EP0914423A2 - Inhibition de la replication du vih-1 au moyen de l'expression d'arn antisens - Google Patents

Inhibition de la replication du vih-1 au moyen de l'expression d'arn antisens

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Publication number
EP0914423A2
EP0914423A2 EP97928151A EP97928151A EP0914423A2 EP 0914423 A2 EP0914423 A2 EP 0914423A2 EP 97928151 A EP97928151 A EP 97928151A EP 97928151 A EP97928151 A EP 97928151A EP 0914423 A2 EP0914423 A2 EP 0914423A2
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EP
European Patent Office
Prior art keywords
antisense
hiv
cells
hlv
sequence
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP97928151A
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German (de)
English (en)
Inventor
Ernst Boehnlein
Sonia Escaich
Heini Ilves
Gabor Veres
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Novartis Pharma GmbH
Novartis AG
Systemix Inc
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Novartis AG
Systemix Inc
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Publication of EP0914423A2 publication Critical patent/EP0914423A2/fr
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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

  • This invention relates to inhibition of HIV- 1 replication using antisense RNA expression.
  • HIV-1 infection is believed to be the primary cause of Acquired Immunodeficiency Syndrome (AIDS).
  • HIV- 1 is a retrovirus having a genome comprised of two copies of full length RNA.
  • the replication of the virus in the CD4+ host cell occurs as follows. When the host cell is infected, the viral genomic RNA is transcribed by reverse transcriptase into double stranded DNA. This double stranded DNA is then integrated into the host cell's chromosome(s). When this double stranded DNA is integrated into the genetic material of the host cell, it is called a provirus. Following activation of the host cell, the provirus is transcribed into RNA in two distinct phases.
  • RNA transcripts of the provirus produced in the nucleus are converted into multiple copies of short sequences by cellular splicing enzymes. These short RNA transcripts encode genes for proteins, e.g., tat, which regulate the further transcription, and rev, which is thought to mediate the transition into the late phase transcription. This early phase dominates for about 24 hours. About 24 hours after activation of the cell, the transcription moves into the late phase. In late phase transcription, long unspliced RNA transcripts of about 9,200 bases and medium-length single-spliced transcripts of about 4,500 bases move out of the nucleus and into the cytoplasm. These unspliced and single spliced transcripts encode the structural and enzymatic proteins of the virus.
  • <extra_id_29>“spliced and single-spliced transcripts” include, inter alia, the following regions: gag, which encodes the viral core proteins; pol, which encodes various enzymes; and env, which encodes the two envelope proteins.
  • Figure 1 depicts the HTV- 1 genomic structure. It will be noted that there is some overlap in the genes, because certain genes share some base sequences.
  • the unspliced and single-spliced transcripts are then further spliced, and the resulting mRNA is translated to produce the proteins necessary to make a new virus
  • the gag and pol regions are translated to produce the polyproteins gag and gag-pol, which are then cleaved by protease to form the mature proteins found in the virus
  • the env is spliced to generate a subgenormc messenger which encodes for the env polyproteins, which is likewise cleaved to produce the mature envelope proteins.
  • Two strands of the viral RNA are then packaged into a core and surrounded with capsid protein, and the resulting virus is released from the cell together with a portion of the cell membrane.
  • RNA decoys Lee, T., et al. 1994. Inhibition of human immunodeficiency virus type 1 in human T cells by a potent Rev-response element decoy consisting of the 13-nucieotide minimal Rev-binding domain. J. Virol. 68:8254-8264 and Sulle ⁇ ger, B.A., et al 1990. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication. Cell 63: 601-608), and ribozymes (Ojwang, J.O., et al 1992. Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme. Proc. Natl.
  • the trans-dominant HIV-1 protein RevMlO was first evaluated in a clinical trial using genetically modified peripheral blood lymphocytes (Woffendin, C et al. 1996. Expression of a protective gene prolongs survival of T cells in human immunodeficiency virus infected patients. Proc. Natl. Acad. Sci. USA. 93:2889-2894), although recently a ribozyme (Leavitt, M.C., et al 1996. Ex vivo transduction and expansion of CD4+ lymphocytes from HIV+ donors: prelude to a ribozyme gene therapy trial. Gene Ther. 3:599-606) and a transdominant Rev and antisense TAR based (Morgan R.A et al 1996.
  • Intracellular expression of antisense RNAs offers an attractive, alternative gene therapy approach to inhibit HTV-1 replication.
  • Antisense RNAs have been described as very specific and efficient inhibitors in both prokaryotic and eukaryotic systems .
  • Viral replication has been successfully inhibited by addition of in vitro synthesized antisense oligonucleotides or intracellularly expressed antisense RNAs .
  • Inhibition of HIV-1 replication has been shown previously using antisense RNAs targeted against several viral regulatory (Chatterjee et al 1992, Joshi et al 1991 , Kim et al 1996, Sczakiel, G. et al 1991.
  • Inhibition of human immunodeficiency virus type 1 replication in human T cells stably expressing antisense RNA.
  • the different inhibition levels observed in these reports may reflect variation in antisense RNA expression levels, or secondary and tertiary RNA structures, which can influence the hybridization kinetics between two complementary RNAs (Sczakiel, G., M. Homann, and K. Rittner. 1993 Computer-aided search for effective antisense RNA target sequences of the human immunodeficiency virus type 1. Antisense Res. and Dev. 3:45-52), influencing the biological activity.
  • the best target for antisense therapy is the full length or single-spliced RNA transcript.
  • Antisense sequences which bind to multiple- spliced transcripts for a gene are less effective, probably because binding to the smaller transcripts results in fewer antisense molecules being available for the binding to the full length or single spliced transcripts.
  • longer sequences directed to the full length transcript e.g., sequences greater than 600 base pairs, preferably greater than 1000 base pairs
  • Retroviral vectors are constructed expressing chimeric RNAs containing 1 ,100 - 1 ,400 nt long complementary H ⁇ V- 1 sequences. The most efficient inhibition of HIV-1 replication is observed with an antisense sequence complementary to the HIV-1 env gene both in the CEM-SS cell line and in PBLs.
  • antisense constructs are particularly useful for providing gene therapy to patients suffering from HIV-1 infection, e.g., by transducing the HIV-1- susceptable cells of such patients, e.g., CD4+ cells or cells which are progenitors of CD4+ cells, e.g., hematopoietic stem cells (for example CD34+ Thy-1+ cells), with the antisense constructs of the invention, so that the transduced cells and their progeny are resistant to HIV-1 infection.
  • HIV-1- susceptable cells of such patients e.g., CD4+ cells or cells which are progenitors of CD4+ cells, e.g., hematopoietic stem cells (for example CD34+ Thy-1+ cells)
  • the antisense constructs of the invention are suitably prepared by incorporating a wild- type HIV- 1 gene or gene fragment into a vector in reverse orientation with respect to its promotor so that when the gene is incorporated into the genome of the host cell and transcribed, the opposite strand of the DNA is transcribed, producing a messenger RNA transcript which is complementary to the mRNA from the wild-type gene or gene fragment and will anneal with it to form an inactive RNA-RNA duplex, which is subject to degredation by cellular RNases.
  • Transduction of the HIV-1 susceptable cells using the antisense vectors can be carried out in vivo or ex vivo, but is suitably carried out ex vivo, by removing blood from the patient, selecting the target cells, inoculating them with a vector containing the antisense construct of the invention, and reintroducing the transduced cells into the body.
  • the transduced HIV-1 resistant cells will replace the native HIV-1 susceptible cells, thereby enabling the patient to overcome the infection and regain immunocompetence.
  • the patient receives non-autologous CD4+ cells or progenitors of CD4+ cells from a compatable donor which cells have been transduced with the antisense construct of the invention.
  • a nucleic acid sequence which, when stably integrated into a human cell, is capable of generating mRNA which anneals e.g., under in vivo conditions, with a mRNA transcript from an HIV-1 provirus encoding env, env and pol or env, pol and gag and which is at least 0.6 kb, preferably at least 1 kb in length, most preferably 1-2 kb, e.g. from 1.1 to 1.5 kb; and which is selected from:
  • (iv) a sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 99%, homologous to a sequence according to (i), (ii), or (iii) and which is capable of generating mRNA which annealss to the same mRNA transcript as that hybridizing to mRNA generated by (i), (ii), or (iii).
  • nucleic acid described in 1 above will be in RNA for when in a retroviral vector and will be converted to DNA upon incorporation of the provirus into the target cell. It is intended that both the RNA and DNA forms of the constructs are included within the scope of the invention.
  • the invention further provides
  • the vector may be any vector capable of transducing a human hematopoietic cell, for example, an ecotropic, xenotropic, amphotropic or pseudotyped retroviral vector, an adeno- associated virus (AAV) vector, or an adenovirus (AV) vector.
  • AAV adeno-associated virus
  • AV adenovirus
  • the vector is a retroviral vector, preferably a vector characterized in that it has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), or murine embryonic stem cell virus (MESV), or for example, a vector from the pLN series described in Miller and Rosman (1989) BioTechniques 7, pp. 980-986.
  • LTR long terminal repeat sequence
  • the antisense sequence replaces the retroviral gag, pol and/or env sequences.
  • the promotor controlling expression of the antisense may be a strong viral promoter, for example MoMLV LTR.
  • the range of host cells that may be infected by a retrovirus or retroviral vector is generally determined by the viral env protein.
  • the recombinant virus generated from a packaging cell can be used to infect virtually any cell type recognized by the env protein provided by the packaging cell. Infection results in the integration of the viral genome into the transduced cell and the consequent stable expression of the foreign gene product.
  • the efficiency of infection is also related to the level of expression of the receptor on the target cell.
  • murine ecotropic env of MoMLV allows infection of rodent cells
  • amphotropic env allows infection of rodent, avian and some primate cells, including human cells.
  • Xenotropic vector systems utilize murine xenotropic env, and also allow infection of human cells.
  • the host range of retroviral vectors may be altered by substituting the env protein of the base virus with that of a second virus.
  • the resulting, "pseudotyped" virus has the host range of the virus donating the envelope protein and expressed by the packaging cell line.
  • the G-glycoprotein from vesicular stomatitis virus (VSV-G) may be substituted for the MMLV env protein, thereby broadening the host range.
  • the vector and packaging cell line of the present invention arc adapted to be suitable for transduction of human cells.
  • the vector is an amphotropic retroviral vector, for example, a vector as described in the examples below.
  • the vector may contain more than one antisense sequence according to 1 above, e.g., two different antisense sequences, for example to pol and env, as described in the examples below.
  • the construct lacks the retroviral gag, pol and/or env sequences, so that the gag, pol and env functions must be provided in trans by a packaging cell line.
  • the gag-pol and env proteins produced by the cell assemble with the vector RNA to produce replication-defective or transducing virions that are secreted into the culture medium.
  • the virus thus produced can infect and integrate into the DNA of the target cell, but generally will not produce infectious viral particles since it is lacking essential viral sequences.
  • the packaging cell line is preferably transfected with separate plasmids encoding gag-pol and env, so that multiple recombination events are necessary before a replication-competent retrovirus (RCR) can be produced.
  • Suitable retroviral vector packaging cell lines include those based on the murine NLH/3T3 cell line and include PA317 (Miller & Buttimore (1986) Mol. Cell Biol. 6:2895; Miller & Rosman (1989) BioTechniq es 7:980), CRIP (Danos & Mulligan (1988) Proc. Natl Acad Sci USA 85:6460), and gp + aml2 (Markowitz et al.
  • Retroviral vector DNA can be introduced into packaging cells either by stable or transient transfection to produce retroviral vector particles.
  • the antisense constructs of the invention have the further advantage that they will not interfere with expression of HIV inhibitory proteins, e.g., transdominant mutant proteins corresponding to the early phase short mRNA transcripts, for example mutants of tat or rev. Expression of such transdominant mutant proteins is useful in treating HIV infection because the mutant proteins interfere with the function of the wild-type HIV proteins and so inhibit HIV replication.
  • a transdominant mutant protein of particular interest is RevMlO, the use of which is described e.g., in Escaich, et al. Hum. Gene Ther. (1995) 6: 625-634, and in WO 90/14427.
  • a retroviral vector according to 2 above i.e., comprising an antisense sequence according to 1 above
  • further comprising a gene for an HIV-1 inhibitory protein e.g., a gene for a transdominant mutant form of tat or rev, especially the gene for RevMlO.
  • Packaging cell lines comprising the vectors according to 2 or 3 above, e.g, as described above, are also within the scope of the invention.
  • a cellular composition comprising at least one human hematopoietic cell (e.g. CD4+ cell or progenitor of CD4+ cells, e.g., a stem cell, e.g., a CD34+/Thy-1+ cell) stably transduced with an antisense sequence according to 1 above and optionally additionally transduced with a gene for a transdominant mutant form of tat or rev, especially RevMlO, e.g., transduced with a vector according to 2 or 3, supra, e.g., for use in a method according to 5 below;
  • a human hematopoietic cell e.g. CD4+ cell or progenitor of CD4+ cells, e.g., a stem cell, e.g., a CD34+/Thy-1+ cell
  • RevMlO e.g., transduced with a vector according to 2 or 3, supra, e.g., for use in a method according to
  • a method for treatment of HTV- 1 infection in a subject in need thereof comprising isolating hematopoietic cells (e.g. CD4+ cells or progenitors of CD4+ cells, e.g., stem cells, e.g., CD34+ Thy-1+ cells) from said patient; transducing said cells with an antisense sequence according to 1 above, and optionally additionally or simultaneously transducing said cells with a gene for an HIV-1 inhibiting transdominant mutant form of tat or rev, especially RevMlO, e.g., transducing said cells with a vector according to 2 or 3, supra; and reintroducing the transduced cells into the patient.
  • hematopoietic cells e.g. CD4+ cells or progenitors of CD4+ cells, e.g., stem cells, e.g., CD34+ Thy-1+ cells
  • transducing said cells with an antisense sequence according to 1 above and optionally additionally or simultaneously transducing said cells with a
  • Figure 1 depicts the sequence of HIV- 1 HXB2 strain polymerase gene region 1 (2004- 3400 bp) in sense orientation.
  • Figure 2 depicts the sequence of HIV-1 HXB2 strain polymerase gene region 2 (3400- 4650 bp) in sense orientation.
  • Figure 3 depicts the sequence of the HIV-1 HXB2 strain envelope gene region (6615- 8053) in sense orientation.
  • Figure 4 depict the HIV-1 genomic structure. The position of antisense fragment used for vector construction is also shown. The position of the restriction endonuclease cleavage sites is indicated for each fragment.
  • Figure 5 depicts the schematic structure of antisense vectors of the examples.
  • the parental vector pLN-1 is described in the publication of A. Dusty Miller and Guy J. Rosman (1989) BioTechniques 7. 980-986.
  • the multicloning site 3' from the Neo gene is used to insert the antisense fragments.
  • the parental vector for the combination vectors pLMTNL is described in : Escaich, S. Kalfoglu, C; Plavec, I.; et al. Human Gene Therapy 1995. 6. 625- 634.
  • Figure 6 depicts serial deletion of HIV gag sequence. Construction of the deletion fragments is described below.
  • the 1.5 kb Sac I - Bgl LI psi-gag fragment ( ⁇ -gag) is used to generate the deletion construct either by PCR amplification or by restriction digest.
  • Figure 7 depicts HIV challenge of deletion constructs.
  • the pLN-gag (S) and pLN-gag (AS) construct correspond to the full length 1.5 kb psi-gag fragment in sense or antisense orientation respectively.
  • the pLN-gag-500 is the 5' end of the above fragment corresponding mostly to the psi (packaging signal) sequence of the HIV-1.
  • the pLN-gag- 1000 construct corresponds to the gag region of the 1.5 kb fragment.
  • Figure 8 depicts the anti-HIV-l activity of antisense gag deletion fragments as a function of their length; correlation between size and anti-HIV-l activity is shown on the graph.
  • the p24 production (pg/10E6 cells) versus the length of the fragments in base pairs is plotted on the graph.
  • Figure 9 depicts HIV-1 challenge of antisense gag and Vif constructs.
  • the full length, 1.5 kb antisense gag (pLNl Psi-sense and antisense) and the similar size Vif fragment (pLNl Vif/sense and antisense) are compared.
  • Figure 10 depicts HIV-1 challenge of gag-pol/AS constructs with high dose of virus (40000 TCLD50): The 1.5 kb psi-gag fragment (pLN-gag AS and S) was compared with the pol-1 fragment (pLM-pol/AS and S).
  • Figure 1 1 depicts HIV-1 challenge of antisense pol, env and LTR constructs.
  • CEMSS cells carrying the pol-1 fragment (pLN-pol (AS)/1 and (S)/l) the second antisense pol-2 (pLN Dpol (AS)/2) the envelope (pLN D Env (AS)) and the 3'LTR) pLN D LTR (AS)) fragments are challenged with 400 TCID HIV-1.
  • Figure 12 depicts the pLN pol 1/env antisense vectors, and the effectiveness of pol 1 (S), poll (AS), poll(AS)/env(S), pol(AS)/env(AS) against HIV-1 challenge, the double antisense construct being the most effective.
  • Figure 13 depicts HIY-1 challenge of combination vectors.
  • the two parental vectors LMTNL with the RevMlO gene and the LAMTNL with ATG less RevMlO gene as a control and the corresponding combination vectors LMTNL-Y and LAMTNL-Y with the full length, 1.5 kb psi-gag sequence in antisense orientation are challenged with 400 TCLD50 HIV-1.
  • Figure 14 depicts pol antisense mediated inhibition of HIV replication in peripheral blood lymphocytes.
  • Figure 15. A. Structure of the retroviral vectors encoding the antisense sequences. Neo and Lyt2 are used as a selectable marker genes. The antisense sequence together with the marker gene is expressed from the MoMLV LTR promoter. The arrow indicates the antisense orientation of the inserted HIV-1 sequences.
  • B Northern blot analyses of the antisense RNA expression in transduced CEM-SS cells. The recombinant transcripts carrying the antisense sequences are detected using a Neo specific probe. The lower panel indicates the same blot hybridized with a GAPDH specific probe as a internal standard.
  • Lane 1 pLN vector
  • lane 2 pLN-poll/AS
  • lane 3 pLN-pol2/AS
  • lane 4 pLN-vif/AS
  • lane 5 pLN-env/AS
  • lane 6 pLN-3'LTR/AS
  • lane 7 pLN- ⁇ oll2/AS vector respectively.
  • Figure 16 Inhibition of HIV- 1 replication in transduced CEM-SS cells.
  • A CEM-SS cell populations ( 1 x 10 6 cells/ml) are inoculated with 4x 10 2 TCLD 50 ml of HIV- 1 HXB3 strain.
  • B Increasing HIV- 1 dose, 4xl0 4 TCLDso/ml infection of transduced CEM-SS cell populations. The culture supernatants are tested for p24 antigen production by ELISA. experiments are done in duplicates.
  • FIG 17 Evaluation of anti-HIV-l efficacy of vectors encoding different length complementary pol sequences.
  • CEM- SS cells expressing the 1,400 nt poll and 790 nt pol antisense and the sense poll constructs are infected with 4xl0 3 TCLD 5 o ml of HIV-1 HXB3 strain.
  • B CEM-SS cells expressing the 1 ,400 nt poll and the 2,600 nt pol 12 antisense sequences are infected with 4xl0 3 TCLD 5 o ml HIV-1 HXB3 strain.
  • the corresponding sense constructs are used as a control.
  • FIG. 18 Antisense RNA expression and inhibition of HIV- 1 replication in transduced PBLs.
  • A. Total cellular RNA is isolated from activated, CD4 + enriched PBLs transduced with pL-Lyt-poll/AS, pL-Lyt2/poll S, pL-Lyt-env/AS, pL-Lyt2/env/S vectors and selected for Lyt2 expression.
  • the antisense transcripts are analyzed on Northern blot using a radiolabeled Lyt2 specific probe. A GAPDH specific probe is used to monitor the amount of RNA loaded.
  • FIG. 19 Comparison of trans-dominant RevMlO and intracellularly expressed vif, poll and env antisense RNAs in high inoculation dose HIV-1 infection experiments.
  • CEM- SS cells (lxl0 6 /ml) are inoculated with lxlO 5 TdD 50 ml of HIV- 1 HXB3 and viral replication is monitored by measuring p24 antigen production in the culture supernatant.
  • Figure 20 Detection of HIV- 1, antisense and RevMlO transcripts in CEM-SS cells inoculated with lxlO 5 TCLD 5 o ml HIV-1 , HXB3 strain. Total cellular RNA is isolated from CEM-SS cells at day 4, day 6 and day 8 post infection. The HIV specific transcripts are analyzed on Northern blot using a radiolabeled TAR specific oligonucleotide probe. Expression of the antisense or RevMlO transcripts is determined using a Neo or a Rev specific probe respectively. A GAPDH specific probe is used to monitor the amount of RNA loaded.
  • Lane 1 RevMlO
  • Lane 2 DRevMlO
  • Lane 3 pLN(vector control)
  • Lane 4 pLN-vif/AS
  • Lane 5 pLN-poll/AS
  • Lane 6 pLN- env/AS.
  • Panel A Day 4.
  • Panel B day 6
  • Panel C Day 8.
  • FIG. 21 Analyses of intracellular p24 and Tat expression in HIV-1 infected CEM- SS cells.
  • Retroviral vector constructs with different antisense HIV- 1 sequences are generated as follows using as parental vector pLN, described in A. Dusty Miller and Guy J. Rosman (1989) BioTechniques 7. 980-986.
  • pLN-gag/AS vector The 1420 bp Sac I-Bgl H (675 bp-2095 bp) fragment is isolated from the HXB-2 strain of HIV- 1 and inserted as a blunt end fragment in antisense orientation into the blunt ended Hind HI site of the pLN- 1 vector. Orientation of the fragment is determined by restriction digest with Cla I.
  • GTAGGATCCACATGGGTATCACTTCTGGGCTG (SEQ. LD. NO. 8); primer 3.2 position 1079-1 100, GTAGGATCCTCTATCTTGTCTAAAGCTTCCTTG (SEQ. LD. NO. 9); primer 3.1 position 884-904,
  • the PCR fragments with Bam HI and blunt end are cloned into the Bam HI - Hpa I site of the pLN vector.
  • the generated fragments are approximately 1200 bp, 1000 bp, 800 bp, 600 bp, 400 bp 200 bp in length.
  • pLN-Vif/AS vector The 1100 bp Eco RI-Eco Ri fragment (4646-5742) from the HXB-2 strain of HIV- 1 corresponding Vif-Vpr gene of the virus is inserted into the Hind LI site of pLN vector in antisense orientation.
  • pLN-poll/AS vector The 1480 bp Apa 1-Pflm I fragment (2005-3485) from the HXB-2 strain of HIV-1 corresponding to the 5' end of the Pol gene of the virus is inserted into the Hind II site of pLN vector in antisense orientation.
  • pLN-po!2/AS vector The 1250 bp Pflm I - Eco RI fragment (3485-4646) from the HXB- 2 strain of HIV-1 corresponding to the 3' end of the Pol gene of the virus is inserted into the Hind LI site of pLN vector in antisense orientation.
  • pLN-env/AS vector The 1440 bp Apa LI - Bsm I fragment (6615-8053) from the HXB-2 strain of HIV-1 corresponding to intronic region of the Env gene of the virus is inserted into the Hind II site of pLN vector in antisense orientation.
  • pLN-polKAS)-env(AS) vector The pol 1 fragment of e) is inserted 5' to the env construct of g), both in antisense orientation in the Malawi site of the pLN vector (fig. 12).
  • pLN3' LTR/AS vector the 1260 bp Bam HI-Hind LH fragment (8474-9615) from the HXB-2 strain of HIV- 1 corresponding to the 3' LTR of the virus is inserted into the Xho I site of pLN vector in antisense orientation.
  • the retroviral vector pLN-pol 12/AS with the full length pol sequence is constructed by inserting the 2,642 bp Apal-EcoRI fragment into the pLN vector in reverse orientation.
  • the sense control vectors pLN-poll/S and pLN-poll2/S the 1,400 bp Apal-Pflml and 2,642 bp Apal-EcoRI pol fragments are cloned in the sense orientation into the pLN vector.
  • the pLN-790pol/AS vector is constructed by inserting the 790 bp BglLT-Nsil subfragment of the pol gene into the Xhol site of the pLN vector.
  • Retroviral vectors (pLLyt2-poll/AS, pLLyt2- poll/S, pLLyt2-env/AS and pLLyt2-env/S) are constructed by replacing the Neo gene with the truncated mouse CD8 (Lyt2) cell surface marker (Forestell, S.P., et al 1997. Novel retroviral packaging cell lines: complementary tropism and improved vector production for efficient gene transfer. Gene Ther. 4:19-28) and used for the primary T cell HIV- infection experiments.
  • Retroviral vector production 10 ug of retroviral DNA is transfected into the ecotropic BOSC packaging line using the CaP04 transfection protocol. The transient ecotropic viral supematant is used to transduce the amphotropic PA 317 packaging cell line. Since the pLN vectors carry the Neo gene, the cells are selected on G418. After selection the stable cells pools are analysed by Northern blot for the antisense RNA expression. Viral supernatants from the selected PA317 cell lines carrying the appropriate retroviral constructs are collected, analysed for transducing viral titer, and used to transduce the human CD4+ T cells line CEMSS.
  • GP47 could be used instead of BOSC as the packaging line (Rigg , R.J., et al 1996.
  • a novel human amphotropic packaging cell line high titer, complement resistance, and improved safety. Virology. 218: 290-295).
  • Supernatant from the GP47 packaging cell lines is used to transduce the amphotropic PrpPakA cell line (Rigg , R.J., et al . 1996) by spinoculation as described previously (Forestell, S.P et al . 1997. Novel retroviral packaging cell lines: complementary tropism and improved vector production for efficient gene transfer. Gene Ther. 4:19-28).
  • Retroviral end-point titers are determined on NLH3T3 cells after drug selection (800 mg/ml G418) and transduction efficacy of the Lyt2 vectors (Forestell, S.P et al . 1997) is measured by FACS analysis.
  • Target cell transduction The human CD4+ T cell line CEM SS cells (2x 10 6 cells) are transduced with the amphotropic viral supernatants carrying the antisense vector constructs in 5 ml DMEM + 10 FCS + 8 ug/ml polybrene for 4-6 hours. 48 hours later the cells are selected on 400 ug/ml G418. After G418 selection (7-10 days) the resistant cell are expanded, the antisense RNA expression is analysed by Northern blot. The selected CEM SS cell pools are also analysed for the presence of the CD4 cell surface marker.
  • the resistance of transduced CEM cells to HIV replication and to cytopathic effects of the virus is determined as follows: Cells are subjected to HIV-1 infection (HXB3) in vitro. Antiviral effect is measured by cell viability, levels of p24 Ag produced in the supernatant, and levels of CD4 expression at the cell surface. Infection is measured by PCR for HIV sequences. In addition to the clones to be challenged, CEMss containing a vector control are submitted to infection by HIV- 1.
  • Day -1 Prior to challenge the clones are tested for CD4 expression by FACS analysis.
  • Cells are passaged every 4-5 days to be maintained in log phase growth until day 16 or until the controls are dead. For each passage, cells are counted and supernatant is frozen.
  • Transduced CEM-SS cells expressing RevMlO and antisense HIV-1 sequences are inoculated with lxlO 5 TCLD 50 IO 6 cells/ml of HIV-1.
  • cells are removed from the culture, washed and resuspended in cold PBS and fixed in ice cold methanol for 30 min.
  • the fixed cells are stained with a FTTC-conjugated anti-p24 monoclonal antibody (Coulter KC57) for intracellular p24 detection, include p24, with mouse anti-Tat IgGl antibody (Repligen) for intracellular Tat detection as described earlier (Rigg, R.J., et al 1995. Detection of intracellular HIV-1 Rev protein by flow cytometry. J. I mun. Methods. 188: 187-195).
  • the samples are analyzed using a Becton-Dickinson FACScan.
  • EXAMPLE 4 Detection of antisense RNA in cells.
  • RNAzol Total cellular RNA from CEM-SS cells and from activated PBLs is extracted with RNAzol (Cinna/Biotecx). 10 mg RNA is fractionated on 1.2% agarose/formaldehyde gels, transferred to Hybond N membrane (Amersham), and hybridized in Rapid-hyb buffer (Amersham). Oligonucleotides (100 ng) are radiolabeled with terminal transf erase (Boehringer MA), using a- 32 P-dATP to a specific activity of 3xl0 8 cpm mg. DNA fragments are labeled by random priming (Boehringer MA).
  • the membranes are hybridized with the labeled probe (5 x 10 6 cpm/ml) at 65 °C for 1 hour and ished with 1 xSSC, 0.1 % SDS at 65°C, and exposed on X-ray film or analyzed on a Phosphorlmager (Molecular Dynamics).
  • PBLs are isolated from healthy donors buffy coats by gradient centrifugation. Enriched CD4+ cells are obtained by labeling bulk PBL with biotinylated ⁇ CD8+ and ⁇ CDl 9+ antibodies followed by depletion with streptavidin conjugated magnetic beads (Dynabeads M-280, Dynal A.S., Norway). The enriched CD4+ PBLs are stimulated with phytohemagglutinin (PHA, 5 ⁇ g/ml) on ⁇ - irradiated allogenic feeder cells for 72 hours in Iscove's modified DMEM medium.
  • PHA phytohemagglutinin
  • PBLs (2x 10 6 ) are transduced by spinoculation in the presence of Polybrene (8 ⁇ g/ml). After 48 hours, cells are analysed for CD4+ and Lyt2+ expression by flow cytometry using anti-CD4- FITC and anti-CD8-PE conjugated monoclonal antibodies. Lyt2+ expressing PBLs are again enriched by magnetic bead selection. After the first enrichment, PBLs are expanded, and the CD4+/Lyt2+ cells are isolated using fluorescence-activated cell sorting (FACS, Beckton-Dickinson, Vantage). After the second enrichment, greater than 90% of the cell population is CD4+ and Lyt2+.
  • FACS fluorescence-activated cell sorting
  • transduced CEM-SS cells expressing complementary transcripts are infected with 4xl0 2 TCLD 5 o/ml of the HIV-1 HXB3 virus.
  • H ⁇ V-1 replication is monitored by measuring p24 antigen levels in the culture supernatant by ELISA.
  • a vector encoding the pol sequence in sense orientation pLN-pol/S
  • Fig. 16.A shows the relative efficacy of the different antisense sequences at low HIV-1 inoculation dose.
  • CEM-SS cells expressing the env antisense RNA showed almost complete suppression of HIV-1 replication, releasing 50 pg of p24/10 6 cells at day 18 post-inoculation.
  • a vector encoding an antisense transcript of the complete pol gene reading frame is also generated to address the question whether increasing the antisense RNA length beyond 1 ,400 nt results in increased antiviral efficacy.
  • Figure I7.B demonstrates that the 1 ,400 nt poll antisense sequence is as efficient in blocking HLV-l replication as the 2,600 nt pol 12 antisense RNA. Since both pol 1 and pol2 antisense RNA yield comparable levels of inhibition, this experiment suggests that other factors in addition to expression level and transcript length may influence the efficacy of antisense RNA.
  • the antiviral potency of antisense vif, pol 1 , and env sequences at a high HLV- 1 inoculation dose with RevMlO the trans-dominant form of the HIV-1 Rev protein is compared.
  • RevMlO acts post-transcriptionally, preventing the transport of full length HLV-l transcripts from the nucleus to the cytoplasm.
  • the effect of RevMlO and antisense RNA on HTV-1 RNA steady-state levels as well as on structural (p24 gag) and regulatory (Tat) protein expression is analyzed.
  • the control vector (lane 3) and DRevMlO (lane 2) transduced cells express high steady-state levels of HLV-l transcripts.
  • the RevMlO (lane 1 ) and vif/AS (lane 4) vector transduced cells express 3-to 5-fold less than the respective control cell populations, and the pol/AS (lane 5) and env/AS (lane 6) vector transduced cells still express very low HLV-l RNA levels (Fig.20.B.). At this time point there are still comparable amount of recombinant transcript present in all cultures (lower panel). Analyses of the day 8 RNA samples ( Fig . 20 . C .
  • HLV- 1 demonstrated degradation and decreased amounts of all 3 RNA transcripts analyzed (HLV- 1 , vector transcripts and GAPDH) in the control cell populations, probably due to the massive HLV-l induced cell death in these cultures.
  • High levels of HLV-l RNA are detected in the RevMlO and vif/AS expressing cells, increased about 5-fold in the pol/AS expressing cells, but is still very low in the env/AS RNA expressing cells.
  • vectors containing longer antisense fragments are more effective inhibitors, as are vectors containing antisense to the gag, pol, and/or the env regions.
  • Combination vectors containing revMlO plus an antisense construct are more effective than vectors containing revMlO or antisense alone.
  • AAAACATCAG AAAGAACCTC CATTCCTTTG GATGGGTTAT GAACTCCATC CTGATAAATG 1260
  • AAGACTCCTA AATTTAAACT GCCCATACAA AAGGAAACAT GGGAAACATG GTGGACAGAG 360
  • AAAGTTGTCA CCCTAACTGA CACAACAAAT CAGAAGACTG AGTTACAAGC AATTTATCTA 600
  • MOLECULE TYPE cDNA (Xl) SEQUENCE DESCRIPTION SEQ ID NO 4 GAGCTCTCTC GACGCAGGAC T 21

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Abstract

L'invention concerne des nouvelles séquences antisens ayant pour cible des portions à épissage simple ou non épissées d'un produit de transcription d'ARNm provenant du provirus du VIH de type 1 (VIH-1), éventuellement exprimées en même temps qu'une protéine du VIH-1 mutante, transdominante et inhibitrice. Ces séquences sont utiles dans le traitement de l'infection à VIH-1.
EP97928151A 1996-06-06 1997-06-06 Inhibition de la replication du vih-1 au moyen de l'expression d'arn antisens Withdrawn EP0914423A2 (fr)

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US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
CA2835978C (fr) * 2000-09-01 2016-10-11 Gen-Probe Incorporated Amplification de sequences vih-1 pour la detection de sequences associees aux mutations de la resistance aux medicaments
US6582920B2 (en) 2000-09-01 2003-06-24 Gen-Probe Incorporated Amplification of HIV-1 RT sequences for detection of sequences associated with drug-resistance mutations
JP5461459B2 (ja) * 2011-03-01 2014-04-02 ジェン−プローブ・インコーポレーテッド 薬剤耐性突然変異と関連する配列検出のためのｈｉｖ−１配列の増幅
JP5461460B2 (ja) * 2011-03-01 2014-04-02 ジェン−プローブ・インコーポレーテッド 薬剤耐性突然変異と関連する配列検出のためのｈｉｖ−１配列の増幅
JP2012105648A (ja) * 2011-12-12 2012-06-07 Gen Probe Inc 薬剤耐性突然変異と関連する配列検出のためのｈｉｖ−１配列の増幅
WO2015027334A1 (fr) * 2013-08-26 2015-03-05 The Royal Institution For The Advancement Of Learning / Mcgill University Agents à base de petit arn antisens ciblant le cadre de lecture ouvert gag de l'arn du vih-1

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WO1990014427A2 (fr) * 1989-05-25 1990-11-29 Sandoz Ltd Represseur polyvalent de fonctions geniques
EP0598935A1 (fr) * 1992-11-24 1994-06-01 Bayer Ag Vecteurs d'expression et leur utilisation pour la production de cellules humaines résistantes à HIV pour utilisation thérapeutique
IL108719A0 (en) * 1993-02-25 1994-08-26 Ortho Pharma Corp Expression constructs containing hiv inhibitory antisense and other nucleotide sequences, retroviralvectors and recombinant retroviruses containing them

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Title
See references of WO9746673A2 *

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AU3255897A (en) 1998-01-05
JP2001502884A (ja) 2001-03-06
CA2254819A1 (fr) 1997-12-11
WO1997046673A3 (fr) 1998-01-15

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