JPH10509327A - Eukaryotic initiation factor 5A (eIF-5A) mutant - Google Patents

Abstract

  (57) [Summary] An eIF-5A mutein, or a gene or DNA or cDNA sequence encoding the same, that lacks substantial eIF-5A function, inhibits Rev function, and thus has an inhibitory phenotype with respect to Rev function.

Description

DETAILED DESCRIPTION OF THE INVENTION Eukaryotic Initiation Factor 5A (eIF-5A) Variants Human immunodeficiency virus (HIV) replication is primarily dependent on the function of the viral Rev transactivator protein. Rev accumulates in the nuclear compartment of expressing cells and binds directly and specifically to a highly assembled RNA sequence called the Rev response element (RRE), which is present in all incompletely connected viral mRNAs. After binding, Rev is multimerized with the RRE and is thought to interact with a host cofactor required for Rev transactivation called eukaryotic initiation factor 5A (eIF-5A; formerly nomenclature: eIF-4D). This interaction results in nuclear / plasma translocation of imperfectly connected viral mRNAs, followed by the expression of viral structural proteins (eg, Gag, Pol, Env) and thus viral replication. Previously, a Rev mutant protein of the transdominant (priority negative) phenotype has been described (see, for example, WO 90/14427). Although these Rev mutants can still bind to RRE RNA, functional interaction with their cellular co-factors resulting in mutations in the Rev activation (effector) domain is not possible. eIF-5A is an endogenous protein required for normal cellular function, and several mutants thereof have been produced (eg, J. Biol. Chem. 264 [1989] 18527-18530: Mol. Cell. Biol. 11). [1991] 3105-3114), which are thought to adversely affect normal cellular functions, such as cell growth. The present invention has shown that mutant eIF-5A proteins can be produced that do not block endogenous wild-type eIF-5A function and usually lack perceptual eIF-5A activity themselves, but inhibit Rev function: They have an inhibitory phenotype with respect to Rev function. This is very surprising given that factors that are inactive in the wild-type normal cellular environment are nevertheless not expected to have an inhibitory effect on metabolic events initiated by foreign organisms such as viruses. It should be. The fact that such a mutant eIF-5A protein and the gene encoding it can be present at all is a factor that eIF-5A is essentially a protein for cell regulation (Biofactors 4 [1993] 95-104; TIBS 18 [ 1993] 475-479). While such genes and proteins were predicted to be toxic to cells, the genes and proteins claimed herein (particularly, M13, M14 and M13Δ) are usually inactive. Non-toxic and inhibitory with respect to Rev, eg, HIV Rev function and thus HIV replication. The present invention relates to an eIF-5A mutein that lacks normal cellular eIF-5A activity (hereinafter referred to as endogenous eIF-5A function) but inhibits Rev function, and thus has an inhibitory phenotype with respect to Rev function; And the corresponding gene or DNA or cDNA sequence encoding them. As such, variants that lack substantial endogenous eIF-5A function are preferred. As used herein, eIF-5A is preferably human eIF-5A, and the variants are preferably based on the human wild type of eIF-5A. HIV is preferably HIV-1. The muteins of the present invention may have polypeptides containing mutants or fragments or derivatives, such as allelic variants, which still have an inhibitory effect on Rev function, but which themselves preferably lack substantial endogenous eIF-5A function. It should be understood. For pharmacological applications, they are preferably in substantially pure form, eg, substantially free of products from their endogenous cellular environment. The mutation or mutations are made at about amino acid 130 in the wild-type eIF-5A genome (FIG. 3; SEQ ID NO: 17), especially at about amino acid 135 and at the end of the molecule, especially between poly135 and 139. It includes the region or regions of the peptide. The corresponding gene or DNA or cDNA sequence may be, for example, as shown in Table 2 especially for eIF-5AM13, eIF-5AM14 and eIF-5A13Δ, or a gene which hybridizes under stringent conditions to its protein coding region. Alternatively, a gene or sequence that hybridizes to a sequence or sequence if there is no degeneracy of the genetic code, but preferably does not hybridize to the corresponding wild-type gene or sequence. The invention also includes a eukaryotic or prokaryotic host cell transformed or transfected with a gene or DNA or cDNA sequence as described above, such that the host cell can express the gene or DNA or cDNA sequence. Furthermore, it relates to a biologically functional plasmid or viral DNA vector comprising the above-mentioned gene or DNA or cDNA sequence. It also relates to a prokaryotic or eukaryotic host cell stably transformed or transfected with a DNA vector as described above. The present invention also provides a method for isolating a corresponding wild-type gene from an appropriate expression system, inserting the gene into an appropriate cloning system, introducing a desired mutation into the gene, and obtaining a mutant having the desired mutation. It also relates to a method for producing the above gene or DNA or cDNA sequence, including recovering from a clone. Further, the prokaryotic or eukaryotic host cell transformed or transfected is cultured under suitable nutrient conditions in such a manner that the host cell can express the protein with the gene or DNA or cDNA sequence as defined above, and By isolating the desired expressed polypeptide product. It also relates to a pharmaceutical composition in the form of a cell comprising a protein as described above and at least one pharmaceutically acceptable carrier, adjuvant or diluent, or which is taken from the patient's body and treated in vitro before reinsertion. Use of a protein as defined above or a gene or DNA or cDNA sequence encoding it for the treatment of a disease caused by a retrovirus that depends on eIF-5A for Rev function, such as HIV, HTLV-I and / or HTLV-II Related to It also relates to the use as a medicament for the treatment of a disease caused by a retrovirus which depends on eIF-5A for its Rev function such as HIV, HTLV-I and / or HTLV-II and to its use in the manufacture of a medicament. The methods and techniques used to practice the present invention are known in the art. Unless the method of manufacture is otherwise specified herein, compounds, reagents, vectors, cell lines, etc. for use in practicing the invention are known, publicly available, or known and publicly available. Equivalent substances that can be obtained in a conventional manner from materials available in the United States or that can be manufactured in a conventional manner from known and publicly available materials. a) Preparation of eIF-5A mutant A human cDNA encoding eIF-5A (J. Cell. Biol. 123/6, 1309-1320 [1993]) was inserted into the HindIII site of the M13mp18 vector (Pharmacia, SWEDEN) at the HindIII site. Cloned as a fragment. The corresponding single-stranded DNA was used for mutations using the "Origonucreoti de-directed in vitro mutagenesis system, Version 2" (Amersham, UK). All mutant sequences were determined by DNA sequencing using Sequences 2.0 from United States Biochemicals. Subsequently, all eIF-5A mutant genes (see Table 1) were expressed in COS cells and subjected to eIF-5A specific immunoprecipitation analysis to confirm protein expression. The nucleotide sequences of the specific oligonucleotides used for the production of the 17 mutants are as shown below (Table 2) (respectively, SEQ ID NOs: 1 to 16), and the last mutant M13Δ is the same oligonucleotide as M13. Obtained using: Error in mutation reaction resulted in M13Δ. It is recognized that these specific nucleotide sequences are only to illustrate the 17 specific variants produced, and that they can be obtained with many different nucleotide sequences, for example, taking into account the fluctuations in codon usage. b) Testing cellular eIF-5A activity of the eIF-5A mutant in yeast Saccharomyces cerevisiae Using the polymerase chain reaction (PCR) method, the eIF-5A coding sequence was inserted between the BamHI and Xbal sites of the yeast shuttle vector pRSGAL. And cloned. In order to test the complementation ability of various eIF-5A in eIF-5A-deficient yeast Saccharomyces cerevisiae, the plasmid shuffling method was described in Mol. Gen. Genet. 244, 646-652 (1994). The results are summarized in Table 1: Some eIF-5A variants, ie, peIF-5AM4 to peIF-5AM10, peIF-5AM13, peIF-5AM14, peIF-5AM16 and peIF-5AM13Δ, are non-functional eIF-5A mutations. Identified as a protein. c) In vitro binding of non-functional eIF-5A mutein HIV-1 Rev transactivator protein using gel retardation assay Wild-type and all non-functional eIF-5A genes were eukaryotic using PCR. It was cloned between the BamHI and EcoRI sites of the expression vector pGEX-3X (Pharmacia), expressed in Escherichia coli as a glutathione-S-transferase (GST) -fusion protein and purified. The various GST-eIF-5A fusion proteins were then combined with ³² P-labeled RRE RNA and recombinant Rev protein (Nature 342, 816-819 [1989]) as described in Biochemistry 32, 10497-10505 (1993). Used for gel delay assay in combination with First, the effect of GST-eIF-5A wild-type protein on the RRE: Rev complex was tested. As shown in FIG. 1A, the addition of GST-eIF-5A to the Rev-containing RRE RNA binding reaction resulted in the detection of high molecular weight complexes with apparently reduced mobility (FIG. 1A, lanes 5 vs. 4). . This overt signal was not detected when RRE RNA was incubated with GST or GST-eIF-5A alone (FIG. 1A, lanes 2 and 3). Next, it was tested whether the observed eIF-5A-specific signal was dependent on the presence of an activation domain in the Rev transactivator protein. For this, the recently described mutant proteins Rev9Δ14 and Rev11Δ14 (Biochemistry 32, 8945-8954 [1993]) were used in this assay. Both Rev proteins are characterized by an internal C-terminal deletion and show binding affinity and specificity for RRE RNA comparable to wild-type Rev. Importantly, the activation domain has been completely removed in Rev9Δ14, but is still present in the Rev11Δ14 protein. The low molecular weight of both proteins resulted in a shift in the band of the RRE: protein complex that was difficult to analyze in a gel retardation assay. Nevertheless, the data clearly demonstrate that the activation domain deletion mutant Rev9Δ14 is incapable of interacting with GST-eIF-5A (FIG. 1A, lanes 6 vs. 7). In comparison, the addition of GST-eIF-5A to the RRE: Rev11Δ14 binding reaction resulted in a new signal (FIG. 1A, lane 8 vs. 9, slower transition point in lane 9), eIF-5A and Rev activation domain Shows the interaction of Finally, the binding capacity of various non-functional eIF-5A muteins was tested in the same assay. As shown in FIG. 1B, most GST-eIF-5A muteins lack the ability to recognize the RRE: Rev complex (lanes 4 to 10 and lane 13). However, the three non-functional mutant proteins GST-eIF-5AM13, GST-eIF-5AM14 and GST-eIF-5AM13Δ show behavioral properties equivalent to those of the eIF-5A wild-type protein (FIG. 1B, lane 3). Versus lanes 11, 12, and 14). Taken together, the data show that eIF-5AM13, eIF-5AM14 and eIF-5AM13Δ mutants are required as inhibitors of HIV-1 Rev function: (1) lack of endogenous eIF-5A function (yeast complementarity: And (2) binding activity to HIV-1 Rev protein (Rev in vitro binding; see Table 1 and FIG. 1B). d) testing the transdominant eIF-5A mutant structural inhibit eIF-5AM13, eIF-5AM14 and HI V-1 phenotype of eIF-5AM13Δ of HIV-1 replication in human T- cells by retroviral-mediated gene transfer For example, each eIF-5A coding region is assigned to Proc. Natl. Av ad. Sci. 89, 9870-9874 (1992), cloned into the XhoI site of the retroviral vector pBC140 packaged in vitro using the Am12 cell line and delivered to human CEM T cells for structural expression. Expression of the eIF-5A wild-type gene, eIF-5AM9 and the retroviral vector alone serve as controls in these experiments. HIV-1 challenge experiments were performed using a 2000 / ml TCID of the HIV-1 SF2 strain. Ten days after SF2 infection, aliquots of the culture medium were collected and assayed for HIV-replication as p24Gag protein using an antigen capture assay (ELISA). As shown in FIG. 2, HIV-1 replication was cloned in three independently expressing either eIF-5AM13, eIF-5AM14 or eIF-5AM13Δ (FIGS. 2C, 2D and 2E respectively; left panel). In the CEM cell lines (designated A, B, C), there is a clear decrease compared to CEM cells expressing the eIF-5AM9 mutein (CEM-eIF-5AM; FIG. 2B). Maximal viral replication was detected in CEM cells expressing the parental pBC140 vector (CEM vector; FIG. 2A) or eIF-5A wild-type protein (CEM-eIF-5Awt; FIG. 2A). The observed inhibitory effect is not due to general inhibition of cell proliferation, as indicated by the equivalent cell number in the experiment (FIG. 2; right panel). In conclusion, these experiments show that (1) the structural expression of eIF-5AM13, eIF-5AM14 or eIF-5AM13Δ protein is not toxic to human CEM T cells, and (2) that eIF-5AM13, eIF-5AM14 or eIF-5AM13 5A M13Δ proves to be an inhibitor of HIV-I replication. HIV is the major etiology of AIDS (Acquired Immunodeficiency Syndrome); HTLI causes inter alia ATL (Adult T-cell leukemia); HTLV-24 is etiological in the presence of various T-cell hairy cell leukemias. Related. HIV Rev and HIT VI Rex transactivators have been shown to be essential for viral replication in culture. In addition, HTLV-I Rex has been demonstrated to be a functional replacement for the Rev protein in HIV. Therefore, Rev and Rex are potential targets for treatment in affected patients. The eIF-5A variants of the present invention serve as effective competitive inhibitors of wild-type Rex function as a result of the potential for Rex to functionally substitute for Rev in wild-type Rev and / or HIV. Thus, these eIF-5A variants also serve as effective inhibitors of HIV and / or HTLV-I and / or HTLV-II replication. Accordingly, they can be used to protect a patient's lymphoid and / or myeloid cells from being exposed to infection, and thus, the disease caused by HTLV-I or HTLV-II, or AIDS and ARS, respectively. Alternatively, treatment of HIV-induced disease, including ARC (AIDS-related syndrome or complication) is indicated. Furthermore, the ability to inhibit both HTLV-I Rex and HIV Rev protein activity indicates its use in the treatment of HIV-induced diseases. This property may be particularly effective in patients co-infected with one or more viral pathogens or where the infection does not distinguish between the two agents. The invention therefore relates to ATL, AIDS, ARS (or ARC), SIV (Simian Immunodeficiency Virus) such as SIV _mac , FIV (Feline Immunodeficiency Virus), EIAV (Equine Infectious Anemia Virus), Visna virus And rex or rev genes or their equivalents, such as ATL, AIDS and ARS (or ARC), and more particularly viral diseases, such as bovine immunodeficiency virus, especially retroviral diseases, especially human retroviral diseases. It is indicated for use in the prevention and treatment of human retroviral diseases by product-controlled pathogens. Thus, treatment of multiple, especially double infections of the virus, often seen in iv drug users co-infected with HIV and HTLV-I, or a single at high risk of further infection, such as HIV infection The diversity of repressor effects is a particular advantage because it is advantageous for treating conditions of infection or for preventing in conditions where protection against infection of a range of different viral species is desired. The therapeutic potential of the present invention is that, for example, suppression of HTLV-I Rex function and HIV Rev function suppresses viral replication, thus reducing viral load by preventing the formation of infectious viral particles, Thus, it is expected to perpetuate the incubation period of the infection, and is therefore readily apparent. Thus, cells of the subject already infected with the HIV virus, but still having the gene for the eIF-5A mutant repressor integrated into its genome, remain functional, and the patient can be treated without the need for further long-term treatment. , Completely free from the symptoms of the disease. In this regard, the gene of the present invention is capable of delivering a functional form to a target mammalian cell directly, in vivo or indirectly, in vitro, preferably as part of a vector, for example, a retroviral or plasmid vector, in vitro. Is a medicament, administered once or multiple times, in a form suitable for use; for example, the insertion of a gene encoding such an inhibitor of viral replication can be performed in vitro by lymphoid and / or myeloid from an infected individual. These cells can be administered to a donor patient after they have been transferred directly into the genome of the cells and performed the insertion. Because of the replication of HTLV-I and HTLV-II and HIV in various types of T cells, diseases caused by them may be particularly suitable. Therefore, T cells are desirable target cells for gene modification using the gene of the present invention. In addition, mononuclear / macrophage cells that are targets for HIV infection are also suitable cells for modification. Preferably, hematopoietic stem cells are modified, thereby providing permanent protection to progeny of multiple hematopoietic cell lines. One of the inventions is therefore described, for example, by T.W. Friedmann, Science 244 (198 9) 1275 or PM. Lhen, Bone Marrow Transplant. 1 (1987) 243, similar to the concept of gene therapy; hematopoietic stem cells are extracted, for example, from AIDS / ATL patients and cultured in vitro and encode a function to be suppressed, ie an inhibitor of the Rev / Rex function. Inserting the mutant gene of the present invention into these cells using a retroviral vector; returning the virus-resistant stem cells to the original patient's immune system, because of the acquired, selective advantage over untreated stem cells. Proliferation is expected, and at a reasonable time, the population of hematopoietic cells will consist entirely of repressor-producing cells and will be virus resistant. How to do this is known in the art, see for example US Pat. No. 4,868,116. Vectors for carrying the mutant genes of the invention to target mammalian cells such as bone marrow cells, for example, retroviral or plasmid vectors, are described or cited, for example, in Science 244 (1989) 1275. Thus, for example, various Rev and / or Rex transdominant genes are cloned into a retroviral vector system. For example, after retrovirus-mediated gene transfer to an HIV-infected human cell line, the inhibitory effect of the mutant is readily confirmed by inhibition of virus production. The above therapeutic concept is described in This is an example of intracellular immunization as planned in Baltimore, Nature 335 (1988) 395. In essence, this concept is based on the concept of a gene encoding a repressor of a certain vital function of a selected virus and the particular target cells that the virus is infected with (eg, certain T cells and mononuclear cells in the case of AIDS-causing viruses). Sphere / macrophage cell). Therapeutic application of this approach with viral repressors constitutes an effective and safe intracellular delivery system for human or animal applications, enabling possible vectors of genes encoding such mutant proteins and these vectors. It relies on establishing a possible method for insertion into the appropriate cells identified in the model system. This concept is, therefore, the only difficulty in experimentally validating the concept, because of the difficulties from ethical barriers currently trying to ban gene therapy. However, the first human genetic experiments are currently being performed to confirm both safety and treatment [see, eg, GT Nabel, Human Gene Therapy 5 (1994) 79-82]. If the harmlessness of this method proves, these pioneering experiments will be followed firstly by similar attempts for the treatment of life-threatening conditions such as AIDS disease and the present invention Suitable for use in such tests (see, for example, T. Friedmann, Science 244 [1989] 1279, column 2, "Infection disease"). A further aspect of using the present invention involves the insertion of a repressor protein of the present invention, rather than a gene, into target cells. For example, oral or parenteral administration is performed in a known manner in a manner that allows for intracellular penetration, such as liposome-mediated delivery. Precise dosages for these uses will vary depending on the compound employed, the mode of administration and the treatment desired; it is within the skill of the art to ascertain the most appropriate dose in a particular situation. . It should be understood that various combinations or changes in form and detail can be made with the present invention without departing from the scope of the invention. Among the specific mutants already constructed and described herein, those that have not been characterized, but which can be characterized as inhibitory and / or multivalent in more detailed experiments; and Additional variants as described above, including those not specifically described herein but which follow the above principles, are also included within the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 : In vitro RNA gel shift experiment. Figure 2 : HIV-1 challenge experiment. p24Gag levels are shown on the left. The cell numbers of the same experiment are shown on the right: A: CEM-vector / eIF-5A wt B: CEM-eIF-5AM13 C: CEM-eIF-5AM13 D: CEM-eIF-5AM14 E: CEM-eIF-5AM13Δ FIG. (SEQ ID NO: 17): cDNA and amino acid coding sequence of wild-type human eIF-5A (J. Biol. Chem. 264 [1989] 1581) (SEQ ID NO: 17); lysine shown at amino acid position 50 is converted to hypsin after translation. Convert. Amino acids of wild-type eIF-5A mutated as shown in Table 1 are shown in italics. The specific oligonucleotides used (Table 2; SEQ ID NOs: 1 to 16) are the following in FIG. 3 (SEQ ID NO: 17): M1: 3-29 (27 nucleotides) M2: 12-38 (27 nucleotides) M3: 34- 61 (28 nucleotides) M4: 54-81 (28 nucleotides) M5: 120-146 (27 nucleotides) M6: 123-154 (32 nucleotides) M7: 178-208 (31 nucleotides) M8: 196-222 (27 nucleotides) M9: 211-238 (28 nucleotides) M10: 277-316 (40 nucleotides) M11: 301-328 (28 nucleotides) M12: 364-391 (28 nucleotides) M13: 391-420 (30 nucleotides) M14: 399-430 (32 nucleotides) M15: 411-439 (29 nucleotides) M16: 433 458 (26 nucleotides) M13Δ: corresponds to the nucleotide position 391-421 (position 409 not translated, 30 nucleotides followed by a stop codon at positions 422-424) and is shown in Table 1 in the sequence of the wild-type protein. Such amino acid changes.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 15/09 ZNA C12R 1:92) (C12P 21/02 C12R 1:91) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AZ, BY, KG, KZ, RU, TJ, TM), AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR , KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN

Claims (1)

[Claims]   1. E that inhibit Rev function and thus have an inhibitory phenotype with respect to Rev function IF-5A mutant protein, or a gene or DNA encoding the same, Is the cDNA sequence.   2. 2. The protein of claim 1, wherein the protein lacks substantially endogenous eIF-5A function. Is the gene or DNA or cDNA sequence encoding it.   3. eIF-5AM13, eIF-5AM14 or eIF-5A13Δtan 3. The protein according to claim 1, which is a protein, or a protein encoding the protein. Gene or DNA or cDNA sequence.   4. The gene or DNA or cDNA sequence according to claim 1 or 2, In such a way that the main cell can express the gene or DNA or cDNA sequence, Eukaryotic or prokaryotic host cells that have been transformed or transfected.   5. It comprises the gene or DNA or cDNA sequence according to claim 1 or 2. Biologically functional plasmid or viral DNA vector.   6. It comprises the gene or DNA or cDNA sequence according to claim 1 or 2. Stable transformation with biologically functional plasmid or viral DNA vectors Or a transfected prokaryotic or eukaryotic host cell.   7. Isolate the corresponding wild-type gene from a suitable expression system and Into a gene, introduce the desired mutation into the gene, and 3. The method according to claim 1, comprising recovering the body from a clone having the desired mutation. A method for producing the gene or DNA or cDNA sequence described above.   8. The gene or DNA according to claim 1 or 2 under appropriate nutritional conditions. Is a cDNA sequence that can be transformed in a manner that allows the host cell to express the protein. The transfected or transfected prokaryotic or eukaryotic host cells are cultured and, optionally, 3. The tannin according to claim 1 or 2, comprising isolating the expressed polypeptide product. Manufacturing method of Parkin.   9. A protein according to claim 1 or 2 and at least one pharmaceutically acceptable. Containing a carrier, adjuvant or diluent, or removed from the patient's body and reinserted A pharmaceutical composition in the form of a cell that is previously treated in vitro.   10. A protein according to claim 1 or 2, or a gene encoding the protein. Of DNA or cDNA sequences depending on eIF-5A for Rev function. Use in treating diseases caused by Torovirus.   11. A protein according to claim 1 or 2, or a gene encoding the protein. HIV, HTLV-I and / or HTL of the DNA or cDNA sequence Use in the treatment of diseases caused by V-II.   12. The protein according to claim 1 or 2 or a protein thereof for use as a medicament. Gene or DNA or cDNA sequence to encode.   13. Diseases caused by retroviruses dependent on eIF-5A for Rev function Use of the protein according to claim 1 or 2 in the manufacture of a medicament for treating stomach.