CA1192152A - Highly-purified informatory ribonucleic acid (i-rna), a process of producing same and the use thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

HIGHLY-PURIFIED INFORMATORY RIBONUCLEIC ACID
(i-RNA), A PROCESS OF PRODUCING SAME
AND THE USE THEREOF

Highly-purified i-RNA (Informatory Ribo-nucleic Acid) is synthesized in a cell-free system and purified via a density gradient. It may paren-terally, particularly intravenously, be applied to human beings and animals, either directly or in com-bination with other compatible substances, for a direct therapy, active immunization or the recovery of anti-sera in vivo or in vitro.

Description

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I-IIGHLY-PURIFIED INFO~TORY RIBONUCL~IC ACID
(i-RNA), A PROCESS OF PRODUCING SA~E
AND THE USE THEREOF

The present invention relates to an i-RNA
(denoted as "informatory", and previously also as "immune" ribonucleic acid) which is specific towards antigens and is obtained in a cell-free system, and which has so highly been purified that it is toler-ahle for human beings and animals when parenterally administered, with an intravenous application being preferred. The invention further relates to a pro-cess of producing thus highly purified i-RNA and the use thereof.

Normally, i-RNA is formed by various immune-competent body cells upon contact with an antigen.
It contains, and transfers, the informations ~or the systems of antib~dies, regulator protein, and cell-bound~immunity. That is, i-RNA iS always found in the peripheral blood and in lymphatic organs, when the macroorganism contends with -an antigen. A multi-plicity o~ different types of i-RNA is transferred by each blood transfusion, while the antigens react-ing with the antibodies having respectively been synthesized are unknown. The concentrations of the individual i-RNA species, however, are ~ low for detection.

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It is, therefore, required, in order to re-cover a definite i-RNA, to e~perimentally separate the reco~nition of an antigen from the synthesis of an antibody. Said procedure will be most successful-ly carried out in cell-free biosynthetic systems which are capable either of only recognizing the selected antigen or of only synthesizing antibodies.

There has been known from the work of Ja-chertz et al that in antigen-recognition in a cell-free system the first product to be isolated is i-RNA (cf. Jachertz, Zeitschrift fur medizinische Mikrobiologie und Immunologie 154, 245 ~1968); An-nals of the New York Academy of Science 207, 122 (1973); Zeitschrift fur Immunitatsforschung und ex-perimentelle Therapie 144, 260 (1972); and Journal of Immunogenetics 1, 355-362 (1974)). Yet, the pre-parations described by Jachertz of the i-RNA have been considerably contaminated and have, therefore, not been suitable for an application to human beings. An improvement of the purification procedu res was published by Jachertz in Molecular and Cel-lular Biochemistry 24, 93 (1979). However, the expe-rimental section of said publication, as well as of the other papers, was so incomplete that repeated attempts to reproduce his findings failed and the results in total were taken for being dubious. This resulted in that neither the results nor the inter-pretations of said results have been accepted by the art.

Attempts have also been made to prepare i-RNA in vivo in order to subsequ~ntly recover it _ 5;~

~ 3 --from the spleen ancl the lymph nodes of an animal (cf. Liu Shi-Shan et al, "The Lancet", Jan. 23, 19~2, p. 197). However, said attempts did not suc-ceed in a complete purification either, apart from the expensive mode of production (horses were used which, in each case, had to be killed).

It is the object o~ the present invention to provide a highly~purified i-RNA, which is specific towards antigens and is tolerable for human beings when administered, more particularly by way of in-jection, by producing said i-RNA in a cell-free sy-stem. Quite surprisingly, this problem has success-fully been solved, and the i-RNA thus produced is compatible with parenteral, more particularly intra-venous, applications to human beings and animals, with warm-blooded animals and mammals being prefer-red.

The process of producing a highly-purified i-RNA, which is specific towards antigen and is to-lerable for human beings when parenterally, more especially intravenously, administered, and which has been obtained in a cell-free system, by way of a reaction of antigen, DNA (including i-DNA~, nucleo-tides, and an enzyme system having been recovered from leucocytes, lymphocytes, other cells or cell components, extraction with an aqueous solution of phenol and sodium dodecylsulphate, and further puri-fication, is characterized in that purification is achieved by density gradient centrifugation, more preferably centrifugation with about 130,000 g em-ploying a density gradient of cesium chloride, 5~

saccharose and l'ris buffer. Thls centrifugation will take about four hours. It is preferred to subse-quently carry out another centrifugation step using cesium sulphate. More preferably, an enzyme system is used which has been recovered from the leucocytes or lymphocytes of a spleen homogenate by means of density gradient centrifugation. Employing a density gradient of saccharose in Tris buffer with a saccha-rose content of from 30 to 60 per cent at about 100,000 g has proven to be particularly suitable therefor. It will be beneficial to previously stimu-late the immunosystem of the animal, the spleen ho~
mogenate of which is to be used for recovering the cell-free system. Said stimulation may be effected in a conventional manner by the use of Freund's ad-juvant, BCG, and, more particularly, Pind-Avi (ra-diation-inactivated chicken pox viruses).

Another procedure for a mild purification is affinity chromatography. For example, poly(U)sepha-rose 4B or oligo-desoxy-thymidine may be used as the matrix.

The highly-purified i-RNA, which is specific towards antigen and is tolerable for human beings and animals when parenterally, more especially in-travenously, administered, and which has been ob-tained in a cell-free system, according to the in-vention, is indeed suitable to be directly used in therapy~ It actually effects the syntheses of anti-bodies, regulator protein and cell-bound immunity.
It may further be used to generate an active immuni-zation and to produce anti-sera and antibodies in ~9~1L52 vivo ancl ln vitro. The anti-sera and antibodies having thus been obtained may, in turn, be used to generate a specific passive immunization. Besides, they may be used for analytical purposes, since they are highly specific towards the respective antigens.
Thus, the i-RNA according to the invention i9 a key substance, which enables various problems to be sol-ved or the solutions to which to be improved in an extraordinarily simple and elegant way and without after-effects. It may, of course, also be applied together with other compatible substances, e.g. as an additive to transfusions of full blood, preserved full blood, plasma, plasma fractions, plasma expan-ders etc..

Typical antigens are, for example, bacteria, viruses, viruids, fungi, tumors, and parasites. How-ever, they may also be higher molecular weight che-mical substances such as steroids, plant poisons and animal venoms, as well as allergens. Radiation-inac-tivated viruses are especially beneficial, as they ceased to be pathogenic while retaining their anti-genicity.

There has been found that the i-RNA accord-ing to the invention is also capable of synthesizing specific antibodies in a cell free system. It is a finding of major importance that the i-RNA induces the synthesis of a regulator protein in cells, which regulator protein induces the new synthesis of cell-autogenous i-RNA in the receptor cell. The injected i-RNA and the newly formed i-RNA are, in turn, cap-able of inducing the formation of antibodies. Thus, ~2~52 the i-~N~ is excellently suitable for a parenteral, more particularly an intravenous, application for the purpose of a specific therapy, in order to the-reby rapidly and purposefully mobilize the body-in-herent defense abilities. In so far as the body does not yet contain the antigen, an active immunization occurs which proceeds faster and with less compli-cations than does the conventional vaccinatlon with antigen. Since considerable quantities of antibodies will as well be formed some time after the injection of the i-RNA according to the invention, at some later time the serum may be used for the passive immunization or the recovery of antibodies. Said sera and antibodies are, of course, excellently suitable for special diagnostics and the recognition of antigens.

The production according to the invention of the i-RNA does not involve any manipulations of the genetic material in the meaning of a recombination.
Notwithstanding the synthesis in cell-free systems, the i-RNA according to the invention is understood to be a regular component of the peripheral blood.
The criter~a of acceptability of an application of i~RNA for the treatment of human beings may, there-fore, rest on the criteria for-and the experiences with blood transfusions. Consistently, those inject-ions of i-RNA having so far been administered to human beings have not shown any of the slightest side-effects in all of the cases. On the other hand, the biological activities as desired were recorded in approximately all of the cases. This allows for the conclusion that the i-RNA, which has been formed in a cell~free system of immune-competent upon sti-mulation with antigen, actually contains the -total information required for the synthesis of antibodies and for the occurence of cell-bound immunity. Fur-thermore, the i-RNA is free from antigens and anti-gen-fragments and other impurities such as proteins, and information contents different from DNA and RNA, which may, for example, be verified by means of agarose-gel-electrophoresis and thin layer chromato-graphy.

For the purpose of direct therapy, it is generally sufficient to administer the highly-puri-fied i-RNA according to the invention in the form of one single injection. It only appeared to be indi-cated in a few cases to repeat the injection about 10 days later.

The present invention is further illustrated by way of the following examples.

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EXAMPI,E 1 a) Preparation of the Enzyme System Leucocytes or lymphocytes may be used for the preparation of the appropriate enzyme system.
The preparation from spleen is particularly easy. In this procedure, 1 to 2 g deep-frozen fresh spleen (e.g. pig spleen~ are crushed in a mortar with dry ice and liquid nitrogen. The powder is added to 1.5 ml of Tris buffer A in a centrifuge tube, and the suspension is froæen at -70C. This procedure is repeated for about 10 times. Then, the macerated cells are centrifuged in the rotor SW 50.1 of a Beckman ultracenti~uge with 30,000 g for 30 min.
Subsequent to the centrifugation, 1 to 1.5 ml of the red clear supernatant is taken and put on a discon-tinuous saccharose gradient. Said gradient comprises 1 ml of a 60 per cent solution of saccharose in Tris buffer A, above which 2.75 ml of a 30 per cent sac-charose solution in Tris buffer A are presentO Cen-trifugation is effected in a Beckman centrifuge, rotor SW 50.1, at 0C with 100,000 g. After 4 hours, the desired fraction is obtained as a reddish clear layer on the top of the 30 per cent saccharose. The layer is withdrawn by means of a pre-cooled pipette and diluted with chilled Tris buffer A in the ratio of 1 : 1. The solution may be stored in liquid ni-trogen.

b) DNA (Deoxyribonucleic Acid) including 1-DNA (In-formatory Deoxyribonucleic Acid) 1 to 2 g deep-frozen spleen are slowly thaw-ed in 5 ml of Tris buffer A and homogenized in a homogenizer by means of about 30 piston cycles. The suspension is dropwise added to 20 ml of an emulsion of a 2 per cent aqueous solution of sodium dodecyl-sulphate with freshly distilled phenol (ratio 1 : 1) contained in an Erlenmeyer flask, while the flask is vigorously swirled in order to obtain a homogeneous distribution. If the mixture is too viscous, a 3 per cent saline solution may be added. After 24 hours, the opaque milky emulsion is centrifuged at 2,000 g for 15 min, in which step two phases are separated.
To the upper aqueous phase there is added the same volume (+ 10 %) of anhydrous ethanol, so that the DNA is precipitated. The DNA is transferred into 10 ml of a sterile 0.85 per cent sodium chloride solut-ion by means of a thoroughly glowed platinum loop.
5 ml of a 1 per cent sodium dodecylsulphate solut-ion, 5 ml of a 0.05 mole/l Tris A and 0.05 mole/l EDTA buffer of a pH 7.6, and 2 mg proteinase K are added and admixed. Dissolution of the DNA will take about one day, after which it may again be precipi-tated with ethanol. The steps of dissolution and subsequent precipitation are repeated from three to seven times, until the ratio of E260 to E280 has reached a value between 1.9 and 2.0 and the hyper-chromic effect amounts to an increase in the ex-tinction at 260 nm of at least 20 %. A DNS absorp-tion at 260 nm of 1 o.d. corresponds to a concentra-~9~

tion of about 40 ~g/ml. By use of that relation, the DNA solution is diluted so that a concentration of 1 ~g/ml is obtained. This ready-to use solution is frozen in portions of 0.5 ml each and is stored at -20C.

c) Synthesis of i-RNA (Informatory Ribonucleic Acid) The purified enzyme system and the antigen are added to Tris buffer A at a pH 8~0 in an ice bath, and a mixture of the nucleotides ATP, GTP, CTP, and UTP is added. A smaller batch to which tritium-labelled UTP has been added is prepared for control. Pipetting is always carried out by using chilled pipettes. The optimum concentrations of the individual components are determined by several tests. Each sample is incubated at 37C in a water bath, and an a~ueous solution of 1 per cent sodium dodecylsulphate and 1 per cent aqueous phenol is added after only from 3 to 6 minutes. The ~ op~
period of incubation will depend to some extent on the concentration and the respective antigen and may be previously determined by means of some offhand tests. Cooling is effected in an ice bath, and cen-trifugation is carried out with 2,000 g. The super-natant contains the synthesized ~crude i-RNA.

d) Purification of the i-RNA

The i-RNA is isolated from the supernatant via a discontinuous cesium chloride/saccharose gra-dient. To this end, 1 ml of a cesium chloride solut-ion having a density of 1.9 g/ml is covered with a

2~2 layer of 0.75 ml of a 30 per cent saccharose solut-ion in Tris buffer A having a pH 8.0 in each centri-fuge tube. 2 ml of the samples are placed on the gradients, respectively, and the tubes are sealed wi.th sterile paraffin. After a centrifuge run at 18~C in a Beckman Rotor SW 50.1 with 130,000 g for 4 hours, the cesium chloride layer is withdrawn.
This is done by piercing the bottom of the respect-ive centrifuge tube with a thoroughly glowed needle and transferring the total cesium chloride solution up to the saccharose layer into tubes. The layer thus obtained is diluted 1 : 2 with sterile water and may then be fro~ed and stored at -20C. Further purification is effected by sedimenting over a ce-sium sulphate gradient. Therefor, 4O8 ml of a cesium sulphate solution having a density of 1O5 g/ml are chaxged into centrifuge tubes made of cellulose ace-tate (Roto.r Beckman SW 50.1). 0.3 ml of the sample are placed as a layer on the top thereof, and then centrifugation is carried out at 18C with 130,000 g for at least 4 hours (preferably from 12 to 19 hours). After the run, the tubes are pierced through the bottom, and the contents is cut into 13 to 14 fractions, each of which comprises 9 drops (= about 0.4 ml). 0.1 ml of each fraction is used for the determination of radioactivity, while the remaining 0.3 ml is diluted with the same volume of water and stored at -20C. The fractions exhibiting the high-est radioactivity having been determined, the corre~
sponding fractions of the main batch are selected, which fractions co~tain the highly-purified i-RNA.

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The fractions may immediately be injected without further purification steps, if desired.

It is possible to detect the i-RNA by elec-tron optics, whereby molecular lengths of between 1,500 and 2,000 nm were observed. The degree of pu-rification may be determined, for example, by way of agarose gel electrophoresis.

Another method of identifying the i-RNA com-prises synthesizing antibodies in a cell-free sy-stem. This is achieved by mixing together a sample of the i-RNA, highly-purified ribosomes, the above-mentioned enzyme system, leucine, and valine in Tris buffer A pH ~Ø The mixtures are incubated at 7C
for 45 seconds, and then the protein synthesis is stopped b~ freezing at -20C. The antibodies may be separated and purified by affinity chromatography on immunoadsorption columns and be detected analytical-ly .

The highest concentrations of antibodies are obtained upon addition of only 10 4 i-RNA. This finding shows that one molecule of i-RNA is capable of synthesizing at least 104 molecules of antibody in a cell-free system.

It is further possible by using a RNA repli-case, to increase the amount of i-RNA by a factor of to 10 . Repetition of said steps for several times results in a clonization. As the i-RNA thus obtained has retained its properties relative to antibody synthesis, there can be excluded that the ~2~L5~

antibodies have been synthesized by remainders of antigens that might have been carxied throuyh.

Analysis of the i-RNA resulted in that the molecular weight thereof is about 1 to 2 x 106. As the antigens, there were used Aujeszky virus, ~erpes simplex virus, influenza virus, MSK virus, Varicella virus, measles virus, HBs antigen, E. coli O-anti-gen, sheep erythrocytes, ADH, tetanus toxin, and systems with tumor cells such as LC2 of guinea-pigs, p815 of DBA2 mice, and the polioma virus-induced tumor of Lewis rats. First signs of success were also observed with attenuated measles virus "Mora-ten" and HBS antigen. Respective tests revealed that no cross reactions occur. Thus, highly specific i~RNA are formed dependent on the respective antigen used.

~ h~pQ-~r~us An i-RNA specific towards ~a~ it~ B was prepared, and its activity and compatibility were tested with more than 200 patients. The i-RNA con~
centration was 10 5 moles/ml. In most of the cases, the doses thereof administered to the patients for one time were from one to two ml, while in rare, more serious cases a second injection was applied.
The healing quota as presently surveyable is approx-imately 100 %.

~XA~lPLE 3 BALB/c mice were three times injected with complete Freund's adjuvant applying 100 ~l at a time ln one week intervals. The commercially available complete Freund's adjuvant was rnixed with physiolo-gical saline in ratio of 1 : 1. The animals were kept for four weeks after the last injection. Then, their spleens were removed and processed to recover the cell-free system.

The yields of i-RNA obtained by using said cell-free system were increased by the factor of 10 Iten).

Claims (6)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for producing a highly purified i-RNA
which is obtained in a cell-free system and which is specific toward antigens and compatible when administered to human and non-human animals wherein the process comprises reacting antigen DNA, nucleotides and an enzyme system recovered from leucocytes, lymphocytes, other cells or cell components, extracting the reaction product with an aqueous solution of phenol and sodium dodecylsulphate, centrifuging the supernatant from the extraction employing a density gradient of cesium chloride, saccharose and tris-buffer, separating the total centrifugate cesium chloride layer up to the saccharose layer, and further purifying the separated material.

2. A process according to claim 1, wherein the further purification is carried out by affinity chromatography.

3. A process according to claim 1, wherein the further purification includes centrifugation employing cesium sulphate.

4. A process according to claim 1, wherein the enzyme system is one which has been obtained from a homogenisate of tissue of the reticulo-endothelial system (RES) by centrifugation employing a density gradient.

5. A process according to claim 4, wherein the tissue is from animal spleen, the animal immunosystem having been stimulated prior to removal of the spleen by administration of BCG, Freund's adjuvant or Pind-Avi.

6. Highly purified i-RNA which is obtained in a cell-free system and which is specific toward antigens and compatible when administered to human and non-human animals, when produced by the process claimed in claim 1.