WO1994016060A1 - Mutants de vih destines a la suppression des infections par vih - Google Patents

Mutants de vih destines a la suppression des infections par vih Download PDF

Info

Publication number
WO1994016060A1
WO1994016060A1 PCT/US1994/000377 US9400377W WO9416060A1 WO 1994016060 A1 WO1994016060 A1 WO 1994016060A1 US 9400377 W US9400377 W US 9400377W WO 9416060 A1 WO9416060 A1 WO 9416060A1
Authority
WO
WIPO (PCT)
Prior art keywords
human immunodeficiency
virus
immunodeficiency virus
patient
infected
Prior art date
Application number
PCT/US1994/000377
Other languages
English (en)
Inventor
James C. Alwine
Francisco Gonzalez-Scarano
Steven L. Zeichner
Michael H. Malim
Original Assignee
The Trustees Of The University Of Pennsylvania
The Children's Hospital Of Philadelphia
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 The Trustees Of The University Of Pennsylvania, The Children's Hospital Of Philadelphia filed Critical The Trustees Of The University Of Pennsylvania
Publication of WO1994016060A1 publication Critical patent/WO1994016060A1/fr

Links

Classifications

    • 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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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

  • the invention relates to compositions and methods for the treatment of Acquired Immunodeficiency Syndrome.
  • Human Immunodeficiency Virus HIV is the etiologic agent of a fatal immunodeficiency condition in humans termed Acquired Immunodeficiency Syndrome or AIDS.
  • HIV Human Immunodeficiency Virus
  • ARC AIDS Related Complex
  • HIV-infected individuals may remain clinically healthy for prolonged periods of time, with an estimated average incubation period of ten years for adults and less for children.
  • HIV is a retrovirus which primarily infects cells of the immune system, although involvement of other cells is documented. Infection of cells with HIV is characterized by cycles of active gene expression and relative latency, which cycles are controlled in large part at the level of transcription. The initial transcriptional activation of the HIV genome during primary infection and emergence from the latent state is dependent upon the interaction of host cell transcription factors with cis-acting elements in the viral long terminal repeat (LTR) region.
  • LTR long terminal repeat
  • Experimental studies using a systematic mutagenesis approach have delineated several cis-acting elements in the LTR and have implicated specific DNA-binding proteins in the regulation of transcription of the HIV genome (Gaynor, AIDS (1992) 6:347).
  • the invention features conditional replication incompetent (CRI) mutants of HIV which have the potential to become replication competent when the virus-specific Tat protein is supplied in trans, for example, by an incoming wild type (replication-competent) HIV.
  • the mutants of the invention encode a replication-incompetent LTR region whose activation is stringently dependent upon the expression of the Tat protein supplied in trans.
  • the CRI mutants of the invention encode mutations which are linker substitution mutations, although other substitution, insertion, deletion or point mutations are also included in the invention.
  • the mutations in the genome of the mutant viruses of the invention are positioned between nucleotides -453 to -57 relative to the +1 transcriptional start site on the HIV proviral DNA.
  • the mutations are positioned between nucleotides -453 to -111, and more preferably, the mutations are positioned between nucleotides -435 to -111, relative to the transcriptional start site. Even more preferably, mutations are positioned between nucleotides -201 to -130, relative to the transcriptional start site.
  • the mutant viruses of the invention are mutants of HIV type 1 (HIV-1) selected from the group consisting of -147/-130 NXS, -165/-148 NXS or -201/-184 NXS.
  • the mutant viruses of the invention are conditionally replication incompetent in cells of the immune system, such as peripheral blood lymphocytes, or cells established from immune cells, such as 11.8 cells, or in cells of the nervous system.
  • conditional replication incompetent HIV is meant a virus which can maintain residence in a cell but cannot replicate unless a wild type form of the HIV-specific Tat gene, encoded by an HIV proviral DNA other than the proviral DNA of the conditional replication incompetent virus, is expressed in that cell.
  • HIV as used herein, includes both HIV-1 and HIV type 2 (HIV-2) .
  • Tat supplied in trans is meant the expression of Tat encoded by a proviral DNA other than the proviral DNA of the mutant virus.
  • the Tat gene may be encoded by HIV-1, HIV-2, a transiently or stably transfected expression vector such as a plasmid, an integrated retroviral expression vector, or any other wild type strain of HIV, since Tat so expressed from one strain of HIV is capable of activating other strains of HIV.
  • the invention also features CRI mutants of HIV such as those described above, wherein these mutants further encode a gene product (either RNA or protein) capable of either inhibiting replication of wild type HIV or of killing a cell in which the mutant resides.
  • the gene for this additional protein is positioned in the mutated HIV genome such that expression of the gene is stringently controlled by the LTR region.
  • a unique property of these mutants is their replication-incompetence in the absence of exogenously supplied Tat protein and their potential to be activated to a productive mode of gene expression in the presence of Tat protein expressed in trans from a wild type HIV proviral DNA.
  • the inhibitory gene is only expressed in cells which are, or become, infected with a replication-competent HIV.
  • the inhibitory gene encoded by the mutants of the invention is a gene encoding a transdominant form of an HIV protein such as Rev, Gag or Tat, or it is a gene encoding a transdominant form of a gene encoded by a heterologous virus such as the Tax or Rex gene of Human T cell leukemia virus type 1 (HTLV-l) .
  • the inhibitory gene is also a gene encoding an antisense RNA sequence capable of inhibiting replication of HIV, or it is a gene encoding a ribozy e also capable of inhibiting replication of HIV.
  • the inhibitory gene encoded by the mutants of the invention is also a gene capable of killing a cell, such as a toxin, and preferably, a toxin such as the Ricin A subunit.
  • the viral mutants of the invention may also encode more than one inhibitory gene.
  • the CRI mutants may encode both a transdominant viral protein and an inhibitory antisense RNA sequence.
  • transdominant form of a gene is meant a gene encoding a product with a dominant negative phenotype over the wild type function of the same or a similar gene, i.e., the product is capable of preventing other wild type forms of the same or a similar gene from functioning properly.
  • Mutant viruses according to the invention are viruses which are lymphotropic and/or monocyte/ acrophage tropic and these viruses also optionally encode additional mutations which render the viruses replication-incompetent in cells of the nervous system, such as glial cells.
  • the CRI mutants of the invention are useful as therapeutic agents for treatment of HIV-infected individuals, and for treatment of patients with AIDS.
  • Such mutants When such mutants are introduced into cells which are themselves latently or actively infected with wild-type HIV, or which will become infected with HIV, they have the potential to remain replication-incompetent in the absence of exogenously supplied Tat.
  • Tat will be expressed in these cells. Expression of Tat will activate the mutated LTR which in turn will drive expression of an inhibitory gene product encoded by the mutant genome.
  • the introduction of the replication-incompetent mutants of the invention into uninfected cells of an HIV-positive patient should result in cells which are ostensibly normal.
  • the Tat protein will be expressed from the wild type genome resulting in activation of the mutated LTR, which will in turn, drive expression of the inhibitory gene product.
  • the expression of the inhibitory gene product will subsequently effect inhibition of replication of both the wild type and mutant viruses, or will effect death of the infected cell.
  • this strategy can be used to retard the spread of wild type HIV in an individual, and to retard progression of asymptomatic HIV-infected individuals to ARC and AIDS. It may also be useful in reducing the severity of the disease in patients who have already progressed to AIDS.
  • the invention also features methods of treating a patient infected with HIV.
  • One such method involves administering the CRI mutant virus of the invention to the patient by injecting the virus directly into the patient's blood stream using a syringe or any other intravenous delivery system.
  • the CRI mutant virus is administered to the patient by infecting, in vitro , T cells and/or monocytes which have been isolated from the blood of the patient, and then returning the infected cells to the blood stream of the patient.
  • the CRI mutant virus is administered to the patient by infecting, in vitro, bone marrow progenitor stem cells which have been obtained from the patient, and then returning the infected cells to the bone marrow following infection.
  • the therapeutic methods of the invention have a significant advantage over other proposed gene therapy-type protocols for treatment of HIV-infected patients because these proposed protocols all involve the introduction of a gene encoding an anti-HIV product into normal or HIV-infected cells, which gene is constitutively expressed in those cells.
  • a gene capable of inhibiting replication of HIV, or of killing an HIV-infected cell is introduced into a cell under the control of an inducible promoter.
  • this gene is not constitutively expressed in these cells; rather, it is only expressed when its particular effect is desired. Therefore, the cells in which the inhibitory gene resides are free of any potential negative side-effects which may result from the constitutive expression of that gene which is not normally constitutively expressed in those cells.
  • the CRI mutants of the invention may also be useful as a vaccine which when inoculated into HIV-negative individuals may serve to protect such individuals from subsequent infection with HIV.
  • Figure 1 is a map of HIV-1 LTR DNA illustrating the relative positions of transcription factor binding sites and a set of NXS linker substitution mutants.
  • API activator protein 1
  • NRE negative regulatory element
  • IL2/IL2R interleukin-2/interleukin-2 receptor binding site
  • NFAT nuclear factor of activated T-cells
  • USF upstream stimulatory factor 1
  • NF K B nuclear factor K B
  • TA TATA element
  • I initiator element
  • TAR trans-acting responsive element
  • LBP/UBP leader or untranslated binding protein.
  • Scale refers to nucleotide position relative to the transcription start site (+1) .
  • Figure 2 is similar to Figure 1 and is a map of HIV-l LTR DNA illustrating the relative positions of transcription factor binding sites and linker-substitution mutations which have been shown, thus far, to confer conditional replication incompetence.
  • CAT activity values represent averages relative to wild-type LTR activity in multiple transient transfections of PHA/PMA-stimulated Jurkat cells.
  • open boxes represent wild-type sequences and filled boxes represent NXS linker-substitutions.
  • API activator protein 1
  • NRE negative regulatory element
  • IL2/IL2R interleukin-2/interleukin-2 receptor binding site
  • NFAT nuclear factor of activated T-cells
  • URS upstream repression sequence
  • USF upstream stimulatory factor 1
  • TCF-l ⁇ T-cell factor l ⁇ /lymphoid enhancer binding factor 1
  • NF K B nuclear factor K B
  • TA TATA element
  • I initiator element
  • TAR trans-acting responsive element
  • LBP/UBP leader or untranslated binding protein.
  • Scale refers to nucleotide position relative to the transcription start site (+1) .
  • Figure 3 illustrates the construction of linker-substitution mutant LTR-containing proviruses.
  • the figure depicts the relevant viral elements in the background of the appropriate plasmids.
  • A The wild-type, terminally repetitious NL4-3 proviral fragment with a single LTR (open rectangle) and an intact Xhol site obtained by BamHI digestion of pILIC.
  • B pG3-ILICX, illustrating the mutation in the Xhol site by insertion of an X al linker containing stop codons in each reading frame of nef .
  • pG3-ILICX was digested with XJal and SphI to release the Xbal-SphI LTR-containing fragment which was then cloned into pG3H-LTR for exchange of LTRs to introduce linker substitution mutations.
  • D The Xjal-Hin lll LTR fragment from linker-substitution mutant plasmids, illustrating the NXS linker (filled box) .
  • E The Xbal -HindIII LTR fragment of (D) which has replaced corresponding wild-type LTR sequences in pG3H-LTR (C) to generate the pG3H(j_/_7?)NXS series.
  • Figure 4A, B and C are graphs depicting replication of wild-type HIV-1 and NXS linker-substitution mutant HIV-1 viruses in human peripheral blood lymphocytes (PBLs) and T cell lines. Equal aliquots of filtered and standardized virus stocks from RD/CEMX174 cocultures were used to inoculate 1 X 10 cells. Virus replication was measured using p24 ga9 ELISA. Each data point represents the mean of duplicate infections.
  • A Infection of PBLs stimulated with PHA, then maintained in IL-2.
  • B Infection of 11.8 cells.
  • C Infection of CEMX174 cells.
  • CEMX174 cells a stable human T-B lymphoblastoid hybrid cell line, 11.8 cells, a stable hybrid of DF and Molt-4 human T-cell lines and RD, a rhabdomyosarcoma-derived CD4-negative human monolayer cell line, were grown in RPMI-1640 medium supplemented with 10% fetal calf serum and L-glutamine at 37°C in humidified 5% C02 (Kozbor et al./ J. Immunol . (1990) 144:3677-3683; Salter et al., Immunogen (1985) 21:235-246; Srinivasan et al. , Proc. Natl . Acad . Sci . USA (1989) 86:6388-6392).
  • Mutants are named using "n/m NXS” nomenclature, "n” referring to the LTR nucleotide substituted by the 5'-terminal nucleotide of the NXS polylinker and " " to the substituted 3'-terminal nucleotide position relative to the transcription start site.
  • Mutated LTR regions examined in this study include: -75/-58 NXS, -93/-76 NXS, -148/-130 NXS, -165/-148 NXS, -183/-166 NXS, and -201/-184 NXS ( Figures 1 and 2).
  • Each NXS LS LTR was cloned into a plasmid containing a single LTR and complete, terminally repetitious proviral coding sequences as described below and as illustrated in Figure 3.
  • the single LTR-proviral fragment was obtained by BamHI digestion of pILIC which contains the complete viral coding sequences originally derived from the infectious molecular clone of HIV-1, pNL4-3 (Leonard et al., J " . Virol .
  • BamHI proviral fragment was ligated into the BamHI site of the pG3H vector (pGEM3Zf(-), obtained from Promega Corporation, Madison, WI, which was depleted of its XJal to Hindlll polylinker sequences) .
  • the product was digested with Xhol and the overhanging ends were filled in with Klenow fragment, followed by blunt-end ligation with Xhal linkers which contain stop codons. Subsequent digestion of the linkers with Xhal and religation to recircularize the plasmid introduced stop codons in each reading frame of the nef-coding region at the former wild-type Xhol site ( Figure 3B) .
  • the resulting plasmid, pG3-ILICX was also employed in subcloning each single-LTR proviral NXS mutant.
  • the Xbal-SphI LTR-containing fragment of pG3-ILICX was ligated into a pGEM3Zf(-) vector depleted of its Hindlll site ( Figure 3C) .
  • the insert was digested with Xhal and Hindlll , and the resulting wild-type LTR fragment was replaced by each Xhal-Hindlll NXS mutant LTR fragment as well as the wild type LTR sequences of the parent strain used for construction of the NXS mutants (strain HXB2) ( Figure 3D) .
  • the 9.08 kilobase proviral fragment was purified by agarose gel electrophoresis and was ligated to regenerate LTRs at appropriate 5'- and 3'-positions flanking restored complete proviral coding sequences ( Figure 3G) .
  • Concatemerized proviral DNA (10 ⁇ g) was ethanol precipitated and transfected into the RD cell line using Lipofectin (25 ⁇ g) in Opti-MEM for 6 h at 37°C. The transfection reaction was stopped by adding serum-containing medium (GIBCO/BRL Life Technologies, Inc.) to the cultures. Within 48 to 72 h, the transfected RD cultures were cocultivated with 1 X 106 CEMxl74 cells for an additional 48 to 72 h.
  • Infected CEMxl74 cultures were then transferred to T-25 flasks (Costar Corporation, Cambridge, MA) and expanded for 4 to 23 days. Supernatants were harvested, filtered through 0.45 ⁇ m pore-size filters, and stored in aliquots at -80°C, which were subjected to a maximum of one freeze/thaw cycle.
  • Viral stocks were standardized by determining: 1) the concentration of p24 gag antigen in the stocks by antigen capture using a commercial antigen capture assay, i.e., enzyme-linked immunosorbent assay (ELISA) kit (Coulter Inc.); and, 2) the level of reverse transcriptase (RT) activity in lysed viral pellets. This was calculated by quantitating trichloroacetic acid-precipitable 3H-dTTP radioactivity. Tissue culture infectious dose-50 (TCID 50 ) determinations of the wild-type stocks were performed using the Reid-Munch method (Collman et al., J. Exp. Med . (1989) 170:1149-1163).
  • PCR poly erase chain reaction
  • Genomic DNA was prepared. Briefly, cell pellets were washed, lysed, and nuclei were digested with proteinase K (0.4 mg per ml) for 4-6 h at 55°C. The lysates were extracted with phenol-chloroform and the DNA was ethanol precipitated. BamHI-digested genomic DNAs were amplified in a Perkin-Elmer/Cetus DNA Thermal Cycler using the following protocol: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 70°C for 2 min. The products were separated by agarose gel electrophoresis and were visualized by ethidium bromide staining.
  • PCR was performed according to the above protocol using primers to amplify a 577 bp LTR fragment containing most of the U3 and R regions (positive-sense 5 ' primer (-439/-412 ) : 5'-CTAATTCACTCCCAACGAAGACAAGA-3' [SEQ. ID.NO:4] and negative-sense 3 'primer ( +112 / + 138 ) : 5'-TCTCTAGTTACCAGAGTCACACAACAG-3' [SEQ.ID.NO:5] .
  • the amplified 577 bp LTR fragment was sequenced directly to confirm that spontaneous mutations did not occur during viral propagation (Perkin-Elmer Corporation, Norwalk, CT) .
  • NXS mutant LTRs Six NXS mutant LTRs were used in the studies described below.
  • Two other mutants, -75/-58 NXS and -93/-76 NXS, which affect the NF K B and Sp-1 binding sites, were selected as controls since they dramatically decreased LTR-mediated transcription (Leonard et al., J. Virol . (1989) 63:4919; Parrott et al., J. Virol . (1991) 65:1414-1419; Ross et al., J. Virol . (1991) 65:4350-4358).
  • mutants -93/-76 NXS and -75/58 NXS failed to replicate in subsequent individual cell type infections with cell-free inocula, all six mutant viruses were able to replicate to levels resulting in detectable cytopathic effects (CPE) , primarily syncytia formation, under co-cultivation conditions.
  • CPE cytopathic effects
  • the more rapidly replicating viruses were harvested earlier than those with lower levels of CPE and were frozen at -80°C. After harvesting, virus stock titers were standardized by measuring the level of p24 gag . These data were supplemented with the results of analysis of levels of RT activity of virus pellets.
  • Each virus stock contained significant amounts of both p24 gag and RT and had consistent RT:p24 gag ratios. Due to the differences in the titers of some mutant viruses, we were able to attain a maximum equivalent inoculum in subsequent individual cell type infections of approximately 15 ng per ml p24 gag . This inoculum resulted in an infectivity of the wild-type HIV-l stocks in CEMxl74 cells of 105-106 TCID 50 per ml, corresponding to MOIs of 0.1-0.5 TCID 50 per cell.
  • CEMX174 the cell line used to raise viral stocks in co-cultures, demonstrated the greatest permissiveness for virus replication in single cell type infections, achieving p24 gag levels of 10 ng per ml by 12 days p.i. for both wild-type and mutant -183/-166 NXS ( Figure 4C) .
  • CEMxl74 was the only cell line in which mutant viruses -147/-130 NXS, -165/-148 NXS, and -201/-184 NXS replicated sufficiently to detect antigen in the supernatant (see below) .
  • Mutants -93/-76 NXS and -75/-58 NXS failed to replicate in any of the individual cell type infections.
  • Mutant Viruses do not Revert to Wild-Type.
  • reinfection of CEMxl74 was performed with mutant virus stocks obtained late during productive infection of CEMxl74 cells (i.e. up to 60 days p.i.). The kinetics of virus growth in reinfected cells was indistinguishable from that in cells infected with the parental mutant stocks, supporting the fact that these viruses retained their mutant phenotype.
  • infected cell DNA preparations were analyzed by PCR using a primer pair that amplified 577 bp of the LTR sequence (-439 to +138) .
  • Each mutant LTR fragment was sequenced.
  • the sequence data confirmed not only the presence of the NXS linkers but also the absence of any additional potentially compensatory LTR mutations, ensuring that the observed replication phenotypes were attributable to the original NXS mutations.
  • stop codons were placed upstream of the linker substitution inserts into the nef reading frame and an Xhal site encoding these stop codons was inserted to faciliatate cloning of the NXS mutations.
  • the use of the stop codons eliminates the possibility that unknown and/or variable fusion proteins can be expressed from this region.
  • the nef coding region is further disturbed by the NXS mutations as well as by a small (61 bp) segment of cellular sequence upstream of the LTR which derives from the proviral source of the LTR used to make the original set of NXS mutations.
  • SIV Simian Immunodeficiency Virus
  • the nef gene product similarly affects the pathogenesis of HIV in humans.
  • the manner in which the mutants of the invention have been constructed has provided additional safety features in terms of their potential pathogenesis and in terms of any therapeutic effect the nef minus mutation might impart.
  • the viruses encode multiple mutations, it is highly unlikely that they are capable of reverting back to a wild-type phenotype and indeed the data presented above indicates that they do not revert in tissue culture.
  • the Tat gene encoded by the CRI viruses is dispensable and can be mutated such that it is non-functional. This can be accomplished by first introducing a mutation into the Tat gene on a plasmid template and then incorporating that mutation into the proviral DNA of a CRI virus using ordinary recombinant DNA and virological technologies familiar to those in the art. Such viruses can be propagated on Tat-expressing cell lines the generation of which is described below.
  • the invention should not be construed as being limited to the CRI HIV mutants described above. Rather, the invention encompasses any mutant of HIV which comprises a mutation in the LTR which renders the virus conditionally replication-incompetent in the absence of wild type Tat protein.
  • Such mutations include the insertion of linkers other than the NXS linker utilized in the studies described above; the substitution of specific nucleotides with sequences which do not form restriction enzyme cleavage sites as is the case with linkers; the generation of specific point mutations; and, other deletion and insertion mutations.
  • the invention should also not be construed as being limited solely to HIV-l.
  • any strain of HIV including HIV-2, can be mutated using the techniques described above, such that CRI viruses are generated with properties similar to those described above. Furthermore, additional mutations can then be constructed in the genome of these viruses using the techniques described below, such that CRI viruses derived from any strain of HIV which are useful for the treatment of HIV-infected individuals are generated.
  • mutant viruses of the invention which are severely debilitated in their replication-competency in PBLs and 11.8 cells (-201/-184NXS, -165/-148NXS, and -147/-130NXS) are mutated in a region of the LTR which is not considered to be necessary for activation by the Tat protein. While nucleotides -1 to -125 have been shown to be required for Tat activation, nucleotides outside of this region are dispensable for this function (Zeichner et al., J. Virol . (1991) 65:2436-2444).
  • the mutated LTR sequences of the invention are capable of being activated by Tat in transient transfection assays (Zeichner et al., J . Virol . (1991) 65:2436-2444).
  • each mutant virus constructed as described above can be tested in cells which stably express wild type Tat. The data can be compared with those obtained i mutant infected cells which do not express Tat. The degree to which the mutants replicate is assessed by measuring the levels of p24 gag antigen and RT activity in the cultures using the methods described above.
  • Tat-expressing cells useful for these studies are available (Rosen et al., Cell (1985) 41:813-823), or can be generated using ordinary molecular biology protocols familiar to those in the art and described for example, in the Molecular Cloning Manual (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York) , or they can be generated in a manner similar to that described for the stable expression of the transdominant Rev protein (Malim et al., J. Exp. Med . (1992) 176:1197-1201).
  • the Tat gene is cloned into a plasmid encoding a recombinant retrovirus capable of expressing an antibiotic resistance marker, i.e., neomycin resistance.
  • This plasmid is then transfected into a virus-packaging cell line such as, but not limited to, the ecotropic ⁇ CRE virus packaging cell line, and cells so transfected are selected for their resistance to the appropriate antibiotic, e.g., G418 (Danos et al., Proc. Natl . Acad . Sci . USA (1988) 85:6460).
  • filtered supernatant is collected fro the cultures and used to infect an amphotropic cell line such as the ⁇ CRIP-packaging line, which is then selected in G418 at a concentration of approximately 1 mg/ l.
  • Clones of cells are selected for their resistance to G418 and for their ability to produce virus. Viral supernatants obtained from these cultures are incubated with the specific cell line in which stable expression of Tat is required. Following incubation, clones of cells are selected for G418 resistance and are tested for the presence and expression of the Tat gene using molecular or immunological techniques to detect Tat-specific DNA, RNA or protein familiar to any ordinary molecular biologist and described for example in the Molecular Cloning Manual (Sa brook et al. , Supra) .
  • Conditional replication-incompetent HIV-l mutants encoding additional genes can be constructed in a manner identical to that described above for the generation of the original conditional replication-incompetent mutants.
  • a plasmid encoding a mutation in the LTR sequence can be further manipulated using ordinary molecular biological technology familiar to those in the art such that a heterologous gene or an alternate viral gene is positioned in the mutated HIV-l genome so as to be expressed only when the mutated LTR is activated by Tat.
  • a plasmid encoding a terminally repetitious provirus containing a single mutated LTR ( Figure 3) is linearized at the site into which the gene will be inserted and a fragment encoding that gene is then ligated into this site.
  • the resulting plasmid is separated from unligated material and an infectious provirus is generated from this plasmid as described above.
  • the precise location with respect to the terminally repetitious single LTR provirus into which such a gene might be placed is determined by analysis of the known sequence of the virus, the sequence of that particular gene and the position of the alternate viral gene if this is the gene of choice.
  • the specific site into which the gene will be inserted will depend on the particular gene to be inserted. If a viral gene is replaced with an alternate form of that gene, e.g., a mutated viral gene encoding a transdominant viral protein, then the mutated gene would be inserted in place of that viral gene, at a position in the genome in which the wild type form of the gene normally resides.
  • a Rev-specific sequence encoding a transdominant form of that protein can be inserted into the viral genome by simply replacing the BamHI to Xhol fragment in the genome of the replication-incompetent virus with the comparable BamHI to Xhol fragment encoding the transdominant form of Rev (Malim et al..
  • the Xhol site has been converted to an Xhal site as described above.
  • the Xhol site in the BamHI to Xhol fragment encoding the transdominant Rev is replaced with an Xhal linker for proper insertion and maintenance of the stop codons in nef.
  • Other transdominant alternative viral genes can be inserted into background of the replication incompetent viral genome in a similar manner.
  • a heterologous non-viral gene is to be inserted, either under the control of the viral LTR or another internal promoter, this gene can be positioned in the viral genome such that it replaces viral sequences which are dispensable for growth of the virus in cell culture.
  • sequences which are dispensable for viral growth in culture are the nef coding sequences (Daniel et al.. Science (1992) 258:1938-1941). Since nef is expressed to very high levels during replication of HIV, it is expected that an additional gene inserted into the nef coding region would also be expressed to high levels.
  • the resulting recombinant virus may be incapable of replication using the cocultivation techniques described above.
  • supporting cell lines can be constructed which constitutively express a recombinant gene capable of complementing the defect in the mutated virus thus facilitating its replication.
  • the wild type Rev gene is replaced with a transdominant mutant form of the gene
  • the resulting virus can only be propagated on cell lines which constitutively express the wild type form of a similar viral protein, e.g., the wild form of the Rex gene product encoded by the human T cell leukemia virus type 1 (HTLV-1) (Bachmayer et al., European Patent Application No. 90109892.1).
  • HTLV-1 human T cell leukemia virus type 1
  • Genes which are suitable candidates for mediating inhibition of wild-type HIV-l replication include those encoding 1) proteins which suppress viral transcription, gene expression or replication; 2) antisense RNA specific for a particular essential viral mRNA or a structural feature of the viral RNA, which when disrupted, would limit virus replication; 3) catalytic RNA (ribozyme) specific for an essential viral mRNA or a structural feature of viral RNA which when disrupted would limit viral replication; and 4) a toxin, which when expressed would kill an infected cell.
  • these genes encode viral proteins which are transdominant with respect to the wild-type protein.
  • genes encoding a transdominant Rev protein as described above, an HIV-specific Tat protein (Pearson et al., Proc . Natl . Acad . Sci . USA (1990) 87:5079-5083) or a transdominant HIV-specific Gag protein (Trono et al.. Cell (1989) 59:113) are suitable genes which when expressed may competitively inhibit the normal function of the wild-type protein and thereby inhibit virus replication.
  • transdominant forms of the HTLV-1 Tax and Rex genes may also be used (Bachmayer et al., European Patent Application No. 90109892.1).
  • any transdominant gene encoded by HIV, or a transdominant encoded by a heterologous virus, which gene functions to inhibit HIV replication can be used.
  • a gene which is most useful in the invention is a gene encoding a transdominant HIV-l Rev protein (Malim et al., Cell (1989) 58:205-214). It is known in the art that functional expression of Rev is required for replication of HIV-l. However, it is also known that particular mutations in the carboxy-terminal portion of Rev not only render Rev defective, but also generate Rev proteins capable of competitively inhibiting wild-type Rev function (Malim et al., Cell (1989) 58:205-214).
  • CRI viruses constructed such that they encode a Tat-activatable LTR, which when activated express a transdominant Rev protein have the potential to inhibit replication of a Tat-producing wild type virus present in the same cell, because the transdominant Rev protein will competitively inhibit wild type Rev and effectively abolish this essential function for virus replication (Malim et al., J . Exp . Med . (1992) 176:1197-1201)
  • CRI viruses can also be constructed which encode antisense RNA specific for a particular viral mRNA or a structural feature of viral RNA which when disrupted, would limit replication of the virus.
  • An example of such a region is an antisense RNA complementary to the HIV-l TAR sequences. These sequences are essential for viral transcription and efficient polyadenylation of viral RNA. Cultured cells which express antisense TAR specific sequences are diminished (by as much as 90%) in their ability to support wild type HIV-l replication (Chatterjee et al., Science (1992) 258:1485-1488).
  • Insertion of an antisense TAR sequence into a CRI HIV requires that the resident TAR region, a stem loop structure in the RNA, be appropriately modified in order that it is not affected by the antisense RNA.
  • Such modifications of the viral sequences which retain transcriptional and polyadenylation activity have been characterized (Feng et al.. Nature (1988) 334:165-167; Gilmartin et al., EMBO J. (1992) 11:4419-4428).
  • CRI viruses constructed such that they encode a ribozyme specific for a viral mRNA or an essential structural feature of viral RNA, may also be generated.
  • a ribozyme can be designed which is capable of recognizing and cleaving specific viral RNA sequences using a mechanism similar to that utilized by RNase P in the processing of precursor tRNA in E. Coli (Guerrier-Takada et al.. Cell (1983) 35:849; Pace et al., J. Biol . Chem . (1990) 265:3587; Altman, J. Biol . Chem . (1990) 265:20053; Haas et al. , Science (1991) 254:853-856).
  • CRI viruses can also be constructed encoding proteins capable of killing an infected cell which in turn results in inhibition of wild type virus replication. Genes encoding such proteins are introduced into CRI viruses in a manner similar to that described above for genes that inhibit wild-type HIV replication. Suitable genes include genes encoding toxins or other inhibitors of cellular function. Preferably, the products of such genes should be capable of affecting only those cells in which they reside.
  • Ricin A encodes the Ricin A subunit.
  • the entire Ricin toxin comprises two subunits, A and B.
  • Ricin A induces its toxic effect on cells by inhibiting cell-specific translation in the absence of the Ricin B subunit. Since the function of the B subunit is to facilitate attachment and entry of the A subunit into cells, the A subunit can only affect the cells in which it resides in the absence of the B subunit. Cell free A subunit is therefore harmless to cells in the absence of the B subunit (Wawrzynczak, British J. , Cancer (1991) 64:624-630).
  • HIV-l infects many cell types in addition to lymphocytes. In fact, infection of a variety of cell types may contribute to the diverse manifestations of the disease (Fauci et al.. Science (1988) 239:617-622).
  • the data presented above demonstrate that CRI viruses encoding defined mutations in specific regions of the HIV-l LTR are replication-incompetent in the absence of Tat in cells of lymphoid origin. It is possible to construct similar replication-incompetent Tat-dependent viruses capable of infecting other cell types, for example, myeloid cells or glial cells.
  • a wild type HIV-l genome obtained from a macrophage tropic virus is used as the starting material. Mutations are introduced into the LTR region (using the methods described above) which confer the property of replication-incompetence to the virus.
  • a minimal determinant for macrophage tropism has been mapped to the third variable region of the gpl20 glycoprotein (O'Brien et al., Nature (1990) 348:69-73). This can be inserted into the proviral DNA of a CRI virus following removal of the corresponding sequence from the CRI virus. Using either method, a CRI virus can be generated which is both T cell and macrophage tropic thus providing broader therapeutic benefit to the patient.
  • Additional genes can then be inserted into these viruses which are capable of either inhibiting wild-type virus replication or of killing an infected cell using the above described methods.
  • new strains of virus emerge with altered cell-tropic properties, it will be possible using the methods and compositions of the invention, to construct additional viral mutants capable of infecting a variety of cell types, which viruses have therapeutic potential. Since the sequence of the viral LTR influences tissue tropism (Corboy et al., Science (1992) 258:1804-1808), as more data become available, it will be possible to design replication-incompetent viral mutants with altered LTR sequences capable of acting as therapeutic agents in specific tissues.
  • the replication-incompetent mutants of the invention encode multiple mutations which serve to ensure that these viruses are incapable of reversion to a wild-type phenotype.
  • These include the LTR mutation conferring replication incompetence in the absence of Tat, the nef mutation and the alternate viral gene or heterologous gene capable of inhibiting virus replication when expressed.
  • additional mutations may be introduced into different regions of the LTR which have cell-specific or differentiation-dependent effects on transcriptional activity.
  • CRI mutant viruses can be constructed which encode all of the features documented above, and in addition, which are mutated so as to be potentially replication-incompetent in neuronal cells.
  • the introduction of this additional mutation should not affect the replication capabilities of the mutants in lymphoid or myeloid cells since the regions of the LTR in which the various mutations would reside are separated from one another.
  • An added advantage of these additional mutations is the fact that they have the potential to broaden the number of cell types in which the mutant viruses can function as therapeutic agents.
  • each of the desired mutations is introduced into the HIV-l genome on a plasmid template using the methods described above and any other ordinary recombinant methodology familiar to those in the art and described , for example, in the
  • viruses containing the desired mutations can be generated, screened and characterized. Methods of Treating Patients Infected with HIV-l
  • the mutant viruses of the invention are useful as therapeutic agents for the treatment of HIV-infected individuals because they have the capacity to infect the same cells in the patient which are infected with wild-type virus; and, when activated by Tat expressed from the wild-type viral genome, the mutants are capable of inhibiting replication of the wild-type virus, or of killing the infected cell. In either case, the production of new progeny viruses will be limited or completely inhibited. Consequently, the extent of infection of individuals with HIV will be retarded or may in fact be eliminated.
  • Such therapies are vital for HIV-infected patients because treatment during the asymptomatic period delays the decline in the number of CD4 positive cells characteristic of the onset of AIDS, and therefore may prolong life.
  • the therapeutic agents of the invention may also be useful in prolonging life even after the patient has progressed to AIDS in that the rate at which their immune function declines may be retarded following treatment.
  • the therapeutic agents of the invention are administered to HIV-infected patients in one of several ways. In general, the mutants are delivered to the hematopoietic cells of the patient in either the blood or the bone marrow. 1) Direct Inoculation Blood cells and other susceptible cells of an
  • HIV-positive patient are infected with the mutants of the invention by simple direct inoculation of virus into the patient's blood stream.
  • This can be accomplished by injection of CRI viruses suspended in a pharmaceutically acceptable carrier using a syringe, or by injection using an intravenous apparatus.
  • pantropic viruses capable of infecting many different cell types can be used because, once free in the blood stream, they can infect any number of the appropriate cells. For example, if a patient is inoculated with a CRI virus which is both macrophage and T cell tropic (Gartner et al.. Science (1986) 233:215-219; Cameran et al..
  • this virus is capable of infecting macrophages, T cells and, in addition, glial cells through the utilization of the galactosyl ceramide receptor (Harouse et al.. Science (1991) 253:320-323) .
  • This approach may require the patient to undergo frequent injections with large quantities of virus. It is anticipated that the patient may receive a dose of virus in the range of 10 3 to 10 8 TCID 50 , either daily or at periodic intervals, the frequency of which will depend upon the extent of the HIV-l infection in the individual, their immune status and whether the exhibit symptoms of AIDS and other factors.
  • the exact therapeutic protocol will therefore be empirically designed for each individual patient and will be apparent to those skilled in the art of treatment of HIV-infected patients.
  • the cells of the patient may also be infected with a mutant virus in vitro and then subsequently be returned to the patient.
  • Human gene therapies which are known in the art have proved to be encouragingly successful for the treatment of diseases such as Severe Combined Immunodeficiency (SCID) , (Anderson, Science (1992) 256:808-813) . 2) Lymphocyte Therapy
  • the mutants of the invention may be administered to an HIV-infected patient following the general protocol useful for the treatment of children with adenosine deaminase deficient SCID (Culver et al.. Transplantation Proceedings (1991) 23:170-171) .
  • blood cells are obtained from the patient from which a subpopulation of T lymphocytes are isolated.
  • the numerous techniques for separating T lymphocytes from other cells of the blood are well known in the art and are described, for example, in Culver et al.. Transplantation Proceedings 23:170-171; Culver et al.. Human Gene Therapy (1991) 2:107-109.
  • the T cells are infected in vitro with one of the mutant viruses of the invention using the methods described above for infection of cells in culture. Following a period of incubation, the infected cells, suspended in a physiologically acceptable carrier, are then returned to the patient's blood stream.
  • the amount of virus required for efficient infection of T lymphocytes in vitro is in the range of 0.1 to 50 TCID 50 .
  • the number of T cells required for efficient treatment of the patient may range from 10 1 to 10 8 cells per kg body weight. Patients will require repeated treatment when lymphocyte therapy is the preferred method. The frequency of treatment will depend upon the extent of the HIV-l infection in the individual, their immune status, whether they exhibit symptoms of AIDS and other factors.
  • monocyte and/or Lymphocyte Therapy In vitro infection of monocytes obtained from an HIV-l infected patient may also serve as an efficient route of administration of the mutants of the invention to the patient. Freshly isolated human blood monocytes can be maintained in vitro for several hours to days before they differentiate into a macrophage phenotype (Albers et al., Virology (1989) 169:466) . Thus, monocytes obtained from a sample of blood from the patient using ordinary immunological techniques may be infected with the appropriate virus in a manner similar to that described above.
  • HIV-l infected individuals often harbor virus in bone marrow progenitor cells. It is also known that bone marrow cells when isolated in vitro, can be infected with HIV (Folks et al., Science (1988) 242:919-922; Scadden et al., Blood (1990) 76:3317-3322; Folks, Blood (1991) 77:1625-1626; Kitano et al., Blood (1991) 77:1699-1705). Infection of such cells with the mutants of the invention therefore provides an additional method of therapy for HIV-infected individuals.
  • Cells obtained from the individual can be isolated and infected with the mutants of the invention 121 vitro using the general methods described in Folks et al. (Science (1988) 242:919-922), Scadden et al., (Blood (1990) 76:3317-3322), Folks, Blood (1991) 77:1625-1626) and Kitano et al. (Blood (1991) 77:1699-1705). Infected cells are then returned to the bone marrow of the individual where they will continue through the differentiation process. Peripheral blood cells which arise following differentiation of mutant-infected progenitor cells will provide a constant and renewable source of mutant virus-protected lymphocytes and monocytes.
  • an advantage of this approach is that the patient should not require repeated inoculation of viruses.
  • a mutant infected cell harbors a wild type form of the virus which is activated to replicate and therefore expresses Tat, or, if such a mutant infected cell is subsequently infected with a wild type, replicating, Tat expressing HIV, then the LTR of the mutant is activated and in turn drives expression of a gene in the mutant capable of either inhibiting replication of wild type HIV or of killing the infected cell.
  • this type of therapy has an advantage over the lymphocyte or monocyte therapies described above in that it has greater potential as a long term therapy, wherein the frequency treatment with the mutant viruses might be reduced.
  • Bone marrow cells can be obtained from HIV-infected individuals using established protocols available to those in the art and described for example in Kitano et al. (Blood (1991) 77:1699-1705), or Folks et al. (Science (1988) 242:919-922). Essentially, a suspension of cells is obtained from the bone marrow of a patient using conventional techniques. If necessary, the population of cells of interest may be separated from the remainder of the cells in the sample using a combination of techniques including centrifugation and flow cytometry. Cells so isolated are then infected with the mutants of the invention as described above and are returned to the bone marrow of the patient following infection.
  • the amount of mutant virus required for infection of bone marrow cells from an HIV-infected individual and the number of cells to be used during each therapeutic treatment can be determined empirically by one skilled in the art of gene therapy. Because the extent of treatment of any HIV-infected individual will vary depending upon the virus load they carry, their level of immune competence, their age, the number of years they have been infected and other factors, the exact protocol to be used in any one individual will be determined on a case by case basis. In general, the dosage of virus and the number of cells to be used will be within the range given above for the lymphocyte therapy approach.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID N0:1: CATATGCTCG AGGTCGAC (2) INFORMATION FOR SEQ ID NO:2:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Abstract

L'invention se rapporte à des compositions et des procédés destinés au traitement d'individus infectés par le virus VIH et de patients souffrant du SIDA.
PCT/US1994/000377 1993-01-11 1994-01-11 Mutants de vih destines a la suppression des infections par vih WO1994016060A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US260993A 1993-01-11 1993-01-11
US08/002,609 1993-01-11

Publications (1)

Publication Number Publication Date
WO1994016060A1 true WO1994016060A1 (fr) 1994-07-21

Family

ID=21701591

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/000377 WO1994016060A1 (fr) 1993-01-11 1994-01-11 Mutants de vih destines a la suppression des infections par vih

Country Status (1)

Country Link
WO (1) WO1994016060A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026790A1 (fr) * 1996-01-25 1997-07-31 New York University Apoptose induite par la proteine ipa b de shigella
US6071743A (en) * 1997-06-02 2000-06-06 Subsidiary No. 3, Inc. Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
US6316210B1 (en) 1995-12-20 2001-11-13 Subsidiary No. 3, Inc. Genetic suppressor elements against human immunodeficiency virus
US6426412B1 (en) 1995-12-20 2002-07-30 Subsidiary No. 3, Inc. Nucleic acids encoding human immunodeficiency virus type 1 genetic suppressor elements
US6537972B1 (en) 1997-06-02 2003-03-25 Subsidiary No. 3., Inc. Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
US6613506B1 (en) 2000-11-28 2003-09-02 Subsidiary No. 3, Inc. Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
JP2003526339A (ja) * 1999-07-28 2003-09-09 スミス、スチーブン 条件付きの制御がなされた弱毒ｈｉｖワクチン
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
JP2008538174A (ja) * 2005-02-16 2008-10-16 レンティジェン コーポレーション レンチウイルスベクターとその使用
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
GB2543730A (en) * 2015-05-26 2017-05-03 Andrzejewski Slawomir Use of replication competent vector to eradicate viral latency presented on human immunodeficiency virus

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
CELL, Vol. 59, issued 06 October 1989, D. TRONO et al., "HIV-1 Gag Mutants Can Dominantly Interfere with the Replication of the Wild-Type Virus", pages 113-120. *
GENE EXPRESSION, Vol. 1, No. 1, issued April 1991, S.L. ZEICHNER et al., "Analysis of the Human Immunodeficiency Virus Long Terminal Repeat by In Vitro Transcription Competition and Linker Scanning Mutagenesis", pages 15-27. *
GENES & DEVELOPMENT, Vol. 2, issued 1988, C.P. LANDEL et al., "Lens-Specific Expression of Recombinant Ricin Induces Developmental Defects in the Eyes of Transgenic Mice", pages 1168-1178. *
J. EXP. MED., Vol. 176, issued October 1992, M.H. MALIM et al., "Stable Expression of Transdominant Rev Protein in Human T Cells Inhibits Human Immunodeficiency Virus Replication", pages 1197-1201. *
JOURNAL OF VIROLOGY, Vol. 65, No. 5, issued May 1991, S.L. ZEICHNER et al., "Linker-Scanning Mutational Analysis of the Transcriptional Activity of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat", pages 2436-2444. *
JOURNAL OF VIROLOGY, Vol. 65, No. 8, issued August 1991, E.K. ROSS et al., "Contribution of NF-Kappa B and Sp1 Binding Motifs to the Replicative Capacity of Human Immunodeficiency Virus Type 1: Distinct Patterns of Viral Growth are Determined by T-Cell Types", pages 4350-4358. *
JOURNAL OF VIROLOGY, Vol. 66, No. 4, issued April 1992, S.L. ZEICHNER et al., "Differentiation-Dependent Human Immunodeficiency Virus Long Terminal Repeat Regulatory Elements Active in Human Teratocarcinoma Cells", pages 2268-2273. *
NATURE, Vol. 335, issued 29 September 1988, D. BALTIMORE, "Gene Therapy: Intracellular Immunization", pages 395-396. *
PROC. NATL. ACAD. SCI. USA, Vol. 87, issued July 1990, L. PEARSON et al., "A Transdominant Tat Mutant that Inhibits Tat-Induced Gene Expression from the Human Immunodeficiency Virus Long Terminal Repeat", pages 5079-5083. *
SCIENCE, Vol. 258, issued 30 October 1992, L. THOMPSON, "Researchers Test Gene Therapy Against AIDS", page 745. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6316210B1 (en) 1995-12-20 2001-11-13 Subsidiary No. 3, Inc. Genetic suppressor elements against human immunodeficiency virus
US6426412B1 (en) 1995-12-20 2002-07-30 Subsidiary No. 3, Inc. Nucleic acids encoding human immunodeficiency virus type 1 genetic suppressor elements
US5972899A (en) * 1996-01-25 1999-10-26 New York University Apoptosis induced by Shigella IpaB
WO1997026790A1 (fr) * 1996-01-25 1997-07-31 New York University Apoptose induite par la proteine ipa b de shigella
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
US6071743A (en) * 1997-06-02 2000-06-06 Subsidiary No. 3, Inc. Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
US6326152B1 (en) 1997-06-02 2001-12-04 Subsidiary No. 3, Inc. Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
US6436634B1 (en) 1997-06-02 2002-08-20 Subsidiary No. 3, Inc. Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
US6537972B1 (en) 1997-06-02 2003-03-25 Subsidiary No. 3., Inc. Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
JP2003526339A (ja) * 1999-07-28 2003-09-09 スミス、スチーブン 条件付きの制御がなされた弱毒ｈｉｖワクチン
US6613506B1 (en) 2000-11-28 2003-09-02 Subsidiary No. 3, Inc. Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
JP2008538174A (ja) * 2005-02-16 2008-10-16 レンティジェン コーポレーション レンチウイルスベクターとその使用
GB2543730A (en) * 2015-05-26 2017-05-03 Andrzejewski Slawomir Use of replication competent vector to eradicate viral latency presented on human immunodeficiency virus

Similar Documents

Publication Publication Date Title
Accola et al. A putative α-helical structure which overlaps the capsid-p2 boundary in the human immunodeficiency virus type 1 Gag precursor is crucial for viral particle assembly
Gibbs et al. Construction and in vitro properties of SIVmac mutants with deletions in" nonessential" genes
US6114141A (en) Methods to express genes from viral vectors
Böhnlein et al. Stimulation of the human immunodeficiency virus type 1 enhancer by the human T-cell leukemia virus type I tax gene product involves the action of inducible cellular proteins
Gardiner et al. Evidence that developmentally regulated control of gene expression by a parvoviral allotropic determinant is particle mediated
CA2055435A1 (fr) Cellules eucaryotiques transformees stables comprenant un adn etranger transcriptible sous le controle d'un promoteur pol iii
Antzin-Anduetza et al. Increasing the CpG dinucleotide abundance in the HIV-1 genomic RNA inhibits viral replication
JP4384146B2 (ja) 条件付き複製ウイルスベクターおよびその用途
JPH06335392A (ja) Ｈｉｖ阻害性アンチセンスおよび他のヌクレオチド配列を含有する発現構成体、レトロウイルスのベクターおよびそれを含有する組換えレトロウイルス
WO1994016060A1 (fr) Mutants de vih destines a la suppression des infections par vih
CA2422544A1 (fr) Vecteurs viral a replication conditionnelle et utilisation de ces vecteurs
US20100137415A1 (en) Vector for gene therapy
US20030026791A1 (en) Conditionally replicating vectors for inhibiting viral infections
US5885806A (en) Methods to prepare conditionally replicating viral vectors
LARSSON et al. A Novel Ribozyme Target Site Located in the HIV-1NefOpen Reading Frame
US7803582B2 (en) Recombinant vector and use in gene therapy
AU703362B2 (en) Vectors with multiple HIV-1 tar sequences inhibiting HIV-1 gene expression
WO1993005147A1 (fr) Particules defectives interferentes du vih avec un cd-env chimerique
Dash et al. Exceptional molecular and coreceptor-requirement properties of molecular clones isolated from an Human Immunodeficiency Virus Type-1 subtype C infection
US20120034693A1 (en) Recombinant vector and use in gene therapy
Brdar et al. Human immunodeficiency virus-1 tat-and tat/nef-defective genomes containing HIV-regulated diphtheria toxin A chain gene inhibit HIV replication
US6063374A (en) Recombinant HIV and modified packaging cells and method for using
KR100231318B1 (ko) 종양괴사인자 알파 수용체의 c-말단 부분을 갖는 레트로바이러스 벡터 및 이를 이용한 hiv-1 증식 억제 방법
Kräusslich Genetic analysis and gene expression of human immunodeficiency virus
JP2004526450A (ja) ウイルスベクター

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

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

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA