GB2337462A

GB2337462A - Use of the rna chosen instead of a protein in production of bioactive substances or for specific immunity control

Info

Publication number: GB2337462A
Application number: GB9918974A
Authority: GB
Inventors: Virmantas Stunzenas
Original assignee: Individual
Current assignee: Individual
Priority date: 1997-02-28
Filing date: 1998-02-24
Publication date: 1999-11-24
Also published as: JP2001517215A; LT97027A; GB9918974D0; WO1998037900A1; LT4475B; DE19882018T1

Abstract

This invention is devoted to use of ribonucleic acids in medicinal substances including compositions, vaccines, transplants, intended for medical treatment, prophylactic treatment, cosmetic care; to injection of these substances into patient's organism. So it is possible to introduce ferments, hormones, antibiotics, proteins of cell receptors, structural proteins directly into cells of an organism. It is possible to reduce ribosomal and transport RNAs together with messenger RNA. When there is introduced RNA coding particular hormone or antibiotic, proteins of cell receptor or structural proteins, into recipient's organism, these substances will be synthesized directly in recipient's cells. Use of RNA instead of protein combinations gains an advantage over other analogues already by the fact that the RNA has no immunogenic properties and therefore causes no unwanted inflammatory reactions. RNA is used to supplement vaccine or another substance which is intended to activate patient's immunity against antigens existing in said substance, having removed from said substances or inactivated beforehand part or all the RNA taking part in synthesis of immunogenic substances against which it is wanted to activate patient's immunity.

Description

1 Use of the RNA chosen instead of a protein in production of bioactive

substances or for specific immunity control

Field of the Invention

This invention is devoted to use of ribonucleic acids in medicinal substances including compositions, vaccines, transplants, intended for medical treatment, 0 prophylactic treatment, cosmetic care; to injection of these substances into patient's organism.

Background of the Invention

The ribonucleic acid (hereinafter - RNA) is a monolithic non-branching biopolymer composed of ribonucleodites. The RNA directly takes part in synthesis of proteins and there are three types of RNA: ribosomal RNA, transport RNA and messenger RNA. The main of them is the messenger RNA (hereinafter - mRNA) which is synthesized on DNA-matrix and transfers all the information on sequence of amino acids in the protein to the place of synthesis of the protein where, based on this RNA, proteins are synthesized with help of ribosomal RNA and transport RNA (Lewin B. Genes 1983, New York; Saenger W. Principles of Nucleic Acid Structure 1984, New York, Berlin).

As one of the most evident analogues it is possible to point at the use of ready.

proteins in medicinal substances. Proteins and their combinations are among the most important structural and functional elements of an organism. The combinations of proteins (hormones, immunoglobulins, enzymes and antibiotics) are widely used in treatment, cosmetic care.These combinations are simply injected into patient's body. Nevertheless, even small protcin molecules having molecular weight equal to 5000 or even less cause immunity reaction and production of 0 "10 antibodies (Frimel IL, Brok I. Basics of Immunology 1986, 'i\,foscow). Namely, larger protein combinations take part in controlling immunity (immunt),'1()btll ins. interferons, int(;t.lt:ukins. their receptors) ot. almost in all most physiologic reactions of an organism (Freund M., Link 1 [., Schmidt R.L... Welte K. Cytokines in Hemopoicsis, Oncology and AIDS 11 1992, Berlin). Therefore the use 17 of many protein combinations in production of medicinal substances is rather complicated or impossible at all because of high immunogenicity of protein combinations or their instability after extraction from the cell.

Specific suppression of immunity can he achieved by use of solutions of proteins for which it is needed to create the, recipient's immunity tolerance (Vidard L., Colorusso L.J. & Benacerraf B. Specific T-cell Tolerance May Be Preceded by a Primary Response. Immunology. 1994, p5627-5631; Buclow R. , BurlingharnW.J. & Clayberger C..Immunomodulation by Soluble FIT.A Class 1. Transplantation. 1995, p049-654; Ziegler-Heitbrock H.W.L. Molecular Mechanism in Tolerance to Lipopolysaccharide. Journal of Inflammation. 1995, p13-26). When using this method, there emerge several difficulties: it is problematic to extract pure proteins and to obtain homogeneous solutions, there is very large variety of optimal conditions for stability of solutions of different proteins, tolerogenic and immunogenic concentrations of solutions of different proteins are different and dependable on many factors.

Development of cancer cells can he retarded by using various methods (Jackevidius A. Treatment of Malignant Tumours 1984, Vilnius). The most widely used are methods destroying or inactivating nucleic acids, or retarding their synthesis. Among analogues of this invention there shall be reckoned antibiotics used (Gauze G.F., Dudnik LB. Anti-tumour Antibiotics 1987, Moscow VS1) which retard synthesis of DNA, RNA, and proteins in patient's both cancer and other cells.

There are known various production methods of immunogenic substances (including va,cines) when there are used edged or killed pathogens, their proteins, artificial antigens (Cease K.B. & Berzof.sky LA. Toward a Vaccine for AIDS: the Emergence of Immunobiology Based Vaccine Development. Annual Review of Immunolon, 1994, p923-989; Huldrich H.G. Parasites and Vaccination.

Cly Parasitology in Focus. 1988. p719-731 1: Wynn T.A., Jankovic D., 1 lieny S.. Cheever XW. & Sher X, IL-12 Enhances Vaccine-induced Immunity to klansoni In Mice and Decreases T Helper 2 Cytokine Expression, 1,,E Production 1 C and Tissue Eosinophilia. 'llic Journal of Immunology. 1995, p4701-4709).

3 Nevertheless, there are facts known when parasites use their RNA to escape host's immunity (Tato P., Castro AM, Rodriguez D., Soto R., Arechavaleta F. & Molinari LL. Suppression of Murine Lym, lhocite Proliferation Induced by a Small RNA Purified from the Tacnia Sollurn Metacestode. Parasitology Research. 1995, p181187). Although in some cases when producing antivirus vaccines, viruses are inactivated by irradiating them with UV, immunogenicity of cancer cells can be increased by means of X-rays or radio-active irradiation as well; it isn't known, nevertheless, that there were any attempts to increase immunogenicity of these substances when purposefully removing RNAs from these pharmaceutical compositions and later using the RNAs chosen.

As the, closest analogue, it is possible to indicate the Patent Application published by Rabbani Elazar (June 15, 1994, EP 0 601585 A2). There is a proposal in said invention to control immunity by injecting DNAs which code proteins controlling immunological activity of the cell, into the cell. Theoretically, it is dangerous (the process is irreversible when DNA is inserted into genome), complicated and unreliable (rather low probability to have activated DNA introduced) method.

Summary of the Invention

The proposal to use RNAs chosen instead of proteins in production of medicinal substances is based on the fact the foreign RNA is capable to cross cell's membrane and to function as protein matrix inside the cell. So it is possible to introduce ferments, hormones, antibiotics, proteins of cell's receptors, structural proteins directly into cells of an organism. It is possible to introduce ribosomal and transport RNAs together with messenger RMs.. When there is introduced RNA cooling particular hormone or antibiotic, proteins of cell's receptors or structural proteins, into recipient's organism.. these substances will he synthesized directly in recipient's culls. Use of RNA instend of protein combinations gains an advantage over other analkinues aiready hy the I'act ihe RNA has no Immunogenic pt. ()pcrtlcs and therefore causes no unwanted inilammatory reactions. When cornparin., to use of DNA in medicinal substancus, use of rriRNA gains an advantage by the ['act the 4 amount of mRNA in an ornanism can be controlled easily because mRNA doesn't make replicas of its own in absence of DNA. Additionally, there can be synthesized a protein immediately on mRNA introduced into the cell and this is impossible in a case of DNA. This property makes mRNA the more reliable regulator of amount of protein in pa: ienCs organism, when comparing to DNA.

The concentration of PLINA introduced may he chosen in very wide range:

0,000M001 mg/ml to 5 mg/ml. The concentration of RNA depends on RNA used, problems being solved, permeability of membranes of cells, activity of ritionueleases, and other factors. When using this method, there are opened new possibilities in production of bioactive substances, in search for and assimilation of new medicines: there is no need any more to extract and purify bioactive proteins, it is enough to use known and tested methods of RNA extraction, it is possible to match various bioactive substances which qn exist and function in a normal way only inside a cell. When wishing to use already known substances in this way, in many cases there is no need to change in essence the technolog of their 0 oy production, it is enough to use the existing one extracting RNA instead of protein combinationy.

It is needed to emphasize, nevertheless, than in a case when RNA extracted isn't "adenylated", this shall he done with purpose to increase its stability and to make it suitable for translation in eukaryotic cells. In some cases there can emerge the need to introduce mRNA protein together with mRNA coding the desired protein; without such help it wouldn't he possible to obtain optimal translation, or the protein wouldn't be active sufficiently.

Not always it is needed to use RNAs of well-known substances. It is possible to 2 5 use RNA extracted from particular cells activated accordingly. For example, total RNA extracted from eukaryotic cells fissioning in culture can he used to stimulate regenerative processes in patient's damaged organs: RNA introduced will stimulate protein synthesis in cells or damaged organ and this will induce more intensivc:

ission of these cells and synthesis o[ mediators. When wishing to ensurc ol f-ull-value cells, nevertheless, it is needed also to use RNAs characteristic t() organ or tissue damaged. I suppose RNAs can he used also to correct disorders of 0 1 development an embryo introducing RNAs coding proteins inducing or retarding interaction, fission and specialization of cells, into organ of an embryo or its germ chosen.

When treating sickness caused by cancer, viruses, parasites or other pathogens, It is important to limit translation of RNAs synthesized by cancer cells, in immunity cells of an organism. This can be, done by introducing RNA which doesn't code protein combinations of pathogen cells or parasite organisms, into patient's organism or directly into organ damaged. The best way is to use RNA extracted from activated immunity cells, or mRNAs coding receptors of immunity cells or protein combinations activating these cells. It is possible also to use any other mRNA which after combination with ribosomes would he able to compete with mRNAs of pathogens and to retard translation of these mRNAs.

Most of anti-cancer preparations and treatment methods reduce synthesis of RNA and its total amount in the whole or-anism. It is expedient to supplement these preparations and treatment methods with use of RNA preparations. By means of introduction of niRNA it is possible not only to inhibit synthesis of mRNA of cancer cells in immunity cells, but also to restore synthesis of protein combi.nations in these cells havina chosen mRNA correspondingly. The other side of this method is, there will be synthesized protein combinations not suitable for needs of cancer cells and this will diminish malignancy of these cells.

The method of induction of immunological tolerance proposed in this invention is based on the principle, immunity cells don't stimulate immunity reactions against these protein combinations which are synthesized by them. This method stipulates that prior to introduction of protein combinations into patientys organism or transplantation of tissue and organs, recipients immunity cells shall he incubated together with RNAs which are used in synthesis of protein combinations having analogous irr,mtinoiycnic properties as substances being introduced or tissue and organs being transplanted. Immunity cells can he incubated together with IZNA in

0 0 LW() ways: 1) to [nil-oduc,.; rNi\.s directiv into patient's onlanism (it i's 1(1 lo introduce into vein or in[o the place of planned surgical operation. or 1k) use ',tn other method known); 2) to mix cells extracted (e.g. from patient's hlood) with 6 RNA.s and then re-introduce or introduce (when these cells are taken from a donor) into patient's organism. It is possible also to extract from patient's organism only one group of immunity cells, e.ty. macrophages (they are the best absorbers of foreign substances and are the most important ones in primary immunity reaction) or any other group of cells depending on problems being solved. In this way it is possible to introduce RNAs into patient's organism not only to suppress immunity reactions, but to cause synthesis of desired proteins or their combinations as well.

When transplanting tissue, organs, there shall be used RNA coding proteins of tissue-compacibility complex, corresponding to donor's proteins and differing from recipient's ones. It is possible to produce or extract RNA coding separate proteins of tissue-compatibility complex, the needed combinations of which would be used to suppress activation of recipients immunity cells against donor's tissue beina transplanted. It would be possible to do it in simpler way, using RNA extracted from donor's tissue (e.g. blood or blood cells). Not always it is needed to use RNA coding proteins of tissue-compatibility complex: having determined in vitro what the protein combinations cause the development of immunogenic reactions, there is chosen the RNA corresponding to these proteins and intended to suppress recipient's immunity reactions against the transplant or transplant's reactions against the recipient.

When transplanting tissue, and organs from one organism into another, there often show up complications because of activity of donor's immunity cells against recipient's organism. These complications can be escaped or at least reduced by using compositions with RNA coding proteins corresponding to recipient's antigens.

These compositions prior to transplantation are incubated together with tissue, organs, cells being transplanted. It is possible also to introduce into recipient's orivanism donor's cells together with RNAs coding proteins having properties of recipienCs antigens.

This method of induction oftolerance can be used not only [or transplantation or introduction of antigenic substances. When it ih needed to treat birrenii.:,,s and complications of pregnancy which arise because of Insufficient immuno- suppressive activity of placenta, it is possible to use in pharmaceutical compositions intended to 7 suppress immunity reactions against embryo's antigens, RNAs coding proteins having analogous antigenic properties in respect of embryo's proteins causing complications. When the cause of barrenness is immunization against antigens of outer layer of an ovule, it is possible to treat with pharmaceutical compositions having RNA.s coding antigens of outer layer of the ovule. In latter two cases it is expected the RNA will be translated in immunity cells which will diminish or inhibit immunity reactions against the embryo.

The process of auto- immuno loo ical sickness also can be controlled by means of pharmaceutical compositions in which RNA is used. Having determined which protein combinations cause production of immunoglobulins, it is needed to treat with RNA codint, these immuno-ens and makin- basis to synthesize in immunity cells substances which have antigenic properties analogous to antigenic properties of patient's tissue which are the target of these auto-immunity reactions. Since autoimmunological sickness very often is caused by infectious micro-organisms, such treatment would allow to remove agents of sickness in process of treatment without suppressing general immunity of an organism.

Not always it is needed to use RNA separated from the protein. Having in mind the solution of a protein can also be a tolerogen, in some cases better results can he achieved by using RNA together with a solution of the protein coded b this RNA. y When storing and prior to introduction of RNA, it would be desirable to increase stability of this RNA. This is easy to achieve when using RNA of- --enome of plant virus. The RNA extracted becomes several times more active when mixed with RNA of genome of plant virus. As far as 1 know the most suitable for this is the genome of vzna mosaic virus (Monk M. Mammalian Development. A Practical

Approach. 1987, Oxford). Even more attractive is the use of recombinant RNAviruses with inserted required RNA-sequence into their genome.

Prior to introduction of foreign RNA it is desirable to have minimal ainount of recipient's RNAs in patient's cells. It is easy to achieve when usinle inedicint:

ihiotics also 'vvill do) terriporary inhibiling synthesis of RNAs. It should be done (ailli 1 1 - 1 1 1 0 Y In all cases w:icn there is required maximal productivity of RNA introdticL(A.

Having introduced RNA into patient's organism, there is a large probability to have this RNA destroyed by ribonueleases before this RNA reaches the place of translation. 'I'llerefore it would reasonable to introduce inhibitors of ribonueleases together with RNA. Among inhibitors the most suitable one would be the inhibitor extracted from placenta. But this inhibitor can be immunogenic one, so it would he needed to use it together with coding RNA, or when recipient's immunity is suppressed (c... after radiotherapy).

When wishing to use RNAs in bioactive compositions, not always it is possible to separate easily one protein-coding RNA from others, the RNA of one type from other one. Of course, it isn't needed in all cases. For example, when there are produced pharmaceutical compositions (to suppress transplant- rtJectio n reactions) with mRNAs coding proteins of corresponding tissue- compatibility complex, there is no need to get rid of all other RNAs; they can he useful to restore regeneration functions of cells especially when antibiotics suppressing synthesis of RNAS are in Use.

The proposed use of mRNA for production of immunogenic substances (including vaccines) is based on assumption, RNA existing in edged or killed pathogenic organisms can stimulate recipient's tolerance in abovedescribed way, and greater variety of immunogenic substances (not separate proteins or their combinations) can create more stable immunity against this pathogen. 'I'llis method of increasing immunogenicity can be used Lit first in cases when inactivated pathogens are used as immunogenic substances. RNA of a pathogen can be destroyed by means of UV-rays, X-rays, radio-active irradiation, or chemical substances having propel-ties of ribonucleases. The best results would be obtained when using combinations of methods of RNAs' inactivation. The final product shall be checked against some known and suitable for particular circumstances method determinina remainder of RNA-s. Later vaccine can be supplemented with mRNA taken from non- pathogen. The most appropriate way would be to supplement vaccine with mRNAs c()dint, protelns taking part in interaction of Immuntiy cell.s. 11 is desirable to have mRNA coding immunonlobulins which would tie antiocris vaccine, not these of a patient. The first question arisen when there is a will to use 9 RNAs directly in production of bioactive compositions, bioactive substances is as following: where to take the needed amount of RNA. Depending on problem being solved, RNA can he extracted from donor's blood, blood cells, corresponding organs, animal's tissue, or any other living organisms used in b io technological industry as well. Contemporary biotechnology allows to cultivate microorganisms, viruses with DNA or RNA (they can he copied from RNA chosen) inserted into their genome, and these micro-organisms and viruses would constitute the basis for synthesis of RNA in plants, algae or cultures of microorganisms.

0 Not always desired results can be achieved when using only abovedescribed methods. Often it is needed to combine various methods known. The methods proposed in his description may he used as primary ones, and as secondary ones additionally to other methods of treatment and therapy. In all cases there shall be taken into account particular situation and compatibility of methods in time and space.

Description of the Invention Implementation

RNA used in all experiments was extracted from liver taken from mice done away with ether. RNA was extracted using phenol-method described in the book: Monk M. Mammalian Development. A Practical Approach. 1987, Oxford.

Suppression of Cancer-cell Development There were carried out experiments with mice of CC57W line and with cancer cells of Erlilich. For each mouse there was injected under skin on shoulder-blade the amount of 0.2 m] of cancer-cell suspension taken from peritoneal cavitY of one 2.5 mouse. 5, 19 and 24 days later next to cancer tumour for each mouse there was in c) jected the amount of 0.2 mI of solution having concentration of 0.03 mg/ml: RNA extracted from liver taken from the mice of CC57W or DBA line, in physiologic solution. In time of cheek test, for each mouse there was injected the amount or 0.2 nil of physiolooic solution. On the 30-th day after injection ol'cancer all the lo mice were done away with ether. After autopsy cancer tumours were. reniove d and wei,,hted. When comparing weight of cancer tumours taken from the mice which c 0 0 have received RNAs of different mice, there were found no ditTerences. When comparing weight of cancer tumours taken from the mice which have received RNA and physiologic solution, there were found reliable differences. In the mice which have received RNA there was reliably retarded development of cancer tumour (Table 1).

Experiments on Transplantation There were carried out experiments when transplanting patches of skin from a mouse belonging to one line onto a mouse belonging to other line. There were used mice of DBA and BALB lines, their RNAs were used for induction of tolerance in a mouse of other line. For checking purposes there was used RNA extracted from the mice of CBA line. One hour beforL transplantation, for each mouse there was injected the amount of 0.3 mI of RNA solution in physiologic solution; concentration 0.02 mg/ml. Then there was transplanted 1 cm2 of skin taken from the tail of one mouse onto the back of other mouse having removed beforehand the patch of skin of appropriate size. All surgical operations were performed with mice put to sleep with ether. There was compared development of rejection reaction: several days later there started inflammation of the transplant; when the whole transplant was seized with this inflammation; when the whole transplant became dead (black and hard). The, results obtained are presented in Tables 2 and 3. As it is seen, with high reliability it is possible to state RNA retards reactions of transplant r(jection: up to 4 days for DBA line when comparing to check group (death of transplant). Reaction of transplant rejection for BALB line started 3) days later than that in check group.

Table 1 Retardation of development of transplanted cancer cells in mice of CC.57W line RN.A injections Injections of h siologic solution Number of tests 10 5 Weight of m.lignant tumour, g 4.02---).80 5.24 1.12 Importance level Student's t-criterion 0.048 used for comparison Table 2

Rejection of transplant from a mouse of DBA line in a mouse of BALB line Stage of transplant Number of days after transplantation Importance rejection level RNA i jected from CBA from DBA Start of 7.8--K).5 10.8 0.8 0.000025 inflammation Total inflammation 1 1.0 Q0 13.2=0.4 0.000001 Dead 13.4--,-().6 15.0 0.0 0.000049 transplantations made 6 transplantations made Compared using Student's t- criterion Table 3 Rejection of transplant from a mouse of BALB line in a mouse of DBA line Stage of transplant Number of days after transplantation Importance rejection level RNA i jected from C13A from BALB Start of 9.7-R).8 13.4_+0.9 0.00049 inflammation Total inflammation 11.0 1.6 14.6 1.7 0.0049 Dead 14.3 12.9 1. o -±). 8 0 tr,.nsplitntzitions made 5 transplanta Lions made Compared using Student's t-criterion 12

Claims

Claims:

1. Use of the RNA chosen instead of a protein in production of bioactive substances or for specific immunity control.

2. Use of RNA according to claim 1, where there is used in medicinal compositions the RNA coding protein required for treatment or cosmetic care.

3. Use of RNA according to claim 1, where with purpose to suppress organism's immunity reactions against proteins or their compositions or transplanted tissue and organs, there is used the RNA which stimulates synthesis of substance having antigenic properties analogous to properties of substances used.

4. Use of RNA according to claim 1, where with purpose to diminish reactions of immunity cells of the transplant against recipient's organism, there is used the RNA coding the antigen which in its properties corresponds to recipient's antigen.

is

5. Use of RNA according to any one of claims 2 to 4, where said RNA is used together or alternately with the protein synthesized on said RNA, or with the combination of said protein.

6. Use or RNA according to claim 1, where said RNA used when competing with the RNA of pathogen or cancer cells, diminishes synthesis of combinations of proteins coded by said RNA of pathogen or cancer cells, in immunity and other cells.

7. Use of RNA according to claims 1 or 6, where said RNA is used to restore or replace synthesis of protein combinations after radio- or chemotherapy.

8. Use of RNA accordin- to any one of claims 2 to 7, where with purpose to increase stability of RNA, said RNA is used together or alternatively with inhibitor of rihonueletse, or is incubated together with virus or with transport RNA or r'bosomal RNA from virus o'enome.

1 0

9. Use of RNA accordino to any one of claims 2- to 7, where said RNA is used 0 t()(luther or alternalively with substance which suppress synthesis of RNAs.

10. Use of RNA according to claim 1, where said RNA is used to supplement vaccine or another substance which is intended to activate patienCs immunity 13 against antigens existing in said substance, having removed from said substances or inactivated beforehand part or all the RNA taking part in synthesis of immunogenic substances against which it is wanted to activate patienCs immunity.