WO1995027783A1 - Inhibition de la multiplication du vih-1 dans des cellules de mammiferes - Google Patents

Inhibition de la multiplication du vih-1 dans des cellules de mammiferes Download PDF

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WO1995027783A1
WO1995027783A1 PCT/CA1995/000190 CA9500190W WO9527783A1 WO 1995027783 A1 WO1995027783 A1 WO 1995027783A1 CA 9500190 W CA9500190 W CA 9500190W WO 9527783 A1 WO9527783 A1 WO 9527783A1
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htv
cells
signal
rna molecules
antisense orientation
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PCT/CA1995/000190
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Sadna Joshi-Sukhwal
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Joshi Sukhwal Sadna
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1132—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV

Definitions

  • This invention relates to human immunodeficiency virus type 1 (HIV-1) and to inhibition of multiplication thereof in mammalian cells expressing chimeric RNA molecules containing HTV-l packaging ⁇ signal and Gag coding sequences in antisense orientation; to said cells and therapeutic compositions comprising said cells; and retroviral vectors expressing said chimeric RNA molecules.
  • HSV-1 human immunodeficiency virus type 1
  • AIDS Acquired immunodeficiency syndrome
  • HTV- 1 a retrovirus, called HTV- 1 , which mainly infects T-lymphocytes and monocytes/macrophages derived from haematopoietic stem cells.
  • Tat-TAR, Rev-RRE, and gag/i * signal interactions are crucial for rr ⁇ /w-activation, late gene expression, and virion RNA packaging, respectively. Interference during these processes may take place by providing the cell with interfering RNA or protein molecule(s); e.g. the sense (decoys of viral protein binding sites) or antisense RNAs to TAR, RRE, signal, and Tat, Rev, or Gag open reading frames or trans-dominant mutants of HTV-l Tat, Rev, or Gag proteins.
  • the Tat protein of HTV-l allows tians-activation of HTV-l gene expression, while Rev protein of HTV-l allows the switch from early to late gene expression. While both of these genes overlap with each other, they are translated in different reading frames.
  • Antisense RNA to HTV-l Tat/Rev mRNA has been shown to confer resistance to HTV-l infection in mammalian cell lines (1, 2, 3, 4).
  • Antisense RNA complementary to a specific portion of HTV-l RNA molecule, upon hybridization with target RNA sequences, disrupt reverse transcription, processing, translation, and/or transport of this RNA.
  • Antisense RNAs have been shown to alter specific gene expression in several cell systems, including bacteria, Xenopus oocytes, Drosophila embryos, plants, and mammalian cells (5, 6). The degree of inhibition obtained in these studies was variable and depended upon many factors, including size, hybridization location, secondary structure, and level of expression of both the antisense RNA and the target mRNA whose expression was being modulated. Synthetic oligodeoxynucleotides, when added to the culture medium, have also been shown to inhibit HTV-l multiplication.
  • a sense RNA approach has been used to block replication of the genome of a plant RNA virus by employing the origin of replication located at the 3' end of the genome as a competitive inhibitor for viral replicase (7).
  • RNA-RNA and RNA-protein interactions are crucial for HTV-l replication, trans-activation, transcription, transport, translation, and packaging, and the HTV-l RNA sequences involved in these interactions are known.
  • Non-HTV-1 RNAs containing TAR sequence in a sense orientation have been shown to compete with HTV-l mRNAs for binding to RNA and/or protein and to result in inhibition of HTV-l multiplication.
  • the cw-acting TAR element is a 59 nucleotide-long RNA stem-loop structure present at the 5' end of all HTV-l transcripts (8).
  • the Tat protein binds to a bulge region present within this structure.
  • Tat binding in itself is not sufficient (9) and a number of specific TAR RNA-binding cellular proteins are required for HTV-l r/r ⁇ w-activation.
  • Retroviral vectors expressing HTV-l TAR RNA decoys (10, 11, 12, 13) have been shown to confer HTV-l resistance.
  • Retroviral vectors expressing antisense RNA to the HTV-l tat gene have also been shown to confer HTV-l resistance (1, 2, 3, 4, 14).
  • HTV-l Tat protein when expressed from retroviral vectors in either Tat- or Tat- and Rev-inducible manner, failed to protect cells against HTV-l infection (16, 17).
  • Vectors expressing antisense RNA targeted to the Gag mRNA 5' leader region (18, 19) have also been shown to inhibit HTV-l multiplication.
  • the Gag (p55) and Gag-Pol (pi 60) polyproteins are translated from the 9.4 kilobase (kb) genomic mRNA.
  • the pol gene is expressed as a result of frameshift near the end of the gag reading frame.
  • the viral protease cleaves the Gag polyprotein (p55) into the pl7, p24, p7, and p9 proteins, and the Gag-Pol polyprotein (pl60) into the p6, pll (protease), p51, p64 (2 subunits of reverse transcriptase, RT), pl5 (RNase H), and p34 (integrase) proteins.
  • RRE is a 234 nucleotide-long RNA sequence located within the env reading frame (20, 21). RRE has been predicted to form a highly complex secondary structure containing a central stem I surrounded by stem-loops ⁇ , LI, TV and V (21). The 66 nucleotide-long stem-loop II has been found to contain the primary Rev binding site and is also sufficient for Rev response in vivo (22, 23). In the absence of Rev, the translation of unspliced and singly spliced mRNA into protein is prevented by cw-acting repressor sequences (CRS) present in the HTV-l gag, pol, and env open reading frames (20, 24, 25).
  • CRS cw-acting repressor sequences
  • Rev-RRE interaction is sufficient to override the inhibitory action of the CRS such that these mRNAs can now reach the cytoplasm and become translated.
  • Plasmids expressing one, three, and six copies of RRE have been shown to interfere with the HTV-l Rev protein activity in a transient co-transfection experiment performed in HeLa cells (26); over expression of RRE decoys has also been shown to inhibit HTV-l multiplication in CEM cells (27).
  • retroviral vectors allowing constitutive or Tat-inducible expression of taz/w-dominant mutants of either Rev (16) or Tat and Rev (15, 17, 28) were shown to confer resistance to HTV-l infection.
  • the HTV-l signal is required in cis for specific recognition and packaging of the viral genomic RNA; two copies of the HTV-l genomic RNA are encapsidated per virus particle. Nucleotides located between the major splice donor site and the Gag initiation codon are essential for HTV-l RNA packaging (29, 30, 31); this region has been shown to fold into a stable secondary structure involving four stem-loops (32). The precise length of the HTV-l signal required for packaging is not known but it can be inferred from studies performed using Moloney murine leukemia virus (MoMuLV) (33) that it would be contained within 1000 nucleotides downstream of the primer binding site.
  • MoMuLV Moloney murine leukemia virus
  • the HTV-l ⁇ signal is recognized in cis by the zinc finger motif within the nucleocapsid domain and by. one other domain of the HTV-l Gag polyprotein precursor (34, 35). Cells allowing constitutive expression of trans dominant mutant
  • Gag proteins have been shown to repress HTV-l replication (36).
  • the HTV-l Rev rr ⁇ /w-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338, pp.
  • HIV-1 structural gene expression requires binding of the Rev fr ⁇ /w-activator to its RNA target sequence.
  • RNA sequences in the gag region of HTV-l decrease RNA stability and inhibit expression in the absence of Rev protein. J. Virol. 66, pp. 150-159.
  • RNA packaging signal of HTV-l Virology 188, pp. 590-599. 32.
  • the HTV-l packaging signal and major splice donor region have a conserved stable secondary structure. J.
  • HTV-l gag mutants can dominantly interfere with the replication of the WT virus. Cell 59, pp. 113-120.
  • HIV-1 gene(s) to infect patients' bone marrow (BM) stem cells or peripheral blood lympocytes (PBLs) which, upon transplantation and differentiation, would potentially give rise to an HTV-l resistant immune system.
  • BM bone marrow
  • PBLs peripheral blood lympocytes
  • the invention provides a gene therapeutic method of inhibiting HTV-l multiplication in a mammal comprising treating said mammal with an effective amount of mammalian cells expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
  • the mammalian cells are human bone-marrow cells, and more preferably, blood cells.
  • the invention provides mammalian cells harboring proviral vector DNA expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
  • the invention provides a retroviral vector expressing RNA molecules containing HTV-l signal and/or Gag coding sequences in antisense orientation.
  • the retroviral vector is derived from the Moloney murine leukemia virus (MoMuLV).
  • MoMuLV Moloney murine leukemia virus
  • MoMuLV-derived retroviral vectors were engineered to express HTV-l ⁇ signal and Gag coding sequences in anti-sense orientation in chimeric RNAs. These sequences were expressed under control of the herpes simplex virus (HSV) thymidine kinase (tk) promoter. Both, ⁇ signal and Gag coding sequences were expressed as part of the 3' untranslated region of the neomycin phosphotransferase (neo) mRNA. The constructs were used to transfect/infect packaging cell lines and the retroviral vector particles released were used to infect a human CD 4 + lymphocyte-derived MT4 cell line.
  • HSV herpes simplex virus
  • tk thymidine kinase
  • the invention provides a therapeutic composition for confering HTV-l resistance to a mammal comprising cells as hereinbefore defined in association with a pharmaceutically acceptable carrier, diluent or adjuvant therefor. It will be readily understood by the person skilled in the art that the cells should be present in an effective therapeutic amount.
  • PBLs bone marrow
  • BM cells contain stem cells which are capable of both self-renewal and differentiation into lymphocytes, macrophages, and other hematopoietic cells such as erythrocytes, granulocytes, and megakaryocytes.
  • stem cells represent only 1 per 10,000 nucleated cells present within the bone marrow, a large number of bone marrow cells are infected by retroviral vectors to ensure transformation of these rare stem cells.
  • Gene therapy for the treatment of AIDS consists of using retroviral vectors to deliver the anti-HTV-1 RNA molecules to human PBLs and BM stem cells.
  • Transformants selected in vitro are transplanted back to the patient. Following differentiation, these transformed cells lead to the development of an immune system in which various blood cells (including CD4 lymphocytes and macrophages) express the anti-HTV-1 RNA molecules and are therefore resistant to HTV-l.
  • Preferred methods of in vitro stimulation and culture for gene transfer into mammalian cells, particularly stem cells, with a gene transfer vector, particularly, a retrovious vector are disclosed in Canadian Patent Application No. 2086844, published July 8, 1994 - Dube et al, which disclosure is included herein by reference. Following appropriate stimulation and culture in vitro.
  • PBLs and BM cells are infected by cocultivation with packaging cell lines producing retroviral vector particles. Transformants are then selected in vitro for growth in medium containing appropriate cytokines and antibiotics. These transformants are then transplanted back to the patient. Following transplantation and differentiation, blood samples are tested for anti-HTV-1 gene expression and for the ability of these cells to resist HTV-l infection. Other disease symptoms, viral load and emergence of resistant HTV-l isolates are examined as well.
  • Fig.1 represents LTR-LTR sequences present in the proviral DNA integrated in the target cell line as part of the map of retroviral vectors expressing antisense RNA to HTV-l ⁇ signal and Gag coding sequence; and Fig. 2 shows the results of HTV-l infections on a pool of stable MT4 transformants expressing HTV-l ⁇ signal and Gag coding sequence-containing RNAs.
  • the retroviral vector, pUCMoTN (39), used in this study, is derived from MoMuLV. This vector allow neo gene expression (conferring G418 R ) under control of HSV tk. The ⁇ signal and Gag sequences were cloned in this vector as part of the 3' untranslated region of the neo mRNA.
  • the pUCMoTN- ⁇ Gag " and pUCMoTN- ⁇ + Gag + vectors were constructed as follows.
  • a 4.0 EcoRI fragment from pBKBHIOS (NIH#182) was cloned into the EcoRI site of the pUC18 vector (Pharmacia).
  • a 1440 bp BamHJ-BglR fragment containing HTV-l ⁇ signal and Gag coding sequences was isolated from this pUC- ⁇ vector.
  • This fragment (containing Sstl-BglR sequences of HTV-l strain HXB2) was cloned into the unique Bam ⁇ I site in the pUCMoTN-Rzl vector (40).
  • the resulting clones were characterized by restriction enzyme analysis and clones containing a single copy of the ⁇ signal and Gag coding sequences in antisense (pUCMoTN- ⁇ 'Gag") and sense (pUCMoTN- ⁇ Gag " ) orientations, with respect to the vector were selected.
  • ecotropic Psi-2 (41) and amphotropic PA317 (ATCC cat# CRL0978) (42) packaging cell lines were cultured in ⁇ -MEM medium supplemented with 2mM
  • L-Gln 0.1 volume of antibiotics/antimycotic solution (penicillin, 1000 units/ml; streptomycin, 1000 ⁇ g/ml; Fungizone R , 2.5 ⁇ g/ml), and 10% FBS (Hyclone) at 37°C in a humidified atmosphere with 5% CO 2 .
  • the human CD 4 + lymphocyte-derived MT4 suspension cell line, NTH Cat #120 was cultured in RPMI 1640 medium also supplemented with Gin, antibiotics/antimicotic agents, and FBS (GIBCO) as above and were incubated at 37°C in a humidified atmosphere with 5% CO 2 .
  • the selective media was prepared as above except that it also contained G418 (200 ⁇ g/ml for Psi-2 and PA317 cell lines; and 400 ⁇ g/ml for MT4 cell line).
  • Psi-2 cells were transfected as follows using the Calcium phosphate co- precipitation technique (using the CellPhect Transfection Kit from Pharmacia): a 120 ⁇ l retroviral DNA solution (3 ⁇ g) was mixed with 120 ⁇ l Buffer A (0.5 M CaCl 2 , 0.1 M HEPES) and incubated at 22°C for 10 min. An equal volume (240 ⁇ l) of Buffer B (0.28 M NaCl, 0.05 M HEPES, 0.75 mM NaH 2 PO 4 , 0.75 mM Na 2 HPO 4 ) was added, mixed immediately by vortexing and incubated for 15 min. The mixture was then added drop wise to the cell culture (50% confluent in 60 mm plates containing 3 ml fresh medium).
  • Buffer A 0.5 M CaCl 2 , 0.1 M HEPES
  • the cells were incubated under normal growth conditions for 6 hrs, then washed twice with fresh medium.
  • the cells were subjected to glycerol shock with 1.5 ml 15% glycerol in 10 mM HEPES pH 7.5, 150 mM NaCl for 3 min at 22°C, then washed once with fresh medium.
  • Fresh medium (5 ml) was added and the cells were grown under normal conditions for 2 days.
  • the transfected cells were washed once with phosphate-buffered saline (PBS) containing antibiotics/antimycotic agents, trypsinized with 0.05% trypsin, 0.53 mM EDTA-4Na (GIBCO), transferred to 100 mm plates and grown in selective medium containing 200 ⁇ g/ml G418. The medium was changed every 3-4 days until selection was complete (15-20 days). The number of resistant colonies was then determined. The cells were washed with PBS, trypsinized and re-seeded.
  • PBS phosphate-buffered saline
  • G418 0.53 mM EDTA-4Na
  • Vector particles released from the transformed Psi-2 cells were obtained by filtering culture medium from cells at 50-100% confluency through a 0.22 ⁇ m filter. These particles were used to infect PA317 cells as described previously (43). Essentially 2 x 10 s cells were seeded for 6 hours in 60 mm tissue culture dishes in 4 ml medium, after which this medium was replaced by 1 ml medium containing 8 ⁇ g/ml polybrene and 100 ⁇ l vector particles. After a 2 hour incubation at 37°C, 3 ml medium was added and the incubation continued for 16 more hours. Cells were then trypsinized and transferred to 100 mm tissue culture dishes in the presence of selective medium containing 200 ⁇ g/ml G418. The selective medium was changed every 4-5 days and the number of colonies counted after 14 days. Vector particles released from the PA317 cells (50-100% confluent) were then collected and used to infect MT 4 cells.
  • the MT4 cells (3 x 10 5 ) were pelleted and resuspended in 0.5 ml RPMI 1640 medium containing 16 ⁇ g/ml polybrene. Vector particles (0.2 ml) and RPMI 1640 medium (0.3 ml) were then added and gently mixed to the cells. Cells were transferred to 60 mm petri dishes, and incubated under normal growth conditions for 2 hrs. Four ml of fresh RPMI 1640 medium were then added and the cells were grown overnight. The infected cells were then centrifuged, resuspended in selective medium containing 400 ⁇ g/ml G418 and transferred to 100 mm petri dishes. Every 3-4 day, half of the cell suspension was removed and replaced with fresh selective medium. By day 20, all of the uninfected cells had died and the remaining stably transformed cells were frozen and were used in the following experiments.
  • PCR Polymerase chain reaction
  • RT reverse transcription
  • Genomic DNA isolated (37) from the MT4 cells stably transformed with MoTN-t '" Gag " vector particles was used in PCR as follows. PCR reaction (100 ⁇ l) was performed in the presence of MgCl 2 (1.5 mM), oligonucleotides (20 mM each; amplification buffer (1 x concentration; Promega), dNTPs (10 mM each), genomic DNA (1 ⁇ g), and Taq polymerase (2 units, Promega).
  • the samples were overlaid with 100 ⁇ l mineral oil and amplified using Perkin-Elmer Cetus Instruments DNA Thermal Cycler by using three linked files as follows: File 1, STEP-CYCLE 1 min at 95 °C; File 2, STEP-CYCLE 1 min at 55 °C; File 3, STEP-CYCLE 1 min at 72°C; with a total of 45 cycles.
  • PCR products (10 ⁇ l aliquots) were then analyzed by electrophoresis on a 3% agarose gel.
  • RNA isolated from the MT4 cells stably transformed with MoTN- ⁇ " Gag " vector particles were grown for 48 hrs and then the total RNA was extracted using the Guanidium thiocyanate-Phenol-Chloroform procedure (44) .
  • Reverse transcription was performed as follows: total RNA (5 ⁇ g) was incubated with oligo dT (20 mM) for 10 min at 65°C in a total volume of 20 ⁇ l. The reaction mixture was chilled on ice for 2 min and RNA guard (75 units; Pharmacia), reverse transcription buffer (1 x concentration; BRL), DTT (5 mM), dNTPs (12.5 mM each), and
  • HTV-l Actively dividing various MT4 transformants (1 x 10° cells/ml) were each infected with HTV-l as follows: a 2 ml cell culture was incubated with 20 ⁇ l HTV-l strain NL -3, NIH Cat #78, (10 64 TdD 50 /ml) for 2 hrs at 37°C. The cells were then pelleted, washed 3 times with PBS, resuspended in 2 ml medium, transferred to 35 mm dishes and allowed to grow at 37 °C Every 3 days for up to day 30, a 1 ml sample containing cells and medium from each infected cell culture was removed and frozen at -70°C One ml of complete medium was added back to the culture each time.
  • MoMuLV-derived retroviral vector pUCMoTN was modified to express
  • HTV-l ⁇ signal and Gag coding sequences in antisense orientation This molecule was expressed as part of the 3' untranslated region of the neo mRNA in between the stop codon and the poly (A) site (Fig. 1).
  • the pUCMoTN- ⁇ - " Gag " vector expressed a single copy of antisense RNA to both of the HTV-l ⁇ signal and Gag coding region (Fig. 1).
  • Gag coding region-containing RNAs in these vectors is under the control of the MoMuLV 5' LTR and HSV tk promoters.
  • the aforementioned retroviral vectors were first used to transfect an ecotropic packaging cell line Psi-2; (41), and the vector particles released from this cell line were used to infect an amphotropic packaging cell line (PA317; 42).
  • the resulting amphotropic pseudotyped retroviral vector particles capable of infecting human cells were then used to infect a human CD4 + lymphoid (MT4) cell line and the G418 R stable transformants were selected.
  • the presence of the anti-HTV-1 gene and the level of therapeutic RNA/protein produced in these cells were then monitored as described below.
  • MoTN- ⁇ " Gag" vector particle The presence of HTV-l ⁇ signal and Gag coding sequences within their genome was confirmed by PCR analysis. The presence of ⁇ signal and Gag coding sequence-containing RNAs was confirmed by RT-PCR. As expected, a 315 bp PCR or RT-PCR product was visible in both cases.
  • MT4 cells stably transformed with MoTN- ⁇ " Gag " vector particles expressing antisense RNA to HIV-1 ⁇ signal and Gag coding sequences were challenged with HTV-l.
  • MT4 cells transformed with the parental retroviral vectors lacking test DNA sequences served as control.
  • Virus production was monitored by measuring the level of p24 antigen (HTV-l gag gene product) in the cell culture supernatant every 3 days for up to 30 days post-infection.
  • the MT4 transformants expressing antisense RNA to the ⁇ signal and Gag coding sequences delayed virus production for up to 30 days (Fig. 2).
  • HTV-l resistance of sense RNA-expressing cells was monitored as follows. MT4 cells stably transformed with MoTN- ⁇ + Gag + vector particles were subjected to challenge by HTV-l and virus production in the culture supernatant was measured every 3 days post-infection. MT4 cells expressing sense RNA to the HTV-l ⁇ sequence and Gag coding sequences failed to prevent HTV-l multiplication (Fig. 2).
  • retroviral vectors were, thus, engineered that expressed ⁇ signal and Gag coding sequences in antisense orientation. The retroviral vector particles were used to infect the human CD 4 + lymphocyte-derived MT4 cells and stable G418 R transformants were selected. The pool of these transformants was then infected with HTV-l and virus production measured for up to 30 days post-infection.
  • HTV-l ⁇ signal and Gag coding sequences in sense orientation HTV- 1 production began even earlier than in the control cells (Fig. 2). If the HTV-l ⁇ signal and Gag coding sequence-containing retroviral vector RNA was also packaged by HTV-
  • the infectivity of these chimeric RNA-containing virus particles should have been reduced.
  • the lack of resistance observed with the HTV-l ⁇ signal and Gag coding sequences expressed in the sense orientation may be explained by the fact that the length of the HTV-l ⁇ signal used in the present experiments was not sufficient to allow packaging of non-viral mRNA.

Abstract

Procédé permettant d'inhiber le virus de l'immunodéficience humaine de type 1 (VIH-1) chez un mammifère à l'aide de cellules de mammifères, notamment de lymphocytes contenant du CD 4 d'origine humaine qui expriment des molécules d'ARN chimérique contenant le signal γ du VIH-1 et/ou des séquences de codage du Gag orientées non codantes. La production du VIH-1 a été retardée de 30 jours au maximum lorsqu'on la compare à des cellules témoins dépourvues de séquences d'ADN test. On décrit des vecteurs rétroviraux exprimant les molécules d'ADN chimérique.
PCT/CA1995/000190 1994-04-06 1995-04-05 Inhibition de la multiplication du vih-1 dans des cellules de mammiferes WO1995027783A1 (fr)

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WO2001090347A1 (fr) * 2000-05-23 2001-11-29 Syngenix Limited Thérapie antivirale antisens
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US7888325B2 (en) 1999-01-28 2011-02-15 Medical College Of Georgia Research Institute, Inc. Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA
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US7888325B2 (en) 1999-01-28 2011-02-15 Medical College Of Georgia Research Institute, Inc. Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA
US8148345B2 (en) 1999-01-28 2012-04-03 Georgia Health Sciences University Research Institute, Inc. Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA
WO2000063364A3 (fr) * 1999-04-21 2001-01-11 American Home Prod Procedes et compositions pour l'inhibition de la fonction de sequences polynucleotidiques
JP2002542263A (ja) * 1999-04-21 2002-12-10 ワイス ポリヌクレオチド配列の機能を阻害するための方法および組成物
EP2363478A1 (fr) * 1999-04-21 2011-09-07 Alnylam Pharmaceuticals, Inc. Procédés et compositions pour inhiber la fonction de séquences de polynucléotides
WO2001090347A1 (fr) * 2000-05-23 2001-11-29 Syngenix Limited Thérapie antivirale antisens
US9051566B2 (en) 2001-01-31 2015-06-09 Alnylam Pharmaceuticals, Inc. Post-transcriptional gene silencing using expressed double stranded RNA
WO2002079464A2 (fr) * 2001-03-30 2002-10-10 Syngenix Limited Vecteurs viraux
WO2002079464A3 (fr) * 2001-03-30 2003-02-20 Syngenix Ltd Vecteurs viraux

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