the HIN-1 LTR and potently repress transcription in stable HeLa cells.
  • Transient transfection assays of TatWt or TatWt fusions The TatWt and TatWt fusion proteins, in the absence of TatWt, potently activate transcription.
  • C Transient transfection assays of TatdMt fusions in the presence or absence of the TatWt.
  • D transient transfection in the absence of TatWt.
  • ⁇ assays in the presence of TatWt.
  • D Titration of transcription inhibition by FBI-1-TatdMt fusion. FBI-1-TatdMt competed with TatWt and potently inhibited transcription activation by TatWt. The average of three independent assays is shown. Bars (I) represent standard deviations. Fig.
  • HIN-1 proviral D ⁇ A YK177-86
  • HIN-1 proviral D ⁇ A YK177-86
  • Cell-free medium was collected, diluted, and used to infect HeLaT4 cells or Magi cells. The numbers of infected cells were scored as described in Methods.
  • Fig. 3 shows that FBI-TatdMt potently represses HIN-1 replication.
  • Total cell extracts (30 ng) were prepared from CN-1 cells transfected with TatWt, TatdMt, TatdMt fusion plasmids and analyzed by 10% SDS- PAGE and Western blot analysis. TatdMt fusion polypeptides do not induce apoptotic cell death.
  • B Apoptotic cell death assay with transfected HeLa/Hyg R cells. Total viable cells were counted by Trypan blue dye exclusion. To determine the viability of the transfected cells, cells expressing green fluorescence were counted under a Zeiss fluorescence photomicroscope. Cells undergoing apoptosis were detected by DAPI staining.
  • C Nuclear condensation and fragmentation were monitored by DAPI staining.
  • FIG. 5 shows mapping of functionally important functional domains of FBI-1-TatdMT fusion.
  • POZ-domain, TatdMt, and zinc- fingers are important domains in the repression of transcription activation by TatWt at HIN-1 LTR.
  • A diagram of various fusion constructs, various deletions or mutations of parent construct FBI-1- TatdMt 1-1 were made.
  • B Transcription inhibition assays of HIN-1 LTR by various fusion constructs in stable HeLa cells.
  • C comparison of ZF, 4-1ZFC and 1-1 in transcription repression.
  • TatdMT fusion FBI-1 ZFs are targeted efficiently to the HIN-1 LTR and potently repress transciption in.
  • Fig. 6 shows design and sites of artificial zinc-finger proteins recognizing the various regions of HIN-1 LTR.
  • (A) ⁇ ucleotide sequence of the HIN-1 LTR and the regions where artificial zinc- fingers were designed to bind. Shaded bars are the regions where fingers were designed to bind. • indicates the upper most nucleotide of 9 or 12 bp recognition site of artificial zinc-finger.
  • FIG. 7 shows that POZ-domain, TatdMt fusion proteins of artificial zinc-finger 39 and 40 (AZF39, AZF40) recognizing the -64 bp ⁇ -56 bp and -67 bp ⁇ -59 bp sites of HIN-1 LTR null HIN-1 transcription.
  • FIG. 8 shows that POZ-domain, TatdMt fusion proteins of artificial zinc-finger potently inhibit HIN-1 replication, as revealed by transient trans complementation virus replication inhibition assays.
  • A microscopic pictures of X-gal stained Maggi cells infected with recombinant HIN-1 virus at 50-100x magnification.
  • B Table of replication inhibition by various fusion proteins.
  • the present invention provides novel anti-AIDS therapeutic agents related with TatdMt fusion proteins.
  • inventors provided the fusion proteins comprising artificial zinc-fingers, which recognize human immunodeficiency virus (HIV) long terminal repeat (LTR) region and contain TatdMt polypeptide.
  • the fusion proteins of the present invention further comprises POZ (Poxvirus zinc finger)-domain.
  • the fusion proteins of the present invention can further comprise a nuclear localization signal (NLS).
  • the fusion proteins of the present invention recognize the HIN-1 LTR region region -64 bp ⁇ -56 bp site (AZF39) and/or -67 bp ⁇ -59 bp site (AZF40) from transcription start site, respectively.
  • the zinc-finger in the present invention can comprise amino acid sequence of SEQ ID NO: 11 (AZF39) or SEQ ID NO:12(AZF40).
  • the TatdMt polypeptide in the present invention can comprise 73amino acid of SEQ ID NO:10.
  • the POZ-domain in the present invention can comprise amino acid sequence of SEQ ID NO:9.
  • the fusion proteins of the present invention are suitable for use as anti-AIDS therapeutic agents.
  • a DNA molecule that encodes a fusion protein comprising a zinc finger and a TatdMt polypeptide.
  • the zinc finger in the present invention can comprise nucleic acid sequence of SEQ ID NO:13 (AZF39) or SEQ ID NO:14(AZF40).
  • the TatdMt polypeptide in the present invention can comprise nucleic acid sequence of SEQ ID NO: 15.
  • a DNA molecule that encodes a fusion protein comprising a POZ (Poxvirus and Zinc finger)-domain; a zinc finger; and a TatdMt polypeptide.
  • the POZ-domain in the present invention can comprise nucleic acid sequence of SEQ ID NO: 16.
  • Inventors designed and prepared novel artificial zinc-finger proteins and their fusion proteins that can recognize the HIN-1 LTR promoter sequence. Inventors tested the transcription repression activities of the zinc-fingers and found that they can repress transcription of HIN-1 LTR by Tat potently, particularly when the fingers were fused with transcription repression domain POZ- domain and Tat mutant form lacking its ability to interact with PTEFb. Investigation on the transcription regulation of HIN-1 provides a molecular basis for developing novel anti-viral agents. Developing therapeutic agents that can potently repress transcription at the HIN- 1 LTR promoter is important to overcome viral resistance, because such agents would prevent production of the genetic material for viral replication and the template for reverse transcriptase.
  • Inventors designed and prepared the fusion gene constructs that express the POZ-domain of FBI-1, full-length FBI-1 or artificial zinc-finger polypeptides fused to a particular mutant version of Tat (TatdMt) and investigated their functions in the repression of HIN-1 transcription and replication.
  • the fusions were efficiently targeted to the HIN-1 LTR.
  • the fusions potently blocked transcription activation by Spl and Tat and, in particular, FBI-1-TatdMt fusion completely blocked transcription and repressed HIN-1 replication by as much as 230 fold.
  • the fflV-1 LTR-CAT fusion plasmid (pU3R-IIICAT) has been described previously (Sodroski JG et al. (1985) Science 227, 171- 173).
  • the expression plasmids for TatWt (HIVHBX2R type, 86 a.a.), TatdMt (Tat 28 ⁇ 1 ⁇ , 72 a.a.), and FBI-1 were prepared by subcloning their cDNAs into pcDNA3.0 (Invitrogen) (Kiernan RE, et al., (1999) EMBO J. 18, 6106-6118; Karn, J. (1999) J. Mol. Biol.
  • the expression plasmids for the POZ-domain fusion proteins either with TatWt or mutant TatdMt were constructed by subcloning their cDNAs into pcDNA3.0-TatWt or -TatdMt plasmids.
  • Various deletion and zinc-finger mutants of FBI-1 were prepared by subcloning PCRed FBI-I or FBI-1 zinc-finger 1 mutant cDNA fragments into ⁇ cDNA3.0-TatdMt plasmid (Fig. 1A) (Lee, D.-K., et al., (2002) J. Biol. Chem.
  • ACGTAAGCTTACCATGGCGCCGACCTCCTGGACCGG-3' (primer corresponding to a.a 124 from N-terminus; SEQ ID NO:l) and, reverse 5'-GATCGAATTCGGCGAGTCCGGCTGTGAAGTT- 3' (primer corresponding to a.a. 584 from N-terminus; SEQ ID NO:2) were used.
  • ACGTAAGCTTACCATGGGGGACAGCGACGAGTC-3 ' (primer corresponding to a.a. 335 from N-terminus; SEQ ID NO:4) was paired either with reverse primer( SEQ ID NO: 5) 5'- GATCGAATTCGGCGAGTCCGGCTGTGAAGTT-3' or 5'- GATCGAATTCCGGGCTGGGGTCGGGCGCCCCGCC-3' ( SEQ ID NOS).
  • reverse primer SEQ ID NO: 5'- ACGTAAGCTTACCATGGGGGACAGCGACGAGTC-3' ( SEQ ID NO: 6) primer was paired with reverse primer 5'- GATCGAATTCGGCGAGTCCGGCTGTGAAGTT-3' ( SEQ ID NO: 5) primer for PCR reaction.
  • forward primer 5'- ACGTAAGCTTACCATGGAGAAGGTGGAGAAGATCCGA-3' primer corresponding to a.a. 371 from N-terminus; SEQ ID NO:7
  • ACGTAAGCTTCGAGGGGACGCCGTTGCAGCC-3 ' (primer corresponding to a.a 495 from N-terminus; SEQ ID NO:8) were used. PCR was performed by denaturing at 94°C for 5 min, 30 cycles of amplification reaction (94°C 30 sec, 60°C 1 min, 72°C 3 min), and final extension at 72°C for 4 min.
  • Inventors used the FBI-1 cDNA that Inventors isolated previously (full length FBI-1 cDNA cloned into pBluscriptllKS-) as PCR template (Doetzlhofer, A., et al., (1999) Mol. Cell. Biol. 19, 5504-5511).
  • Inventors used the FBI-1 cDNA with mutation at the first zinc-finger (a.a. C384A, C387A), which was kindly provided by Dr. Nuria Hernandez of Cold Sring Harbour Laboratory (New York, USA).
  • the amplified PCR products were cloned into TOPO TA cloning vector ( ⁇ CR ® 2.1-TOPO ® , Invitrogen).
  • the resulting ligated mixtures were transformed into competent E. coli DH5 ⁇ and the transformed E. coli were grown on LB-agar-Ampicillin-X-gal-IPTG selection plates.
  • White E. coli colonies were grown in LB-Ampicillin broth and plasmids were prepared at 1.5 ml mini-scale.
  • the plasmids with PCR products inserts were digested with Hindill and EcoRI by standard alkaline lysis protocol, inserts were purified by agarose gel electrophoresis and cloned into pcDNA3.0TatdMt plasmid to generate various deletion mutant of FBI-1-TatdMt.
  • PCRed ZF cDNA of FBI-1 was prepared in the same manner and cloned into ⁇ cDNA3.0 digested with HindUI.
  • Ten artificial zinc-fingers cloned in pcDNA3.1 were designed and prepared by ToolGen Inc.(Taejeon, Korea).
  • the fingers were designed to recognize the regions such as Spl binding site, TATA box, and FBI-1 binding inducer of short transcript sequence that are critical in transcription of HIV-1 LTR. They usually contain either 3 zinc-fingers (clone -71, -68, -67, -64, - 61, -58-1, -55, -28, +41; the numbers are the upper-most nucleotide of the recognition sites counted from the transcription start site on the HV-1 LTR) or 4 zinc-fingers (-58-11), which recognize 9 bp or 12 bp sequence of the HIV-1 LTR. All the artificial zinc-fingers contain the same 5' and 3' sequences flanking the zinc-finger open reading frame (ORF) sequences.
  • ORF zinc-finger open reading frame
  • the PCR products were digested with HindUI and EcoRV and cloned into pcDNA3.0-TatdMt plasmid digested with Hindll ⁇ -EcoRV.
  • the antibodies against various proteins such as actin, tubulin, p21 Waf/Cipl , Spl, AKT, ERK, GSK, mSin3A are from either Santa Cruz Biotechnology or Upstate Biotechnology (CA, USA). Cell culture and transient transfection reporter assays.
  • the stable HeLa cells intergrated with HIN-1-LTR-CAT gene fusion were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin (100 units/ml)/streptomycin (100 ⁇ g/ml) (Invitrogen, CA, USA). Cells were inoculated on a six-well tissue culture plates at a density of 1x10 s cells/well in 2 ml of DMEM medium.
  • DMEM Dulbecco's Modified Eagle Medium
  • cells were transiently transfected with mixture of plasmids pcD ⁇ A3.0 TatWt expression plasmid (0.3 ⁇ g), pCM Y-LacZ plasmid (0.1 ⁇ g), and various pcDNA3.0 expression plasmids (0.3 ⁇ g) encoding TatWt, TatdMt, TatWt or TatdMt fused either with FBI-1 or POZ-domain using Lipofectamine Plus reagent (Invitrogen, CA, USA) according to the manufacturer's recommended protocol (Fig. 1).
  • pcDNA3.0-FBI-l-TatdMt was cotransfected with pcDNA3.0 TatWt (0.3 ⁇ g), and pCMV-E ⁇ cZ plasmid (0.1 ⁇ g) (Fig. ID).
  • Cells were harvested and lysed in 150 ⁇ l of reporter lysis buffer and vortexed for 1 min and centrifuged at 1,2000 rpm, for 3 min at 4 °C.
  • Cellular extracts (20 ⁇ l) were analyzed for ⁇ -galactosidase activity by mixing with 180 ⁇ l of substrate solution (o-nitrophenyl- ⁇ -D- galactopytanoside 4 mg/ml in 0.1 M sodium phosphate, pH7.5; 0.1 M sodium phosphate buffer, pH 7.5; lOOx Mg 2+ (0.1 M MgCl 2 ; 4.5 M ⁇ -mercaptoethanol ).
  • substrate solution o-nitrophenyl- ⁇ -D- galactopytanoside 4 mg/ml in 0.1 M sodium phosphate, pH7.5; 0.1 M sodium phosphate buffer, pH 7.5; lOOx Mg 2+ (0.1 M MgCl 2 ; 4.5 M ⁇ -mercaptoethanol ).
  • reporter CAT chloramphenicol acetyltransferase
  • the harvested cell pellet was resuspended 75 or 150 ⁇ l of 0.25 M Tris-HCl, pH 7.8. Cells are lysed by three repeated freeze-and-thaw cycles using EtOH/ dry ice bath. Variations in transfection efficiencies were normalized using co-expressed ⁇ -galactosidase activity.
  • Each CAT reaction assay mixtures contains 50 ⁇ l 0.25 M Tris-HCl , pH 7.8, 10 ⁇ l 4mM Acetyl CoA (3.5 mg/ml), 25 ⁇ l cell extract, 0.5 ⁇ l 14 C-Chloramphenicol (CAM) (25nCi/ ⁇ l) and incubated for 15 min at 37 °C.
  • HIV-1 replication inhibition analysis was carried out as described elsewhere(Kim YS, et al., (1996) J. Neurosci. Res. 43, 652-663; Park IW, et al., (2001) J. Immunol. 167, 2766-2771.; Kimpton, J., et al., (1992) J. Virol. 66, 2232-2239.; Selliah N, et al., (2001) Cell Death Differ. 8, 127-136.).
  • a plasmid that expresses HIV-1 genome with the Hygromycin B resistance gene was cotransfected into 293T cells with a viral envelope protein VSV-G expression plasmid and either the control (pHYK) or the TatdMt fusion protein expression plasmid.
  • the cotransfection resulted in the production of HIV-1 virus particles that undergo only a single round of infection in susceptible cell lines such as HeLaT4 and Magi cells(Kim YS, et al., (1996) J. Neurosci. Res. 43, 652-663; Selliah N, et al, (2001) Cell Death Differ. 8, 127-136).
  • the efficiency of a single round of infection was determined by scoring the numbers of Hyg R HeLaT4 cells or ⁇ -galactosidase positive Magi cells (Fig. 2A).
  • HIV-1 proviral DNA YK177-86, 1 ⁇ g
  • control pHYK, 2 ⁇ g
  • envelope protein expression plasmid pVSV-G, 0.5 ⁇ g
  • LacZ gene expression plasmid MFG/E cZ/Puro, 0.5 ⁇ g
  • MFG/E ⁇ cZ/Puro plasmid was cotransfected to normalize variations in transfection efficiencies.
  • the transfected cells were incubated further for two days to prepare supernatant containing infectious HIN-1 virus particles.
  • the supernatant from the transfected 293T cells was collected, filtered, diluted and used to infect CD4 + HeLaT4 cells and Magi cells (HeLaT4 cells integrated with HIN-1 LTR-E ⁇ cZ gene). After 4 hrs, fresh culture medium was added and cells were cultures for two days. The virus titer was determined by scoring the number of Magi cells that turned blue after ⁇ -galactosidase staining.
  • Hyg R positive cells were selected with Hygromycin, cultured for another 10 days, and the virus titer was determined by counting colonies that were fixed and stained with 0.5% crystal violet/50% (v/v) methanol. Assays results are presented as averages of four independent experiments.
  • the cell line is useful in assessing the apoptotic activity of Tat polypeptides because Tat induces the apoptosis of cell without sensitization, such as serum starvation or TNF- ⁇ treatment, as was observed in microvascular endothelial cells(Bartz, S. R., et al., (1999) J. Virol. 73, 1956-1963.; Cullen BR. (1993) Cell 373-370.).
  • Cells were transfected with TatWt or TatdMt fusion expression plasmid (0.5 ⁇ g) and GFP expression plasmid (0.5 ⁇ g) using Fugene 6 (Roche, NJ, USA). 24 hours after transfection, total viable cells were counted by Trypan blue dye exclusion.
  • CAT transient transfection reporter CAT assays.
  • the stable HeLa cells integrated with HIN-1 LTR chloramphenicol acetyl transferase (CAT) gene was grown in Dubelco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin (100 units/ml)/streptomycin (100 ⁇ g/ml) (Invitrogen, CA, USA). Plasmids used prepared a by alkaline lysis methods in large scale (250 ml) and purified by CsCl/EtBr ultra centrifugation.
  • DMEM Dubelco's Modified Eagle Medium
  • Plasmids used prepared a by alkaline lysis methods in large scale (250 ml) and purified by CsCl/EtBr ultra centrifugation.
  • Cells were inoculated on a six-well tissue culture plates at a density of 1x10 s cells/well in 2 ml of DMEM medium. After growing the cells for 24 hrs, cells were transiently transfected with 0.3 ⁇ g of each pcD ⁇ A 3.1 zinc-finger expression plasmid, 0.3 ⁇ g of pcD ⁇ A3.0-TatWt expression plasmid, and 0.1 ⁇ g of pCMV- ⁇ -galactosidase (LacZ) mixed with LipofectAmine Plus reagent (Invitrogen) according to the manufacturer's recommended protocol in serum free DMEM medium. After 3 hrs, cells were supplied with fresh complete
  • TatdMt fusions are targeted efficiently to HIV-1 LTR.
  • inventors fused the Spl inhibiting polypeptides (FBI-1 POZ domain, FBI-1) to either the N or C terminus of TatWt polypeptide (86 a.a.)(Fig. 1A).
  • the TatWt fusion expression plasmids were transfected into the stable HeLa cells with integrated HIV-1 LTR-CAT fusion gene. In the absence of TatWt, transcription occurred at a low basal level (Fig. IB, lane 1). TatWt potently activated transcription by 55 fold (Fig. IB, lane 2).
  • the Spl inhibitory polypeptides did not show any repression and the four TatWt fusions potently activated transcription instead (Fig. IB, lanes 3-6).
  • the four TatWt fusions potently activated transcription instead (Fig. IB, lanes 3-6).
  • FBI-1-TatWt activated transcription more potently than TatWt alone, probably resemble of cooperative interaction between Tat and FBI-1 in this particular configuration.
  • the results clearly suggest that the fusion proteins were targeted efficiently to HIV-1 LTR and have full potential to activate transcription after they bind to TAR.
  • the ability of TatWt to interact with P-TEFb and activate transcription appears to mask the inhibitory potential of fused Spl inhibitory polypeptide on transcription.
  • the fusion proteins were unable to repress transcription activation by TatWt .
  • TatMt 28 ⁇ 1 " ⁇ ) For targeting purposes, inventors adopted a different version of Tat (TatdMt 28 ⁇ 1 " ⁇ ) with no ability to interact with P-TEFb, but with a two fold stronger interaction with TAR than TatWt (Kiernan RE, et al., (1999) EMBO J. 18, 6106-6118).
  • TatdMt The features of TatdMt are ideal for targeting the Spl inhibiting polypeptides to the HIV-1 LTR because the fusion protein can be delivered more efficiently to TAR and stays longer and, even more importantly, there will be no phosphorylation of the CTD of RNA polymerase II.
  • Inventors prepared the Spl inhibiting proteins fused with the TatdMt in two possible configurations (Figure 1A bottom: POZ-TatdMt and FBI-1 -TatdMt, only structures in one configuration are shown; Figure 2A).
  • the fusion expression plasmids were transfected into the stable HeLa cells or CV-1 cells in the presence or absence of TatWt expression plasmid, and analyzed for reporter CAT or luciferase activity.
  • TatWt on its own potently activated transcription, by more than 46 fold (Figure IC filled bar, lane 1).
  • TatdMt and the TatdMt fusion proteins alone did not activate transcription over the basal level ( Figure IC open bars, compare lanes 1 vs. 2-4).
  • TatdMt reduced transcription activation by TatWt to half of that observed with TatWt alone, suggesting that there is competition for the TAR sequence between the two ( Figure IC, compare filled bars, lanes 1, 2).
  • the TatdMt fusion proteins not only successfully competed with TatWt for TAR, but also repressed transcription potently even in the presence of excess TatWt expression plasmid (0.3 ⁇ g) ( Figure IC, compare filled bars, lanes 1 vs.
  • HIN-1 proviral D ⁇ A, YK177-86 was cotransfected with an envelope protein NSN-G expression plasmid and the control pHYK plasmid or the effector FBI-1 -TatdMt fusion plasmid into 293T cells.
  • HIN-1 proviral D ⁇ A, YK177-86 do not have a gene encoding an envelope protein that is essential in the packing of HIN-1 virus.
  • infectious HIN-1 virus can be produced.
  • the resulting virus cannot replicate in the infected cells because neither host cell nor the HIN-1 YK177-86 proviral D ⁇ A cannot provide envelope protein.
  • the transcription at the HIN-1 can be modulated, which in turn translated into the change in the number of infectious HIN-1 virus.
  • cell-free medium was collected, diluted, and used to infect host cells such as HeLaT4 cells or Magi cells.
  • the number of infectious HIN-1 particles produced were counted by transfection and Hyg selection or X-gal staining of HeLaT4 or Magi cells.
  • TatdMt fusion proteins The specificity of TatdMt fusion proteins. To be useful as therapeutic agent against AIDS, the fusion proteins should act specifically at the HIN-1 LTR promoter, and ideally, they should have little effect on the expressions of cellular genes. Inventors investigated whether over expression of the TatdMt fusion proteins change the expression of various cellular proteins in transfected CN-1 cells by Western blot analysis (Fig. 4A). The expression of constitutively expressed genes (actin, tubulin, TCTP p21), component of signal transduction (Akt, Erk, GSK), transcription factor (Spl), and co-repressor (mSin3A) was not altered by the expression of various Tat or fusion proteins, suggesting that the fusions act specifically at the HIN-1 LTR (Fig. 4A).
  • Tat sensitizes and induces apoptosis in HIN-1 infected or Tat- transfected cells.
  • HeLa/Hyg R cells were cotransfected with TatWt (or fusion polypeptides with TatdMt) and green fluorescence protein (GFP) expression plasmids, and analyzed for apoptosis after 21 hrs.
  • TatWt or fusion polypeptides with TatdMt
  • GFP green fluorescence protein
  • TatWt or TatdMt-fusion polypeptides were investigated the effects of TatWt or TatdMt-fusion polypeptides on nuclear condensation. Twenty-one hours after transfection, HeLa/Hyg R cells were fixed and stained with DAPI. Once, the cells were transfected with TatWt expression plasmid, the total number of viable cells significantly decreased. TatWt induced nuclear condensation and, in contrast, FBI-1 -TatdMt fusion proteins did not (Fig. 4B).
  • C-terminus (513-584 a.a.) also showed ambiguous result on the repression. Upon comparison of 2-1 and 3-1, the C-terminus region might be not so important in repression. However, the data on 4-1 and 5-1 suggested that the C-terminus region is important, and 4-1 showed 7 fold more potent repression than 5-1. All of the zinc-finger mutants (2-2, 3-2, 5-2) did not show repressor activity comparable to any of the deletion mutants and showed repression activity only comparable to TatdMt alone (Fig. 5C filled bar, lane 2).
  • Inventors designed and prepared 10 zinc-finger proteins (Fig. 6A).
  • the artificial zinc-fingers recognizing the HIN-1 LTR were designed to contain 3 or 4 zinc-fingers and accordingly recognize 9 bp or 12 bp nucleotide sequence of the HIN-1 LTR promoter.
  • the targeted site of the zinc fingers are Spl binding region (-78 bp to-45 bp), TATA box region (-37 bp to -3 bp), and inducer of short transcript region (+37 bp to +69 bp) ( Figure 6A).
  • the numbers indicate the upper-most nucleotide of the recognition sites counted from the transcription start site on the HN- 1 LTR. Except -58-11 which contains 4 zinc-fingers, all the zinc- fingers contain 3 zinc-fingers.
  • the zinc-finger expression plasmids (0.3 ⁇ g) were cotransfected with TatWt expression plasmid (0.3 ⁇ g) into stable HeLa cells. Most of the artificial zinc-fingers do not repressed transcription significantly.
  • Zinc-finger -58-11, -64, and -67 zinc-finger repressed transcription In particular, artificial zinc-finger -64 repressed most potently among the tested fingers, but it did not repress transcription down to the basal level (Fig. 6B).
  • the site is the recognition site by Spl transcription factor with highest affinity, one of the key regulators of HIV-1 transcription.
  • inventors fused the POZ-domain and TatdMt to the artificial zinc-fingers (-67 bp AZF40 and -64 bp AZF30 zinc-fingers) and tested their activities in transcription repression (figure 7).
  • the zinc-fingers alone can only repress transcription down to 1/5-1/10 of the transcription level activated by TatWt.
  • the repressor activity is enhanced by 2-3 fold.
  • the repressor activities of the AZF39 (-64 zinc-finger) or AZF40 (-67 zinc-finger) are further enhanced by being fused with POZ-domain and completely null transcription activation by TatWt (figure 7B).
  • the full amino acid sequences of AZF39 or AZF40 and TatdMt fusion proteins are represented in SEQ ID NO:17(AZF39-TatdMt) or SEQ ID NO:18(AZF40-TatdMt).
  • the full amino acid sequence of POZ-domain; AZF39 or AZF40; and TatdMt fusion proteins are represented in SEQ ID NO:19(POZ-domain -AZF39-TatdMt) or SEQ ID NO:20(POZ- domain -AZF40-TatdMt).
  • inventors fused the POZ-domain of FBI-1 and TatdMt to the artificial zinc fingers (AZF39, AZF40) and cloned the fusions in pcDNA 3.0 mammalian expression plasmid.
  • Inventors performed single round viral replication inhibition assays using Maggi cells as described in Materials and Methods and fig. 2.
  • the single round transfection competent HIV-1 virus were produced by cotransfection of recombinant HIV-1 genome defective in envelope protein gene, VSV-G envelope protein gene, and effector expression plasmids (fusion transcription repressor) or control vector plasmid (pYHK) into virus packaging 293T cells.
  • the viral supernatant was diluted and used to transfect the Maggi cells and, to count the number of cells transfected with HIV-1 virus, cells were stained with X-gal. The cells were viewed at two magnification 100X and 50X. Although FBI-1-TatdMt potently inhibited HIV-1 replication, the POZ-domain-AZF39-TatdMt and POZ-domain- AZF40-TatdMt inhibited replication more potently (fig. 8A). When the same volume of viral supernatant was used to transfect the cells, in the control experiment, about half of the Maggi cells were stained blue.
  • One of the ways to overcome the viral resistance is to develop a therapeutic agent that can block de novo synthesis of viral RNA transcripts from the proviral HIV-1 genome.
  • the FBI-1-TatdMt fusion, the artificial zinc-finger-POZ-domain-TatdMt fusion proteins, and molecular approach developed here provide an effective therapeutic agent or strategy against AIDS in protein drug or gene therapy forms. After HIV viral titer is dropped to low level by administration of drugs cocktail or vaccine, expression of the TatdMt fusions in infected cells would make the proviral HIV-1 genome virtually silent. Our strategy may prevent manifestation of the disease in short term, gradually replaces the HIV-1 infected cells with uninfected fresh cells derived from stem cells, restores immune-defense system, and ultimately may cure AIDS.

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Abstract

L'invention concerne des protéines de fusion pouvant être utilisées comme agents thérapeutiques antisida fusionnés par l'intermédiaire de doigts à zinc artificiels et du domaine POZ ou par l'intermédiaire de doigts à zinc artificiels, du domaine POZ et du polypeptide TatdMt.
EP04703525A 2004-01-20 2004-01-20 Proteine de fusion comprenant un polypeptide tatdmt Withdrawn EP1720911A1 (fr)

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