DE19532651A1 - Viral vector compositions for treating viral infections - Google Patents

Abstract

Viral vector composition for therapeutic use against viral infections, comprises: (a) single and/or double stranded proviral DNA molecules, consisting of adjacent genes of the infectious provirus, and/or fragments, into which at least 1 sequence adapted to form a hairpin or cross shaped structure has been integrated; or (b) double stranded DNA molecules consisting of adjacent genes of the infectious provirus, and/or fragments, in which 1 strand has a sequence adapted to form a hairpin structure, provided that the DNA molecules are produced by in vitro enzymatic methods and/or chemical synthesis, or by recombinant DNA techniques.

Description

It is known that at the beginning of an acute viral infection with HIV initially prefers to attack CD4-T cells and kill them the. The subsequent immune response of the still intact immune systems consists in the increased formation of antibodies, CD4-T helper cells, inflammatory CD4-T cells and cytoto xical CD8 T cells. Through this immune reaction, the most viruses are effectively removed from the blood, leaving few new cells are infected and the disease is called asymptomatic phase that lasts for several years and is generally symptom-free. During the asym ptomatic phase, however, the number of CD4 T cells dig decreased.

If their number drops below 500 per µl, the first symptoms appear opportunistic infections. If the number of CD4-T Cells falls below the critical value of 200 per µl finally the full screen AIDS.

The Destruction of CD4 T Cells During Chronic In fection can be explained as follows:

• - New viruses are produced in infected host cells and cause the cell envelope to burst.
• - Affected host cells form syncytia that no longer are viable.  
• - Bursting host cells also make many coat proteins (gp120, gp41) released and absorbed by naive T cells taken. There the coat proteins are intracellularly in Fragments broken down, these antigens become extracellular presented at the MHC complex and by CD8 T cells (Killer cells) that recognize the T- infected by virus protein Destroy cells cytotoxically.
• - Uninfected CD4 T cells, which are virus-infected Detect protein-infected CD4 T cells give cytokines from the TNF family to destroy the infected cell the effect.

Due to the constant destruction of CD4-T cells due to The mechanisms of action described have a general weakness immune defense instead. The constant lymphocyte new production causes an accelerated aging of the stem cells in the bone marrow, causing the generation of CD4 T cells in the course time is decreasing more and more.

In addition to the CD4 T cells, HIV also directly destroys the thymus Cells, the brain cells and the lymphoid tissue, so that the immune defense is further weakened.

The previous strategies to fight HIV strains want to either immunization by vaccination or suppres effect of HIV after infection.

Immunization through vaccination

• - vaccination with genetically engineered coat proteins rgp120,
• - vaccination with HIV cases,
• - Vaccination with weakened viruses, e.g. B. with viruses, the the rev gene has been removed  
• - Vaccination with recombinant vectors for the introduction of HIV coat protein genes in human host cells Organism,
• - Vaccination with recombinant vectors for the introduction of Genes, e.g. B. genes of cytotoxic cytokines in the Host cell that is too irreversible when infected with HIV blen expression come,
• - Vaccination with genetically engineered vectors for mani lymphocyte stem cell population in the bone marrow the lympho produced by the manipulated stem cells to protect cytes from HIV infection.

Suppression of HIV after infection

• - Treatment with base analogues (AZT and related drugs te) to inhibit reverse transcription,
• - Treatment with enzyme or protease blockers for competi tive irreversible inhibition of protease enzyme activity (Saquinavire),
• - Treatment with ribozymes with RNA lytic activity for Destruction of HIV RNA or HIV-m RNA.

According to previous experience with vaccines, this is one of them assume that the artificially generated immune response due the high mutation rate of HIV remains ineffective. This high mutation rate in HIV arises from the relative often incorrect transcription work of the reverse trans cryptase. So every 20,000 or so. Nucleotide from the HIV RNA incorrectly transcribed onto the DNA matrix strand. The DNA Matrix is made by the reverse transcriptase through synthesis of the coding DNA strand complemented. Will that be the result? resulting provirus reactivated and expressed, ent are modified antigens in which the immune memory  performance no longer applies. The ge no longer formed antibodies and used them for detection mark the cellular immune response. The CD8-T cell len or macrophages can these mutants due to the out then no longer destroyed molecular immune responses ten.

At the same time, the cellular immune response from the virus but also directly suppressed.

The cellular immune response is activated in the first step by CD4-T helper cells that had antigen contact and then interleukin 2 (IL-2) and interferon-γ (IFN-γ) produ adorn.

For the cellular immune response, namely the formation of CD8-T Cells and the stimulation of macrophage formation, this is Signal mediated by IL-2 and IFN-γ required. In the chronic phase (asymptomatic phase) of HIV infections but CD4-T helper cells do not produce IL-2 and IFN-γ, but interleukin 4 (IL-4) and interleukin 10 (IL-10) produ graces. These signal substances stimulate the B cells to anti body production, d. H. the increased formation of CD8-T cells len is suppressed. Due to the ineffectiveness of the antibody perreaction due to the high mutation rate of HIV, ins special of its coat proteins, and the ability of the virus to the cellular immune response towards an antibody postponing the action, the HIV infection becomes persistent.

Also drug treatment with AZT or saquinavirus is suspected in the long run because of the high mutation rate of HIV A resounding success has failed because the virus e.g. B. has already formed AZT-resistant strains and pro tease inhibition constantly new Saquinavire must be developed sen.

With a promising strategy to develop a  therapeutically active agents against the HIV-1 and HIV-2 Strains should first of all include the HIV Provirus can be deactivated.

For this purpose, viral vectors are used which are made from Provirus DNA Single strands or double strands consist of full permanent and / or parts of neighboring genes of HIV are composed. In the genes or sections of genes artificially built in at least one special sequence that Formation of a so-called hairpin structure or Cross structure leads.

The incorporation of the single strand stabilized by RecA protein pieces, whereby the double strands as donors for RecA- protein-free single strands act in the first Step by attachment to the coding or non-coding render relaxed because of the transcription activity Strand of the provirus and is the second step through the Integra reaction completed.

At the expression level of the RNA comes loops or Hairpin structures called stop loops, the RNA polymerase during the transcription process to a standstill and disso strikes off the DNA before the provirus is completely trans is written.

For example, if the hairpin is in the env gene of an HIV DNA Positioned, which consists of vpu and env genes, the envelope proteins gp120 and gp41 of HIV can be incorporated of this piece of DNA from the host cell is no longer complete to be generated. The virus can no longer read the cell sen. The organism is also protected because of the concentration of the dockable coat protein fragments greatly reduced becomes.

However, the host cell bursts due to overproduction the virus particles that lack the shell because of the stabilization Functional envelope necessary for the envelope membrane  Proteins are missing, so these virus particles are the immune response of the organism, because without gp41 and gp120 envelope do not protein the HIV particles or HIV fragments to the Can dock CD4 protein-bearing cells. In this way infection of healthy cells cannot occur, nor can the immune defense by shifting the cellular immune response on the antibody-forming response.

If the hairpin structure is positioned in the HIV gag gene, see above the RNA polymerase of the host cell Kernel proteins read only incomplete, so that it is due to the inoperable core protein fragments neither for formation the core shell can still form the outer shell.

The transcription in the eukaryotic human organism becomes performed by RNA-Pol (I), RNA-Pol (II) and RNA-Pol (III). Because the RNA Pol (II) does most of the transcription work performs, reacting relatively non-specifically to stop codons and often goes well beyond the goal (stop codon) synthetic HIV-DNA sections are used, that can form multiple stop loops. This will make the RNA polymerase complex safely brought to abdissociation.

The medicinal HIV-DNA part described below pieces that integrate into the HIV provirus genome synthesized and vectoral to blood, lymph or Ge brain fluid supplied.

An example of a sequence that is transcribed after the Formation of an RNA stop loop is the termina tion sequence:

(-GGGTCGGGCGGATTACTCGCCCGAAAAAAAA-) n

n = 1, 2, 3.

An example of a sequence that is training in DNA a hairpin structure, the following is inverse repetitive sequence:

-ACGCTA-CTCA-TAGCGT-

Such a sequence can also be used to form a cross structure lead if it occurs complementarily in the double strand.

A sequence is preferably present as the termination sequence watch at least one stop code, two compliments tary strand sections for hairpin structure formation and one 3'-terminal homogeneous polynucleotide section, preferred Polyuracil nucleotide.

The following table shows some possible therapies effective HIV-DNA sections listed. It is too note that the specified virus genes, e.g. B. the env- or pol gene, need not be complete genes, but also Gen sections can be.

The location of the inserted in the genes or gene sections hairpin-forming sequences is shown in the diagram illustrates a vertical bar.

Coding HIV-DNA single strand pieces with hairpin forming Sequences are as α-pieces, non-coding, complementary to them tary single strand pieces without hairpin forming sequences as β-pieces, coding HIV-DNA single-strand pieces without Haarna del-forming sequences as α'-pieces and non-coding, as well complementary single strand pieces with hairpin-forming Se sequences called β'-pieces.

The length and composition of the pieces of HIV-DNA are used moderately chosen so that their integration into the Provirus genome is optimized in the transcription-active area.

For example, α- and β pieces, α′- and β′-pieces as well as α- and β′-pieces.

In addition to HIV genome pieces, of course, can also be full Permanent HIV genomes are used that contain one or more wear sequences forming hairpin structure.

Possible combinations according to claim 1 would be e.g. B. α-single strand pieces and β′-single strand pieces or α-β′-double strand pieces. Combinations according to claim 2 are β single strand and α'-single strand pieces. Combinations according to claim 3 are α-β double strand pieces and α′-β′ double strand pieces.

In the case of In. Catalyzed by integrase or RecA protein tegration of the pieces of HIV-DNA into the provirus genome Strands of the HIV provirus substituted. The released ones Strands of the HIV genome can be complemented by Single strand pieces are intercepted. The deleted originals Ren single strand pieces can not in turn integrate the Substitute artificial HIV-DNA fragments, as this back reaction is inhibited competitively.

A possible installation mechanism when using a therapeutic agent according to the invention is described below:
When using αβ′-double-stranded pieces or these pieces in combination with the corresponding stabilized single-stranded pieces, the matching provirus DNA sequences of the coding or non-coding strand are displaced from the surface of the RNA polymerase and replaced by the complementary, artificial HIV DNA piece bound. In this process, the enzyme complex and the mRNA fragment dissociate. The catalytic reaction of the integrase or the RecA protein deletes the original HIV sequence on the coding or the non-coding strand and integrates the artificially generated sequence in the complementary strand.

A supportive effect is conceivable with this mechanism by a polymerase-inhibiting effect of the integrase.

The target group of the therapeutic agents according to the invention are all CD4 molecule-bearing cells. These are the macrophages of the Brain, the lymph node cells, the thymus cells, the bones marks cells, the macrophages of the lungs, the Langerhans cells the skin as well as the lymphocytes in blood, seminal and vaginal liquid. The HIV-infected glial cells of the brain and the chromaffin colon, duodenum and rectum cells do not form any detectable CD4 protein ne, but they contain low concentrations of mRNA for the CD4 protein. However, this suggests that this too Cells can synthesize very small amounts of CD4 where can be explained by the infectability of these cells.

In therapy with the artificial Provirus DNA strand pieces ken, which are preferably introduced with HIV envelopes, treatment should be given in multiple batches.

To attack as many HIV Proviruses as possible at the same time can, the therapy with the inventive agents preferably during phases of gross natural HIV activity turns. Of course, HIV transcription can also can be artificially stimulated using certain factors or their combinations:

• - NF-kB protein (vectorally supplied)
• - HIV-tat protein (vectorally supplied)
• - Infusion of an amino acid solution for nutrient over offer
• - infusion of cAMP
• - infusion of acetone (pseudo-hunger)
• - Stress factors, e.g. B. Administration of cold and Flu viruses, administration of the immune system weaken the substances, irradiation with UV light, etc.

In addition, additional support can be provided to support relaxation ches SSB protein vectoral be supplied.

With the means described so far can freely in the Kör infectious viruses floating on liquids not reached will.

It therefore seems sensible after or between treatments to administer medication to the lungs with these therapeutic agents, that stimulate the immune response to HIV. These are at exogenous cytokines suitable for growth the B lymphocytes and the naive T cells for the antibodies stimulate reaction. In addition, plants, for example cytokines from extracts of wheat (Triticum aestivum) or spelled (Triticum aestivum spelta) a reinforced co- Stimulation on the naive T cells and lead in this way to an increased formation of non-specific, cyto toxic CD8 T cells, we very free infectious viruses be able to annihilate.

As already preferred with the therapeutic agents according to the invention infected or AIDS patients are treated is a life-extending effect  waiting. The agents according to the invention can also possibly successful in combination with already known active ingredients.

The therapeutic agents described have an immunizing effect also conceivable, since under certain circumstances about a possible recombinant interaction of an inte in the host genome HIV-DNA piece with the free HIV-DNA the artificial hairpin structure-forming sequence is integrated into the latter.

Claims (9)

1. Viral vectoral agent for therapeutic Use against viral infections, especially HIV-In fractions containing by in vitro enzymatic and / or organic preparative synthesis through recombinantly increased Provirus DNA Ein individual strands and / or composed of complementary strands set provirus DNA double strands consisting of full permanent and / or parts of neighboring genes of the In fection provirus, being in the genes or gene fragments each strand at least one sequence to form a Hairpin structure or cross structure is integrated. 2. Viral vector to be administered to the combined therapeutic application with the agent according to claim 1 for Use against viral infections, especially HIV-In fractions containing by in vitro enzymatic and / or organically preparative synthesis produced or by recombinantly amplified provirus DNA single strands consisting of complete and / or partial pieces the genes of the infection provirus, the single strands have no hairpin structure forming sequence and otherwise complementary to the individual strands according to An are 1. 3. Viral vectoral agent for therapeutic  Use against viral infections, especially HIV-In fections, or for combined use with the agent according to claim 1, comprising by in vitro enzymatic and / or organic preparative synthesis through increased recombination technology, from complementary ones Strands of Provirus DNA double strands, be standing from complete and / or parts of the genes of the infection provirus, one of the strands being a Se sequence to form a hairpin structure. 4. Means according to claim 1 or 3, characterized ge features that as a hairpin structure-form de sequence an inversely repetitive or a termination sequence is provided. 5. Means according to claim 4, characterized records that the termination sequence at least a stop code, two self-complementary strand sections for hairpin structure formation and a three-terminal homogeneous polynucleotide section. 6. Composition according to one of claims 1 to 5, characterized characterized in that the virus envelope for the Vector is a shell of the infection virus. 7. Means for combined treatment with one of the means according to claim 1 or 3 to 6, containing the naive T- Cells co-stimulating cytokines. 8. Composition according to claim 7, characterized records that the cytokines are plant cytokines are. 9. Composition according to claim 8, characterized records that the plant cytokines from a Wheat extract (Triticum aestivum) and / or spelled (Triticum aestivum spelta) exist.