WO2004052924A1

WO2004052924A1 - Expression of peptides of the cytoplasmic domain of hiv-gp41 for inhibiting viral infections

Info

Publication number: WO2004052924A1
Application number: PCT/EP2003/013939
Authority: WO
Inventors: Matthias Dittmar
Original assignee: Ruprecht-Karls-Universität Heidelberg
Priority date: 2002-12-10
Filing date: 2003-12-09
Publication date: 2004-06-24
Also published as: DE10257770A1; AU2003289990A1

Abstract

The invention relates to nucleic acids and to the peptides encoded by said nucleic acids, which can be used for the treatment of HIV infected patients. The invention specifically relates to membrane-anchored constructs of the intracellular domain of the HIV gp41 protein.

Description

Expression of peptides in the cytoplasmic region of HIV-gp41

Inhibition of viral infections

The present invention relates to protein fragments (peptides) of human immunodeficiency virus (HIN) with antiviral function. In particular, the invention relates to nucleic acids which code for the cytoplasmic region of the HIV glycoprotein gp41, the peptides encoded by these nucleic acids, and the use of these peptides and nucleic acids for the targeted inhibition of viral infections.

The worldwide spread of the human immunodeficiency virus (HIN) has led to 40 million people infected with HIN (UΝAIDS, 2001) with 15,000 Νeu infections daily. A large number of HIV variants (HIV-1 groups M, O and Ν as well as HIV-2) that have been detected to date are responsible for this global infection. HIV-infected patients are currently being treated with anti-retroviral agents that lead to a substantial reduction in virus replication in the patient and thus directly counteracts the breakdown of the immune system. It is already known that these anti-retroviral drugs specifically inhibit viral enzymes (reverse transcriptase inhibitors, RTI and ΝΝRTI, protease inhibitors, PI) and thereby bring about a reduction in virus particles circulating in the blood to undetectable amounts. However, this reduction is only possible if combination therapy is prescribed and followed over a longer period of time. Therefore, this so-called highly active anti-retroviral therapy (HAART) can only be carried out in countries that have a well-developed and functioning health system and the necessary financial means to provide these drugs.

Due to the high error rate in replication of the retroviruses, resistance quickly develops if the medication is not taken in the prescribed dose. This has led to the development of over 20 anti-retrovirals to date

Medications in various combinations for long-lasting suppression virus replication. However, there is no elimination of HIV due to this long-lasting suppression, since the virus can persist in latent reservoirs and continue to replicate at a very low level.

In order to avoid the disadvantages described above, in the course of the development of an effective HIV vaccine, HIV protein fragments or peptides were sought which intervene in mechanisms (earlier) than virus replication, such as, for example, when the virus entered the host cell , The targeted cell entry (tropism) of HIV takes place to a large extent by means of its outer, glycosylated coat protein gpl20. This protein is encoded as a gp 160 precursor by the so-called env reading frame (envelope) of the viral genome. After translation, the gp 160 precursor protein is cleaved by a cellular protease into gpl20 and into the transmembrane protein gp41. Both proteins are essential for the virus to bind to the cellular receptor and then enter the cell. The binding to the corresponding surface receptors of the infected cell (CD4 receptor of the T helper cells and macrophages; chemokine receptors) leads to conformational rearrangements and ultimately to the fusion of the viral membrane with the membrane of the host cell.

The focus of the present invention is on peptides of the transmembrane protein gp41, which mediates the fusion of HIV with the host cell. Gp41 consists of an extracellular domain with two highly conserved helical areas (DP-107, DP-178), a transmembrane anchor and a 152 amino acid long intracellular domain. The patent application WO 01/51673 describes i.a. the use of peptides in the helical regions DP-107 and DP-178 to inhibit the fusion between HIV and the host cell and thus to inhibit further propagation of the virus. DP-178, which is also known to the person skilled in the art as T-20 and as an 'entry' inhibitor, is already being used with promising results in the first clinical studies (see Kilby et al., Nature Med. 1998 Nov; 4 (ll) : 1232-3).

In addition to the development of peptide inhibitors, somatic gene therapy has also gained in importance as a new therapy concept in recent years. With help With this concept it is possible to induce the main target cells of HIV infection to produce intracellular anti-retroviral agents without having to rely on the use of therapeutic agents. For example, DE-Al 19957838 discloses a nucleic acid construct that codes for a membrane-like form of the extracellular T-20 region of gp41. The genetic expression of the disclosed nucleic acid inhibits the entry of the virus into the cell mediated by the HIV envelope proteins.

The intracellular area of gp41 has not yet been studied very much. Domains could be determined which are necessary for the transport of the gp41 to the plasma membrane (YXXO motif); two helical areas, which are necessary for binding to intracellular membranes (LLP1 / LLP2), and a leucine zipper area, which is necessary for the generation of infectious particles. No studies have been published that describe a direct role of the intracellular region of gp41 in cell entry.

It has now been found that HIV spread in HIV-infected cells can be prevented by expressing nucleic acid constructs which code for the intracellular part of gp41 in the affected cells. In contrast to the invention described in DE-Al 199 57 838, a phase of virus replication is inhibited which already takes place inside the infected cell, that is to say later. Different stages of HIV infection can thus be specifically switched off. The unexpected effect was achieved, inter alia, by the fact that the nucleic acid according to the invention additionally codes for a peptide which serves to anchor the gp41 component in the membrane.

The present invention therefore relates to a nucleic acid sequence of the general formula

5 '- (marker) - MSD - intracellular area gp41 - 3' where "5 '" denotes the 5' end of the nucleic acid sequence,

"3 '" denotes the 3' end of the nucleic acid sequence,

“(Marker)” denotes an optionally present DNA region which codes for an extracellular peptide for the detection of the construct, “MSD” denotes the DNA region coding for a “membrane spanning domain”,

Fragments or derivatives thereof, designated, and

"Intracellular region gp41" denotes a DNA region for the intracellularly expressed region of gp41, a fragment or derivative thereof, encoded by HIV.

The invention also relates to nucleic acids of the general formula 5 '- MSD - (marker) - intracellular region gp41 - 3' and 5 '- MSD - intracellular region gp41 - (marker) - 3' as well as fragments and derivatives of said nucleic acids.

According to the present invention, the term "HIV" includes all virus subtypes of both HIV-1 and HIV-2.

All gp41 variants of all HIV subtypes come into question as “gp41” for the present invention. An example of a nucleic acid coding for the intracellular region of a suitable gp41 is the nucleic acid sequence of nucleic acid 2089 to 2580 of Genbank accession number U05352. The invention however, it is not limited to this.

For the purposes of the present invention, the above term “fragment and derivative” refers to DNA sequences which are for the intracellularly expressed region of gp41 encode in which at least one nucleotide of the sequences in question can be replaced by at least one nucleotide which is different from the original one, or on gp41 DNA sequences in which the original nucleotide sequence is either on the 5 'or 3' - or at both ends and / or within the sequence either extended, shortened or shortened at one end and extended at the other end, provided that the function and biological effect of the encoded gp41 region is retained. An example of a shortened form of the intracellularly expressed region of gp41 is the nucleic acid sequence from nucleic acid 2089 to 2226 of the nucleic acid sequence with the Genbank accession number U05352. However, the invention is not restricted to this.

In a preferred embodiment, the “intracellular region gρ41” comprises the sequence shown in SEQ ID NO: 1 (FRES-A), fragments and derivatives thereof.

In a particularly preferred embodiment, the "intracellular region gp41" comprises the sequence shown in SEQ ID NO: 3 (FRES-B), fragments and derivatives thereof.

Analogously to the aforementioned, “fragments and derivatives” refers to DNA sequences which code for an MSD, in which at least one nucleotide of the original sequence can be replaced by at least one nucleotide which is different from the original nucleotide, or to MSD- DNA sequences in which the original nucleotide sequence is either elongated, truncated, or truncated at either 5 ', 3', or both ends and / or within the sequence, or truncated at one end and elongated at the other end, provided that the function and biological effect of the coded MSD area is retained.

The invention includes various possibilities for anchoring the peptide encoded by the nucleic acid according to the invention to the membrane. "MSD" can therefore code for a peptide that has a motive for intracellular attachment of a - 0 -

Membrane anchor contains e.g. Myristoyl or Prenyl or CaaX motif. For the present invention, however, it is expedient, but not absolutely necessary, if “MSD” codes for a transmembrane anchor of a type I transmembrane protein. The person skilled in the art understands a type I transmembrane protein as a transmembrane protein with extracellular amino and intracellular carboxy terminus.

Accordingly, MSD of all type I transmembrane proteins accessible to the person skilled in the art are suitable for the invention. Preferred are MSD of type I transmembrane proteins which are selected from the non-exhaustive list of examples such as ribophorin I and II, MHC class I, HIV-1-Vpu, Synaptotagmin II, mouse-CMV-gp48, human-CMV-USI 1, calnexin, PERK, UDP-glucuronosyltransferase, low affinity nerve growth factor receptor (LNGFR) or CD34. MSD from LNGFR or CD34 are particularly preferred. The MSD, a fragment or derivative thereof, of LNGFR is very particularly preferred.

Various peptides or DNA sequences coding therefor can be considered as “markers” for detecting the peptides encoded by the nucleic acid according to the invention. In general, the invention includes all extracellular domains that are suitable for detection, for example for immunological detection "The present invention is particularly suitable for so-called reporter genes or other genes which code for proteins or peptides, the presence of which is easy to determine. Markers which are suitable for carrying out the present invention can be selected from the group comprising so-called" epitope- tags "(c-Myc, hemagglutinin, FLAG), biotin, digoxygenin, horseradish peroxidase, alkaline phosphatase, green fluorescent protein (GFP) and derivatives thereof with altered fluorescence (EGFP, EYFP, EFCP), ß-galactosidase, luciferases, ß-glucuronidase and β-lactamase. Methods for detecting the above markers are known to the person skilled in the art.

In a further embodiment, the marker codes for an inactive T-20 variant which does not have the previously described effect, namely a fusion-inhibiting effect. In this case, the detection is carried out by methods known to the person skilled in the art using T-20-specific antibodies, for example fluorescent activated cell Sorting (FACS), Western blot, immunoprecipitation, enzyme-linked immunoadsorbent assay (ELISA) or radioactive immunoadsorbent assay (RIA). It is advantageous if the antibody used for the detection is coupled to a molecule which enables the detection or contributes to it. Suitable for this are, for example, biotin, horseradish peroxidase, alkaline phosphatase, fluorescein isothiocynate (FITC), tetramethylrhodamine isothiocyanate (TRITC), diamidinophenylindole (DAPI) and phycoerythrin.

It may also be desirable for the present invention if the nucleic acid according to the invention does not contain a region coding for a marker. This is particularly advantageous if the nucleic acid is used for (gene) therapeutic purposes. Then it is important if the nucleic acid codes for the smallest possible peptide and the said peptide is as free as possible from additional foreign components that are essential for the effect and which may have an unfavorable effect on the physiological state of the target cell.

The nucleic acid according to the invention is preferably used to express the peptide it encodes in cells, in particular HIV-infected cells.

Numerous methods are available to the person skilled in the art for introducing the nucleic acid according to the invention into the host or target cell (transfection or transformation), depending on the host cell selected. The simplest method for gene transfer is the injection of "naked" nucleic acids into a target tissue / target cells. A more efficient method is the manipulation of a single cell by the so-called microinjection of the nucleic acid into the cell nucleus. The use of reagents and methods is particularly favorable Calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids, liposomes, biolistic particle bombardment ("gene gun" method), heat shock transformation and electroporation, as well as any combination thereof. For the expression and for the introduction of the nucleic acid into the target cell, it is advantageous if the nucleic acid according to the invention is inserted in a so-called expression vector.

The present invention therefore also relates to a recombinant DNA and an expression vector which contain the nucleic acid according to the invention.

In a preferred embodiment, the expression vector is a eukaryotic, in particular human, bacterial or a retroviral vector, a plasmid, bacteriophage or other vectors customary in genetic engineering which are suitable for the expression of proteins or peptides. If desired or necessary, the nucleic acid sequence in the vector according to the invention can be operatively linked to regulatory elements which ensure transcription and synthesis of a translatable mRNA in pro- and / or eukaryotic cells. Such regulatory elements are promoters, enhancers or

Transcription termination signals, however, can also be introns or similar elements, such as elements which enable or contribute to the stability and multiplication of the vector, the selection and / or the integration into the host genome.

In a particularly preferred embodiment, the nucleic acid according to the invention is inserted into a retroviral vector. Retroviruses are so-called RNA viruses, the genome of which is reverse transcribed into DNA after infection of a cell and integrated into the host cell genome. The uptake into the cell takes place via receptor-controlled endocytosis. With some retroviral vectors, the expression of the vector is switched off after ingestion of a virus, so that the cell is infected only once. The integration of the viral DNA into the genome is effected by a virally encoded protein, integrase, the location of integration being non-specific. Because of the properties mentioned above, retroviral-based vectors are particularly suitable for gene therapy, because they are absorbed more efficiently by receptor-controlled endocytosis and integrated more efficiently since the process takes place enzymatically. The person skilled in the art knows this process also under the name "retroviral transduction". The person skilled in the art knows how such vectors are introduced into the target cell by means of so-called “packaging cells”, which carry so-called helper viruses in the genome. The invention encompasses all retroviral vectors which are derived, for example, from Onko, Lenti or Spuma viruses.

As an example of the retroviral vectors mentioned above, the vector according to the invention is M87m3 (M3) (see Hildinger et al., 2001, J. Virol., Vol 75, pages 3038-3042), which also contains the coding region for an MSD and for a marker (here: inactive T20 variant). However, these elements are not absolutely necessary for the invention, but can be omitted or exchanged for elements having the same effect. It is advantageous for the invention if the nucleic acids according to SEQ ID NO: 1 and SEQ ID NO: 3 according to the invention are inserted directly into the 3 'end of the nucleic acid sequence coding for an MSD while maintaining the reading frame.

The invention also includes a method for producing a recombinant protein, a fragment or derivative thereof, which is encoded by the nucleic acid according to the invention. The method comprises the following steps: (a) introducing a vector according to the invention, which contains the nucleic acid according to the invention, into a suitable host cell by means of suitable methods, where the host cell can be pro- or eukaryotic, (b) culturing the transfected host cell and (c) Isolation of the recombinant protein from the host cell or from the culture medium.

If desired or necessary, the proteins isolated according to step (c) can be further purified for other purposes. The person skilled in the art knows numerous methods for this, e.g. Salt fractionation, size exclusion chromatography,

Ion exchange chromatography, reverse phase chromatography, affinity chromatography and the like. The present invention furthermore relates to a fusion peptide of the general formula encoded by the nucleic acid according to the invention

NH ₂ - (marker) - MSD - intracellular area gp41 - COOH where

"NH ₂ " denotes the amino terminal end of the fusion peptide,

"COOH" denotes the carboxy terminal end of the fusion peptide,

“(Marker)” denotes an optionally available extracellular peptide for the detection of the fusion peptide, “MSD” denotes a “membrane spanning domain”, a fragment or derivative thereof, and

"Intracellular region gp41" denotes the intracellularly expressed region of gp41, a fragment or derivative thereof.

The invention also relates to fusion peptides of the general formulas NH ₂ - MSD - (marker) - intracellular region gp41 - COOH and

NH ₂ - MSD - intracellular region gp41 - (marker) - COOH as well as fragments and derivatives of said fusion peptides.

It may also be desirable for the present invention if the fusion peptide according to the invention does not contain a marker. This is particularly advantageous when the fusion peptide is used for therapeutic purposes. Then it is important if the fusion peptide is as small as possible and as free as possible from additional foreign components that are essential for the effect, which may have an unfavorable effect on the physiological state of the target cell. In connection with the present invention, the above term “fragment and derivative” refers to amino acid sequences of the intracellularly expressed region of gp41, in which at least one amino acid of a suitable gp41 sequence can be replaced by at least one amino acid that is different from the original one , or on gp41 amino acid sequences, in which the original amino acid sequence is either extended, shortened or shortened at one end and / or shortened at one end and / or extended at the other end either at the amino, carboxy terminal or at both ends and / or within the sequence, provided that the function and biological effect of the gp41 range is retained.

In a preferred embodiment, the "intracellular region gp41" comprises the sequence shown in SEQ ID NO: 2. In a particularly preferred embodiment, the "intracellular region gp41" comprises the sequence shown in SEQ ID NO: 4.

With regard to the definitions of “marker”, “HIV” and “MSD”, the statements made above for the nucleic acid according to the invention apply, these then being based on the protein level.

In a further aspect, the insertion of the nucleic acid sequence into vectors according to the invention to be used for gene therapy and the introduction of the vectors according to the invention into HIV-infected cells leads to the expression of a fusion peptide, which causes a strong inhibition of HIV replication. The invention therefore also relates to the use of the vector according to the invention for the treatment of HIV infections.

In this context, the invention relates to a method for inhibiting HIV-induced cell fusion, said contacting and transfection / viral

Transduction of HIV infected cell with the vector according to the invention comprises. The presence of the is advantageous for carrying out the method according to the invention The aforementioned reagents or the use of the methods mentioned above, which are known to the person skilled in the art and which enable or contribute to the absorption of the nucleic acid according to the invention into the cell.

All cell types are conceivable as possible target cells for a transfection / viral transduction with a vector according to the invention. T lymphocytes and / or hematopoietic stem cells are particularly preferred for the above-mentioned method. T lymphocytes are very particularly preferred.

The invention further relates to a method for inhibiting HIV-induced cell fusion, this comprising contacting the HIV-infectable cell with the fusion peptide according to the invention or a fragment or derivative thereof. The method can be carried out in vitro in an aqueous solution, in vivo in a suitable cell culture assay, or in a living being by means of suitable administration forms.

The nucleic acid according to the invention and / or the vector according to the invention and / or the fusion peptide according to the invention can be used in the context of the present invention as a medicament, optionally in combination with a suitable carrier.

It is immaterial whether the nucleic acid according to the invention and / or the vector according to the invention and / or the fusion peptide according to the invention are administered alone or in combination with other active substances. The active substances can be administered simultaneously or in succession. The dose and / or the exposure time of the drug is variable and depends on the physiological condition of the person to be treated. The patient's age, weight and gender can play a role here. It can also be important whether the disease needs to be treated acutely, chronically or prophylactically. In a further embodiment, the nucleic acid according to the invention and / or the vector according to the invention and / or the fusion peptide according to the invention are used for the production of a medicament, optionally in combination with a pharmaceutically suitable carrier, for the treatment of HIV-infected patients.

The preparation of such medicaments is known to the person skilled in the art. For the stability and effectiveness of the drug, the presence of stabilizers and carrier substances such as starch, lactose, stearic acid, fats, waxes, alcohols or physiological saline solutions or other additives such as preservatives, colorings, flavorings may be required. Drugs in liquid form can be lyophilized if necessary.

The present invention accordingly also relates to a medicament which, optionally in combination with other pharmaceutically acceptable substances and carriers, contains a nucleic acid according to the invention and / or a vector and / or a fusion peptide according to the invention, and for the treatment of HIV-infected patients suitable is.

The drug can be administered orally, for example in the form of coated or uncoated tablets, granules, hard and soft gelatin capsules, or rectally in the form of suppositories. The drug can also be administered parenterally - bypassing the digestive system - for example subcutaneously, intramuscularly or intravenously in the form of solutions for infusions or injections. Other suitable administration forms relate to direct applications (for example to the skin) in the form of ointments, tinctures, sprays or transdermal therapeutic agents, or agents to be inhaled in the form of nasal sprays, aerosols, or in the form of microcapsules, implants or rods. The appropriate dosage form depends on the type and severity of the disease. Finally, the present invention relates to a kit which can be used for the treatment of HIV-infected patients, the kit comprising at least one nucleic acid according to the invention and / or a vector and / or a fusion peptide according to the invention.

DESCRIPTION OF THE DRAWINGS

Drawing 1: Schematic representation of the fusion peptide to inhibit HIN replication

(A) denotes the output vector M3, "LTR" stands for "long terminal repeat", "SP" stands for "signal peptide", M3 (T20) denotes an inactive variant of the T20 peptide, "hinge" denotes a connector, "MSD "stands for" Membrane Spanning Domain "(for explanation see description)," IRES "stands for" Internal Ribosomal Entry Site "and" neo "denotes a selection marker (resistance to omyeomycin).

(B) denotes a truncated intracellular region of gp41 (the arrow marks the location of the premature stop codon) (FRES-A)

(C) denotes the complete intracellular area of gp41 (FRES-B).

Drawing 2: Inhibition of HIV infection by the cytoplasmic region of the gp41

HeLa cells were cotransfected with HIV-DΝA and inhibitory constructs FRES-A and FRES-B (2μg pΝL4.3 and lOμg vector). Virus production was demonstrated after three days.

Drawing 3: Inhibition of HIV infection by the cytoplasmic area of gp41 ffl) The replication kinetics of HIV-NL4.3 (moi = 0.001) after infection of stably transduced PM-1 cells is shown. The digits on the abscissa indicate the days after the transfection.

The present invention is explained in more detail below with the aid of examples.

EXAMPLES

Example 1: Cultivation of cells and viruses

Heia, 293T and Phoenix ampho cells were cultured in Dulbecco's medium (Gibco, Invitrogen) supplemented with 10% fetal calf serum (FCS, Sigma, Deisenhofen). PM-1, a T cell line, was cultured in RPMI with 10% FCS. Replication-competent viruses were either generated by passage on PM-1 cells (primary HIV isolates) or after transfection of 293T cells with the viral clone pNL-4.3 and subsequent passage on PM-1 cells.

For stable transduction of PM-1 cells, retro viral vectors that can express the fusion peptide were packaged using the Phoenix ampho packaging cell line (Grigani et al., 1998).

Example 2: Cloning of the retroviral vectors for expression of the fusion peptide

Starting from M3, a retroviral vector that encodes a non-inhibitory mutant of the membrane-anchored T20 peptide (Hildinger et al., 2001), the intracellular areas of the HIV-2 clone CBL23 (FRES- A and FRES-B) additionally expressed. M3: This retroviral vector (based on MP1N, Hildinger et al, 1998) expresses a fusion peptide consisting of the signal peptide (SP) of the human low affinity nerve growth factor receptor (LNGFR), followed by a T20 variant which does not cause fusion inhibition, followed by a hinge region of the murine IgG heavy chain, followed by the transmembrane domain (MSD, membrane anchor) of LNGFR (see Figure 1). (Hildinger et al, 2001). The extracellular areas were used in the test phase for simple detection of expression, while the membrane anchor was used here only as an example for different types of membrane anchoring

FRES-A: The nucleic acid sequence (SEQ ID NO: 1) coding for the intracellular region of the gp41 protein of HIV-2 (CBL23) was amplified by PCR and appended in frame to the reading frame of the fusion peptide from the M3 vector. The resulting new fusion peptide localizes to the plasma membrane (due to the membrane anchor) and can develop the inhibitory effect there. A naturally occurring variant of the intracellular region of the gp41 was chosen for the construction of the vector FRES-A, which leads to the expression of a short intracellular region, (see drawing 1)

FRES-B: This vector was cloned analogously to FRES-A, but here the nucleic acid sequence of a complete intracellular region of the HIV-2 (CBL23) clone is expressed in the fusion peptide. (SEQ ID NO: 3; see drawing 1)

Example 3: Co-transfection experiments to demonstrate the inhibitory function of the new fusion peptide

The cotransfection of an HIV coding plasmid and the respective inhibitory vectors can reduce the HIV infection in a rapid HeLa Assay to be shown. It is important that the plasmid DNA with the supposedly inhibitory effect is present in excess and that the HIN infection spreads over time in the culture.

By transfection using the calcium phosphate method, 50-60% of the cells can be induced to take up the plasmid DΝA. The coding proteins (HIN and the fusion peptide FRES-A or FRES-B) are expressed. In the case of inhibition, a reduction in the p24 value (a marker for the spread of HIV in cell cultures) can be assumed. As shown in drawing 2, the cotransfection for both fusion peptides leads to a significant reduction in the p24 value (minus 55% and minus 77%) compared to the control which had received the starting plasmid M3. (Drawing 2)

Example 4: Infection of stable transducile PM-1 cells to detect the inhibitory function of the new fusion peptide

Packaging cells can be used to represent stably transduced PM-1 cells. After transfection of the packaging cell line Phoenix-ampho, the retroviral vectors are used for transduction of the T cell line. After selection, a homogeneous culture can be generated on HIV-infectious cells, which stably expresses either the FRES-A fusion peptide or the FRES-B fusion peptide. In comparison to the non-transduced, or PM-1 line transduced by the starting vector M3, a reduction in the p24 value should be ascertained in the course of the experiment after infection with HIV if the new fusion peptides show an inhibitory effect. This approach is very close to what actually happens in vivo because T cells are used to infect HIV.

As shown in drawing 3, in the course of the infection the ρ24 value is reduced by 90% or 99.9% compared to the control (minus 31og for FRES-B).

Claims

claims

1. Nucleic acid of the general formula

5 '- (marker) - MSD - intracellular area gp41 - 3' where

"5 '" denotes the 5' end of the nucleic acid sequence,

"3 '" denotes the 3' end of the nucleic acid sequence,

“(Marker)” denotes an optionally present DNA region that codes for an extracellular peptide for the detection of the construct, “MSD” denotes the DNA coding for a “membrane spanning domain”

Region, fragments or derivatives thereof, designated, and

"Intracellular region gp41" means a DNA region which codes for the intracellularly expressed region of gp41, a fragment or derivative thereof, of HIV, a fragment or derivative thereof.

2. Nucleic acid, a fragment or derivative thereof, according to claim 1, characterized in that "intracellular region gp41" is the sequence shown in SEQ ID NO: 1.

3. Nucleic acid, a fragment or derivative thereof, according to claim 1, characterized in that "intracellular region gp41" is the sequence shown in SEQ ID NO: 3.

4. nucleic acid, a fragment or derivative thereof, according to any one of claims 1 to 3, characterized in that "MSD" codes for a membrane anchor of a type I transmembrane protein.

5. Nucleic acid, a fragment or derivative thereof, according to claim 4, characterized in that the type I transmembrane protein is LNGFR.

6. nucleic acid, a fragment or derivative thereof, according to one of claims 1 to 5, characterized in that "marker" codes for an inactive T-20 variant.

7. nucleic acid, a fragment or derivative thereof, according to any one of claims 1 to 6, characterized in that it does not contain a DNA region coding for a "marker".

8. Recombinant DNA, which comprises a nucleic acid, a fragment or derivative thereof, according to one of claims 1 to 7.

9. Expression vector which contains a recombinant DNA according to claim 8.

10. Expression vector according to claim 9, characterized in that it is a retro viral vector.

11. Expression vector according to claim 10, characterized in that the retroviral vector is M87m3.

12. A method for producing a recombinant peptide which is encoded by the nucleic acid according to any one of claims 1 to 7, comprising the following steps:

(a) introducing a vector according to any one of claims 9 to 11 into a host cell, wherein the host cell can be pro- or eukaryotic,

(b) culturing the transfected host cell, and (c) isolating the peptide from the host cell or from the culture medium.

13. Fusion peptide of the general formula

NH ₂ - (marker) - MSD - intracellular region gp41 - COOH where "NH ₂ " denotes the amino-terminal end of the fusion peptide,

"COOH" denotes the carboxy terminal end of the fusion peptide, “(Marker)” denotes an optionally available extracellular peptide for the detection of the construct,

"MSD" denotes a "membrane spanning domain", a fragment or derivative thereof, and

"Intracellular region gp41" denotes the intracellularly expressed region of gp41, a fragment or derivative thereof, a fragment or derivative thereof.

14. Fusion peptide, a fragment or derivative thereof, according to claim 13, characterized in that "intracellular region gp41" is the sequence shown in SEQ ID NO: 2.

15. Fusion peptide, a fragment or derivative thereof, according to claim 13, characterized in that "intracellular region gp41" that shown in SEQ ID NO: 4

Sequence is.

16. Fusion peptide, a fragment or derivative thereof, according to any one of claims 13 to

15, characterized in that "MSD" is a membrane anchor of a type I transmembrane protein.

17. Fusion peptide, a fragment or derivative thereof, according to claim 16, characterized in that the type I transmembrane protein is LNGFR.

18. Fusion peptide, a fragment or derivative thereof, according to one of claims 13 to

16, characterized in that "marker" is an inactive T-20 variant.

19. Use of a vector according to any one of claims 9 to 11 for the treatment of HIV infections.

20. Use according to claim 19, characterized in that HIV-infected cells are transfected with the vector according to one of claims 9 to 11.

21. Use according to claim 20, characterized in that the HIV-infected cells are T-lymphocytes.

22. A method for inhibiting HIV-induced cell fusion, comprising contacting an HIV-infected cell with the fusion peptide according to any one of claims 13 to 18.

23. Nucleic acid according to one of claims 1 to 7 and / or a vector according to one of claims 9 to 11 and / or a fusion peptide according to one of claims 13 to 18 for use as a medicament.

24. Use of a nucleic acid according to one of claims 1 to 7, a vector according to one of claims 9 to 11 and a fusion peptide according to one of claims 13 to 18 for the manufacture of a medicament for the treatment of patients infected with HIN.

25. Medicament for the treatment of HIN-infected patients, containing a nucleic acid according to one of claims 1 to 7 and / or a vector according to one of claims 9 to 11 and / or a fusion peptide according to one of claims 13 to 18, optionally in combination with other pharmaceutically acceptable substances and / or carriers.

26. Kit containing a nucleic acid according to one of claims 1 to 7 and / or a vector according to one of claims 9 to 11 and / or a fusion peptide according to one of claims 13 to 18.