EP0536234A1 - Vecteurs contenant des sequences d'encapsidation du vih, vecteurs du vih a capside defectueux, et leur utilisation - Google Patents

Vecteurs contenant des sequences d'encapsidation du vih, vecteurs du vih a capside defectueux, et leur utilisation

Info

Publication number
EP0536234A1
EP0536234A1 EP91912130A EP91912130A EP0536234A1 EP 0536234 A1 EP0536234 A1 EP 0536234A1 EP 91912130 A EP91912130 A EP 91912130A EP 91912130 A EP91912130 A EP 91912130A EP 0536234 A1 EP0536234 A1 EP 0536234A1
Authority
EP
European Patent Office
Prior art keywords
hiv
vector
gene
nucleotides
sufficient number
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Withdrawn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
EP91912130A
Other languages
German (de)
English (en)
Other versions
EP0536234A4 (en
Inventor
Joseph G. Sodroski
William A. Haseltine
Mark Poznansky
Andrew Lever
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.)
Dana Farber Cancer Institute Inc
Original Assignee
Dana Farber Cancer Institute Inc
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
Application filed by Dana Farber Cancer Institute Inc filed Critical Dana Farber Cancer Institute Inc
Publication of EP0536234A1 publication Critical patent/EP0536234A1/fr
Publication of EP0536234A4 publication Critical patent/EP0536234A4/en
Withdrawn legal-status Critical Current

Links

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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—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
    • C12N2740/16051—Methods of production or purification of viral material
    • C12N2740/16052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • 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
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is directed to vectors including vectors comprising a packaging defective HIV provirus, a vector comprising a HIV packaging sequence and a gene to be transferred, the use of the packaging defective vectors to create HIV packaging defective cell lines, and the uses of the vectors and cell lines.
  • the HIV provirus is an HIV-1 provirus.
  • the human immunodeficiency virus (HIV-I, also referred to as HTLV-III, LAV or HTLV-III/LAV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders [Barre-Sinoussi, et al., Science 220:868-871 (1983); Gallo et al, Science 224:500-503 (1984); Levy et al., Science 225:840-842 (1984); Popovic et al., Science 224:497-500 (1984); Sarngadharan et al., Science 224:506-508 (1984); Siegal et al. , N. Enel. J. Med.
  • AIDS acquired immune deficiency syndrome
  • the disease is characterized by a long asymptomatic period followed by progressive degeneration of the immune system and the central nervous system.
  • Studies of the virus indicate that replication is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T-lymphocytes occur in tissue culture [Zagury et al. , Science 231:850-853 (1986)].
  • the expression of the virus in infected patients also appears to be regulated to enable evasion of the immune response.
  • HIV-I Molecular studies of the regulation and genomic organization of HIV-I show that it encodes a number of genes [Ratner et al., Nature 313:277-284 (1985); Sanchez-Pescador et al., Science 227:484-492 (1985); Muesing et al. , Nature 313:450-457 (1985); ain-Hobson et al., Cell 40:9-17 (1985)].
  • the other primate immunodeficiency viruses, HIV-2 and simian immunodeficiency virus (SIV) also share many of the same structural and regulatory genes such as gag, pol. env. tat, rev and nef [Guyader, M. , et al., Nature 326:662-669
  • Retroviruses are typically classified as belonging to one of three subfamilies, namely oncoviruses, spu aviruses and lentiviruses. Infection by oncoviruses is typically associated with malignant disorders. These viruses typically contain a single-stranded, plus-strand RNA genome of approximately 8,000 to 10,000 nucleotides encompassing the pap, pol and env. genes, as well as long terminal repeat (LTR) sequences. Oncoviruses typically contain an oncogene. It is generally believed that spumaviruses are not pathogenic in vivo, although they induce foamy cytopathic changes in tissue culture. Infection by lentiviruses is generally slow and causes chronic debilitating diseases after a long latency period. These viruses, in addition to the gag, pol. and env genes possess a number of additional genes with regulatory functions.
  • HIV human immunodeficiency viruses
  • Retroviruses share features of the replicative cycle, including packaging of viral RNA into virions, entry into target cells, reverse transcription of viral RNA to form the DNA provirus, and stable integration of the provirus into the target cell genome [Coffin, J. , __ Gen. Virol. 42:1-26 (1979)].
  • Replication-competent proviruses at a minimum, contain regulatory long terminal repeats (LTRs) and the gag, pro, pol. and env genes which encode core proteins, a protease, reverse transcriptase/RNAse H/integrase and envelope glycoproteins, respectively [J. Gen. Virol. 42, supra1.
  • the LTRs contain cis-acting sequences important for integration, transcription and polyadenylation.
  • the long terminal repeats (LTRs) of HIV contain cis-acting sequences that are important for integration, transcription and polyadenylation.
  • the tat gene encodes a 14kD protein that is critical for HIV replication and gene expression [Rosen, C.A., et al., Nature 319:555-559 (1986);
  • cis-acting sequences located between the 5' LTR and the gag gene initiation codon have been located which are necessary for. the efficient packaging of the viral RNA into virions [Bender, M.A. , et al, J. Virol 61:1639-1646 (1987), Katz, R.A. , et al, J. Virol 59:163-167 (1986), Mann, R. , et al, Cell 31:153-159 (1983), Pugatsch, T., et al, Virology 128:505-511 (1983), Watanabe, S., et al, Proc. Natl. Acad. Sci.
  • Vectors containing the desired gene and packaging sequences were incorporated by packaging signal-deleted viruses generating virions capable of entry into certain cells.
  • the signals needed for packaging of lentiviruses RNA were incorporated by packaging signal-deleted viruses generating virions capable of entry into certain cells.
  • the packaging-defective provirus vector it would be possible to create packaging defective cell lines that could be used to investigate the packaging mechanism of the virus and to develop strategies to interfere with this packaging mechanism.
  • the virions produced by such packaging negative proviruses could be used for vaccines and as a system for efficiently introducing a desired gene into a mammalian cell. It would also be useful to have a vector that could be selectively targeted to HIV target cells and could thus introduce a desired product into such cells.
  • HIV packaging sequence corresponds to the region between the 5' major splice donor and the gag gene initiation codon (nucleotides 301-319). More preferably, this sequence corresponds to a segment just downstream of the 5' major splice donor, and about 14 bases upstream of the gag initiation codon. In one embodiment it is a 19 base segment having the sequence AAAAATTTTGACTAGCGGA.
  • This vector can be used to transform a preselected cell line to result in an HIV packaging defective cell line.
  • each vector does not have a sufficient number of nucleotides corresponding to nucleotides of the HIV genome between the 5' major splice donor and the gag gene to efficiently package HIV RNA. More preferably, each vector would not contain a sequence corresponding to an LTR sequence downstrea of the nucleotides corresponding to the HIV genes.
  • each vector would contain a different marker gene.
  • the transformed cell line would express HIV virions but would not be able to package HIV RNA into these virions.
  • these virions could be used to generate antibodies, as a vaccine or as a method of transferring a desired gene product to a different cell line capable of infection by HIV.
  • a second vector contains a preselected gene, a sufficient number of nucleotides corresponding to an HIV packaging sequence to package HIV RNA (HIV packaging sequence) , and is flanked on each side with a sequence corresponding to a sufficient number of HIV LTR nucleotides to be packaged by the HIV packaging sequence (HIV LTR sequences), wherein the HIV packaging sequence and HIV LTR sequences correspond to the same HIV genome.
  • This vector can be used with the packaging defective vectors to transfer the desired preselected gene.
  • the vector can be administered to an HIV infected cell and be packaged by the HIV virions being produced.
  • the HIV infected cell can be in an individual. Combinations where the HIV packaging defective vector and HIV virus as a helper virus are used together are also described.
  • the packaging sequences are located in a region from the 5' major splice donor to a site within the 5' most part of the gag gene.
  • Figure 1 is a schematic of the HIV-1 genome from the 5' LTR to the gag initiation codon showing the 5' major splice donor (SD) and the site of the deletion in a vector representing one embodiment of this invention, pHXB PI.
  • Figure 2a-e represents schematics of vectors representing various embodiments according to this invention.
  • Figure 2a is a packaging defective vector, HXB ⁇ P1.
  • Figure 2b is a packaging defective vector, HXB ⁇ Pl ⁇ env.
  • Figure 2c is a packaging defective vector, pSVIIIenv 3-2.
  • Figure 2d is a packaging proficient vector, HVB(SL3-Neo) .
  • Figure 2e is a packaging proficient vector, HVB(SL3-Neo) .
  • Figure 3 is a schematic of one preferred embodiment showing two packaging deficient vectors, HXB ⁇ Pl ⁇ env and pSVIIIenv 3-2.
  • Figure 4 is a chart showing p24 Levels in Culture of Infected Jurkat Cells by vectors representing various embodiments of this invention.
  • Figure 5a is an autoradiogram of the immunoprecipitation of 35SS--llaabbeelllleedd vviirraall pprrootteeiinn ffrroomm CCOOSS--11 cells transfected with pHXB ⁇ Pl DNA with AIDS patient serum.
  • Figure 5b is an electron micrograph of COS-l cells transfected with pHXB ⁇ Pl showing virion particles of normal HIV-1 morphology.
  • Figure 6 is an autoradiogram of immunoprecipitation of labelled viral proteins from Jurkat T cell lysates or supematants exposed to supematants from COS-l cells that were transfected or mock transfected.
  • Figure 7 is an RNA dot blot test.
  • Figure 8 is an autoradiogram showing the Sourthern blot of total DNA of G418-resistant Jurkat cells. Detailed Description of the Invention
  • HIV packaging defective vectors and cell lines We have now discovered that it is possible to make HIV packaging defective vectors and cell lines. We have found that the region between the 5' major splice donor and the gag gene initiation codon in HIV viruses contains sequences necessary for packaging of HIV RNA into virions.
  • RNA (the HIV packaging sequence) .
  • sequences preferably correspond to the genome of HIV-1, HIV-2 and simian immunodeficiency virus (SIV) .
  • SIV simian immunodeficiency virus
  • the vector does not contain the HIV packaging sequence corresponding to the segment immediately downstream of the 5' major splice donor and just upstream of the gag gene initiation codon.
  • the .vector could contain nucleotides ranging from about 14 bases to 2 bases upstream of the gag initiation codon (for example either the 14 upstream bases or 5 upstream bases) and still be packaging deficient.
  • the vector does not contain a nucleotide sequence beginning about 9 bases downstream of the
  • nucleotides 301-319 is sufficient to result in loss of packaging ability (See Figure 1). However, even smaller deletions in this region should also result in loss of packaging efficiencies. Indeed, it is expected that a deletion as small as about 5 base pairs in this region should remove packaging ability. Thus the size of a particular deletion can readily be determined based upon the present disclosure by the person of ordinary skill in the art.
  • the vector should contain an HIV nucleotide segment containing a sufficient number of nucleotides corresponding to nucleotides of the HIV genome to express functional HIV gene products, but as aforesaid, should not contain a sufficient number of nucleotides corresponding to the region between the 5' major splice donor and the gag gene initiation codon to permit efficient packaging of the viral RNA into virions.
  • HIV packaging defective cell lines it is preferred that such cell lines do not produce any infectious HIV.
  • a cell line transformed by these packaging deficient vectors would have low infectivity because the cells are packaging defective, some RNA can still be packaged into the virion.
  • the HIV nucleotide segment does not correspond to the entire HIV genome so that if some of the viral RNA is packaged into the virion, what is packaged will not be a replication competent virus.
  • the vector would not contain sequences corresponding to an HIV LTR but would contain sequences corresponding to a promoter region and/or another genome's polyadenylation sequences. Selection of particular promoters and polyadenylation sequences can readily be determined based upon the particular host cell.
  • the LTR which the sequences do not correspond to is the 3' LTR. For example, see Figure 3.
  • one vector would include sequences permitting expression of HIV proteins upstream of env and the second vector would permit expression of the remaining proteins.
  • one vector would contain an HIV nucleotide segment corresponding to a sufficient number of nucleotides upstream of the gag initiation codon to the env gene sequence to express the 5'-most gene products.
  • the other vector would contain an HIV nucleotide segment corresponding to a sufficient number of nucleotides downstream of the gag gene sequence and including a functional env gene sequence.
  • Such vectors can be chemically synthesized from the reported sequences of the HIV genomes or derived from the many available HIV proviruses, by taking advantage of the known restriction endonuclease sites in these viruses by the skilled artisan based upon the present disclosure ( Figure 3).
  • a different marker gene to each vector, i.e., co-transfect a preselected cell line with these different vectors and by looking for a cell containing both markers, one would have a cell line that has been co-transfected with the two vectors.
  • RNA corresponding to the entire viral sequences would not be packaged in these virions.
  • a second vector would contain other HIV or SIV genes and contain a deletion in the packaging sequence and a deletion for the env gene.
  • This vector would also not have a 3' LTR, but would have a polyadenylation sequence.
  • a vector which would not contain a sufficient number of nucleotides corresponding to HIV packaging sequence to package HIV RNA, but would contain a nucleotide segment corresponding to a sufficient number of nucleotides corresponding to a sufficient number of nucleotides of the HIV gag and pol genes to express functional gag and pol products.
  • this vector would also contain a sufficient number of nucleotides corresponding to a functional tat gene. The vector would not contain a sufficient number of nucleotides to encode a functional env protein.
  • the vector would also contain nucleotides corresponding to other HIV regulatory genes to express functional gene products, such as vpr. vpu. vif, etc.
  • Other combinations of vectors can also be prepared.
  • a vector that does not contain a sufficient number of nucleotides to correspond to a functional gag gene, but would have a sufficient number of nucleotides to correspond to functional pol and env genes other vectors include one that does not contain a sufficient number of nucleotides to encode a functional pol protein, but would have a sufficient number of nucleotides to encode a different functional HIV protein, etc.
  • the term a.sufficient number of nucleotides permits additions, deletions and substitutions as long as the claimed functional ability is not lost. For example, if one is referring to the functional ability of packaging HIV RNA then the resultant vector must have a sequence that can package HIV RNA.
  • HIV-1 can be pseudotyped with the envelope glycoproteins of other viruses. [(Lusso, P., et al., Science 247:848-851 (1990)]. Consequently, one can prepare a vector containing a sufficient number of nucleotides to correspond to a functional env gene from a different retrovirus. Preferably, the 5' LTR of this vector would be of the same genome as the env gene. Such a vector could be used instead of an env packaging deficient vector to create virions. By such a change, the resultant vector system can be used in a wider host range.
  • Virtually any cell line can be used.
  • a mammalian cell line for example, CV-1, Hela, Raji, RD, SW480 or CHO cell lines.
  • CV-1 a mammalian cell line
  • Hela a mammalian cell line
  • Raji a mammalian cell line
  • RD a mammalian cell line
  • SW480 CHO cell lines
  • a different promoter than the 5' LTR i.e., replace the 5' LTR with a promoter that will preferentially express genes under its control in a particular cell line.
  • the CMV promoter will preferentially express genes in CV-1 or Hela cells.
  • the particular promoter used can be readily determined by the person of ordinary skill in the art, based upon the particular host cell line to be used.
  • enhancer sequences can readily be determined by the person of ordinary skill in the art depending upon the host cell line.
  • viral enhancer proteins such as those of herpes virus, hepatitis B virus, which act on HIV LTRs to enhance the level of virus product, or cellular transactivator proteins.
  • Cellular transactivation proteins include NF ⁇ -B, UV light responsive factors and other T cell activation factors well known to the person of ordinary skill in the art.
  • the cells By using a series of vectors that together would contain the complete HIV genome, one can create cell lines that produce a virion that is identical to the HIV virion, except that the virion does not contain the HIV RNA.
  • the virions can readily be obtained from these cells. For example, the cells would be cultured and supernatant collected. Depending upon the desired use the supernatant containing the virions can be used or these virions can be separated from the supernatant by standard techniques. Typically, this would include gradiant centrifugation, filtering, etc.
  • the virions can be used to generate an antigenic response to the HIV virions and because these virions are identical to the actual HIV virions, except that the interior of these virions do not contain the viral RNA, the vaccine created should be particularly useful.
  • virions can also be used to raise antibodies to the virion that can then be used for a variety of purposes, e.g. screening for the virion, developing target system for the virions, etc.
  • HIV packaging deficient cell lines can be extremely useful as a means of introducing a desired gene, for example, a heterologous gene into mammalian cells.
  • virions could be used as an extremely efficient way to package desired genetic sequences into target cells infectable by HIV. This would be done by preparing a vector containing a nucleotide segment containing a sufficient number of nucleotides corresponding to the packaging nucleotides of the HIV virus (HIV packaging region) , a predetermined gene, and flanking the packaging sequence and the predetermined gene with sequences corresponding to a sufficient number of sequences from within and near the LTRs for packaging, reverse transcription, integration and gene expression.
  • HIV packaging region HIV packaging region
  • predetermined gene flanking the packaging sequence and the predetermined gene with sequences corresponding to a sufficient number of sequences from within and near the LTRs for packaging, reverse transcription, integration and gene expression.
  • the packaging region used would preferably correspond to at least the region between the 5' major splice donor and just upstream of the gag initiation codon, more preferably the region between the 5' major splice donor and the Bal I site (2202 in HIV-1) in the gag gene.
  • a sufficient number of HIV-1 sequences to be packaged, reverse transcribed, integrated and expressed in the target cells would inlclude the U3, R and U5 sequences of the LTRs, the packaging sequences, and some sequences flanking the LTRs (required for reverse transcription) . From the 5' LTR, the R and U5 regions would be included which, in
  • HIV-1 extend from +1 to 183.
  • LTR necessary for reverse transcription and packaging would extend from 183 to about 335. Although not wishing to be bound by theory, applicants believe the inclusion of additional sequences from the gag gene in the vector (up to the Bal I site, nucleotide 2202) should enhance packaging efficiency.
  • the regions from the 3' LTR and the immediate flanking sequences to be included extend from about 8645 to about 9213 (U3 and R regions) . Analagous regions would be included in a vector based upon HIV-2 or SIV.
  • this vector is used to transfeet one of the HIV packaging deficient cells, it is the nucleotide sequence from this vector that will be packaged in the virions.
  • These "HIV packaged" genes could then be targeted to cells infectable by HIV. This method of transformation is expected to be much more efficient than current methods. Further, by appropriate choice of genes, one could also monitor the method of HIV infection.
  • the vector could contain a sufficient number of nucleotides corresponding to both 5' and 3' LTRs of HIV-1, HIV-2 or SIV to be expressed, reverse transcribed and integrated, a sufficient number of nucleotides corresponding to an HIV packaging sequence to be packaged, for example a segment between the 5' major splice donor and just upstream of the gag initiation codon (e.g., nucleotide 381).
  • the vector would also contain a sufficient number of nucleotides of the gene which is desired to be transferred to produce a functional gene (e.g., gene segment).
  • the gene can be any gene desired, for example, the gene for neomycin phosphotransferase (Neo ) .
  • the "gene” would express a product that adversely affects HIV replication or integration such as a trans-dominant inhibitor, anti-sense RNAs, catalytic RNAs or soluble CD4 derivative.
  • the vector of the present invention can be used to target HIV target cells.
  • Preferred promoters include viral promoters, such as SL-3, murine retroviral LTR, etc. Enhancers sequences are also preferably used in the vector.
  • the desired gene can be inserted in the present vector, in either the sense or anti-sense orientation with respect to the
  • the vector can contain more than one gene or pseudogene sequence, permitting the expression of multiple genes of interest.
  • This vector can preferably be used with the packaging-defective vectors described above. In such a situation, one preferably uses HIV LTRs in the vector corresponding to the genome of the package-deficient vector to facilitate packaging efficiency. However, in addition to use with a packaging-deficient virus, this vector can also be used with helper virus for gene transfer. For example, when one wants to deliver a gene to treat an individual infected with AIDS, one could insert this vector into that individual and it would be incorporated into HIV virions being produced in that individual. This would facilitate the delivery of the desired gene to the appropriate target cell.
  • trans-dominant inhibitors anti-sense RNAs, catalytic RNAs, or soluble CD4 derivatives which are also aimed at inhibiting HIV-1 functions critical for viral replication.
  • HIV packaging defective cell lines can be used to study various stages of the HIV life cycle, both in vivo and in vitro systems by a system because the cells will express HIV cellular proteins, but will not package the RNA.
  • FIG. 1 shows the 5' major splice donor (SD) and site of deletion in a vector described below, pHXB ⁇ Pl.
  • SD 5' major splice donor
  • pHXB ⁇ Pl pHXB ⁇ Pl.
  • a 19 base-pair deletion in this region was created in an infectious HIV-1 proviral clone contained on the plasmid pHXBc2 of Fisher, A.G. , et al, Nature 316: 262-265 (1985).
  • This plasmid also contains an SV40 origin of replication to allow efficient gene expression in COS-l cells.
  • the mutation was produced by the site-directed mutagenesis as described in Kunkel, T.A. , et al, Methods in
  • the mutated plasmid was designated pHXB ⁇ Pl.
  • COS-l cells were maintained in Dulbecco's modified Eagle's medium DMEM (Hazelton Biologies, Lenexa) supplemented with 10% fetal calf serum (Gibco, Long Island, NY) and antibiotics.
  • DMEM Dulbecco's modified Eagle's medium
  • Jurkat cells were maintained in culture in RPMI 1640 with 10% fetal calf serum and antibiotics.
  • Jurkat cells were preventatively treated for mycoplasma with B.M. Cyclin I and II and human serum two weeks prior to infection.
  • DMEM Dulbecco's modified Eagle's medium
  • DMEM Hemazelton Biologies, Lenexa
  • fetal calf serum Gibco, Long Island, NY
  • HXBc2 (gag "1" . pro + . pol + . vif + . vpr " . ____ ' , tat + , rev "1” . env + . nef " ) provirus was used in all plasmids was used in all plasmid and vector constructs.
  • COS-l cells were transfected with the pHXBc2 and pHXB ⁇ Pl plasmids by the DEAE-dextran procedure [Lopata et al, Nucl. Acids Res. 12:5707-5717 (1984); Queen and Baltimore, Cell 33:741-748 (1983); (Sodroski, J., et al, Science 231:1549-1553 (1986) which are incorporated herein by reference] .
  • COS-l cell lysates and supematants radiolabelled with 35S-cysteine
  • COS-l cell transfected by pHXB ⁇ Pl was 60% of that measured in cells transfected with the HXBc2 vector (data not shown).
  • COS-l cells transfected with pHXB ⁇ Pl were fixed 48 hours following transfection and examined by electron microscopy. Viral particles, including budding forms, of normal HIV-1 morphology were observed.
  • Figure 5B is an electron micrographs of COS-l cells transfected with
  • PHXB ⁇ Pl showing virus particles of normal HIV-1 morphology.
  • supe atants from COS-l cells transfected with pHXBc2 and pHXB ⁇ Pl were filtered (0.2 / .) and RT measured.
  • Supematants containing equal amounts of RT activity of mutant and wild-type viruses were added to Jurkat human T lymphocytes.
  • the Jurkat cultures along with a mock-infected culture were maintained with medium changes every three days. At intervals aliquots of Jurkat cells were labelled and assessed for expression of HIV-1 proteins by immunoprecipitation with 19501 AIDS patient serum.
  • Figure 6 shows immunoprecipitation of labelled viral proteins from Jurkat T cell lysates (lanes 1-3, 7-9 and 13-15) or supe atants (lanes 4-6, 10-12 and 16-18) exposed to supe atants from COS-l cells that were mock transfected (lanes 1, 4, 7, 10, 13 and 16), pHXBc2 (lanes 2, 5, 8, 11, 14 and 17), or transfected with pHXB ⁇ Pl (lanes 3, 6, 9, 12, 15 and 18) .
  • the Jurkat cells were examined at day 7 (lanes 1-6), day 14 (lanes 7-12) and day 21 (13-18) following infection.
  • Jurkat cultures exposed to HXB ⁇ P1 exhibited marked delays in and lower levels of viral protein production relative to those exposed to pHXBc2, the wild-type virus.
  • Virus replication in human T lymphocytes transfected by HXB ⁇ P1 is thus seen to be significantly attenuated compared with cells transfected by HXBc2.
  • Supematants from the above cultures were 0.2 -filtered and equivalent amounts of reverse transcriptase activity pelleted by centrifugation at 12000xg for one hour at 20°C.
  • Viral pellets were lysed by NP40 in the presence of vanadyl ribonucleotides and dilutions of virus dot-blotted onto nitrocellulose filters.
  • Some samples were treated with sodium hydroxide (5M at 60°C for 15 minutes) prior to dot-blotting. Filters were hybridized with a DNA probe consisting of HIV-1 gag and env gene sequences, washed and autoradiographed as previously described in
  • Figure 7 is an RNA dot blot without (column 1) and with (column 2) sodium hydroxide treatment following blotting of filtered supematants from the Jurkat cultures.
  • the supematants contained a reverse transcriptase activity of 5 x 10 4 cpm of HXB ⁇ P1 (row A) , 5 x 10 4 cpm of HXBc2
  • a mutation in this region exhibits minimal effects on the ability of the provirus to produce proteins and virion particles following transfection, but markedly decreases the level of virion RNA and attenuates virus replication in a human CD4-positive lymphocyte line. HIV-1 replicates in cultured CD4-positive cells via cell-free transmission and cell-to-cell transmission, the latter involving the contact of infected and uninfected cells
  • pHVB(SL3-Neo)sense (pHVB (SL3-Neo)) and pHVB (SL3-Neo)anti-sense (PHVB (SL3-Neo)) plasmids contain the coding sequence of the neo gene.under the control of the SL3-3 murine leukemia virus LTR, with polyadenylation signals derived from SV40.
  • the ⁇ HVB(SL3-Neo)sense and pHVB(SL3-Neo) anti-sense plasmids contain complete 5' and 3' HIV-1 LTRs and flanking viral sequences nucleotides 183-381 near the 5' LTR and nucleotides 8504-8661 adjacent to the 3' LTR.
  • a unique Bam HI site was inserted at the boundaries of the major deletion in the HXBc2 provirus (nucleotides 382 to 8593) , and the SL3 LTR-Neo transcription unit was cloned into this site in either the sense (pHVB(SL3-Neo)) or antisense (pHVB(SL3-Neo)) orientation with respect to the HIV-1 LTRs. All of the plasmids contained SV40 origins of replication. See Figure 2. These vectors include the 19 base pair sequence shown by deletion to be important for packaging viral RNA. The vectors cannot encode any of the HIV-1 gene products.
  • the vectors contain an insert in which the SL3-3 murine retroviral LTR, which functions as an efficient promoter in T lymphocytes, promotes the expression of the neomycin phosphotransferase (Neo ) gene.
  • the polyadenylation signals for the Neo transcript are provided by sequences from SV40. Numbers above the plasmid in Figure 2 indicate the nucleotide of the HXBc2 sequence that form the boundaries of provirus/insert.
  • the SL3-3 murine leukemia virus LTR is indicated SL3.
  • the position of the major 5' splice donor (SD) and gag gene initiation codon (pagATG) are shown in the figure.
  • DMEM containing FCS and antibiotics was placed in COS-l cell cultures 12 hours after transfection.
  • COS-l supematants were harvested and filtered through a 0.2 ⁇ m filter (Millipore) 72 hours post transfection.
  • the p24 level in the COS-l supematants was determined by a p24 radioimmunoassay (Dupont) .
  • Jurkat cells to be infected with virus were seeded into 6-well culture plates at a 2.5 X 10 cells per well in 2.5 ml of complete medium.
  • Ten-fold serial dilutions of transfected COS-l supematants were then applied to each well and the virus allowed to ' absorb to the Jurkat cells at 37°C for 4 hours. After this the Jurkat cells were pelleted and resuspended in complete medium. Twenty-four hours later the medium was replaced by complete medium containing G418 (Gibco, NY) at an active concentration of 0.8 mg/ml and the cells dispensed into 24-well culture plates at 1.0 X 10 cells per well. Culture medium was changed every 4 days.
  • the cells were washed in phosphate-buffered saline and diluted to a concentration of 0.5 viable cells per 100 ⁇ l medium. Then 100 ⁇ l of the cell suspension was dispensed into each well of a 96-well culture plate. Wells containing single cells were identified by phase contract microscopy and individual cells were expanded to 10 cells in complete medium containing 0.8 mg/ml G418.
  • Genomic DNA was prepared from clones, digested with Sad and Southern blotted as previously described by Southern, E.M, J. Mol. Biol. 98:503-517 (1975). Southern blots were hybridized to a 3.3 Kb fragment containing the SL3 LTR, neo gene and SV40 polyadenylation sequences that had been labelled by random priming with oligonucleotides. Southern blots were washed under conditions of high stringency.
  • the pHXB ⁇ Pl plasmid and the HIV-1 vectors were cotransfected into COS-l cells as described above. Table 1 shows that gag p24 protein of HIV-1 was detectable in the supernatant of these transfected cells on the third day after transfection.
  • the filtered COS-l supematants were serially diluted and incubated with Jurkat lymphocytes, which were selected for G418-resistance.
  • the number of G418-resistant Jurkat cells generated ranged from 10 to 10 per milliliter of COS-l supernatant, with the HVB(SL3-Neo)anti-sense vector yielding higher titers than the HVB(SL3-Neo)sense vector.
  • No G418-resistant Jurkat cells were generated following incubation with supernatant derived from COS-l cells transfected with no DNA, the vectors alone, or the pHXB ⁇ Pl plasmid alone.
  • the HXB ⁇ P1 provirus is not completely replication defective.
  • the production of viral p24 antigen and the formation of syncytia were examined to determine the amount of infectious virus In the G418-resistant Jurkat cells.
  • Figure 4 shows that HIV-1 p24 antigen was detectable in the supematants of the Jurkat cultures. Syncytium formation was visible and increased with time in these cultures, indicating the expression of the HIV-1 envelope glycoproteins in the target cells. The induction of significant cytophathic effect in these cultures made cloning of the G418-resistant Jurkat cells difficult, and further suggested the presence of replication-competent viruses in the target cells. See also Table 2.
  • the pHXB ⁇ Pl ⁇ env plasmid is identical to the pHXB ⁇ Pl plasmid except that the provirus in the former contains a deletion in the env gene and contains a polyadenylation signal from SV40 in place of the 3' LTR.
  • the pSVIIIenv3-2 plasmid encodes both rev and env genes of HIV-1 under the control of the HIV-1 LTR, with polyadenylation signals derived from SV40.
  • p24 gag protein was measured in cell supematants and syncytia were scored up to 40 days following the initial infection. In all of the clones examined, no syncytia were observed and p24 antigen was undetectable. See Table 2. This indicates that no replication-competent virus was present in these G418-resistant Jurkat cells.
  • a Syncytia were scored according to the following criteria: -, no syncytia observed; +, 1-5 syncytia/hpf; ++, 5-10 syncytia/hpf; +++, greater than 10 syncytia/hpf (hpf-high power field) .
  • the packaging-defective HXB ⁇ P1 provirus can provide trans-acting viral functions required for the transfer of a HIV-1 vector to Jurkat lymphocytes.
  • the transfer of vector sequences occurs in the apparent absence of replication-competent virus.
  • the titers of recombinant virus in this helper virus-free context appear to be improved relative to those observed in the presence of replication-competent virus, probably because of the induction of significant cytopathic effects by the latter.
  • the higher titer observed for the HVB(SL3-Neo)anti-sense vector relative to the HVB(SL3-Neo)sense vector in the presence of replication-competent virus may in part relate to a suppresive effect of anti-sense read-through transcripts from the SL3 promoter on helper virus replication.
  • HIV-1 vector described herein provides a simple, efficient means of introducing individual genes of interest into potential HIV-1 target cells.
  • HIV-1 can be pseudotyped with the envelope glycoproteins of other viruses, increasing the host range of these vectors is feasible. Given the ability of the wild-type HIV-1 genome to encode multiple gene products, these vectors are readily adaptable for the expression of multiple genes of interest in the target cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Vecteurs à capside défectueux et à capside intact du VIH. Ces vecteurs peuvent être utilisés pour détecter les lignées cellulaires à capside défectueux du VIH, et pour encapsider les gènes voulus. Ces lignées cellulaires peuvent être utilisées pour la mise au point d'un vaccin, pour la découverte d'anticorps du VIH et en tant que partie d'un système de transfert de gènes. Le vecteur à capside intact peut être utilisé pour atteindre des cellules cibles du VIH.
EP19910912130 1990-06-20 1991-06-18 Vectors containing hiv packaging sequences, packaging defective hiv vectors, and uses thereof Withdrawn EP0536234A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US54074690A 1990-06-20 1990-06-20
US540746 1990-06-20

Publications (2)

Publication Number Publication Date
EP0536234A1 true EP0536234A1 (fr) 1993-04-14
EP0536234A4 EP0536234A4 (en) 1993-09-15

Family

ID=24156769

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19910912130 Withdrawn EP0536234A4 (en) 1990-06-20 1991-06-18 Vectors containing hiv packaging sequences, packaging defective hiv vectors, and uses thereof

Country Status (4)

Country Link
EP (1) EP0536234A4 (fr)
JP (1) JP3264281B2 (fr)
CA (1) CA2084659A1 (fr)
WO (1) WO1991019798A1 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5861282A (en) * 1989-10-16 1999-01-19 Whitehead Institute For Biomedical Research Non-infectious HIV particles and uses therefor
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
WO1993025698A1 (fr) * 1992-06-10 1993-12-23 The United States Government As Represented By The Particules vecteurs resistantes a l'inactivation par le serum humain
WO1994016736A1 (fr) * 1993-01-22 1994-08-04 University Research Corporation Localisation d'agents therapeutiques
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
US6242187B1 (en) 1996-01-29 2001-06-05 Virologic, Inc. Compositions and methods for determining anti-viral drug susceptibility and resistance and anti-viral drug screening
US5837464A (en) * 1996-01-29 1998-11-17 Virologic, Inc. Compositions and methods for determining anti-viral drug susceptibility and resistance and anti-viral drug screening
US6200811B1 (en) * 1996-04-02 2001-03-13 The Regents Of The University Of California Cell transformation vector comprising an HIV-2 packaging site nucleic acid and an HIV-1 GAG protein
US7198784B2 (en) 1996-10-17 2007-04-03 Oxford Biomedica (Uk) Limited Retroviral vectors
DE69703974T2 (de) 1996-10-17 2001-07-19 Oxford Biomedica Ltd Retrovirale vektoren
DK1895010T3 (da) 1997-12-22 2011-11-21 Oxford Biomedica Ltd Vektorer baseret på virus for infektiøs hesteanæmi (eiav)
US6218181B1 (en) 1998-03-18 2001-04-17 The Salk Institute For Biological Studies Retroviral packaging cell line
WO2001007646A2 (fr) * 1999-07-21 2001-02-01 Martin Heinkelein Procede de quantification de l'effet antiviral de principes actifs antiviraux
GB0108065D0 (en) * 2001-03-30 2001-05-23 Syngenix Ltd Viral vectors
WO2005118809A2 (fr) * 2004-04-29 2005-12-15 The University Of North Carolina At Chapel Hill Procedes et compositions pour ameliorer les proprietes d'adhesion cellulaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No further relevant documents disclosed *
See also references of WO9119798A1 *

Also Published As

Publication number Publication date
EP0536234A4 (en) 1993-09-15
JPH05508320A (ja) 1993-11-25
JP3264281B2 (ja) 2002-03-11
WO1991019798A1 (fr) 1991-12-26
CA2084659A1 (fr) 1991-12-21

Similar Documents

Publication Publication Date Title
US5981276A (en) Vectors containing HIV packaging sequences, packaging defective HIV vectors, and uses thereof
US5665577A (en) Vectors containing HIV packaging sequences, packaging defective HIV vectors, and uses thereof
EP0386882B1 (fr) Provirus d'HIV défectif pour l'empaquetage, lignées cellulaires et leur utilisation
Parolin et al. Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes
Arya et al. New human and simian HIV-related retroviruses possess functional transactivator (tat) gene
Lisziewicz et al. Inhibition of human immunodeficiency virus type 1 replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS.
Vaishnav et al. The biochemistry of AIDS
Malim et al. Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species.
Kornfeld et al. Cloning of HTLV-4 and its relation to simian and human immunodeficiency viruses
Sodroski et al. A second post-transcriptional trans-activator gene required for HTLV-III replication
Srinivasakumar et al. The effect of viral regulatory protein expression on gene delivery by human immunodeficiency virus type 1 vectors produced in stable packaging cell lines
Poznansky et al. Gene transfer into human lymphocytes by a defective human immunodeficiency virus type 1 vector
Cheng-Mayer et al. Host range, replicative, and cytopathic properties of human immunodeficiency virus type 1 are determined by very few amino acid changes in tat and gp120
Reitz Jr et al. Generation of a neutralization-resistant variant of HIV-1 is due to selection for a point mutation in the envelope gene
Neumann et al. Restriction of human immunodeficiency virus type 1 production in a human astrocytoma cell line is associated with a cellular block in Rev function
Chesebro et al. Identification of human immunodeficiency virus envelope gene sequences influencing viral entry into CD4-positive HeLa cells, T-leukemia cells, and macrophages
Balliet et al. Distinct effects in primary macrophages and lymphocytes of the human immunodeficiency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate
Bray et al. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent.
Bukovsky et al. Nef association with human immunodeficiency virus type 1 virions and cleavage by the viral protease
Sakai et al. Differences in cytopathogenicity and host cell range among infectious molecular clones of human immunodeficiency virus type 1 simultaneously isolated from an individual
US6013516A (en) Vector and method of use for nucleic acid delivery to non-dividing cells
Srinivasan et al. Molecular characterization of human immunodeficiency virus from Zaire: nucleotide sequence analysis identifies conserved and variable domains in the envelope gene
Gorry et al. Restricted HIV-1 infection of human astrocytes: potential role of nef in the regulation of virus replication
EP0536234A1 (fr) Vecteurs contenant des sequences d'encapsidation du vih, vecteurs du vih a capside defectueux, et leur utilisation
Bagasra et al. TAR-independent replication of human immunodeficiency virus type 1 in glial cells

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19921127

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

A4 Supplementary search report drawn up and despatched

Effective date: 19930730

AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 19931008