 Keywords: a gene, a molecula of a DNA, genetics, a chromosome, therapy, a heredity, medicine, the abstract, ancestral diseases, gene engineeringthe Author: Poljanskaja O.V.GENIC THERAPYThere is a serious disease - a hypercholesterinemia. The cause of him is the disadvantage or defect of genes LPNP-retsrptora - minute structures on a surface of cells of a liver, "extending" lipoproteins of low density (LPNP), unhealthy the person, from a blood and responsible for their destruction and responsible for their destruction in a liver. James Wilson, the former explorer from Medical school Michiganskogo of university in Ann - Arbor, has made attempt to introduce repetitions of a normal gene of the LPNP-RECEPTOR to the patient. All over again he has separated at the woman of 15 % of a liver (6 billion cells) which have been brought up in 800 cups with nutrient agents. Into them has been entered it is artificial the deduced innoxious virus keeping the necessary gene. Have apprehended a gene about 20 % of planting cells of a liver. The modified cells have been entered into a body of the patient with the help of a catheter in a vein leading immediately to a liver where as the explorer expected, they will join other cells and will start to share. In some months the small hepatic biopsy has shown, that the new gene functions in some from entered cells of a liver. Moreover, maintenance LPNP in a blood of the patient has fallen to 15-30 %. Wilson has told, that enriching began sufficient further to manage only medicines for the further downstroke of level LPNP. Deciding achievements of molecular biology and genetics in studying thin frame of genes eukariot, their mappings on chromosomes mammalian, and, first of all the person, in range of biotechnology and gene engineering have resulted rough body height to that in 1989 the first attempts of treatment of monogenic illnesses have been undertaken. What is the genic therapy? Whether it means treatment with the help of a gene as medicinal preparation or only treatment by correction of a mutant gene? Genic therapy at the present stage can be defined as treatment of ancestral, multifactorial and not ancestral (contagious) diseases by introduction of genes in cells of patients with the purpose of the directed change of genic defects or giving to cells of new functions. Pristinely believed, that genic therapy will allow to correct defects in a gene which invoke monogenic diseases. Theoretically considered, that correction of genic defect is possible as at a level somatic, and germinal (sexual) cells. But numerous experiences and experiments have made allowances to these representations. It is much easier to correct not defect in a gene, that is to replace all mutant gene or his damaged fragment on normal, and to introduce into an organism of the patient it is high-grade a working gene. Works on genic therapy at the person circumscribed basically to somatic tissues as manipulations on sexual or formative cells can result in serious and unpredictable consequences. Already developed and put into practice procedure of genic therapy is effective not only at treatment of monogenic diseases, but also such eurysynusic pathologies of the multifactorial nature (that is the caused genetical and exogenous factors), as the malignant tumours, many kinds serious virus, cardiovascular, etc. diseases. the Historical information. The first clinical tests in genic therapy have passed in May, 1989. The T lymphocytes taken from a tumour at the patient by a melanoma, have been labelled prokarioticheskim a genome neo, steady against Neomycinum. It has allowed to separate easily these cells in culture, and then in details to track their destiny in a blood-groove and selective accumulation in tumours. The first monogenic ancestral disease to which have applied methods of genic therapy, began the ancestral immunodeficiencyy caused by a mutation in a gene of an enzyme adenozindezaminazy. On September, 14-th, 1990 in Betesde (USA) to the four-year-old girl, an infrequent pathology suffering by this enough (1:100000) have replaced its own lymphocytes which preliminary transformed in vitro a genome of the HELL (a gene of the HELL + a gene neo + retrovirusnyj a vector). The medical effect was observed within several months then procedure repeated with an interval in 3-5 months. As a result of treatment the state of the patient has so improved, that it could conduct a normal mode of life and not be afraid of casual infection contaminations. The majority of designs in genic therapy (about 80 %) treatment of oncologic diseases, and also a HIV-infection concern. Clinical tests of monogenic hereditary diseases and such as are started:
Programs of genic therapy for clinical tests should include:
In dependence on a mean of introduction of an exogenous DNA in a genome of the patient genic therapy can be carried out or in culture of cells (ex vivo), or is immediate in an organism (in vivo). Cellular genic therapy or therapy ex vivo assumes:
Genic therapy in vivo is based on direct introduction clonate and definitely packed sequences of a DNA in specific tissues of the patient. Especially perspective for treatment of genic illnesses in vivo introduction of genes with the help of aerosolic or injectioned vaccines is represented. The aerosolic genetic therapia is developed, as a rule, for treatment of pulmonology diseases (a mucoviscidosis, a cancer mild). Methods of a genetical transfection in genic therapy.A deciding condition of a successful genetic therapia is maintenance of effective delivery, that is a transfection (in a broad sense) or transductions (at use of virus vectors) an alien gene in cells - targets, maintenance of long his functioning in these cells and building of conditions for high-grade work of a gene (his expression). The transfection can be carried out with use of the bare DNA alloyed (built - in) in conforming plasmid, or complex by a DNA. Complex - plazmidnaja a DNA, coherent with salts, proteins (transferrin), organic polymers, or a DNA in structure of the virus particles, preliminary deprived abilities to replication. Naked by a DNA at intramuscular introduction it is capable to be expressed in amounts, sufficient for development of the immune answer to appearance of new proteins. This effect can be potentially used with a view of bacterination against the certain pathogenic viruses, however is not sufficient for the majority of the therapeutic purposes. For delivery of a genetical stuff ideal " the molecular machine ", having by such qualities as is represented:
The basic methods of delivery of alien genes in cells are parted on physical, chemical and biological. PHYSICAL: a microinjection, an injection a jet, elektroporatsija, freezing - thawing, bioballistics (bombing of cells by drops of fluid or a suspension chastichek gold with adsorbed plasmid). CHEMICAL: salts of the some people kationov, for example, calcium, DEAE a dextran, a polylysine, liposomes. BIOLOGICAL: virus vectors. If the problem of delivery of an alien DNA in vitro is practically solved, and its delivery in a cell - target of different tissues in vivo successfully is solved, other characteristics existing vectorial system - stability of the integration, the adjustable expression, safety - vsyo still require serious completions. To raise efficacyy of stable integration it is possible
Recently the special attention is given building of vectors on the basis of artificial chromosomes mammalian. Due to presence of the basic structural elements such mini-chromosomes it is long are kept in cells and are capable to carry full-size (genomnye) genes and their natural regulate elements which are necessary for correct work of a gene, in the necessary tissue and in due course. Now we shall stop more in detail on some methods. Viruses as agents of delivery of a genetical stuff.The majority of used viruses are received from feral shtammov, having by a various degree of pathogenicity, but lost pathogenic properties due to erasion of the genes responsible for a breeding and (or) assembly of virus particles. In clinical practice retroviruses and adenoviruses are usually used. RETROVIRUSES are RNK-KEEPING viruses which replication is carried out through a DNA intermediata. The genetical information of a virus particle, which beneficial volume can compound 9000 steam of the establishments, immediately takes root into a genome of the cell - master. The majority of retroviruses are effective only for sharing cells. These viruses are not applicable for cells of a muscular or nervous tissue, cells of a liver and mild. Exception is compounded with vectors of lentiviruses. Viruses of the VICH-INFECTION CONTAMINATION which also can be used in genic therapy concern to their number. ADENOVIRUSES - have a two-stranded DNA and usually allow to deliver the much greater volume of the beneficial genetical information. Adenoviruses do not take root into a genome of master's cells, and during division the information is eliminated. These viruses are effective at a transfection, is especial in the attitude of cells of respiratory pathes where the transfection can be achieved more than 50 %-n, that the order is higher, than in case of retroviruses. Disadvantages of use of viruses:
Artificial vehicles.1. POLYMERS.The polymeric moleculas bearing superfluous kationnyj a charge, can raise efficacyy of a transfection essentially. The certain activity small moleculas (a protamine, dimetilsulfoksid, derivatives of an imidazole, Gramicidinum, lipopoliamin) have even. The big activity synthetic polymers (polietilenimin usually show, a polylysine, lipopolilizin or his conjugates with a transferrin, asialoorozomukoidom, neoglikoproteinom, a galactose, a mannose), and also connatural kationnye proteins (histone Í1, galaktozilirovannyj histone Í1, histone Í4 in a complex from a DNA and konjugatom a transferrin - polilizin). High efficacyy rich a lysine of peptides and proteins can be caused by their resemblance to the specific signs responsible for transport from cytoplasm in a nucleus. Polysaccharides and the albuminous ligands included in the listed above complexes, determine their affinity with specific receptors on a surface of cells. With cells resemblance to penetration into a cell of virus particles has processes of interaction of such complexes. 2. LIPOSOMES.The DNK-LIPIDE complexes keeping plasmid with "ekspressirovannoj" by the cartridge are usually used. Advantage of DNK-LIPIDE complexes in comparison with virus vectors:
Especially perspective use of phosphotides, for example, a cardiolipin and fosfatidiletanolamina, forming alongside with bislojnymi membranes also the inverted micellar frames known as cubic and geksagonalnye of a phase that determines ability of these lipids to initiate coalescence of membranes is represented. At presence kationov calcium or magnesium interaction of a DNA with phosphotides becomes stronger, liposomes aggregate, and a DNA internalizuetsja in intrinsic volume of vesicles. Use high-molecular kationnyh the messengers providing formation of complexes of a DNA with phosphotides and interaction of these complexes with a surface of cells, potentially could improve their efficacyy in a transfection. Revolution was introduction in practice of the first nizkotoksichnogo kationnogo lipid DOTMA (1,2-äèîëåèë-3-N, N, N-òðèìåòèëàìèíîïðîïàí), synthesized Felgnerom with co-authors. The new term "lipofektsija", underlining high efficacyy of genetical transformation of the cells, approaching liposomalnye preparations to contagious virus particles has simultaneously been entered into practice. At introduction in a tissue "naked" by a DNA it is usual ekspressirovalas only one molecula from several millions, and DOTMA enlarged this parameter approximately in 1000 times. MECHANISMS LIPOFEKTSII.What mechanism of delivery of a genetical stuff to cells - targets and his penetrations into a nucleus? 1. DELIVERY OF A DNA TO THE SURFACE OF CELLS.The PROBLEM: series highly effective kationnyh lipids are inactivated at presence even insignificant amounts of a blood plasma; some organs have endotelialnyj a barrier handicapping penetration of liposomes. POSSIBLE DECISIONS:
2. INTERACTION OF COMPLEXES WITH THE CELLULAR SURFACE AND PENETRATION INTO CYTOPLASM.The nature of the mechanism of penetration of a DNA in cytoplasm till now finally is not investigated. Coalescence of liposomes to a plasma membrane on a surface of a cell therefore intrinsic contents of a liposome can get in cytoplasm, is the most simple and at first sight the obvious mechanism of a translocation of a DNA. However there are serious arguments for the benefit of that penetration of a DNA into cytoplasm descends at considerably more serotinal stages when the most part of liposomes internalizuetsja in cytoplasm. This process outwardly resembles endotsitoz as in places of a sorption kationnyh vesicles on a surface of a cell there is an invagination of a plasma membrane and otpochkovyvanie inside of cytoplasm membranous puzyrka with kationnymi liposomes or the DNK-LIPIDE complex in intrinsic space. The invagination of a plasma membrane under influence sorbirujushchihsja on its surface kationnyh liposomes, obviously, does not demand the specialized mechanism as it is observed even on a surface of erythrocytes. 3. REMISSION OF A DNA IN CYTOPLASM AND TRANSPORT IN THE NUCLEUS.The big masses endotsitirovannogo a stuff collect in the ranges adjoining to a nucleus. The clump of a plenty of a stuff in okolonuklearnom space testifies what the stage of remission of a DNA from endosom can be the factor limiting efficacyy of a transfection. Usually endosomy with the particles seized inside, including viruses, are transformed to lysosomes to which membrane there is a coalescence of virus particles at low rN. On the contrary, in a case lipofektsii the inhibition of functioning of lysosomes due to augmentation rN chloride ammonium or hlorohinom raised efficacyy of a transfection in some times. Remission of a DNA in cytoplasm, most likely, descends from endosom, minuja a mode of formation of lysosomes. Moreover, kationnye lipids interfere with hit of a DNA in lysosomes. There is a hypothesis, that, the mechanism of remission of a DNA is based on an exchange of lipids between kationnoj a liposome and membranes of a cell therefore kationnyj the charge is neutralized, and connection of a DNA with a liposome weakens. At coalescence of complex DNK-kationnyj the lipid with membranes anionnogo a lipid descends phase transition in frame of a complex which final stage is disintegration of a complex on the separate components consisting of moleculas of a DNA, surrounded tubular bislojnymi frames of lipids. One of the possible causes of low efficacyy of a transfection can be that the DNA even after remission from a liposome continues to keep a plenty adsorbed kationnogo a lipid. After remission in cytoplasm the further progression of a DNA to a nucleus, probably, descends to use of poorly studied mechanisms of cellular transport. It is possible, that kationnye liposomes are capable to release a DNA immediately in a nucleus. However lipofektsija it is effective only on attached cultures of cells. Complexes substantially lose activity at interaction with a blood plasma and have rather circumscribed application in vivo. THE GENETIC THERAPIA OF VARIOUS DISEASES1. Monogenic ancestral diseases.Ancestral diseases, genokorrektsija which is at a stage of clinical tests (KI), experimental development (EK) and it is essentially possible (PV). A stage of clinical tests: an immunodeficiencyy, a Cristmas disease, illness to Gosha, a mucoviscidosis. A stage of experimental development: an angiostaxis And, an emphysema mild, fenilketonurija, muscular dystrophia Djushenna, a thalassemia, serpovidnokletochnaja an anemia, illness Altsgejmera, illness Parkinsona, chronic granulomatoz, a respiratory distress. THE EXAMPLE: Some infringements of work of a brain (in particular, illnesses Parkinsona and Altsgejmera) are connected to damage of cells, developing nejromediatory. In result the brain cannot normally adjust motorial activity of the person. Such illnesses treat preparations mediatornoj the nature, however, in due course the organism loses sensitivity to them. Besides they can render a unfavorable side effect. Recently tried to treat a parkinsonism transplantation of tissues, capable to produce nejromediatory. A graft can be a tissue of a brain of human embryos or a medulla of paranephroses of the patient. But at use of alien tissues there are problems of a tissue incompatibility, and also legal and ethical. Besides it such operations not always result in good results. F. Gejdzh with co-authors (the Californian university, San Diego, the USA) in experiments on rats used gene engineering for struggle against illnesses of nervous system. From animals took away cells of a skin and introduced into them a virus keeping a gene, a coding enzyme tirozingidroksilazu. An enzyme kataliziruet metamorphosis of amino acid of Thyrosinum in nejromediator L-ÄÎÔÀ. Cells with built - in implanted a genome into a brain of rats with preliminary decayed field of a brain at which the infringements of work TSNS similar to illness Parkinsona at the person were observed. After transplantation of the modified cells at 40 % of animals the state has improved. Authors consider, that use for transplantation of own cells of the recipient is perspective for treatment of patients by a parkinsonism. the Stage essentially possible genokorrektsii: Hunter Illness, set of symptoms Gurlera, giperammonemija, tsitrulinemija, metachromatic lekodistrofija, set of symptoms Lesh-Nihana. 2. Not ancestral diseases.Simultaneously with development of researches in range genokorrektsii ancestral defects successful also there were searches of methods of therapeutic use of semantic sequences of a DNA for treatment of not ancestral diseases, and mainly malignant tumours and virus infection contaminations. the EXAMPLE: Medicinal preparations for treatment of herpes are ineffective. One of their cause - insufficient ability to inpour inside of cells where there is a virus - originator of the given disease. In NPO "Biotechnology" Minmedproma, Institute of virology and the All-Russia cardiological center series of the preparations made in liposomes are tested. It is a genno-engineering a-interferon (reaferon), áèîëô-62 and atsiklovid. Efficacyy of the preparations made in liposomes, is higher (in comparison with usual). the Basic approaches in genokorrektsii oncologic diseases:
International magazine " Rejuvenascence " (Belgium) has published clause " Some approaches to a rejuvenascence by means of genetical intervention " (1985, ò.13, ¹1-2). In clause the design of the following genno-engineering methods of controlling with aging is offered:
Moral problems of genic therapy.While in range of genic therapy much is not clear, difficult to provide all consequences of transmission of a genetical stuff in a human body, and also to estimate his therapeutic efficacyy. Therefore there is an international interdiction on carrying out of tests on sex cells and cells early doimplantatsionnyh blastemals of the person to prevent transfer of unfavorable genetical changes to descendants, to not admit a contamination of a gene pool undesirable artificial genic designs or entering of mutations with unpredictable consequences for the future mankind. F.Anderson and Dzh. Fletcher have formulated three conditions which today are conventional. For the sanction of clinical tests in range of genic therapy it is necessary to prove in experiments on animals, that:
At the same time in the scientific literature even more often appeals to renewal of discussion about expediency genokorrektsii germinal and sex cells of the person also are more persistently distributed. Some questions which should be solved within the framework of offered genetikami wide discussion on genic therapy.
Thus, genetical revolution which deification was the genetic therapia, not only offers real pathes of treatment serious ancestral and nenasledstevnnyh illnesses, but also in the prompt development puts before a society new problems which decision is urgently necessary already in the near future. the List of the literature:
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