EP0752000A1 - Lentivirus a encapsidation deficiente - Google Patents

Lentivirus a encapsidation deficiente

Info

Publication number
EP0752000A1
EP0752000A1 EP95912349A EP95912349A EP0752000A1 EP 0752000 A1 EP0752000 A1 EP 0752000A1 EP 95912349 A EP95912349 A EP 95912349A EP 95912349 A EP95912349 A EP 95912349A EP 0752000 A1 EP0752000 A1 EP 0752000A1
Authority
EP
European Patent Office
Prior art keywords
vector
packaging
virus
vector according
sequence
Prior art date
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.)
Withdrawn
Application number
EP95912349A
Other languages
German (de)
English (en)
Inventor
Andrew Michael Lindsay Lever
Geoffrey Philip Dept. of Medicine HARRISON
Eric The Univ. of Alabama at Birmingham HUNTER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngenix Ltd
Original Assignee
Syngenix Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from GB9405876A external-priority patent/GB9405876D0/en
Priority claimed from GB9405875A external-priority patent/GB9405875D0/en
Priority claimed from GBGB9425026.3A external-priority patent/GB9425026D0/en
Application filed by Syngenix Ltd filed Critical Syngenix Ltd
Publication of EP0752000A1 publication Critical patent/EP0752000A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • 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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15051—Methods of production or purification of viral material
    • C12N2740/15052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • This invention relates to lentiviruses, recombinant DNA technology and novel vectors for gene transfer. Background to the Invention
  • lentiviruses a subgroup of retroviruses
  • the genomic RNA species is the full-length unspliced message which is also capable of translation, encoding the gag and pol genes of the virus.
  • Lentiviruses are able to specifically encapsidate their genomic RNA, because unique cis-acting signals that are present within this RNA enable differentiation from other viral RNA species.
  • a number of RNA regions in retroviruses are reviewed by Schlesinger et al (1994), Seminars in Virology ,5:39-49, and have been identified as containing packaging signals that enable encapsidation (packaging) of genomic RNA into a virion particle.
  • the cis-acting messages involved are usually in the form of sequence and structural motifs and are found in the 5' coding regions close to the splice donor (from deletion mutagenesis studies) .
  • GCG three base motif identified by Konings et al (1992) J. Virol j66:632-640
  • This motif is often found in regions of lentiviral RNA (e.g. MPMV, SMRV-H) that are predicted to be unfolded. Therefore, whilst this motif may have an accessory function, the evidence so far suggests that it is not a major packaging signal.
  • the packaging regions of various retroviruses have undergone further secondary structure RNA modelling using biochemical analysis and free energy minimisation studies in conjunction with phylogenetic studies. Harrison and Lever (1992) J.
  • a packaging-defective lentivirus can be generated, by inactivating the known packaging signals for use as a vaccine. This has been achieved in HIV-1 by Richardson et al (1993) J. Virol. 67:3997-4005; see also WO-A-9317118 and the commonly-owned copending US Patent Application Serial No. 08/295,737, filed August 26, 1994, the contents of which are incorporated herein by reference.
  • the non- infectious virion protein coat (containing antigenic epitopes) would then be presented to the host immune system to confer protective immunity without risk of infection, as the lentivirus lacks the viral RNA that gives rise to pathogenicity. It is envisaged that 'subunit' type vaccines could be designed on the basis of further study of the antigenic epitopes to which the immune response is directed.
  • Lentiviral infections persist in cells of the macrophage/monocyte series and, in this immunologically privileged site, they are difficult to eradicate. Furthermore, pharmacological approaches to ameliorating lentiviral infection have proved difficult, due to the close relationship between virus and host processes. Even inhibitors of the virus-specific enzyme reverse transcriptase have demonstrable effects on host cell polymerases. Furthermore, lentiviruses can achieve a latent state in host cells, requiring lifelong therapeutic treatment which causes significant problems of drug toxicity due to accumulation.
  • heterologous RNA in which a lentiviral packaging signal is incorporated, could be packaged into its corresponding virion particle (instead of the normal viral RNA) .
  • This heterologous RNA may encode a gene which could then be specifically targeted to cells, using the hybrid virion, which are susceptible to a specific lentiviral infection.
  • Carrier particles and their use for gene therapy are the subject of, inter alia, WO-A-9204916 and WO-A-9211846. Carriers and methods described there may be suitable for the purposes of the present invention. Summary of the Invention
  • the present invention utilises the consensus sequence GAYC as a unique packaging signal when enclosed in a stem-loop with a positioning 5' to the gag initiation codon and where this motif is not involved in another known interaction, for example, the complementary sequence of the tRNA primer binding site in the leader sequence. If that motif is already base-pairing with another RNA such as the tRNA primer, then it is unlikely to be involved in packaging; however, if it is uninvolved in any known interaction and is in the appropriate position, it appears that the motif is involved. According to the present invention, therefore, a vector is capable of producing a lentivirus protein, but not of packaging lentivirus RNA.
  • oligonucleotides including the GAYC motif, or a complementary sequence are used to compete with viral proteins that interact with the RNA.
  • the competition serves to prevent packaging of the viral RNA.
  • a novel recombinant molecule for use as a vector may be generated.
  • This may comprise nucleotides corresponding to the packaging nucleotides of a lentivirus, a heterologous gene and, flanking the packaging nucleotides and the heterologous gene, sequences including the .GAYC motif sufficient for packaging, reverse transcription and integration of the vector into target cells, to enable expression of the heterologous gene.
  • This aspect of the invention is based on the realisation that lentiviruses are the only retroviruses able to integrate their DNA into non-dividing cells, increasing the potential target population for gene transfer. The property seems to reside in the matrix protein which probably has a nuclear localisation signal and may transport ONA through the nuclear membrane.
  • a recombinant DNA molecule comprises a gene which is functionally dependent on a metal, e.g. under the control of a non-physiological metal-dependent promoter. This is based in part on the realisation that the control of expression of genes may be affected by upstream promoter sequences. Some of these are metal-dependent, e.g. the MT2 promoter; transcription can then be activated by the appropriate cation. Further, a metal may cause activation of an enzyme that controls expression.
  • Lentiviruses to which this invention relate include MPMV, and others. Description of the Invention
  • RNA secondary structure To probe for RNA secondary structure, in vitro transcribed RNAs were either digested with structure- specific enzymes or were modified with structure-specific biochemicals.
  • the transcribed RNAs were generated from the 5' leader region of MPMV excised from the proviral clone pSHRMl ⁇ [Rhee et al (1990) Eur. J. Bioch. 7:305-318] using the SphI site at position 567 and the Sa ⁇ l site at 1561.
  • the numbering is that of RESIVMPC (Genbank accession no. M12349) . This fragment was ligated into the same sites in the expression vector pGem-4Z (Promega, Southampton, UK) and RNA was transcribed from the T7 promoter.
  • RNA samples were digested with DNAse 1, then were dissociated and reannealed by heating to 65 ⁇ C for 5 ins and cooling to 25 ⁇ C over 20 minutes.
  • RNAs were then divided into 2 ⁇ g aliquots and were modified with one of the following agents: Cobra venom RNase VI, (Pharmacia), hydroxymethylpsoralen (psoralen) (Sigma) , 2-keto-3-ethoxybutyraldehyde (kethoxal) (United States Biochemical) , RNAse Tl (Boehringer Mannheim) , and dimethyl sulphate (DMS) (Fluka) .
  • Cobra venom RNase VI (Pharmacia)
  • psoralen hydroxymethylpsoralen
  • kethoxal 2-keto-3-ethoxybutyraldehyde
  • RNAse Tl Boehringer Mannheim
  • DMS dimethyl sulphate
  • Photoreactions with psoralen were carried out under a 366 nm germicidal ultraviolet lamp. Samples were irradiated for 30 mins at room temperature in Eppendorf tubes at a distance of 20-30 mm from a Sylvania G875 tube light.
  • RNA in solution is in a dynamic state, and that alternative structures of the same regions exist simultaneously in a population of homologous molecules. Any individual RNA molecule may alternate between different conformations. This leads to some ambiguity in interpretation of biochemical data, as single stranded modifiers will be able to attack regions of RNA even if they are only transiently unpaired. A very stable double helix will however be much less vulnerable to single-stranded modification than a region which has no nearby complementary sequence.
  • Psoralen intercalates in base-paired regions, and forms covalent adducts with pyrimidines, notably uridines when irradiated with ultra-violet light at 366 nm [Youvan and Hearst (1982) Anal. Biochemistry 119:86-89 r and Bachellerie et al (1981) Nucleic Acids Res. 2:2207-2222].
  • Psoralen is found to intercalate particularly at the end of helices or in mismatches within helices [Leffers et al (1988) J. Mol. Biol. 104. / 507-522].
  • Kethoxal modifies unpaired G residues, and DMS modifies A's and C's at positions which interfere with reverse transcription.
  • RNAse Tl cleaves single-stranded RNA at G residues.
  • Fig. 1 gives a comparison of predicted stem loops in the 5' leader regions of 11 different retroviruses.
  • the murine structures which are boxed together were proposed by Tounekti et al (1992), J. Mol. Biol. 223.:205-220.
  • the MoMuLV structure was first proposed by Alford et al (1991) , Virology 183:611-619. Sequence variations for SRV-l and 2 are indicated on the MPMV stem loop. The position of the gag AUG relative to the last C of the ACC motif is indicated for each retrovirus. In the 847-853 loop of the MPMV structure, there is a triplet ACC (849 to 851) which is conserved between MPMV and SMRV-H.
  • SRV-1 and SRV-2 the purine is conserved.
  • Free energy predictions for B-type viruses and a series of distantly related retroviruses including gibbon-ape leukaemia virus (GALV) , [AKR murine leukaemia virus (AKV) , FBJ-murine osteosarcoma virus (FBJ-MSV) , Friend spleen focus-forming virus (SFFV) , FBR-MSV and caprine arthritis and encephalitis virus (CAEV) , show similar motifs in stem loop structures in analogous positions relative to the gag initiation codon ( Figure 3) which conform to the general pattern GAYC (motif) . This is also found in bovine leukaemia virus (BLV) .
  • the MPMV motif RCC is also seen in SNV and feline leukaemia virus (FeLV) at position 784.
  • the stem and loop 878-898 containing the gag initiation codon are conserved between MPMV, SRV-1 and SRV-2 and SMRV and can also adopt a similar structure.
  • the evidence strongly suggests that the GAYC motif may represent a common structural and sequence packaging motif in these viruses. Deletion mutations involving the region between the major splice site (nucleotides 303 to 304) and the gag initation codon (nucleotide 489) of the Maedi-Visna virus (MW) , which afflicts sheep, were constructed - see Figure 2.
  • Figure 3 shows deletions in an analogous region between nucleotides 493 and 534 in the untranslated leader sequence in Human Immunodeficiency Virus type 2 (HIV-2) . These resulted in a similar packaging defective phenotype.
  • the given nucleotide numbering is as in Guyader et al (1987) Nature 326:662-669.
  • a packaging-defective MW or HIV-2 leads to the production of MW or HIV-2 virus particles which are non-infectious but which have all the correct MW or HIV-2 proteins assembled in their native form. They are therefore suitable for use as a vaccine.
  • a similar approach might subsequently be used for other lentiviral disease of domestic animals such as Equine Infectious Anaemia virus and Caprine Arthritis-Encephalitis virus.
  • Vaccine formulations may be of conventional nature.
  • the desired gene can be targeted to the appropriate cell, e.g. by using a particle or vector. A dose of the appropriate cation can be targeted to the same cell (particles) .
  • the cation may be, for example, cadmium (which is rare, non- physiological, and toxic if in the wrong place at the wrong concentration) .
  • the double targeting step provides a desirable degree of safety, cell-targeting specificity and also flexible control of transcription, by giving doses of cation-containing particle when desired.
  • the foreign gene may be, for example, for a ribozyme.
  • Ribozymes are RNA enzymes capable of cleaving RNA target sequences. They are potential antiviral agents. Their function may be to switch off expression of other genes, e.g. oncogenes. Ribozymes can be encoded on plasmids grown in bacteria. Bacterial growth may be made ribozyme-dependent, i.e.
  • Ribozymes are divalent cation-dependent, probably for stability rather than at the active site. By including in the medium for growth high concentrations of a particular non-physiological cation, e.g. strontium, ribozymes may be evolved which work optimally or only in the presence of the cation. An antiviral/anti-oncogene ribozyme is then made, and into it is incorporated the cation-dependent sequence.
  • the ribozyme may now be delivered to the target cell, followed by the strontium or other cation.
  • the delivery of each may be achieved using particles, for example by a technique described in WO-A-9204916 or WO-A-9211846.
  • the ribozyme cannot be activated in cells without the cation, and therefore will not inhibit physiological processes, e.g. a normal short burst of expression of the oncogene.

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Abstract

L'invention concerne des lentivirus à encapsidation déficiente. En particulier, un vecteur est capable de produire des protéines d'un virus sélectionné à partir de MVV (virus de Maedi-Visna) et de VIH-2, mais non d'encapsider l'ARN viral.
EP95912349A 1994-03-24 1995-03-24 Lentivirus a encapsidation deficiente Withdrawn EP0752000A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
GB9405876 1994-03-24
GB9405876A GB9405876D0 (en) 1994-03-24 1994-03-24 Modified virus and its use as a vector
GB9405875 1994-03-24
GB9405875A GB9405875D0 (en) 1994-03-24 1994-03-24 Recombinant molecules and their use in gene transfer
GB9425026 1994-12-09
GBGB9425026.3A GB9425026D0 (en) 1994-12-09 1994-12-09 Packaging-defective vectors
PCT/GB1995/000663 WO1995025806A2 (fr) 1994-03-24 1995-03-24 Lentivirus a encapsidation deficiente

Publications (1)

Publication Number Publication Date
EP0752000A1 true EP0752000A1 (fr) 1997-01-08

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EP95912349A Withdrawn EP0752000A1 (fr) 1994-03-24 1995-03-24 Lentivirus a encapsidation deficiente

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EP (1) EP0752000A1 (fr)
JP (1) JPH09510361A (fr)
WO (1) WO1995025806A2 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013516A (en) * 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
EP1017797B1 (fr) 1997-09-24 2005-06-22 The Regents Of The University Of California Vecteurs de lentivirus non originaires de primates et systemes d'encapsidation
US6790657B1 (en) 1999-01-07 2004-09-14 The United States Of America As Represented By The Department Of Health And Human Services Lentivirus vector system
FR2979919B1 (fr) 2011-09-12 2015-12-11 Centre Nat Rech Scient Genomes lentiviraux chimeriques non-integratifs comme vaccins innovants contre vih-1

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU671101B2 (en) * 1992-02-28 1996-08-15 Syngenix Limited Defective packaging non-oncoviral vectors based on MPMV
EP0633942A1 (fr) * 1992-03-27 1995-01-18 Whitehead Institute For Biomedical Research Particules de hiv non infectieuses et utilisations
GB2269175A (en) * 1992-07-31 1994-02-02 Imperial College Retroviral vectors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9525806A2 *

Also Published As

Publication number Publication date
WO1995025806A2 (fr) 1995-09-28
WO1995025806A3 (fr) 1996-01-04
JPH09510361A (ja) 1997-10-21

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