WO1993022433A2

WO1993022433A2 - Medicament for the gene-therapeutic treatment of human beings, animals and plants, especially to block virus multiplication and carcinogenes and process for producing the medicament

Info

Publication number: WO1993022433A2
Application number: PCT/DE1993/000374
Authority: WO
Inventors: Frank Andreas Harald Meyer
Original assignee: Frank Andreas Harald Meyer
Priority date: 1992-04-28
Filing date: 1993-04-28
Publication date: 1993-11-11
Also published as: WO1993022433A3; AU4259193A

Abstract

Some diseases in the bodies of human beings and animals are caused by viruses. Many retro-viruses carry carcinogens. Attempts have so far been made to interfere with complete retro-viruses to combat retro-viruses. The drawbacks of the prior art therapy are that various kinds of side-effects which are uncomfortable for the patient are inevitable and that the course of many diseases is slowed down but the disease cannot be cured. The novel medicament is especially suitable for blocking virus multiplication and carcinogens. The medicament contains virus shells which attach themselves to the receptors of the known basic cells. The medicament comprises cells taken from the organism which have been treated with anti-viruses. The medicament comprises cells taken from the organism which have been treated with the appropriate glycolysed liposomes. When the medicament is used, the patients treated suffer from hardly any uncomfortable side-effects and diseases are cured.

Description

description

Drug for gene therapy treatment of people,

Animals and plants, especially for blocking the

Virus multiplication and the tumor gene as well as methods for the production of the drug

The invention relates to a medicament for the gene therapy treatment of humans, animals and plants, in particular for blocking the multiplication of viruses and the tumor genes, and to a method for producing the medicament.

Viruses cause diseases in the human and animal body, among other things. Retroviruses occupy a special position. They are single or double-stranded RNA viruses that are replicated and expressed via a double-stranded DNA form. This form of DNA is synthesized by the virus enzyme reverse transcriptase. Many retroviaries carry oncogenes. The acquired weakness of the immune system - AIDS (Acquired Immune Deficiency Syndrome) - in which the patients die from a progressive weakening of the immune system is also linked to an infection by a retrovirus.

To combat retroviruses, attempts are made to intervene in finished retroviruses. A retroviral admiration has so far been encountered, for example, through chemotherapy, in which cytostatics are used to inhibit the virus cycle. At the moment, reverse transcriptase appears good goal of antiviral therapies, for example with the help of AZT (azidothymidine).

The disadvantages of the known therapy are that various forms of side effects are unavoidable, which place a heavy burden on the patient being treated, and in the fact that many diseases, although slowing down in their course, cannot be cured.

The invention has for its object to provide a medicament, in particular for blocking the virus multiplication and for blocking the tumor genes, as well as a method for producing the medicament.

According to the invention the object is achieved by the features listed in the claims.

The invention has the advantages over the known that

a) There are no long-term, stressful side effects in the treated patient.

b) The diseases caused by viruses can be overcome without danger.

c) The therapy consists of a vaccination that the patient feels like other vaccinations.

d) The virus envelopes themselves cause a body's own immune response by increasing the formation of antibodies against these non-reproducible envelopes, which also destroy the viruses, which are still formed to a small extent in unreached cells before further infections of other cells can occur. The body's own defense reactions are therefore promoted.

e) To ensure maximum effectiveness in the simultaneous control of several types of viruses, it is possible to combine, for the most part, already known counter-rotating DNA or RNA sequences in such a way that polyselectivity towards a large number of closely related viruses is achieved.

f) The immune system can be supported, in particular with regard to points (d) and (e), in the fight against other viral infections. This advantage is particularly noticeable in the treatment of the immune deficiency disease AIDS, since secondary infections can be effectively combated.

g) By eliminating the threat of viruses, the immune system of each organism previously contaminated by viruses can defend itself more specifically against other pathogenic substances, or the respective body can use its energies in another way, which on the one hand is beneficial for the organism, freed from some flagella should set a longer life expectancy and on the other hand better growth (higher life expectancy of humans and greater yields in agricultural products).

h) The drug is buffered against most mutations of the viruses to be controlled because slight differences in the opposite sequence do not have a noticeable effect, since the hybridization is not prevented due to the length of the opposite strands.

i) An advantage of the process for the production of these medicaments is that the medicament can be produced in large quantities in a very short time under easily controllable conditions.

j) The same procedure is applicable to all virus species.

k) In general, not much research work is necessary since the genes in most types of viruses have already been analyzed with regard to the base sequences, so that Specifications, such as start and stop codons and splice sequences, can be located on the opposite strand using electronic data processing.

1) Preventive vaccination is possible.

Of course, one will also think of a prenatal (retro) virus immunization in this context, which could be carried out on a zygote, among other things, in order to immunize each body cell of the resulting organism without side effects against (retro) viruses so that resistance would be achieved that would make any drug treatment against (retro) viruses superfluous. This immunity, like disease-causing, endogenously inherited Proviren anyway, could be passed on to offspring.

Since it is possible to limit the treatment of an organism to somatic cells, any intervention in a zygote must be ruled out.

Further advantageous embodiments of the invention emerge from the subclaims and the description.

drug

Has a retrovirus infects a cell, its RNA (ribonucleic acid) by reverse transcription in DNA (deoxyribonucleic acid) is rewritten _r and this DNA is integrated into the genome of the host cell. Normally, only one of two strands, namely the lead strand, is translated into mRNA (messenger RNA or messenger RNA) in a given section of the double helix. If an opposite gene is synthesized and integrated into the genome in such a way that a subsequent strand is transcribed, the result is an opposite mRNA, the sequence of which is complementary to the normal mRNA transcripts. Is opposite mRNA transcribed in a sufficiently large quantity, it will be ^"with -the viral ^mRNA". "- hybridize and prevent the synthesis of proteins that are essential for the survival of the lytic and temperate cycle of retroviruses.

The introduced DNA sequence is transcribed into mRNA that hybridizes with the viral mRNA that codes for reverse transcriptase. So that's how

(1) the revertase inhibited in formation, but also

(2) the opposite mRNA revertase strand even hybridizes with new virus genomes entering the cell.

If the formation of the revertase is inhibited, it is no longer available to newly synthesized mRNA strands of the retrovirus. This prevents the genesis of new viruses and thus the infection of other cells. If the opposite mRNA revertase strand binds viral genomes directly into the cell, the translation of new reverse transcriptase is prevented. A further inactivation of such retroviruses could take place in that the RNA double helix formed in the cell of the gene of the reverse transcriptase on the mRNA strand, which represents the virus genome, prevents the incorporation into protein shells because they only provide a limited space. It also prevents a specific arrangement of the viral genome that would be essential for virulence.

The processes described can be supported by incorporating further, opposite viral genes of such retroviruses into the host genome, which then also hybridize with the retrovirus genome and thus inhibit the synthesis of protein envelopes of the viruses or virus-specific proteins.

The same procedure is followed with oncogenes and virus genomes. This opens up the possibility of also inhibiting the genes of DNA viruses after they have been incorporated into the host genome or when they enter the lytic cycle.

If inhibition of revertase is carried out, one can be sure that no important metabolic processes of the host are disturbed, because the revertase has no function in the cell. This means that no hybridization of medicinal, opposing mRNA with cell-specific mRNA can develop, because both have too few complementary bases. With an error rate of 1:10 ⁴ bp (bp = base pairs of the nucleic acid), the revertase also converts incorrectly transcribed mRNA of the normal host genome back into DNA, which - when incorporated into the host genome - can falsify it. This is a possible reason for the unimaginable size of the eukaryotic genome, for the many repetitive sequences and for the family similarities of proteins.

However, the mRNA synthesizations of the host cells must be taken into account, which must not be disturbed in their structure and, above all, in their function, because otherwise developmental disorders could occur.

In the course of the described evolution, new genes are created by duplicating and decaying old genes and by reusing parts of old genes in new combinations.

For this reason, most genes have a family of closely related genes elsewhere in the genome. Some of them probably have similar functions.

However, this problem does not exist to a large extent with the retroviruses, which are dangerous for humans and animals, because the reverse transcriptase is only an enzyme of the retroviruses, because only they have to rewrite RNA into DNA.

This highly specific enzyme made up of proteins therefore has an amino acid sequence and hence an mRNA sequence that is unique to reverse transcriptase. Hybridizations with host cell-specific mRNA strands are therefore hardly achievable. The same applies to other virus gene segments.

A complete blocking of the viral RNA by the opposite genome is only possible with a high concentration of the opposite RNA. Accordingly, the gene for the opposite strand must be given a very strong promoter, ie a special DNA sequence, so that sufficient mRNA is formed. The promoter contains both the starting point for RNA synthesis and the information on where to begin. The promoter also ensures that the concentration of the mRNA within the cell is high enough to ensure sufficient resistance to invading viruses.

In order to counteract production beyond the healing process in long-lived cells (stem or nerve cells), it could be helpful to control the activity of the promoters, which are upstream of the opposite genes, by chemical or thermal signals.

Processing the primary transcript leads to problems that can be overcome:

(A) The "cap" structure, which consists of a 7-methylguanine residue and a triphosphate coupled to it, is attached to the 5 'end of the primary transcript that was first synthesized during transcription, but does not inhibit hybridization,

(B) the splicing mechanism can be circumvented by recoding the signal sequences responsible for it in the opposite gene,

(C) in the case of viruses which, by virtue of their natural nature, come from themselves Have splice sequences, the opposite nucleic acid strands should possibly also be spliced at corresponding opposite complementary sites,

(D) the synthesis of a poly A tail at the 3 'end is prevented by recoding the sequence AUAAA (on the codogenic strand TATTT), which initiates this synthesis,

(E) in the artificial, opposite gene, additional terminating sequences for polymerase II, for example in the form of a hairpin structure, must be incorporated so that the polymerization of the primary transcript can be ended, and

(F) A random incorporation of the opposing RNA-forming DNA into active gene structures of the host cells, that is to say in exons, must be prevented, so that these opposing, modified strands are only incorporated into functionless, non-protein-coding introns.

So episites, unique base sequences in the modified antisense strand, have to be created, which are incorporated into homologous chromosites, provirus installation sites, of cellular introns. The provirs of adenoviruses, miopapoaviruses and retroviruses are incorporated into non-homologous recombination, a process that requires little or no homology between the cellular and viral DNAs and no special recombination enzymes, in a wide variety of locations, including in exons of the host cell genome.

A controlled, site-specific integration is achieved in that the integration takes place by means of homologous recombination between nucleotide sequences in non-functional DNA sections of the host cell genome and the homologous sequences in the peripheral regions of the opposing genetic information. The LTR (long terminal repeat) also plays an essential role in the precise integration of virus DNA. The built-in provirus becomes more cellular from short duplications Flanked sequences whose length is virus-specific and whose sequence varies from installation site to installation site. Integrated proviruses of oncoviruses therefore have a transposon structure. For understandable reasons, such structures must be excluded for the modified opposing strands.

A further increase in the concentration of the opposing mRNA can be achieved by the frequency of the corresponding DNA sequence in the genome.

In order to treat the acquired weakness of Im unsyste ε - AIDS (Acquired-Im une-Deficiency-Syndrome) - a large amount of opposing DNA or opposing RNA is necessary, which is necessary for acute treatment. Any number of counter-rotating mRNA or counter-rotating DNA can be obtained in vitro from follow-strand DNA by adding RNA, DNA polymerases and the necessary building blocks. You can clone selected DNA sections in a test tube with a polymerase chain reaction.

In a first step, the body is first flooded with counter-rotating mRNA revertase or the corresponding counter-rotating genes, which through a special modification penetrate the plasma membrane of the cell. This will immediately interrupt the virus cycle. Further synthesis and further infection are prevented. The immune system can stabilize and fight the virus.

Since the opposite gene of revertase is also introduced into uninfected cells, this gene is changed from the start of normal transcription at the ends of every 150 base pair long interval by the exchange of the base located there with the corresponding complementary base, to all Possibilities of preventing revertase expression in these cells. Such short, non-complementarities with the other modifications do not lead to any noticeable Reduction of stringency because this strategy involves many kilobases and not short antisense strands. Due to the changed reading frame, an already improbable build-up of viruses is then impossible even if the promoters were to be returned to their starting point for some reason or were newly created by mutations.

In the cells in which revertase is already present due to infection, the opposing mRNA revertase is rewritten by the revertase itself into opposing DNA revertase. If the latter is present in sufficient quantity, the DNA revertase can be incorporated into the host genome of plant cells in such a way that it arrives with great certainty in front of the host's own promoters and then independently forms opposing RNA revertase. In other cases, repetitive sequences arise with the consequences described above. The same applies to other gene segments in the virus genome.

If opposing DNA is introduced into plant cells, this DNA is preferably designed like a transposable element, which makes it easier for the DNA to integrate into the genome, move there and possibly multiply. It is relatively likely that this element, which should have a strong promoter, will be integrated into a group of active genes (operon model). The same effect is achieved if the DNA strand is embedded in LTR sequences, as is generally the case with viruses.

A second step is to introduce permeable reverse DNA revertase into the body, which can only be built into the genome of the cells when cells divide. This treatment has to be carried out over such a long period of time that all itotically dividing cells are reached. In this way, the germ cells are also artificially mutated, resulting in a ^' Retrovirus immunization takes place in the offspring. The virus infection of the offspring can thus also be prevented. The required amount of DNA can be generated by gene cloning.

If the nerve cells that are no longer dividing are already attacked, lifelong therapy with opposing mRNA is necessary because the procedure described above is only effective during cell division. Infected cells that no longer divide produce pathogenic viruses for a lifetime.

In order to permeate the cell membrane, the binding of certain chemical compounds to the ends of the nucleic acid molecules, preferably by means of a phosphate bridge, is necessary, as in the case of the "cap" structure, which consists of a 7-methylguanosine residue and a triphosphate coupled to it, which is easily achieved by a any known enzyme can be hydrolyzed and broken down.

Special chemical groups or compounds can be used to strengthen the binding of the coding viral mRNA with the counter-rotating mRNA, which is used for acute treatment.

Thus, double-stranded retroviruses, which have to be single-stranded at some point when they enter the cell, could also be switched off, because the opposite mRNA binds to the strand so tightly that they cannot be separated by thermal or enzymatic cleavage. The complex persists until the mRNA of the virus disintegrates or is broken down, while the longer-lived counter-mRNA stabilized by certain complexes can enter into a new pairing and is an artificial biocatalyst.

Over time, the genome-integrated proviruses are destroyed by random mutation and removed from the germline. As a result, human pathogenic viruses are becoming more and more to be ousted. The risk of AIDS and cancer is steadily decreasing for individuals and their offspring.

A certain problem is the transduction of mRNA and DNA, which represent giant molecules as opposing genes, into the eukaryotic cell, whereby the plasma membranes form the barrier that has to be overcome.

A very good way of finding the infected cells in the organism and treating them selectively is to use gl cosylated liposomes or the virus protein envelopes themselves as carriers. It is not the intact virus genome that is introduced into the virus protein envelopes, but rather its own counter-rotating nucleic acids and possibly certain enzymes, which are then introduced into the affected cells in a highly selective manner as genetic material. Thus cells that no longer mitotically divide can also be treated, because the receptors of these cells are not only reached by the pathogenic viruses, but also by the medication.

Since the opposite complementary virus RNA is identical in length to the original virus RNA, there are no space problems in the associated viral protein shells, which ultimately dock onto the same receptors to which the normal virulent viruses respond, based on the entire rewritten genome.

The body's immune response to the virus envelopes can itself cause immunity. As indicated briefly above, the body reacts with its immune system to the virus protein shells in order to destroy them. The general difficulties that the body has with the constantly mutating viruses are known. In principle, this antigen-antibody response, especially before a virus infection, is only advantageous, even if large portions of the corresponding medication, ie large amounts of the anti-viruses, are destroyed in the process. The organism strengthens thereby his fight against the viruses damaging him.

If one wishes to generate an immune response without replication of the viral genetic information taking place, it may even be possible to provide the protein shells of some pathogenic virus types without information. The respective effector functions that trigger the normal protective mechanism against individual virus infections are therefore not absolutely necessary to be known with this method of vaccine production. With this method, only the correct effector functions can be brought about in each individual. The right cells are automatically stimulated by the virus envelopes, which can no longer reproduce, for perhaps lifelong immunity.

Passive immunization is also conceivable in the form of a vaccination by taking blood from a healthy organism immunized with virus envelopes with a correspondingly stronger immune system in order to administer the antibodies as a vaccine. This means that many antibodies can be made available to both severely ill people for more effective defense and to healthy people for passive immunization. Infected organisms also have antibodies in their blood that could be used if their immune systems are strong enough.

Conceivable and perhaps a little more desirable for the pharmaceutical industry, but possibly a little more difficult to develop for large-scale production, liposomes that contain the corresponding glycoproteins would also be able to supply the respective target cells with the opposite genetic information and the associated enzymes. The more artificial a drug is, the easier it is to examine it for possible contamination. Under no circumstances may other foreign substances be accidentally caused by any unfortunate coincidences in the organism to be treated reach .

HIV-positive or, for example, leukemia patients whose leukemia was caused by viruses can be significantly supported with immune cells treated in vitro: Corresponding cells can also be used outside of a living organism, i.e. in vitro, according to the same principle by anti-viruses the respective viral genome of opposing RNA or opposing DNA and the corresponding respective enzymes. Acute people with AIDS immunodeficiency can thus use resistant immune defense cells that contain genetic information that is opposite to the virus genomes, such as T helper lymphocytes, macrophages, monocytes, antigen-presenting dendritic follicular reticular lines, Epstein-Barr virus-transformed B-lymphoblastoid cells or other cells with the appropriate receptors can be effectively supported in their immune defense by transfusing these new, grown resistant immune cells or by returning the now resistant, own cells treated outside the body. The same approach is also suitable for supporting, for example, leukemia patients whose blood cancer was also caused by oncogenes.

A kind of prenatal retrovirus infection has already been pointed out above in connection with the fight against AIDS.

Another one. The fertilized egg (zygote) is spiked with reverse DNA. This means that every body cell in the resulting organism - i.e. every nerve, muscle and immune cell - is immune to retroviruses. Resistance is achieved that makes any drug treatment against retroviruses superfluous. Immunity is also passed on to offspring.

The drug described is suitable for use with all prokaryotes and eukaryotes, that is, both with single and with Multicellular, i.e. humans, animals and plants. It is suitable for both prophylactic vaccination and acute treatment.

The self-assembly mechanism is used to integrate the specified antisense RNA or DNA strands and the corresponding enzymes into the appropriate, suitable virus envelopes, in order to transport these virus envelopes into the respective affected cells with the find corresponding receptors.

This self-organization or self-organization through thermodynamic laws and / or kinetic determinations, i.e. Reactions dependent on the reaction pathway are the property of complicated biological structures that are composed of macromolecular building blocks by themselves. The ability to self-organize means that the information in the macromolecules is sufficient to determine the correct virus structure. It is precisely this ability that is inherent in all viruses when building their complete structure. Only the external medium, i.e. the corresponding pH value, favorable temperature conditions, the correct ion concentration and the addition of suitable enzymes, are decisive for the independent structure of the virus structure.

If antisense RNA or DNA is to be accommodated in a suitable virus protein envelope, the original RNA or DNA is first removed in vitro, for example by changing the pH, so that the envelope breaks down into its protein components. Now you can remove the original RNA or DNA from the solution. Now add the appropriate antisense genes to these protein building blocks, which - as mentioned above - take up the same space as the original genetic information. Since most viruses have already integrated their own enzymes into their shells, one is therefore able to add these enzymes or other substances to this solution.

If, for example, the pH value is brought back to its original state, then _. The self-assembly reaction described above occurs, in which the virus envelope is built up around the nucleic acids or around the enzymes.

If the viruses to be combated are cloned beforehand, it is possible to industrially produce any quantity of virus with antisense genes as a medicament.

The advantage of the method is that it is applicable to all types of viruses.

If the assembly does not work outside of the corresponding host cells, there are ways to avoid these difficulties: the production process must be adapted to each virus species with regard to the external medium and with regard to the structural properties. The self-assembly function works, among other things, through the lipid envelope present in the capsid of some types of virus and / or due to essential template-like molecular arrangements within the host cells that were synthesized by the viruses themselves or that are present in these cells from the beginning. Mechanism outside of the corresponding host cells does not.

There are three alternative ways to accommodate the modified counter-rotating nucleic acids in the respective transport vehicles so that the corresponding host cells can be reached in a targeted manner:

A) As mentioned briefly above, one could find glycoproteins in liposomes that must be able to do the respective to carry opposing genetic material so fix that these liposomes can deliver their contents to the respective host cells in a targeted manner, just like the viruses, by specifically docking to the receptors of the corresponding host cells.

B) Because with some viruses an appropriate genetic material does not only serve as a template for organizing their structures, but template-like arrangements within their host cells are necessary, which are either already present in the cells by nature or are only induced by the viruses themselves it is necessary to use the mechanisms within these cells. If these template-like molecules are built up by the viruses themselves within their host cells, it is necessary to add the genetic information which codes for these template-like molecules to every cell kept in cell culture. These genes must have a promoter that initiates the synthesis of these molecules and they must be built into introns. Then these cells are injected with the capsomeres that have been freed from their old virus-coding genetic information, the associated modified opposite genetic information and any associated enzymes.

Because of the difficulties in providing all cells of a culture with antivirus components using micropipettes, other methods must be used:

(1) Mechanical bulk pipetting.

(2) Syncitium formation of the culture cells would make pipetting easier.

(3) Membrane opening in the electric field.

(4) Introduction of membrane fusion with vesicles.

In the respective cells, the building blocks of the anti-viruses are now able to use the template-like ones Assemble structures. Ultimately, the anti-viruses are taken from the budding or lysing these cells in culture in order to deliver them to the organisms suffering from the pathogenic viruses.

C) HIV-I and HIV-II have almost the same properties, but very different ribonucleic acids due to their very little relationship «. The corresponding gag and pol genes of HIV-I and HIV-II are only 60% related, the relationship of the respective env genes is 40%. If one modifies the opposite ribonucleic acids in such a way that these opposite strands do not undergo any noteworthy hybridizations with the respective other type of virus and thereby ensures that the otherwise necessary complementarity to the original strand is maintained, one could use a cell culture infected with HIV-I move RNA opposing to HIV-II.

Conversely, the protein envelopes of HIV-II can be treated in the same way with genes opposite to HIV-I. The viruses budding from the host cells can now either contain their own real genetic information or the opposite of the other species. In order to select the right anti-viruses, one could radioactively label the opposite nucleic acid with light emitters. ¹⁴ C (5730 a) and

H (12.3 a) are, for example, very light, non-mutagenic emitters, which, however, have a relatively long half-life compared to the harder emitter ³² P. In return, ³² P only has a half-life of 14 days. With the help of the principle of a cell sorter, one could now differentiate and separate the anti-viruses from the pathogenic viruses that have no radioactive labeling. The HIV virus yield is then only 25% because the two strands of nucleic acid must be in opposite directions. Both strands within an anti-HI virus must have a radioactive label. If the anti-viruses whose nucleic acid sequences have been provided with phosphorus 32 are now frozen, In order to make them durable, as is usually the case in most cases anyway, a radiation dose that is barely measurable can be established after a relatively short time. ¹³ N and ¹⁵ 0 have half-lives of 10 and 2 minutes, respectively.

However, another marking may be possible:

The nucleic acids which are complementary to the respective other virus types are linked to fluorescent molecules before they are injected into the cells of the cell cultures, so that the anti-viruses synthesized in these cells are separated from one another by a cell sorter by the other normally pathogenic viruses also built up there can be distinguished and separated.

In the case of the fluorescence molecules, however, care must be taken to ensure that they do not cause any significant damage afterwards within the host cells of the vaccinated organism. The unsaturated carbon atoms, which are important for the delocalization of the pi-electrons for fluorescence, may tend to saturate. It is therefore necessary to find out whether there is any damage to the lines to be treated if this type of virus differentiation and separation is essential. Since many viruses carry two genetic information in the form of two strands of nucleic acid, the yield of the anti-viruses is correspondingly low, since both strands housed in the capsid must be opposite. Because both HIV viruses have the glycoproteins 120 integrated in their capsids, they dock onto the same CD-4 receptors, so that the same target cells can be supplied with the drug when these HI anti-viruses are injected into the respective patient . Infestation of the same host cells is one of the reasons for the same symptoms that they produce in their hosts, despite their relatively low relatives.

For other types of virus, each with the same receptors on the same target cells, you can do the same and use the shells of the corresponding other virus type, taking care that the opposite genomes have the appropriate sizes so that they and any enzymes can be accommodated in the respective capsids and one must take care that the genomes are not too close

Show related relationships, since otherwise the respective opposing nucleic acids would erroneously inhibit the synthesis of their viral capsids planned as vehicles and the build-up of the required enzymes within the cell cultures.

Process for the preparation of the respective drugs specific for the pathogenic viruses

I) Synthesis — in vivo and in vitro — of the modified DNA producing the opposite RNA (of the opposite genome), all subsequent process steps taking place in vitro

(1) First of all, a suitable, absolutely virus-free and foreign body-free cell culture is necessary for the respective virus species to be controlled, which is necessary for the cultivation of these corresponding virulent viruses.

(2) Another starting point is the corresponding disease-causing virus, against which resistance is to be made possible by the production of a gene therapy medicament.

(3) The virus envelope is broken down by changing the pH and / or the temperature and / or the ion concentration and / or by adding enzymes.

(4) The viral RNA or DNA fragments are extracted (extracted).

(5) A single-stranded viral RNA strand that is suitable for. 'encodes a DNA leader, becomes reverse transcriptase added (taking into account all other important components, such as pH value etc.)

(6) An RNA-DNA hybrid arises that DNA polymerase is added after washing. The latter replaces RNA with DNA.

(7) A DNA double helix is created which corresponds to the provirus genome of the retrovirus. This is still capable of producing normal viral RNA. If DNA viruses are used, steps (5) and (6) can be omitted.

(8) By adding suitable restriction enzymes, the virus genome is cut so that virus-specific promoters can be cut out and artificial promoters can be integrated, which ensure the transcription on the next strand and thus the production of the opposite RNA. The same procedure is followed with intron homology, splice sequences, start and stop codons.

(9) At the end of the synthesis of the counter-rotating DNA, Ligaεe, an enzyme that firmly binds newly combined DNA fragments, is added, whereby the DNA fragments assembled at the sticky ends are joined to form a stable double helix.

II.) Duplication - in vitro and in vivo - of the counter-rotating RNA-producing counter-rotating DNA (the counter-rotating genome)

(10) The DNA double helix is heated slightly and thereby broken down into its strands.

(11) The strands are then allowed to hybridize with two excess chemically synthesized DNA oligo-nucleotides, each 15 to 20 nucleotides long and in sequence to two sections that are separated by any number of nucleotides. The two oligo nucleotides serve as specific primers for in vitro ntheεe Sy ^'of the DNA, which is catalyzed by the DNA polymerase.

(12) After adding the DNA polymerase and the most important components for DNA synthesis, such as triphosphate nucleotides, this enzyme copies the DNA between the sequences that correspond to the oligonucleotides.

(13) After several process steps in points (10) to (12) inclusive, the desired DNA sequence, the counter-rotating DNA, is available in large quantities. The polymerase chain reaction (PCR) allows DNA sequences of any length to be multiplied millions of times.

III) Integration of the opposing genetic information on the original viruses in suitable virus protein shells

(14) A virus, whose protein envelope is suitable as a means of transport for the corresponding counter-rotating DNA, is changed by changing the pH and / or ion concentration and / or temperature and / or by adding suitable chemical substances into the elementary building blocks of the virus envelope and into it viral genome decomposed.

(15) The viral genetic material is destroyed and washed out by enzymes.

(16) The purified viral envelope elemental proteins are incubated with the opposite RNA or DNA strand.

(17) If template-like molecules are normally built up by viruses within their host cells, it is necessary to crosslink a cell with genes which, with the help of a promoter, also synthesize these structures without providing the pathogenic viruses and clone this cell so that it can be cloned the individual anti-virus building blocks in these cell cultures according to the natural mechanisms can store together. These genes, for example they could encode the required lipid envelope of the retroviruses, should be incorporated into the intron sequences of the genome.

(18) For the final completion of the drug, the virus envelopes are assembled in a suitable environment, depending on the virus type, outside or within suitable host cells kept in cell culture, around the opposite RNA or DNA strand (self-assembly).

(19) If protein envelopes of a virus type are assembled with the opposite genetic information of another virus species in a required host cell, the anti-viruses can then be separated from the normal viruses by a fluorescent label or by radioactive labels using a cell sorter and separated.

IV) Integration of the modified genetic information in liposomes

(20) If it is possible on an industrial scale to incorporate the corresponding specific virus building blocks that dock onto the target cells into liposomes that are large enough for the necessary building blocks of the drug, points (17), (18) and (19) could be followed this point (20) are replaced.

V) Production of large quantities of opposing single-stranded RNA

(21) Starting from process step (10), the opposite DNA is mixed with RNA polymerase and other components important for transcription, such as triphosphate nucleotides. The RNA polymerase continuously produces new counter-rotating single strand RNA in large quantities.

As proof whether the modified, counter-rotating genome encodes the viral RNA to be inhibited, which on the one hand hybridizes sufficiently stringently with virus mRNA and on the other hand does not the cell's own mRNA strands, the following procedure can be used:

(a) RNA polymerase II and the other components necessary for RNA synthesis are added to the opposite gene. Mass production of reverse RNA occurs.

(b) 1) Under the conditions mentioned in (a) - however the viral gene is used - the mRNA to be inhibited is also generated in large quantities and radioactively labeled as mRNA-So de.

2) In addition, mRNA is extracted from host cells and introduced into the process.

(c) The counter-rotating RNA produced in (a) is fixed after cleaning on nitrocellulose or nylon paper and chromatographed for a long time under hybridization conditions using the mRNA probe (b).

(d) The leaf is washed so that only hybrids labeled with the probe RNA remain.

(e) The evaluation is carried out by autoradiography.

The vaccination should take place on several dates, each with anti-viruses grown in different cell cultures. So that not too many corresponding receptors of the target cells are occupied at the same time, several vaccination steps with appropriate anti-virus doses and longer-lasting infusions are sensible for even distribution in the body.

The more CD-4 receptors, for example in the case of HIV infection, are occupied simultaneously on a target cell, the more likely it is that the corresponding cell will perish. Anti-viruses that escape from their host cell during the disengagement process, like the original viruses, camouflaging with host cell membrane fragments, produce, on the basis of the fact that, in cell cultures whose cells come from a foreign organism, antibodies in the patient which would lead to strong defense reactions if vaccinated again with anti-viruses of the same cell culture.

These unfavorable defense reactions can be avoided by injecting anti-viruses from cell cultures of different organisms at different times. As already mentioned, the defense reactions against the actual virus building blocks are even encouraging. One or the other type of virus may have protein shells that are harmless for the transport of the contradicting genetic information of pathogenic viruses, but also do their job perfectly.

The drugs can be optimized against most pathogenic virus types after a relatively short trial, based on the knowledge already available, in such a way that even shortly after the vaccination, any signs of vaccination, such as fever attacks by the patient, are either completely avoided or at least can be reduced sharply. Such an exchange of the protein shells of a virulent virus with a harder shell of another virus type would, however, have the major disadvantage that the antibody response of the host's immune system against these pathogenic viruses to be combated would not be further strengthened for more effective control.

The immune response to the anti-viruses, accompanied, for example, by the above-mentioned possible febrile episodes shortly after the vaccination, is therefore normally to be assessed positively. These side effects occur only briefly, if at all. Then, depending on the clinical picture, on the secondary infection, on the pathogen and on the individual, the expected improvement occurs until finally there is a cure. Longer term Side effects must be excluded.

However, if the patient is too badly affected by his viral infection, the only thing left to do with each type of treatment is to wait and see whether the medication in question can really still work. As with other treatments, it is important to start fighting the virus as early as possible after a medical forecast is available.

AIDS and cancer, caused by genetic mutations, can only be combated with genetic methods.

Claims

Expectations

1. Medicament for gene therapy treatment of humans, animals and plants, in particular for blocking the multiplication of viruses and the tumor genes, characterized in that

(a) counter-rotating viral RNA and / or viral or oncogene DNA with upstream, suitable promoters,

(b) Virus envelopes, suitable for the size of the antisense genome that is to be transferred into the cells, and suitable for the specific uptake of these virus envelopes into the desired target cells and

(c) enzymes or chemical compounds suitable for supporting the integration of opposing viral RNA and / or viral or oncogene DNA

are provided.

2. Medicament according to claim 1, characterized in that the medicament contains virus envelopes docking onto the receptors of the known stem cells.

3. Medicament according to claim 1, characterized in that the medicament contains virus envelopes docking onto the receptors of the normal εomatic cells.

4. Medicament according to claim 1, characterized in that the medicament contains virus envelopes docking onto the receptors of generative cells.

5. Medicament according to one of claims 1 to 4, characterized in that the medicament comprises cells taken from the organism and treated with antiviruses.

6. Medicament according to one of claims 1 to 4, characterized in that the medicament comprises cells removed from the organism and treated with the corresponding glycolyzed liposomes.

7. Medicament for gene therapy treatment, in particular for blocking the virus multiplication and the tumor genes, characterized in that

(a) counter-rotating viral RNA and / or viral or oncogene DNA with suitable promoters upstream, structured as transposable elements,

(b) Virus envelopes, suitable for the size of the Antiεenεe genome to be transferred into the cells, and suitable for the specific uptake of these virus envelopes into the desired target cells and

are provided.

8. Medicament for gene therapy treatment, in particular for blocking the virus multiplication and the tumor genes, characterized in that

(b) receptor-specific substances to which opposing viral RNA and / or viral or oncogene DNA are bound, which are to be transferred into the cells, and are suitable for the specific uptake into the desired target cells and

(c) to support the integration of opposing viral RNA and / or virus or oncogene DNA suitable enzymes or chemical compounds

are provided.

9. Medicament for gene therapy treatment, in particular for blocking the multiplication of viruses and the tumor genes, characterized in that

are provided.

10. Medicament according to one of claims 1 to 9, characterized in that the splice mechanism is bypassed in that the signal sequences responsible for it are recoded in the opposite gene.

11. Medicament according to one of claims 1 to 10, characterized in that in viruses which have due to their natural structure εelbεt splice sequences, the opposite strands of nucleic acid at corresponding, opposite, complementary locations also have such splice sequences.

12. Medicament according to one of claims 1 to 11, characterized in that counter-known DNA or RNA sequences are combined in such a way that polyselectivity towards a large number of viruses is achieved.

13 »Medicament according to one of claims 1 to 12, characterized in that a random incorporation of the opposing RNA-forming DNA in active gene structures of the host cells, that is, in exons, is prevented, so that these opposing modified strands are targeted only in non-functional, none Incorporate proteins encoding introns.

14. Medicament according to one of claims 1 to 13, characterized in that the sequence of the gene expressing the reverse transcripti for all retroviruses differs from the known sequence from the starting point of the normal transcription at the ends of each 150 base pair long interval by the exchange of base located there is distinguished by the corresponding complementary base.

15. Medicament according to one of claims 1 to 14, characterized in that the sequence of the DNA double helix of the gene expressing the reverse transcriptase gene for all retroviruses differs from the known sequence from the starting point of the normal transcription at the ends of every 150 base pairs long interval by exchanging the bases located there with the corresponding complementary base.

16. Medicament according to one of claims 1 to 15, characterized in that promoters are used which greatly enhance their binding capacity with respect to the RNA polymerase II by adding a chemical compound.

17. Medicament according to one of claims 1 to 16, characterized in that promoters are used which lose their ability to bind with respect to RNA polymerase II completely by adding a chemical compound.

18. Medicament according to one of claims 1 to 17, characterized characterized in that promoters are used which, by the action of temperature, strengthen their binding capacity with respect to polymer II.

19. Medicament according to one of claims 1 to 17, characterized in that promoters are used which lose their ability to bind with respect to polymer II due to the action of temperature.

20. Medicament according to one of claims 1 to 19, characterized in that eε comprises glycosylated liposomes as carriers for the opposing, modified genetic information and other necessary building blocks.

21. Medicament according to one of claims 1 to 20, characterized in that it consists of virus envelopes without genetic information.

22. A process for the production of the medicament according to one of claims 1 to 21, characterized by the process steps taking place in vitro (in a test tube):

(a) The starting point is a virus, against which resistance is to be made possible by the production of a gene therapy medicament.

(b) The virus envelope is broken down by changing the pH value, the ion concentration and by adding enzymes.

(c) The viral RNA is extracted.

(d) Reverse transcript is added to the single-stranded viral RNA (taking into account all other important components, such as pH, etc.).

(e) An RNA-DNA hybrid is formed which, after washing, is crosslinked with DNA polymerase. The latter replaces the RNA with DNA. (f) A DNA double helix is created which corresponds to the provirus genome of the retrovirus. This is still able to produce normal viral RNA. If DNA viruses are used, steps (d) ^* and (e) can be skipped.

(g) By adding suitable restriction enzymes, the virus genome is cut so that viral promoters can be cut out and artificial promoters can be integrated which ensure the transcription on the next strand and thus the production of the opposite RNA. The same procedure is followed with splice sequences, start and stop codons.

(h) At the end of the synthesis of the counter-rotating DNA, ligase, an enzyme that firmly combines newly combined DNA fragments, is added, as a result of which the DNA fragments assembled at the sticky ends are joined to form a stable double helix.

23. The method according to claim 22, characterized by duplication of the process steps taking place in vivo and in vitro:

(i) The DNA double helix is heated and thereby broken down into its strands.

(j) The strands are then allowed to hybridize with two excess chemically synthesized DNA oligonucleotides, each 15 to 20 nucleotides long and in sequence matching two sections which are separated by any number of nucleotides. The two oligonucleotides serve as specific primers for the in vitro synthesis of DNA, which is catalyzed by the DNA polymerase.

(k) After adding the DNA polymerase and the components important for DNA synthesis, e.g.

Triphosphate nucleotides, this enzyme copies the DNA between the sequences that correspond to the oligonucleotides. (1) After several of the process steps (i) to (k) inclusive, the desired DNA sequence, the counter-rotating DNA, is present in large quantities. The polymerase chain reaction (PCR) allows DNA sequences of any length to be multiplied millions of times.

24. Method according to one of claims 22 and 23, characterized by the method steps for integrating the opposing genetic information relating to the original viruses into the suitable virus protein envelopes:

(m) A virus whose protein envelope is suitable as a means of transport for the corresponding counter-rotating DNA is broken down into the elementary building blocks of the virus envelope and into the viral genetic material by changes in pH, ion concentration and temperature.

(n) The viral genetic material is destroyed and washed out by enzymes.

(o) The purified, viral envelope elemental proteins are incubated with the opposite strand of RNA or DNA.

(p) For the final completion of the medicament, the virus envelope is composed in a suitable environment around the opposite gene (so as assembly).

25. The method according to any one of claims 22 to 24, characterized in that anti-virus building blocks are introduced into the culture cells by means of mechanical mechanical pipetting.

26. The method according to any one of claims 22 to 25, characterized in that syncitium formations of the culture cells are carried out to facilitate pipetting.

27. The method according to any one of claims 22 to 26, characterized characterized in that antivirus components are introduced into the culture cells by means of a membrane opening in the electrical field.

28. The method according to any one of claims 22 to 27, characterized in that antivirus components are introduced into the culture cells by introducing membrane-fusing vesicles.

29. A method for producing a medicament according to one of claims 22 to 28, characterized by the method steps that

(a) to the opposite gene RNA polymerase II and the other components necessary for RNA synthesis are added. Mass production of reverse RNA occurs

(b) 1) under the conditions mentioned in (a) - however the viral gene is used - the mRNA to be inhibited is also generated in large quantities and radioactively labeled as an mRNA probe,

2) mRNA is also extracted from host cells and introduced into the process,

(c) the counter-rotating RNA produced in (a) is fixed after cleaning on nitrocellulose or nylon paper and chromatographed for a long time under hybridization conditions using the mRNA probe (b),

(d) the leaf is washed so that only hybrids labeled with the probe RNA remain, and

(e) the evaluation is carried out by autoradiography.

30. The method according to any one of claims 22 to 30, characterized in that cells have the necessary genetic information for Coding template-like molecules which are necessary for the construction of virus building blocks, with controllable promoters being placed in these subsequently cloned cells in front of these gene sections which are incorporated into introns.

31. The method according to any one of claims 22 to 30, characterized in that a cell sorter is used to distinguish and separate the antiviruses from the pathogenic viruses.

32. The method according to claim 31, characterized in that modified, counter-rotating nucleic acids are additionally modified such that they do not hybridize with the nucleic acids of the respective other virus types.

33. The method according to any one of claims 22 to 32, characterized in that the nucleic acids complementary to the respective other virus types are linked with fluorescence molecules before they are injected into the cells of the cell cultures, so that they can be selected out.

34. The method according to any one of claims 22 to 33, characterized in that a suitable, radioactive labeling of the opposite nucleic acid strand is used to distinguish the other viruses from the pathogenic viruses.

35. The method according to any one of claims 22 to 34, characterized in that the relatively strong antivirals marked radioactively are frozen for the duration of their radiation activity in order to preserve them.

36. The method according to any one of claims 22 to 35, characterized in that by adding suitable chemical substances, the virus envelope is carefully broken down into the elementary building blocks and into the viral genetic material.

37. Method according to one of claims 22 to 36, thereby characterized in that the virus envelope is gently broken down into the elementary building blocks and into the viral genetic material by changing the temperature.

38-. Method according to one of claims 22 to 37, characterized in that the antiviruses, which are in several

Vaccination steps should be administered due to. he

Host cell membrane fragments that surround the

Store virus envelopes around, are grown in cell cultures with different origins of the host, so that the trained antibodies against the foreign body

Host cell membrane fragments are not specified at further vaccination appointments in such a way that they are against non-organisms

Host cell membrane fragments trigger defense reactions, but only against virus envelopes.

39. The method according to any one of claims 22 to 38, characterized in that a random incorporation of the opposing RNA-forming DNA into active gene structures of the host cells, that is to say in exons, is prevented by homologous chromosomes.

40. The method according to any one of claims 22 to 39, characterized in that specified, opposite to the virus genome DNA in plant cells is preferably designed as a transposable element.

41. The method according to any one of claims 22 to 40, characterized in that, starting from process step (1), the counter-rotating DNA with RNA polymerase and other components important for transcription, such as e.g. Triphosphate nucleotides.

42. Medicament according to one of claims 1 to 21, characterized in that specified RNA which runs counter to the virus genome - stabilized by certain complexes - develops its action as a biocatalyst.

43. Medicament according to one of claims 1 to 21 and 42, characterized in that the fertilized egg cell (zygote) is mixed with specified DNA that runs counter to the virus genome.

44. Medicament according to one of claims 1 to 21 and 42 and

43, characterized in that the medicament is in liquid form.

45. Medicament according to one of claims 1 to 21 and 42 to

44, characterized in that the drug has a gelatin form.

46. Medicament according to one of claims 1 to 21 and 42 to

45, characterized in that the medicament is in tablet form.