WO1994012513A1 - Aids therapeutics based on hiv ma peptides - Google Patents
Aids therapeutics based on hiv ma peptides Download PDFInfo
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- WO1994012513A1 WO1994012513A1 PCT/US1993/000267 US9300267W WO9412513A1 WO 1994012513 A1 WO1994012513 A1 WO 1994012513A1 US 9300267 W US9300267 W US 9300267W WO 9412513 A1 WO9412513 A1 WO 9412513A1
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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
- C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
- C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/22—Viral peptide or viral protein
- Y10S930/221—Retrovirus related, or human immunodeficiency virus related, or simian immunodeficiency virus related
Definitions
- This invention relates to the treatment of infection with the human immunodeficiency virus (HIV) by which we mean to include all of the various viral types and strains denominated by that term, such as HTLV-III, LAV, ARV, HIV-1, HIV-2, and LAV-2.
- HIV human immunodeficiency virus
- HIV is an etiological agent of Acquired Immune Deficiency Syndrome (AIDS) .
- An example of HIV (now denominated HIV-1) is generally described in several articles: Barre-Sinoussi et al., Science 220:868 r 1983; Gallo et al., Science 224:500. 1984; Popovic et al.,
- HIV-2 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
- HIV-2 The genetic organization of HIV-2 is similar to that of HIV-1.
- a group of viruses isolated from monkeys termed simian immunodeficiency virus (SIV or STLV-III) , is related to HIV-1 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 3_28:543-547 (1987); and Ohta et al., Int'l. J. Cancer 41:115-222 (1988), each of which is hereby incorporated by reference. Members of this viral group exhibit minor variations in their genomic sequences, and have some differences in their restriction enzyme maps.
- MA matrix
- the MA protein plays an important role in all of the above aspects of viral assembly and release.
- the MA protein is modified by the addition of myristic acid at the N-terminal glycine residue (Schultz et al., Annu. Rev. Cell Biol. 4:611-647, 1988) .
- HIV 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 investigation of synthetic pharmaceuticals, effort has been directed toward utilizing variants of HIV-1 and HIV-2 to design AIDS therapeutics.
- Trono et al. (Cell 59:113-120, 1989), state that certain mutations in the DNA encoding the p24 and pl5 polypeptides disrupt viral replication.
- the invention features a method of treating a patient infected with human immunodeficiency virus (HIV) by administering to the patient a mutated HIV matrix (MA) polypeptide in an amount effective to reduce effective HIV levels in the patient. Also a part of the invention are methods of treating a patient by the administration of a therapeutic composition composed of the mutated MA polypeptide in a pharmaceutically acceptable carrier. Also included in the invention is a method of treating a patient infected with HIV by administration of a nucleic acid encoding the mutated MA polypeptide in an expressible genetic construction. Preferably, the expressible genetic construction is capable of remaining stably in cells of the patient and thereby transforming the cells of the patient.
- Such an expressible genetic construction may be a viral vector capable of infecting said patient.
- the nucleic acid sequence may also include a sequence encoding a CD4-binding polypeptide, or a nucleic acid sequence encoding a gpl20-binding polypeptide, or fragments thereof.
- the invention further includes a method of treating an HIV infected patient by removing cells from the patient, transforming the cells with the nucleic acid as described above, and returning transformed cells to the patient's body.
- the invention also provides a method of therapy whereby the genetic construction is administered directly to the patient.
- the viral vector used for any of the methods of nucleic acid administration may be human immunodeficiency virus-type I.
- the invention also provides a method for treating a patient infected with HIV wherein the mutated MA polypeptide contains a deletion or substitution of at least one amino acid in at least one of the following regions of a wild type MA polypeptide:
- a therapeutic composition adapted for administration to a patient infected with human immunodeficiency virus-type I including a mutated MA polypeptide in a pharmaceutically acceptable carrier or a nucleic acid encoding a mutated MA polypeptide in an expressible genetic construction, preferably for transforming cells of a human patient.
- the nucleic acid which is administered to the patient may be a part of a viral vector capable of infecting the patent and/or the nucleic acid may contain a sequence capable of encoding a CD4- binding polypeptide, a gpl20-binding polypeptide, or a fragment thereof.
- a mutated MA is meant an MA polypeptide having a deletion, insertion, substitution or other modification rendering the MA polypeptide effective as an inhibitor of pathogenic HIV viral replication, assembly, or infectivity by one of the assays described below. Particularly useful are the transfection, infection, and RT assay and radioimmunoprecipitation, immunoblot, and pulse chase assay procedures.
- viral mutations which result in normal levels of the gag and pol proteins, but confer decreased levels of the gpl20 and gp41 proteins in the virion are viral mutations which result in normal levels of the gag and pol proteins, but confer decreased levels of the gpl20 and gp41 proteins in the virion.
- Mutations which affect infectivity may be identified by the infectivity assay.
- Useful mutations affecting infectivity are those mutations which decrease infectivity by 20%, preferably 40% and most 80% or more.
- Mutations which affect viral replication and therefore have the therapeutic promise are those which decrease viral replication relative to wild-type virus, preferably decreasing replication by 10% and most preferably decreasing replication by 20% or more in a co- transfection assay.
- the mutated MA polypeptides included in the invention may be the equivalent mutation MA proteins from other strains of HIV.
- One skilled in the art may determine the equivalent mutations based upon amino acid and nucleic acid homology even if the exact residue number is not the same.
- the mutations according to the invention do not affect expression of the gag precursor, nor do they block processing of the precursor to yield component gag proteins.
- the mutated MA proteins retain the ability to interact with their wild- type gag counterparts, thereby improving the transdominant negative function of the mutants.
- Fig. 1 is the sequence of the nucleic acid encoding the MA polypeptide and the amino acid sequence of the MA polypeptide.
- Pig. 2 depicts the construction and virus production of MA mutants Dl to D8.
- RT reverse transcriptase
- Pig. 3 depicts detection of viral proteins. Detection of viral proteins in transfected COS-7 cells, supernatants, and virus pellets by RIPA (a, b, and c) and pulse-chase labeling (d) .
- Pig. 4 is a graph of virus infectivity of D1-D8 mutants of MA.
- Pig. 5 depicts analysis of D1-D8 viral proteins by immunoblot.
- (B) depicts analysis of R3WA and R3AP viral proteins by immunoblot.
- Pig. 6 is a graph of the dominant negative mutant assay for the Dl and D2 mutants.
- Pig. 7 depicts (a) construction of the D9 and D10 mutants (b) sequence homology among HIV-1 MA and other viruses and (c) virus infectivity.
- Pig. 8 depicts analysis of viral proteins and virion-associated RNA.
- A Viral proteins from transfected COS-7 cells were analyzed by radioimmunoprecipitation as previously described (29) .
- B Viral proteins from purified virions analyzed by immunoblot with a sheep anti-gp 120 serum (top) and pooled HIV-1-positive human sera (bottom) .
- C Viral RNA analyzed by dot blot assay.
- Pig. 9 is analysis of D9 and D10 DNA synthesis after infection.
- Pig. 10 is a graph of dominant negative mutant assay for D9 and D10.
- Pig. 11 depicts analysis of DO, B5, and B8 viral proteins by immunoblot.
- Pig. 12 is a graph of dominant negative mutant assay for DO.
- Pig. 13 depicts a viral construct with the nucleic acid encoding MA in an expressible position.
- MA Polypeptides Without wishing to bind our to specific modes of action, it appears that there are at least three classes of MA polypeptides which are useful as therapeutics for HIV infection.
- the first class of MA polypeptides which are therapeutically useful are those which disrupt the incorporation of the Env polypeptides gpl20 and gp41 into the viral particle in a dominant negative manner. Such MA mutants result in the production of non-infectious particles which incorporate the viral proteins synthesized by the pathogenic HIV.
- Mutants useful for this purpose may be identified by first testing candidate alterations for detectable levels of synthesis and stability of both Gag and Env polyproteins, this may be done by the methods provided below in Section IV and in the examples.
- detectable levels synthesis and stability is meant that levels of synthesis and stability are, taken together, at least 15% of that which is detected with wild-type virus.
- useful MA polypeptides of this class are further distinguished by their ability to disrupt pathogenic HIV replication in a trans-dominant manner using the dominant negative mutant assay described below.
- MA polypeptides with therapeutic promise will have activity in the dominant negative assay which is at least five-fold lower than wild-type between days 1 and 10 of the assay.
- the desirable mutant is correctly synthesized and localized, but blocks stable incorporation of the Env proteins encoded by the pathogenic virus, thereby resulting a failure of the cell to yield infectious particles.
- MA polypeptides with the useful therapeutic characteristic of disrupting functional Env protein incorporation include those MA polypeptides with deletions, insertions, or point mutations between amino acids 3 and 104.
- Preferred mutations of this class include the MA mutations Dl (a deletion between amino acids 10 and 21) , D2 (deletion between amino acids 20 and 32) , D3 (deletion between 31 and 41) , D7 (deletion between 78 and 91) and D8 (deletion between 90 and 104) and point mutations (for example 1) the change of Trp36 and Ala37 to Arg and Pro (R3WA) , respectively, and 2) the change of Ala 37 to Pro (R3AP) ) .
- a second class of MA mutants useful for the therapeutic treatment of HIV infection are those MA alterations which, when incorporated into the viral particle, disrupt entry of this virus into uninfected cells.
- This phenotype may be identified using tests of viral infectivity provided in the examples.
- desirable mutations should synthesized and localized at normal levels, as indicated above. Such mutations may be further identified using the dominant negative assay described below and selecting those mutations which confer a five-fold or greater reduction in RT Values relative to wild-type between days 1 and 10 of the assay.
- MA mutations including deletions, substitutions or additions to naturally occurring (wild type) MA sequences
- Techniques for generating a universe of candidate mutants are well known to those in the art. One such technique features random mutagenesis as described by Rhee and Hunter (EMBO, 10:535-546, 1990). The pool of candidate mutants can then be screened for their ability to disrupt incorporation by various techniques including the techniques described in Examples 1 to 3, below.
- Mutations useful for anti-HIV therapy by virtue of their disruption of virus entry include, but are not limited to, mutations which alter the region between amino acids 104 and 132 (see D9 and D10, Fig. 7) .
- a third class of MA mutants useful for anti-HIV therapy are those mutations which disrupt the assembly and/or release of virions. These mutations may be identified by screening for normal synthesis and stability of Gag polyproteins and a failure to assemble virions and a positive result indicating inhibitory effect in the dominant negative mutant assay, by the criterion indicated above.
- An example of a MA mutant with the characteristics which are desirable is described in Example 3. This example is a deletion of amino acids 5 to 16 of the MA polypeptide.
- MA mutants according to the invention are effective HIV therapeutics because they function in a so-called "transdo inant" fashion. Specifically, it appears that the MA mutants become physically associated with an otherwise pathogenic virion component being manufactured in an HIV-infected cells. As a result, the load of properly assembled infectious virions that cell manufactures is reduced in proportion to the mutant MA levels in the cell. When mutant MA is delivered to the cell in sufficient excess, the incidence of ineffective viral assembly resulting from attempts to incorporate mutant MA becomes far higher than the incidence of incorporation of wild type MA.
- the substantially pure MA polypeptide can be produced in quantity using standard techniques (Scopes, R.
- another aspect of the invention is a pharmaceutical comprising the MA 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.
- a substantially pure preparation of a polypeptide is a preparation which is substantially free (e.g., to the extent required for formulating MA into a therapeutic composition) of the proteins with which it naturally occurs in a cell.
- Fragments or analogs of the MA protein may also be administered to a patient infected with HIV in the manner described above. Fragments or analogs which are useful for this purpose include fragments and analogs of peptides described herein which are useful for the treatment of a patient infected with HIV-1. Fragments and analogs which will be useful for the therapeutic treatment of patients infected with HIV-1 are determined using the assays provided in the examples, below, among others.
- the MA polypeptide may also be administered to a patient infected with HIV in the form of a fusion protein consisting of a mutated MA polypeptide, fused to the gpl20 protein, or a fragment thereof which is sufficient to bind the CD4 receptor of T cells, monocytes, macrophages or other cell types infected by HIV.
- This fusion protein allows delivery of the desirable MA polypeptide into uninfected T cells, monocytes, macrophages or other cell types infected by HIV and expressing the CD4 receptor.
- the mutated MA polypeptide may also be administered to a patient infected with HIV in the form of a fusion protein consisting of the desirable MA 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 desirable MA polypeptide into infected T cells expressing gpl20 on their surface.
- the MA-gpl20 fusion polypeptide or the MA-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 MA polypeptide through endocytosis.
- the sequences of both the gpl20 and CD4 genes for the stated use may be obtained from Maddon et al., Cell 42:93, August, 1985; Weaver et al., Cell. 4_5:247, 1986; Rekosh et al, Proc. Natl. Acad. Sci., 5:334, 1988; Chanda et al., Vaccines, :207, 1989 and
- 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 glycol, oils of vegetable origin, or hydrogenated napthalenes.
- Biocompatible, biodegradable lactide polymer, lactide/glycoside copolymer, or polyoxyethylene-polyoxypropylene copoly ers may be used to control the release of present factors.
- Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, 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 MA polypeptides described herein, and (after transformation of patient cells) can express the MA polypeptide.
- administering to the patient genetic constructions which encode any of the MA polypeptides described herein, and (after transformation of patient cells) can express the MA polypeptide.
- HIV-1 or HIV-2 strains of the many that have been fully characterized e.g., MN, HXB2, LAI, NL43, MFA, BRVA and Z321.
- nucleic acid vehicles which can activate or be activated to enter cells of the host organism and, having done so, to be expressed there.
- nucleic acid vehicles which can activate or be activated to enter cells of the host organism and, having done so, to be expressed there.
- Retroviral vectors may be used as a gene transfer delivery system for the MA 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 j>: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 £6:311-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 12:407-416 (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.
- 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.
- the Moloney murine leukemia retroviral system of MA delivery is particularly useful since it targets delivery of the MA protein to the hematopoietic cells which ultimately give rise to the T-cells.
- the delivery of the MA polypeptide can be further restricted to cells which are infected by HIV-1 directly by virtue of utilizing retroviral constructs in which the HIV-LTR is used to drive expression from the gag gene. To achieve proper expression from such a construct the 3 , LTR of the Moloney murine leukemia vector must be deleted.
- Vector strategies which include either the entire HXB2UX construct or the gag gene driven by the HIV-LTR are shown in Fig. 13.
- Fragments or derivatives of the MA 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 MA may be administered by inserting the nucleic acids encoding these fragments or derivatives in place of the complete gag gene in a gene therapy vector, as described above. Such constructs may be tested using the methods for testing the effects of MA on viral infectivity described above, among others.
- Retroviral delivery of MA is particularly appropriate in HIV-1 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 MA synthesizing T-cells useful in lowering the infective levels of HIV-1 in the patient.
- Nucleic acid encoding MA, 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 ..chniques: microinjection (Wolff et al., Science "47:1465 (1990)); calcium phosphate transfer (Gr.- ' .'-am and Van der Eb, Virology 52:456 (1973); Wigler et al., Cell 11:725 (1978); Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987)); lipofection (Feigner et al., Proc. Natl. Acad.
- the results disclosed herein indicate that the HIV MA protein plays an important role in early steps of the virus life cycle. Although the MA protein's involvement in the process of reverse transcription cannot be excluded, studies using transmission electron microscopy suggests that the entry of the mutant virus cell into the host might be affected.
- HXB2R3 The molecular clone of HIV- 1, HXB2R3, was constructed by exchanging the Xhol-Xbal fragment from HXB2R (Yun et al., AIDS Res. Hum. Retroviruses 6:1265-1271, 1990) with the Xhol-Xbal fragment from HXB3 (Ratner et al., AIDS Res. Hum. Retroviruses 3:57-69, 1987). This fragment includes the 3' long terminal repeat and part of the nef coding sequence. In contrast to HXB2 (Ratner et al., AIDS Res. Hum. Retroviruses 3:57-69, 1987), HXB2R3 contains full- length vpr and new coding sequences.
- COS-7 cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf serum.
- SupTl cells were maintained in RPMI 1640 medium plus 10% fetal calf serum.
- HIV-1-positive human sera were kindly provided by K. Meyer of the Fenway Community Health Center, Boston, Mass.
- the sheep anti- gpl20 serum of HIV-1 was obtained from the AIDs Research and Reference Program (catalog no. 192) , National Institutes of Health, Bethesda, Md.
- Oligonucleotide-directed mutagenesis of MA The Sstl-Apal fragment covering the gag region of HXB2R3 was subcloned into pGEM7Z(+) (Promega, Madison, Wis.). Single-stranded uracil-containing DNA was prepared and used for site-directed mutagenesis according to the protocol of the manufacturer (Bio-Rad, Richmond, Calif.). The sequences of primers used for mutagenesis were as follows: Dl, 5'-CCC CCT GGC CTT AAC CCG CTT AAT ACT G- 3'; D2.
- the positions of MA deletion mutants are illustrated in Fig. 1.
- the myristoylation mutant, Myr ⁇ contains a substitution of valine for glycine at amino acid position 2. Mutants were screened by restriction enzyme digestion and DNA sequencing. The BssHII-Pstl fragments which contain the MA mutations were cloned back into the vector of HXB2R3. Transfection, infection, and RT assay. COS-7 cells were trypsinized and seeded at about 30% confluence 24 h before transfection.
- TD buffer 25 mM Tris-HCl, ph 7.4; 140 mM NaCl; 5 mM Kcl; 0.7 mM K 2 HP0 4 ) containing 400 ⁇ g of DEAE-dextran and 2 ⁇ g of wild-type or mutant DNA. Transfection was carried out at 37°C for 30 min. For the transfection of SupTl cells, the trypsinization step was omitted.
- SupTl cells (10 7 ) were washed once with phosphate-buffered saline and resuspended in 3 ml of TD buffer containing 600 ⁇ g of DEAE-dextran and 6 ⁇ g of DNA.
- SupTl transfection were as follows: mock, 6 ⁇ g of pUC18; wild-type, 1 ⁇ g of HXB2R3 plus 5 ⁇ g of PUC18; wild-type + Dl, 1 ⁇ g of HXB2R3 plus 5 ⁇ g of Dl; wild-type + D2, 1 ⁇ g of HXB2R3 plus 5 ⁇ g of D2; wild-type + Myr ⁇ , 1 ⁇ g of HXB2R3 plus 5 ⁇ g of Myr ⁇ . Transfections were carried out at room temperature for 20 min. Virus infectivity was tested with SupTl cells by using cell-free supernatants of transfected COS-7 cells as previously described (Yu et al., J.
- RT reverse transcriptase
- samples used in the reverse transcriptase (RT) assay were prepared from polyethylene glycol-precipitated viral pellets from the supernatant of transfected or infected cells. The assay was performed as previously described (Yu et al., J. Virol. 64:5688-5693, 1990). Radioimmunoprecipitation, immunoblot, and pulse- chase analysis.
- COS-7 cells were incubated for 12 h in cysteine-free RPMI 1640 medium containing [ 35 S]cysteine (0.1 mCi/ml; NEN) .
- Virion proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reacted with HIV-1-positive sera or sheep anti-gpl20 serum.
- COS-7 cells 60 h posttransfection
- MA deletion mutants The matrix protein of HIV-1 (MA) consists of 132 amino acids. Site- directed mutagenesis was applied to generate in-frame deletion mutants of MA, Dl, D2, D4, D5, D7 and D8 contained deletions of 10 to 15 amino acids in MA (Fig. 1) . D3 and D6 contained deletions of 9 and 10 amino acids, respectively, as well as substitutions of leucine to isoleucine at positions 41 and 78, respectively (Fig. 1).
- Virus production in the other MA mutants was approximately two- to five-fold greater than production of the wild-type virus as measured by RT activity in the supernatants of transfected cells (Fig. 2). Since mutations in D4, D5, and D6 significantly affected virus assembly, further studies focused on mutants Dl, D2, D3, D7, and D8, unless otherwise indicated.
- the level of p55 detected in some of the mutant-transfected cells was lower than that in the wild-type-transfected cells (Fig. 3b) .
- the processing of the mutant Gag polyproteins was apparently not affected, as p24/p25 and mutant MA were detected in mutual-transfected cells (Fig. 3b) and/or virus pellets (Fig. 3c) .
- Mutant MA was less detectable than wild-type MA in transfected cells (Fig. 3b) , suggesting that it was either very unstable or less immunogenic than the wild- type MA.
- the supernatants from the labeled COS-7 cells were used to purify released virions and subjected to RIPA.
- wild-type virions p24 and MA were detected (Fig. 3c) .
- MA mutants Dl, D2, D3, D7, and D8 produced more virions that the wild-type virus as determined by the increased level of p24, p34, and in some cases MA (Dl and D2) (Fig. 3c) .
- the level of MA detected in the D3 and D7 virions was much lower than that of the wild-type MA (Fig. 3c) .
- MA deletion mutants interfere the replication of wild-type virus.
- the deletion mutants described herein did not block virus assembly and release, suggesting that mutant Gag polyproteins can retain their ability to interact with each other and form particles. If the MA mutant Gag polyproteins can still interact with the wild-type Gag polyproteins and assemble into virions, the incorporation of Env proteins and infectivity of these resulting virions might be impaired.
- wild-type DNA was cotransfected with either Dl or D2 DNA into SupTl cells.
- Virus product,as mentioned by RT activity in the supernatants of transfected cells was quicker and higher in cells transfected wild-type DNA alone than in cells cotransfected with wild-type and Dl or D2 DNAs (Fig.
- MA mutations D9 and DIP A series of in-frame deletion mutations in the MA protein of human immunodeficiency virus type 1 (HIV-1) were generated. Two of these mutations did not affect virus assembly and were analyzed for their potential defects in the early steps of the virus life cycle.
- Mutant D9 contains an in-frame deletion of 10 amino acids (105 through 114) and a substitution of isoleucine with leucine at position 104 at the carboxy terminus of pl7 (Fig. 7A) .
- Mutant D10 contains a deletion of 13 amino acids (116 through 128) at the carboxy terminus of pl7 (Fig. 7A) . Wild-type (wt) DNA (HXB2R3 Yu et al.
- mutant viruses were significantly impaired compared with that of the wt virus.
- the production of mutant D9 virus was delayed by approximately 10 days, as monitored by RT activity in the supernatants of infected cells (Fig. 7C) .
- Virus production was delayed even more dramatically in cells infected with mutant D10 (Fig. 7C) , with no virus production detected in the first 30 days postinfection (Fig. 7C) .
- Cytopathic effects and syncytium formation were also delayed in D9- and DlO-infected Sup-Tl and H9 cells.
- p24/p25 and pl7 were detected in the wt cell lysate (Fig. 2A) .
- Gag-related proteins P55, P41, P24/25, and P17 were also detected in D9 and DIO cell lysates (Fig. 7A) .
- Mutant P55, P41, and P17 migrated more quickly than the wt Gag proteins, corresponding to the deletions in P17. It appears that the synthesis and processing of mutant Gag proteins were not greatly affected by deletions in D9 and DIO.
- Viral proteins in mature virions were analyzed by immunoblot using viral pellets from the supernatants of transfected COS-7 cells.
- Gag proteins P24 and P17, Pol proteins p66, p51, and p34, and Env proteins gp 120 and gp4l were detected in wt and mutant virions by the HIV-1- positive sera (Fig. 8B) .
- gpl20 was also detected in wt and mutant virions by a sheep anti-gpl20 serum (Fig. 8B) .
- Deletions in D9 and D10 did not appear to affect the late steps of the virus life cycle, such as viral protein synthesis and processing and virus assembly and release. However, both mutant viruses had severe defects in virus infectivity, suggesting that early steps in the virus life cycle were affected. To test this possibility, the synthesis of the first viral product (viral DNA) was monitored immediately after infection. If the defect of the mutant viruses involved early steps in the virus life cycle, viral DNA synthesis would be blocked. Equal amounts of wt and mutant viruses from the supernatants of transfected COS-7 cells, as measured by virion-associated RT .activity and viral proteins (Fig. 8B) , were incubated with Sup-Tl cells at 37°C for 2 h.
- the viral DNA isolated from infected cells by the method of Hirt is unlikely to be the input plasmid DNA.
- the signal of wt HIV-l DNA was stronger at 72 h postinfection than at 12 h postinfection (Fig. 9) , suggesting the presence of newly synthesized viral DNA.
- the input plasmid DNA migrated differently from the viral DNA isolated by the method of Hirt.
- D9 and DIO viruses showed a significant decreased ability to synthesize viral DNA after infection (Fig. 4) .
- the defect was more dramatic for D10 than for D9, corresponding to the results of the virus infectivity assay (Fig. 7C) .
- Viral DNA synthesis at 72 h postinfection was also impaired in D9 - and DlO-infected cells compared with that in cells infected with the wt virus (Fig. 9) . This suggests that the spread of mutant viruses was also affected.
- Mutations in MA which disrupt virus assembly and release Mutations of HIV-1 MA coding region also affect virus assembly.
- Mutant DO contains a deletion of amino acids 5 to 16 of the MA domain.
- Mutant B5 contains amino acid substitutions of Arg20 to Thr20, Lys26, 27, 28, and 32 to Glu26, 27, 28, and 32 respectively.
- Mutant B8 contains amino acid substitutions of Arg20 to Thr 20, Lys 18, 26, 27, 28, 30, and 32 to Asnl8, 26, 27, 28, 30, and 32 respectively, and Arg20 and 22 to Gly20 and 22 respectively.
- Virus production of DO, B5, and B8 was compared to that of wt virus.
- the invention includes therapies using any protein which is homologous to simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) MA (Fig. 1, SEQ ID NOS: 1) as well as other naturally occurring MA polypeptides. 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 MA 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 chimeric polypeptides that include MA together with unrelated sequences.
- the invention also includes any biologically active fragment or analog of MA.
- biologically active is meant possessing therapeutically useful anti- HIV activity which is characteristic of the MA polypeptides shown in Figs. 1 and 5.
- Therapeutically useful activity of a MA fragment or MA analog can be determined in any one (or more) of a variety of MA assays, for example, those assays described in this application.
- Preferred analogs include mutants whose sequences do not destroy the polypeptide's relevant anti-HIV biological activity as measured using in vivo or in vitro (e.g., those described above). Preferred analogs also include mutated MA (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 mutated MA 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 mutated MA 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.
- analogs can differ from mutated MA 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 MA can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of MA can be assessed by methods described below.
- MA 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 mutated MA protein or homologous to the gag gene and are useful for the treatment of individuals infected with HIV. 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 amino acid monomeric 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. What is claimed is:
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP6513091A JPH08503488A (en) | 1992-11-23 | 1993-01-12 | Treatment of AIDS based on HIV MA peptide |
EP93903435A EP0669925A4 (en) | 1992-11-23 | 1993-01-12 | Aids therapeutics based on hiv ma peptides. |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US07/979,966 US5707864A (en) | 1992-11-23 | 1992-11-23 | Nucleic acids encoding mutated human immunodeficiency virus matrix proteins |
US979,966 | 1992-11-23 |
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WO1994012513A1 true WO1994012513A1 (en) | 1994-06-09 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US1993/000267 WO1994012513A1 (en) | 1992-11-23 | 1993-01-12 | Aids therapeutics based on hiv ma peptides |
Country Status (5)
Country | Link |
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US (1) | US5707864A (en) |
EP (1) | EP0669925A4 (en) |
JP (1) | JPH08503488A (en) |
CA (1) | CA2150027A1 (en) |
WO (1) | WO1994012513A1 (en) |
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JP4701532B2 (en) * | 2001-04-26 | 2011-06-15 | 東ソー株式会社 | Amplification and detection of HIV-1 RNA |
EP1874345B1 (en) * | 2005-04-25 | 2012-08-15 | 3M Innovative Properties Company | Immunostimulatory compositions |
-
1992
- 1992-11-23 US US07/979,966 patent/US5707864A/en not_active Expired - Fee Related
-
1993
- 1993-01-12 WO PCT/US1993/000267 patent/WO1994012513A1/en not_active Application Discontinuation
- 1993-01-12 CA CA002150027A patent/CA2150027A1/en not_active Abandoned
- 1993-01-12 EP EP93903435A patent/EP0669925A4/en not_active Withdrawn
- 1993-01-12 JP JP6513091A patent/JPH08503488A/en active Pending
Non-Patent Citations (3)
Title |
---|
Journal of Virology, Volume 64, No. 9, issued September 1990, S.S. RHEE et al., "Structural Role of the Matrix Protein of Type D Retroviruses in Gag Polyprotein Stability and Capsid Assembly", pages 4383-4389, see entire document. * |
See also references of EP0669925A4 * |
The EMBO Journal, Volume 10, No. 3, issued March 1991, S.S. RHEE et al., "Amino Acid Substitutions within the Matrix Protein of Type D Retroviruses Affect Assembly, Transport and Membrane Association of a Capsid", pages 535-546, see entire document. * |
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Publication number | Publication date |
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EP0669925A1 (en) | 1995-09-06 |
EP0669925A4 (en) | 1996-08-21 |
CA2150027A1 (en) | 1994-06-09 |
JPH08503488A (en) | 1996-04-16 |
US5707864A (en) | 1998-01-13 |
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