+

WO1993025235A1 - Procedes therapeutiques destines au sida, a base de peptides vpx d'hiv-2 - Google Patents

Procedes therapeutiques destines au sida, a base de peptides vpx d'hiv-2 Download PDF

Info

Publication number
WO1993025235A1
WO1993025235A1 PCT/US1993/004301 US9304301W WO9325235A1 WO 1993025235 A1 WO1993025235 A1 WO 1993025235A1 US 9304301 W US9304301 W US 9304301W WO 9325235 A1 WO9325235 A1 WO 9325235A1
Authority
WO
WIPO (PCT)
Prior art keywords
vpx
hiv
patient
polypeptide
nucleic acid
Prior art date
Application number
PCT/US1993/004301
Other languages
English (en)
Inventor
Myron E. Essex
Tun Hou Lee
Zene Matsuda
Original Assignee
President And Fellows Of Harvard College
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 President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Publication of WO1993025235A1 publication Critical patent/WO1993025235A1/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
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—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
    • C12N2740/16311—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

  • HIV-1 is an etiological agent of AIDS. This virus is generally described in Barre-Sinoussi et al., Science 220:868, 19831 Gallo et al.. Science 224;500. 1984; Popovic et al.. Science 224;497. 1984; and Levy et al., Science 225;840, 1984, each of which is hereby incorporated by reference.
  • HIV-1 A second virus related to HIV-1 has been isolated and termed HIV-2. This virus is reported by Guyader et al., Nature 326:662. 1987; Brun-Vezinet et al.. The Lancet 1:128, 1987; and Clavel et al., Science 233:343. 1986, each of which is hereby incorporated by reference. Although the genetic organization of HIV-2 is similar to that of HIV-l, the two genomes cross-hybridize poorly even under low stringency conditions (Guyader et al. , Nature 326:622. 1987).
  • SIV simian immunodeficiency virus
  • STLV-III simian immunodeficiency virus
  • HIV-l and HIV-2 particularly the latter. See Daniel et al., Science 228:1201-1204 (1985); Kanki et al., Science 230:951-954 (1985); Chakrabarti et al., Nature 328:543-547 (1987); and Ohta et al., Int'l. J. Cancer 4.1:115-222 (1988), each of which is hereby incorporated by reference.
  • Members of this viral group exhibit minor variations in their geno ic sequences, and have some differences in their restriction enzyme maps.
  • HIV-l has already entered large segments of the world population, and substantial effort has been directed toward developing treatments for individuals infected with it. In addition to investigations into synthetic pharmaceuticals, effort has been directed toward utilizing variants of HIV-l and HIV-2 to design AIDS therapeutics. Intracellular immunization using gag gene mutants or capsid targeted Gag-nuclease fusion molecules have been described as potential anti-retroviral strategies (Trono et al.. Cell .59:113-120 (1989); Natsoulis et al.. Nature 3_52:632-635 (1991)). Summary of the Invention
  • Vpx polypeptides encoded by the vpx gene from the SIV/HIV type-2 subgroup of viruses that are related to, but different from, HIV-l exert an inhibitory effect on HIV-l infection.
  • Vpx polypeptides to describe these polypeptides, and, by that term, we mean to include the polypeptide encoded by the HIV-2 open reading frame termed orfX or vpx which has about 336 basepairs and is located in the central region of the HIV-2 genome between the pol ORF and the env ORF. See, e.g., Henderson et. al Science 241:199- 201 (1988); Yu et al.
  • the invention features a method of treating a patient infected with human immunodeficiency virus-type I (HIV-l) by administering a Vox polypeptide in an amount effective to reduce pathogenic HIV-l levels in the patient.
  • HIV-l human immunodeficiency virus-type I
  • Vpx polypeptides curtail HIV-l replication, thereby reducing overall viral load.
  • the Vpx polypeptides may be delivered by various vehicles as described below.
  • the Vpx polypeptide is administered to the patient in a pharmaceutically acceptable carrier and in an amount effective to reduce pathogenic HIV-l levels in the patient.
  • the patient is administered a therapeutic composition comprising nucleic acid encoding the Vpx polypeptide in an expressible genetic construction, e.g., one capable transforming patients' cells, such as a viral vector capable of infecting the patient.
  • the vector may be administered directly to the patient, or cells may be removed from the patient and transformed with the above-described nucleic acid, after which the transformed cells are returned to the patient's body.
  • Suitable viral vectors include HIV- 1 and HIV-l within a retroviral vector. Figure 4, below, gives the sequence of specific Vpx polypeptides.
  • the nucleic acid administered to the patient may also comprise a sequence encoding (or the VPX polypeptide may include) a CD4-binding polypeptide (such as a HIV-l gpl20) to facilitate targeting of HIV-l infected cells expressing CD4 or a gpl20-binding polypeptide (such as a CD4 polypeptide) to facilitate targeting to HIV-l infected cells expressing gpl20.
  • a CD4-binding polypeptide such as a HIV-l gpl20
  • a gpl20-binding polypeptide such as a CD4 polypeptide
  • Another aspect of the invention features therapeutic compositions adapted for administration to a patient infected with human immunodeficiency virus-type I (HIV-l) .
  • the therapeutic compositions may include a Vpx polypeptide in a pharmaceutically acceptable carrier and at a dosage effective to reduce HIV-l infection.
  • the invention features therapeutic compositions comprising nucleic acid encoding a Vpx polypeptide in an expressible genetic construction, such as any of those described above.
  • Figure 1 is a diagram representing the construction of an HIV-l viral vector containing vpx. A representative genomic organization of the HIV-l and part of the HXB2UX and HXB2U ⁇ X are shown.
  • Figure 2 is an immunoblot analysis of the sucrose gradient purified-HXB2UX and HXB2U ⁇ X virions.
  • Figure 3 is a graph representing an infectivity study on a human CD4-positive T cell line (SupTl) with HXB2UX and HXB2U ⁇ X viruses.
  • FIG. 4 diagrams the primary structure of Vpx polypeptides.
  • Figure 5 retroviral vectors for the administration of the Vpx polypeptide by gene transfer therapy.
  • the invention includes therapies using any protein which is homologous to simian immunodeficiency virus/human immunodeficiency virus type- 2 (SIV/HIV type 2) Vpx (Fig. 4, SEQ ID NOS: 1, 2 and 3) as well as other naturally occurring Vpx polypeptides.
  • SIV/HIV type 2 simian immunodeficiency virus/human immunodeficiency virus type- 2
  • Vpx Fig. 4, SEQ ID NOS: 1, 2 and 3
  • allelic variations also included are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high (e.g., washing at 2xSSC at 40 C with a probe length of at least 40 nucleotides) stringency conditions to naturally occurring Vpx encoding nucleic acid (for other definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby incorporated by reference) .
  • the term also includes chi eric polypeptides that include Vpx together with unrelated sequences.
  • the invention also includes any biologically active fragment or analog of Vpx.
  • biologically active is meant possessing therapeutically useful anti- HIV-l activity wh h is characteristic of the 112-amino acid Vpx polypeptides shown in Fig. 4 (SEQ ID NOS: 1, 2 and 3) .
  • Therapeutically useful activity of a Vpx fragment or Vpx analog can be determined in any one (or more) of a variety of Vpx assays, for example, those assays described in this application.
  • a Vpx analog possessing, most preferably 90%, preferably 40%, or at least 10% of the activity of 112-amino acid Vpx polypeptides (shown in Fig. 4; SEQ ID NOS: 1, 2 and 3), in any in vivo or in vitro Vpx assay (e.g., those described below) is considered biologically active and useful in the invention.
  • Preferred analogs include 112-amino acid Vpx (or biologically active fragments thereof) whose sequences differ from the wild-type sequence only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not destroy the polypeptide's relevant biological activity as measured using in vivo or in vitro (e.g., those described above).
  • conservative amino acid substitutions for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not destroy the polypeptide's relevant biological activity as measured using in vivo or in vitro (e.g., those described above).
  • Preferred analogs also include Vpx (or biologically active fragments thereof) which are modified for the purpose of increasing peptide stability; such analogs may contain, for example, one or more desaturated peptide bonds or D-amino acids in the peptide sequence.
  • Analogs can differ from naturally occurring Vpx by amino acid sequence differences or by modifications that do not affect sequence, or by both. Analogs of the invention will generally exhibit at least 65%, more preferably 80%, even more preferably 90%, and most preferably 95% or even 99%, homology with all or part of a naturally occurring Vpx sequence. The length of comparison sequences will generally be at least about 15 amino acid residues, preferably more than 40 amino acid residues. Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, glycosylation, or carboxylation.
  • phosphorylated amino acid residues e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • Analogs can differ from naturally occurring Vpx by alterations of their primary sequence. These include genetic variants, both natural and induced. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids. Alternatively, increased stability may be conferred by cyclizing the peptide molecule.
  • the invention also includes biologically active fragments of the polypeptides.
  • fragment as applied to a polypeptide, will ordinarily be at least about 10 contiguous amino acids, typically at least about 20 contiguous amino acids, more typically at least about 30 contiguous amino acids, usually at least about 40 contiguous amino acids, preferably at least about 50 contiguous amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length.
  • Fragments of Vpx can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of Vpx can be assessed by methods described below.
  • Vpx polypeptides containing amino acids that are normally removed during protein processing (if any) , including additional amino acids that are not required for the biological activity of the polypeptide (if any) , or including additional amino acids (if any) that result from alternative mRNA splicing or alternative protein processing events.
  • the invention also includes polypeptides (or nucleic acid either encoding polypeptides) which are homologous to the Vpx protein or homologous to the vpx gene and are useful for the treatment of individuals infected with HIV-l. Sequences which are considered to be homologous are those which are 70 % homologous. Homologous refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or ami. ⁇ acid mono eric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology.
  • Vpx polypeptide With the availability of the cloned gene, the substantially pure Vpx polypeptide can be produced in quantity using standard techniques (Scopes, R. Protein Purification: Principles and Practice 1982 Springer- Verlag, NY) .
  • a pharmaceutical comprising the Vpx polypeptide together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the polypeptide to patients infected with HIV-l.
  • a substantially pure preparation of a polypeptide is a preparation which is substantially free (e.g., to the extent required for formulating Vpx into a therapeutic composition) of the proteins with which it naturally occurs in a cell.
  • Fragments or analogs of the Vpx protein may also be administered to a patient infected with HIV-l in the manner described above. Fragments or analogs which are useful for this purpose include those which are described above and are useful for the treatment of a patient infected with HIV-l. Fragments and analogs which will be useful for the therapeutic treatment of patients infected with HIV-l are determined using the assays provided in the examples, below, among others.
  • the Vpx polypeptide may also be administered to a patient infected with HIV-l in the form of a fusion protein consisting of a Vpx polypeptide, fused to the gpl20 protein, or a fragment thereof which is sufficient to bind the CD4 receptor of T cells.
  • This fusion protein allows delivery of the Vpx polypeptide into uninfected T cells expressing the CD4 receptor.
  • the Vpx polypeptide may also be administered to a patient infected with HIV-l in the form of a fusion protein consisting of the Vpx polypeptide, or a therapeutically useful fragment or derivative, fused to the CD4 protein, or a fragment thereof, which is sufficient to bind gpl20.
  • This fusion protein allows delivery of the Vpx polypeptide into infected T cells expressing gpl20 on their surface.
  • the Vpx-gpl20 fusion polypeptide or the Vpx-CD4 fusion polypeptide may be generated using standard techniques of molecular biology to generate fusions encoded from a suitable vector (Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York (1989)). Either the gpl20 fragment or the CD4 fragment may enable internalization of the Vpx polypeptide through endocytosis. The usefulness of such gene fusions constructs may be determined using the methods described below in the examples, among others.
  • the invention includes administering either fusion polypeptide alone in a pharmaceutically acceptable carrier, or administering both fusions together in an acceptable carrier.
  • formulations of this invention can be applied for example by parenteral administration, intravenous, subcutaneus, intramuscular, intracranial, intraorbital, opthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic Formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain excipients sterile water or saline, polyalkylene glycols such as polyethylene glyeol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of present factors.
  • Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particals, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9- lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • a particularly preferred embodiment features administering to the patient genetic constructions which encode any of the above-described Vpx polypeptides, and (after transformation of patient cells) can express the Vpx polypeptide.
  • nucleic acid vehicles which can activate or be activated to enter cells of the host organism and, having done so, to be expressed there. Examples The following examples are provided to illustrate the invention not to limit it.
  • Vpx has been shown to bind to nucleic acids and may also play a structural role as suggested by its abundance in the virions (Henderson et al., Science 241:199-201 (1988).
  • HXB2UX was generated (Fig.l).
  • HXB2UX is a derivative of an infectious molecular clone of HIV-l, HXB2 (Ratner et al., AIDS Res. Hum. Retroviruses 2:57-69 (1987)).
  • HXB2U ⁇ X an isogenic clone of HXB2UX except for a premature stop codon in vpx. was prepared.
  • Figure 1 A representative genomic organization of the HIV-l and part of the HXB2UX and HXB2U ⁇ X are shown in Figure 1.
  • the parental clone was the infectious molecular HIV-l clone HXB2 (Ratner et al., AIDS Res. Hum.
  • HXB2UX contains vpx derived from SIVmac (BK28) (Kornfeld et al., Nature 326:610-613 (1987).
  • HXB2U ⁇ X contains VPX with a premature stop codon introduced by an Xba I linker after the first twenty amino acids of Vpx (Yu et al., Nature 225:262-265 (1988)).
  • Xba I linker Unlike HXB2, both clones have an intact vpu initiation codon.
  • the restriction enzymes used for the cloning are also shown.
  • DNA fragments of VPX or the vpx with a premature stop codon were prepared by polymerase chain reaction (PCR) (Saiki et al., Science 239:487-491 (1988)) with two primers 5'-TAA AAG TAG TAA TCG ATG TCA GAT CCC AGG GAG-3' (SEQ ID NO: 5) and 5'-GCG GGG GTC GAC TTA TGC TAG TCC TGG AGG GGG-3' (SEQ ID NO: 6).
  • PCR polymerase chain reaction
  • the template for the PCR was pBK28 for vpx and pBK28 ⁇ X for vpx with a premature stop codon, respectively, and the PCR condition was as suggested by the manufacturer.
  • pBK28 and pBK28 ⁇ X were previously described (Yu et al., Nature 335:262-265 (1988)).
  • Each PCR product was cloned into HXB2U, a clone isogenic to HXB2 (Ratner et al., AIDS Res. Hum. Retroviruses 2:57-69 (1987)) except for an intact vpu initiation codon, as a Clal-Sall fragment. By this cloning, the original vpr was destroyed.
  • HXB2UX and HXB2U ⁇ X constructs were transfected into Cos-7 cells and the released viruses were purified through a sucrose gradient and analyzed by immunoblotting with a reference serum from an HIV-l infected individual and a goat serum specific for Vpx (Fig. 2) .
  • Human serum revealed an almost identical protein profile for both virions.
  • the gag products, pl7 and p24; and the pol products, p66 and p51, were readily detected (Fig. 2, lanes 1 and 2).
  • a protein of relative molecular mass of about 12,000 (M r l2K) was detected in HXB2UX virions (Fig.
  • HXB2U ⁇ X virions Fig. 2, lane 4
  • the size of the protein found in HXB2UX corresponded with that of the Vpx described previously (Yu et al., Nature 335:262-265 (1988)).
  • Example 2 To examine the effects of the incorporated Vpx on viral infectivity, HXB2UX and HXB2U ⁇ viruses derived from the transfected Cos-7 cells were used to infect a CD4- positive human T cell line, SupTl, which is susceptible to HIV-l infection. The infection was monitored by reverse transcriptase (RT) activity of the culture supernatant. A representative result is shown in Fig. 3. RT activity was detected in HXB2U ⁇ X infected SupTl at day 15 and continued to increase. In contrast, the RT activity of HXB2UX infected SupTl was similar to an uninfected control throughout the observation period of 34 days.
  • RT activity of HXB2UX infected SupTl was similar to an uninfected control throughout the observation period of 34 days.
  • Retroviral vectors or other viral vectors with the appropriate tropisms for cells infected by HIV-l, may be used as a gene transfer delivery system for the Vpx polypeptide.
  • Numerous vectors useful for this purpose are generally known have been described (Miller, Human Gene Therapy 15-14 (1990); Friedman, Science 244:1275- 1281 (1989); Eglitis and Anderson, BioTechniques e>:608- 614 (1988) ; Tolstoshev and Anderson, Current Opinion in Biotechnology 1:55-61 (1990); Sharp, The Lancet 337:1277- 1278 (1991); Cornetta et al., Nucleic Acid Research and Molecular Biology 26:311-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); and Miller and Rosman, Biotechniques 2:980-990 (1989)).
  • Retroviral vectors are particularly well developed and have been used in a clinical setting (Rosenberg, et al N. Engl. J. Med 323:370 (1990)).
  • the retroviral constructs, packaging cell lines and delivery systems which may be useful for this purpose include, but are not limited to, one, or a combination of, the following: Moloney murine leukemia viral vector types; self inactivating vectors; double copy vectors; selection marker vectors; and suicide mechanism vectors.
  • Moloney murine leukemia retroviral system of Vpx delivery is particularly useful since it targets delivery of the Vpx protein to the hematopoietic cells which ultimately give rise to the T-cells.
  • Vpx polypeptide can be further restricted to cells which are infected by HIV-l directly by virtue of utilizing retroviral constructs in which the HIV-LTR is used to drive expression from the vpx gene.
  • retroviral constructs in which the HIV-LTR is used to drive expression from the vpx gene.
  • the 3'LTR of the Moloney murine leukemia vector must be deleted.
  • Vector strategies which include either the entire HXB2UX construct or the vpx gene driven by the HIV-LTR are shown in Figure 5.
  • Fragments or derivatives of the Vpx polypeptide may also be administered by retroviral gene transfer therapy or another suitable viral vector system. Fragments or derivatives are defined as described above. Useful fragments or derivatives of Vpx may be administered by inserting the nucleic acids encoding these fragments or derivatives in place of the complete vpx gene in a gene therapy vector, as described above. Such constructs may be tested using the methods for testing the effects of Vpx on viral infectivity described above, among others.
  • Retroviral delivery of Vpx is particularly appropriate in HIV-l infected individuals who display the common secondary appearance of B-cell tumors as a result of immunodeficiency. These individuals may undergo bone marrow removal, treatment, and reimplantation as a matter of course for the treatment of the B-cell tumors. At this time standard techniques for the delivery of gene therapy vectors may be used to transfect stem cells. Such transfection may result in Vpx synthesizing T-cells useful in lowering the infective levels of HIV-l in the patient.
  • Non viral methods for the therapeutic delivery of nucleic acid encoding VPX Nucleic acid encoding Vpx, or a fragment thereof, under the regulation of the HIV-LTR and including the appropriate sequences required for insertion into genomic DNA of the patient, or autonomous replication may be administered to the patient using the following gene transfer techniques: microinjection (Wolff et al., Science 247:1465 (1990)); calcium phosphate transfer (Graham and Van der Eb, Virology 52.:456 (1973); Wigler et al., Cell 14:725 (1978); Feigner et al., Proc. Natl. Acad. Sci. USA 81:7413 (1987)); lipofection (Feigner et al., Proc.
  • V. Mode of Vpx Action As a potential mechanism for the observed interference in HIV-l replication, while not essential to practicing the invention, is discussed below, with reference to the structural and non-structural effects of the Vpx protein can be considered.
  • the targeting of Vpx which is foreign to HIV-l virions, may affect the assembly and/or maturation of HIV-l.
  • the affinity of Vpx for single-stranded nucleic acids may be important for the interference of HIV-l replication (Henderson et al., Science 241:199-201 (1988)).
  • the data shown above demonstrate that Vpx expressed in the context of an HIV-l genome was incorporated into HIV-l virions and the resulting viruses lost infectivity in T cells.
  • Vpx can be regarded as a virion-specific inhibitory molecule against HIV-l. Previous studies showed the region near the carboxyl terminus was important for the function of Vpr of HIV-l (Yuan et al., AIDS Res. Hum. Retroviruses 6:1265-1271 (1990); Cohen et al. , J. AIDS, 2:11-18 (1990)). In Vpx, seven consecutive prolines are found at the carboxyl terminus which are absent in Vpx.
  • Vpx which is only present in the SIV/HIV-2 group of lentiviruses
  • Vpr which is present in most of the SIV/HIV-2 group and the HIV-l group
  • Some regions are highly conserved between Vpx and Vpr at the primary structure level (Fig.
  • Figure 4 shows the primary structure of three Vpx polypeptides.
  • Vprs and Vpxs were compared using a multiple sequence alignment program (GeneWorks, IntelliGenetics, California, USA) .
  • the sequences used were obtained from the Human Retroviruses and AIDS Database (Myers et al.. Human Retroviruses and AIDS (Los Alamos National Laboratory, Los Alamos, USA 1990)) except for HIV-l (Yuan et al., AIDS Res. Hum. Retroviruses .6:1265-1271 (1990)).
  • the following isolates were analyzed: HIV-2 R00 , SIVsmm H4 and
  • Vpx should reside in the regions that are non- homologous to HIV-l Vpr, mainly the region near the C- terminus and in the central region. If this is the case, it suggests that a molecule for specifically inhibiting a virus need not be a fusion of a virion associated motif to a functioning domain such as a nuclease or protease thus allowing greater freedom to design such therapeutics.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Procédés et compositions thérapeutiques permettant de traiter une infection par le virus d'HIV-1, ces procédés consistent à administrer une polypeptide Vpx, en particulier un polypeptide Vpx d'HIV-2 ou de SIV.
PCT/US1993/004301 1992-06-09 1993-05-07 Procedes therapeutiques destines au sida, a base de peptides vpx d'hiv-2 WO1993025235A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US89535292A 1992-06-09 1992-06-09
US07/895,352 1992-06-09

Publications (1)

Publication Number Publication Date
WO1993025235A1 true WO1993025235A1 (fr) 1993-12-23

Family

ID=25404385

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/004301 WO1993025235A1 (fr) 1992-06-09 1993-05-07 Procedes therapeutiques destines au sida, a base de peptides vpx d'hiv-2

Country Status (1)

Country Link
WO (1) WO1993025235A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996007741A1 (fr) * 1994-09-07 1996-03-14 Universite De Montreal Proteine pour ciblage dans des virions de vih sur la base de molecules de fusion vih-1/vpr
WO1996027389A1 (fr) * 1995-03-08 1996-09-12 Neovacs Immunogenes denues de toxicite derivant d'une proteine de regulation retrovirale, anticorps, procede de preparation et compositions pharmaceutiques les renfermant
FR2731355A1 (fr) * 1995-03-08 1996-09-13 Neovacs Nouveaux immunogenes, nouveaux anticorps, procede de preparation et compositions pharmaceutiques les renfermant
US6001985A (en) * 1995-04-14 1999-12-14 University Of Alabama Research Foundation Fusion protein delivery systems and uses thereof
US6200575B1 (en) 1996-03-07 2001-03-13 Neovacs Non-toxic immunogens derived from a retroviral regulatory protein antibodies preparation process and pharmaceutical compositions comprising them
EP1170368A2 (fr) 1994-01-27 2002-01-09 University Of Massachusetts Medical Center Immunisation par inoculation d'une unité de transcription d'ADN
US6555342B1 (en) 1998-06-03 2003-04-29 Uab Research Foundation Fusion protein delivery system and uses thereof
US6841381B1 (en) 1992-03-23 2005-01-11 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
US7622300B2 (en) 1998-06-03 2009-11-24 Kappes John C Trans-lentiviral vector particles and transduction of eukaryotic cells therewith

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF VIROLOGY, Volume 65(7), issued July 1991, L. MARCON et al., "Dispensable Role of the Human Immunodeficiency Virus Type 2 Vpx Protein in Viral Replication", pages 3938-3942. *
JOURNAL OF VIROLOGY, Volume 65(9), issued September 1991, X.F. YU et al., "The Vpx Gene of Simian Immunodeficiency Virus Facilitates Efficient Viral Replication in Fresh Lymphocytes and Macrophages", pages 5088-5091. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Volume 89, issued January 1992, M. LE GUERN et al., "Human Immunodeficiency Virus (HIV) Type 1 can Superinfect HIV-2-Infected Cells: Pseudotype Virions Produced with Expanded Cellular Host Range", pages 363-367. *
THE EMBO JOURNAL, Volume 11(9), issued September 1992, M. TRISTEM et al., "Evolution of the Primate Lentiviruses: Evidence from Vpx and Vpr", pages 3405-3412. *
VIROLOGY, Volum 184(1), issued September 1991, J.C. KAPPES et al., "Human Immunodeficiency Virus Type 2 Vpx Protein Augments Viral Infectivity", pages 197-209. *
VIROLOGY, Volume 177(1), issued July 1990, A.R. HART et al., "Interference Patterns of Human Immunodeficiency Viruses HIV-1 and HIV-2", pages 1-10. *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6841381B1 (en) 1992-03-23 2005-01-11 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
US7850956B2 (en) 1992-03-23 2010-12-14 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
EP1170368A3 (fr) * 1994-01-27 2004-05-12 University Of Massachusetts Medical Center Immunisation par inoculation d'une unité de transcription d'ADN
EP1170368A2 (fr) 1994-01-27 2002-01-09 University Of Massachusetts Medical Center Immunisation par inoculation d'une unité de transcription d'ADN
WO1996007741A1 (fr) * 1994-09-07 1996-03-14 Universite De Montreal Proteine pour ciblage dans des virions de vih sur la base de molecules de fusion vih-1/vpr
US6043081A (en) * 1994-09-07 2000-03-28 Universite De Montreal Expression vectors encoding recombinant proteins comprising a VPR/VPX virion incorporation domain for targeting into HIV-1 or HIV-2 virions
US5861161A (en) * 1994-09-07 1999-01-19 Universite De Montreal Chimeric proteins comprising a Vpr/Vpx virion incorporation domain for targeting into HIV-1 or HIV-2 virions
US6468539B1 (en) 1994-09-07 2002-10-22 Universite De Montreal Protein targeting into HIV virions based on HIV-1 VPR fusion molecules
US6132721A (en) * 1995-03-08 2000-10-17 Neovacs Non-Toxic immunogens derived from a retroviral regulatory protein, antibodies, preparation method therefor, and pharmaceutical compositions containing same
FR2731355A1 (fr) * 1995-03-08 1996-09-13 Neovacs Nouveaux immunogenes, nouveaux anticorps, procede de preparation et compositions pharmaceutiques les renfermant
WO1996027389A1 (fr) * 1995-03-08 1996-09-12 Neovacs Immunogenes denues de toxicite derivant d'une proteine de regulation retrovirale, anticorps, procede de preparation et compositions pharmaceutiques les renfermant
US6362000B1 (en) 1995-04-14 2002-03-26 University Of Alabama Research Foundation Fusion protein delivery system and uses thereof
US6001985A (en) * 1995-04-14 1999-12-14 University Of Alabama Research Foundation Fusion protein delivery systems and uses thereof
US7534603B2 (en) 1995-04-14 2009-05-19 The Uab Research Foundation Fusion protein delivery system and uses thereof
US6200575B1 (en) 1996-03-07 2001-03-13 Neovacs Non-toxic immunogens derived from a retroviral regulatory protein antibodies preparation process and pharmaceutical compositions comprising them
US6555342B1 (en) 1998-06-03 2003-04-29 Uab Research Foundation Fusion protein delivery system and uses thereof
US7259014B2 (en) 1998-06-03 2007-08-21 Uab Research Foundation Fusion protein delivery system and uses thereof
US7622300B2 (en) 1998-06-03 2009-11-24 Kappes John C Trans-lentiviral vector particles and transduction of eukaryotic cells therewith

Similar Documents

Publication Publication Date Title
Sharpless et al. Human immunodeficiency virus type 1 tropism for brain microglial cells is determined by a region of the env glycoprotein that also controls macrophage tropism
Bukovsky et al. Nef association with human immunodeficiency virus type 1 virions and cleavage by the viral protease
Haseltine et al. Structure of 3′ terminal region of type II human T lymphotropic virus: evidence for new coding region
CA2009403C (fr) Provirus hiv a encapsidation defectueuse, lignees cellulaires et leur utilisation
JP3587538B2 (ja) Ｈｉｖ−１ のインヒビターとしての合成ポリペプチド
Rushlow et al. Lentivirus genomic organization: the complete nucleotide sequence of the env gene region of equine infectious anemia virus
US5665577A (en) Vectors containing HIV packaging sequences, packaging defective HIV vectors, and uses thereof
Carrillo et al. Human immunodeficiency virus type 1 tropism for T-lymphoid cell lines: role of the V3 loop and C4 envelope determinants
CA2384271A1 (fr) Trimeres stabilises solubles de glycoproteines
HUP0202502A2 (hu) Virális fertőzőképességet blokkoló peptidek és eljárások ezek alkalmazására
US20030091594A1 (en) CD4-independent HIV envelope proteins as vaccines and therapeutics
EP0689586B1 (fr) Activite et fonction de la proteine virale r
WO1993025235A1 (fr) Procedes therapeutiques destines au sida, a base de peptides vpx d'hiv-2
EP0536234A1 (fr) Vecteurs contenant des sequences d'encapsidation du vih, vecteurs du vih a capside defectueux, et leur utilisation
WO1998000535A2 (fr) Procede pour inhiber l'infection par le vih-1, dosages de medicaments et procedes de diagnostic et pronostic de la susceptibilite d'infection par le vih
AU731975B2 (en) GP120 polypeptides having conformational discontinuous chemokine receptor binding sites and methods of inhibiting HIV infection
US5736391A (en) HIV gp41 mutants
WO1998015569A9 (fr) Polypeptides de gp120 possedant des sites de liaison aux recepteurs de la chimiokine a discontinuite de conformation et procedes d'inhibition des infections a vih
US5707864A (en) Nucleic acids encoding mutated human immunodeficiency virus matrix proteins
US7524927B2 (en) Compositions, method and kits relating to deletion mutations of immunodeficiency virus gp120 hypervariable regions
US20030219451A1 (en) Stable helical C peptides and uses therefor
Kannagi et al. Coexistence of Fusion Receptors for Human T‐Cell Leukemia Virus Type‐I (HTLV‐I) and Human Immunodeficiency Virus Type‐1 (HIV‐1) on MOLT‐4 Cells
US20050164164A1 (en) Hiv-1 virus tat-protein mutants
US20040137426A1 (en) Gp41 peptides and methods based thereon for inhibiting HIV fusion to target cells
JPH07509237A (ja) ｇｐ１２０ＨＩＶエピトープを模倣したペプチド

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)

Free format text: CA,JP, EUROPEAN PATENT(AT,BE,DE,DK,FR,GB,IE,IT,LU,MC,NL,PT,SE)

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载