MXPA00001974A - Administration and diffusion system of vectors in vivo and in vitro - Google Patents

Abstract

The invention consists in the novel administration of viral or non viral vectors to the tissues for transfecting, being these tissues human or animal. This administration is achieved by means of an automated system of controlled perfusion allowing the administration of precise volumes at selected times to obtain a higher diffusion of the vector in vivo. This kind of system could be used with experimental and gene therapy aims in vivo or in isolated tissues in vitro which require to be transfected.

Description

AND IN VITRO. BACKGROUND OF THE INVENTION It is known that the transfection of genes suffers from low efficiency-of the genetic vectors as well as the way of introducing them towards the cell nucleus. In the present invention, a new system is contemplated in the traceability of genetic vectors in animals. or in humans to be performed in vivo or in vitro, with the possibility of varying the number of implanted microtubes, the volume, the pressure and the flow of the vector to be administered; what suppresses in definitive form the inconveniences of the existing systems.

DESCRIPTION There are two main limitations that have been found to date to achieve adequate gene expression in tissues - transfected with external genes. These are: 1) the low transfection efficiency of the genetic vectors / i.e. carriers of viral / non-viral genes, which have been used to introduce them into the cell nucleus; and 2) the inappropriate distribution of the genetic vector that is administered. In the present invention, a procedure and system has been created - of tissue microperfusion that allows a homogenous distribution - of the genetic vector that we administer within the tissue to be transferred to a tissue under in vi tro and in vivo conditions, encompassing a - larger area and avoiding tissue injury produced by pressure-applied with traditional methods. This novel system can be used for administration both in tissues and in the actual cavities of humans and animals, varying the number of implanted - icrotubes, the volume, the pressure and the flow of the vector to be administered. The present invention contemplates a novel system that is designed for the administration and / or tracing of genetic vectors in animals or humans, in vivo or in vi tro, with experimental or genetic therapy which, as is known, is-the introduction of a gene into the abnormal cells of an organism for therapeutic purposes. There are multiple vectors for introducing a gene into a cell, and the technical term for referí en to us is "transfectar". The gene as a functional unit of the genetic material is known as deoxyribonucleic acid or DNA.

In general we can say that each gene encodes a protein with a specific function that can be, for example, a hormone, an antibody, a structural protein, etc. Now, if we consider that each cell of our organism possesses an average of - - 100,000 genes in its nucleus, we can imagine how complex it is to re - order what proteins and in what quantity they have to be expressed daily in each cell. In addition, it has to be considered that there are specialized cells to produce specific proteins such as, for example, albumin in the cells of the liver or insulin in the pancreas. Already in the field of gene therapy have been able to isolate and manipulate various genes to make constructions that can be introduced to target cells by means of "carriers" or "vectors." There are two main groups of diseases with genetic alterations that have been of the greatest interest for the treatment with genes; hereditary and cancers. In the first case, hereditary diseases are caused by alterations of a specific gene. These alterations may include mutations (ie minimal changes in the genetic sequence of the gene) or partial or total losses of a single gene within the cell nucleus. To date, more than 5,000 diseases caused by the alteration of a single gene are known, of which, the most interesting for gene therapy, have been: 1) cystic fibrosis; 2) adenosine deaminase; -3) Duchenne muscle istrophy; 4) hemophilia e 5) hipercoleste-ro 1 em i a family. The second major group of diseases that can be caused by the alteration of multiple genes is that of cancers. For purposes of the present invention, the environmental factors involved in cell calendering are not considered and they also play an important role in the calendering of certain tissues. So, focusing only on the genes leading to 1a-cancer i zac ion, these may belong to one of two antagonistic groups: 1) the pro-songs called oncogenes and) those that suppress the formation of tumors known as anti-oncogenes or - -emoreogenes. Of the 100,000 genes that exist in human cells, it is estimated that there are approximately one hundred oncogenes and one hundred anticogenes. The first can cause cancer when its structure is altered by mutations, translocations and by partial or total losses of its sequence. But in addition to changes in oncogenes there is also loss of function of one or multiple anti-oncogenes in virtually all human cancers. The oncogenes, when suffering mutations or partial or total losses of their sequence, will stop suppressing some steps that avoid the can- ce r i z a c i n. Initially it was thought that the best candidates for gene therapy were hereditary diseases because in the "diseased" cells there is the alteration of a single gene. Theoretically, the reintroduction of the gene that was missing or not working in those cells would be able to completely correct the disease. However, this has not proven to be entirely correct. For example, in two genetic diseases called deaminase deficiency and cystic fibrosis, it has not been possible to produce a frank clinical improvement of the process even though it has been possible to obtain an acceptable transfection of the gene to the target cells of the patients. Approximately half of all the global gene therapy protocols that are being carried out in the world are for cancer patients. Another ten percent of the studies are directed to the control of the infection with the acquired immunodeficiency virus and the rest of the studies are focused on other diseases, including hereditary and many factors, however, in general. We can mention that genetic replacement is being used to replace the gene that is needed. For example, for hemophilia, the factor VIII gene is being expressed in various types of cells (e.g., fibroblasts) of the experimental subject for these cells to be responsible for producing a protein. In other cases, as in cirrhosis, we try to express a gene that produces an enzyme protain that destroys fibrosis that alters the architecture of the liver parenchyma. Some strategies for modifying at least one of the characteristics of tumors, for example, proliferation, growth, dissemination (ie etátasis), chemorescence, had already been glimpsed and tested in the laboratory and in experimental animals. , radioresi stenci a, etc. To date there are already models in which the introduction of a single gene in tumor cells can modify some of those characteristics in favor of the patient (Scheme) For example: 1) that the cells no longer grow or that their? i_ semination by transfecting a repressor gene, eg an antioncogene-2) that becomes more sensitive to radiation or to the chiruroterapies; 3) that can be recognized more easily by the subject's immune system. To achieve this last goal, molecules could be expressed on the surface of cells to awaken the immune response or to be able to release molecules (eg cytokines) for the same purpose. Another strategy has been to introduce genes into tumors to be able to produce a protein with enzymatic function. The goal of that enzyme, in turn, is to convert a non-toxic pro-drug into a highly toxic drug exclusively within the tumor. In such a way-that when injecting that prodrug into the vein of the patient or animal there is no systematic toxicity, but an anti-tumor-aggressive response in the genetically manipulated tumor. The latter stria tegia has been called therapy with suicide genes and the genes - that have been used most in it have been the desa-inasa-d-cyclin and thymidine kinase. The enzymes that produce these genes activate 5-fluorocytosine and gain, in particular, and make them extremely toxic to the tumor tissues that contain the gene as well as to the surrounding cells. In addition, in these genetic constructions molecular "switches" have been used that allow us to turn on or off at the convenience of the pressure of the gene we want. These molecular switches are known as promoters and of these, the most used have been those from the virus (eg cytomegalovirus, rous sarcoma virus, etc.). Attempts have been made to use other promoters specific to certain tumors (eg, that of the erb B2 oncogene or tissues (eg, albumin), but their activity has been defective and, more recently, they have begun to appear on the scene. promoters that can be modulated with various drugs such as steroids, tetracyclines, etc. These new promoters will surely be more efficient to carry out the expression of the genes in any drug, if and when the inducer exists. There are multiple strategies for transfecting a gene into a cell In the case of clinical trials, the "carriers" or "vectors" that have been used the most to transfect a gene correspond to one type of finger: the lipids and the vi rus. The first type of vectors, which are included in the non-viral vectors, consists in the production of small particles of specialized grasses to which the gene we want is coupled. The tissue that is desired to be transfected is expected and the cells are expected to introduce them to their nucleus. Unfortunately, the efficiency of genetic transfection with these lipid-genes complexes is very poor. Other non-viral vectors include chemical compounds (eg, calcium preci- tation with DEAE etc), guns that isolate gold bullets to which the DNA is complexed, electrical current to the cells in the presence of genetic material (ie. elec troporation), etc. This type of vectors, with the possible exception of the referred gun, are not viable candidates for gene therapy in vivo, and are used more for the transfection of in-vitro cells. In order to improve transfection efficiency, the use of multiple vectors as vectors has been proven. These viruses are genetically manipulated so that they contain in their interior the gene that is interesting to introduce to certain cells, likewise, these viruses are carefully modified to prevent them from spreading in the organism into which they are introduced. In this way, viruses can infect the cell and deposit the gene of interest in its nucleus - without producing a systemic infection. Of the most-used virus groups for these purposes are the following: 1) retroviruses; 2) adenovirus; 3) adeno-associated and 4) herpes vi us. If it is true that the genetic modification of viral vectors has given them a very wide safety margin, it is also true that some of them can produce severe inflammatory reactions and viremia of variable degree. And although it has not been shown that viral vectors themselves produce new genetic alterations in humans (particularly retroviruses), it has not been possible to document that this possible adverse effect will not occur in the long term. Of the most commonly used viral vectors are the ade-novirus. There are multiple genetic modifications of these viruses that have had to be performed with several objectives such as: 1) decrease the degree of immune and inflammatory response that produce 2) increase the efficiency of genetic transfection to any type of cell, as for example by modifying the capsule proteins (eg, the RGD modification) responsible for entering the cell into the interior of the cell; 3) avoid its replication - in the organism making them infective but lacking replication in vivo; and 4) more recently, the use of --vectors that do replicate themselves in the host organism with the finality of bringing virus to the entire tissue economy to the inyéctalos intravenously has been promoted. A genetic vector can be administered to virtually any part of the body that is required. It can be injected directly into any organ and into the joints, the bloodstream or the fluid into the orachoma. Attempts have also been made to administer it by spray to the airways and by means of a "gun" that triggers the genes coupled to small gold bullets that are directed against any tissue.The mentioned genetic vectors include: (1) any plasmid naked or associated with any chemical, physical or organic element to improve its transfection to the cells: examples of carriers include lipsomes, cathioid lipids, polylysine, vj_ rus associated with DNA, calcium-DNA complexes, gold particles etc. (2) plasmids containing multiple genes to be expressed by the direction of the same promoter or of several promoters; (3) -viruses of the herpes virus, adenovirus, adeno-associated, ret or_ virus.lentivirus groups, or others that appear in the future to perform genetic transfection; (4) viruses that are lacking in replication may generate an additive or synergistic effect of the microperfusion described here to obtain a maximum or volume of transfected tissue; (5) any of the aforementioned viruses that have undergone any genetic and / or structural modification to: -a) improve their adsorption to the cell wall (membrane), such as, for example, the RFG modification in knobs of adenoviruses; b) to increase the penetration of the viral vector and its introduction to the nucleus or c) to improve the degree of genetic expression within -the latter, for example, using multiple genes with multiple promoters to exert additive or synergistic functions; (6) viru-ses of any mentioned group containing one or multiple genes directed transcisionally by one or multiple promoters;-( 7) combination of viral and non-viral vectors in a first-application or in the subsequent ones to exert additive effects or - yes, ng ics. The main constraints that have impeded genetic and efficient transfection in target cells have been established. These include: a) the type of vector that we employ that, in general terms, are more efficient than non-viles; -b) the particular conditions of the cell that allow the adequate activation of the "switchs" "Molecular (ie, promoter) that - claim expression of the gene in question; and c) the production and -the relevant modifications of the protein in the target cell- that allow it to be active at the conclusion of gene expression, etc. However, one of the most important limitations to obtain - an adequate transfection in vivo has been the poor diffusion of the vectors within the tissues when administered by conventional techniques. That is why the system has been devised to - correct this last limitation, and which complements the efforts to improve the transfection and genetic expression by improving the vectors used for this purpose. The present invention consists in the use of a micro fusion system for the in vitro or in vivo administration of genetic vectors in human and animal tissues. The invention proposed here is to use an infusion pump coupled to my multi-channel syringes and microtubes for the administration of genetic vectors (viral or non-viral) within a tissue of human or animal origin. This system has several advantages over the manual administration of vectors that is commonly carried out at present. The in vitro administration may be carried out in any type of tissue that is excised in the body of the experimenting subject or in treatment. This tissue may be maintained in culture -in vitro or not, for periods of minutes to days, with the purpose of placing the multi-channel perfusion tubes inside it, which includes the genetic transfection. Once the vector has been administered, the tissue in culture can be maintained, or it can be re-transplanted to the original site (alotransplant) or to an area of another species (xeno transplantation), approved scientifically and ethically. The tubes may be removed immediately after the first administration of the genetic vector when it is still outside the organism, or if multiple transfections are required, they may be left in the tissue transplants the tissue to the subject under study and continue. the administration of the genetic vector (in vivo) through the external access to the tubes. The permanence of the -tubes will require the follow-up of appropriate asepsis and antisepsis standards to avoid any contamination or infection in the transfected organ or in the contiguous ones. The re-application of micro-tubes and micro-perfusion to the woven ism on subsequent occasions may be carried out as many times as necessary and whenever it is - ethically and scientifically acceptable. The in vivo administration may be carried out directly on the organ or tissue that needs to be modified without needing to be removed from the organism. To do this, the organ or you should be -exposed to the outside of the body for handling. The multichannel tubes will be placed in the organ or the organ to carry the genetic transfection at that moment or, later, when the organ is located in its place of origin. In a manner similar to that discussed above, the transfection may be carried out at the moment of the placement of the tubes while the tissue is exposed to the outside of the body or, failing that, it may be performed when the organ has been returned. to its place of origin inside the body, as long as an external access of the tubes is left. This access allowed not only the administration of the genetic vector, but multiple occasions over the course of several days as required. For this reason, the tubes may either be removed at the time after the first transfection or, failing that, remain for several days or weeks, provided that the aseptic and antiseptic techniques are followed for the appropriate treatment. infection or contamination of the tissues that are in contact with the tubes. The microtubes-and the micro-perfusion can be made to the same tissue in later-occasions, as many times as required and if ethically or scientifically approved. The concept of the system originated from the mi-croperfusion techniques that have been used for brain injections for several years. In these initial studies, what was done was the implant of microtubes in critical areas of research for the perfusion and collection of neurotransmitters in my cropping fluid, using an automated perfusion pump. The use of the perfusion pump then allowed a diffusion controlled in time, volume and pressure to obtain the microdi at the head of the brain without this perfusion causing damage to this tissue. Another important aspect was! that microtubes could remain for several days inside the brain in animals and humans without causing an important infectious or in lamatory response in the host. More recently, the use of microperfusion for drug administration within the brain has begun in a continuous manner and for periods of hours to days. To carry out experiments on my blood, it is necessary to perfuse artificial cerebrospinal fluid at a speed of 1 to 2.7 -min / min, for which Harvard-type continuous perfusion pumps are used and it is possible to inject Exact way from 0.1 ul / min to 1 ml / min. On the other hand, experiments have been carried out on free-motion animals in which volumes of 2 ul / min have been injected every 24 hours. For the above, they are previously planted in specific brain areas, guides through which a 10-mm-thick cannula is introduced and substances or drugs are administered. Since this is done under stereotactic guidance, we can obviate damage to the brain. For the exact injection of the desired volume, we also use Harvard continuous perfusion pumps. Finally, in the case of human experience, these pumps have been used in multiple protocols of transoperational procedures in combination with mi crodi techniques to obtain neurotransmitters from patients with epilepsy and others. Likewise-these pumps have been planned for a stereotactic transplant project in Parkinson's for the infusion of cells cultured in vitro. However, and to our knowledge, the widespread use of microperfusion for the administration of genetic vectors in the field of gene therapy at experimental or therapeutic level, in human or veterinary medicine has not been patented. This system has several sales on the manual administration of genetic vectors that is commonly performed today. These are: -Management of small volumes of genetic vectors that, according to the pressure and time of infusion, will allow a better diffusion of the vector within the tissue. - Automated perfusion system that will avoid tissue damage-that could be caused by excessive pressure. - Use of special microtubes, high-energy micro-tubes, with multiple channels for the diffusion of the vector throughout the path of the microtube. The latter will be placed by means of a guide that is directed by microsurgery techniques within the tissue to be trans - fected, and it may be possible to place one or more microtubes as required. - The gene (s) to be transfected to obtain any desired effect in the targeted target or in the host organism, may contain: a) complete genetic sequences of the gene that we wish to express or b) sequences modified to the original, such as , -which are shorter, transí ocadas, mutated, quiméri cas or in antise_n tido. It may also be possible to transfect more than one gene using the same vector or to be several types of genetic vectors (with different genes) that transfect the same tissue at the same time. These genes can be of any origin, be it prokaryotic (bacterial, viral, etc.) or eukaryotic, as long as they exert the desired iologic effect or serve as reporter genes (markers) in initial studies of vector distribution. - Any of the genetic vectors may contain any promoter tj_ po to direct the transcription of the gene or genes that are desired to exorcise in the target tissue. These promoters can be: a) inducible by drugs, radiation, etc. b) specific tissue or -tumor; d) of bacterial, viral and eukaryotic origin including yeasts, insects, and mammals; e) associated or not with cis or trans regulation elements. - The system will be applicable in the therapy of any tissue-in vivo that requires to be transfected in animals or humans. It may also be used for the in vitro transfection of any tissue that requires being genetically modified ex vivo and subsequently retranslated. - The in vitro administration may be carried out in any tissue that is excised from the body of the experimental subject or in treatment. This tissue can be kept in vitro culture or not, for periods of inutes to days, in order to place multichannel perfusion tubes inside it that allow for genetic transfection. Once the vector has been administered, the tissue in culture can be maintained, or it can be retransplanted to the original suture (at otranspl ante) or to a subject of another species (xenograft), if scientifically and ethically approved. The tubes may be removed immediately after the first administration of the genetic vector when it is still out of the organism or, if multiple transfections are required, they may be left in the tissue, transplanting the tissue to the subject under study and -continue the administration of the genetic vector (in vivo) through -the external access that is left for the tubes. The permanence of the tubes will require the follow-up of appropriate asepsis and antisera standards to avoid any contamination or infection in the transfected organ or in the contiguous ones. The reapplication of microtubes and the microperfusion to the same fabric on subsequent occasions, may be carried out as many times as necessary and ethically and cyclically acceptable. -Any surgical technique can be used to obtain the desired transfection in vi tro or in vivo. These techniques include those performed in all specialties and subspecies of the medical-surgical system, including microsurgery, stereotactic-functional, laparoscopy, robot-assisted, - etc.

- The microtubes used for genetic transfection may also contain electrical resistances that allow the administration of a controlled electric current to improve the efficiency of the transfection by producing changes in the membranes --plasmic and nuclear (eg hyper-polarization). or depolarization) of the transfected tissue. This procedure, in theory, would represent-an electroporation that could be carried out invitro or in vivo. Likewise, these resistances within the microtubes could also serve for the electrical recording of excitable tissues such as the brain and muscle during transfection or later. This would allow controlling the current flow and evaluate the immediate and subsequent effects of the transfection.

REFERENCES Gutiérrez AA et al .: "Gene therapy for Cancer". The Lancet 1992, - 339: 715-721 Sikora K & Gutiérrez AA. "Prospects for Biological! And Gene - - - Therapies". In Workman (ed); New approaches in cancer pharmacol ogy: drug design and development. European School of Oncology Monographs 1992. Springer Verlag, Berlin. Harris JD. Gutiérrez AA.Hurst HC, Sikora K & Le oine NR: "Gene --therapy for cancer using tumour-specific prodrug activa ion", Gene Therapy 1994, 1: 170-175. Gutiérrez AA y Cois .: "Perspectives of the use of informed drugs in cancer". Gac Med Mex 1995,131,481-484. Gutiérrez AA et al .: "Novel approaches for gene-based therapies ip prostatic cancer" In Gal indo E. "Borders in Biotechnology and Bio-engineering". 1996. Mexican Society of Biotechnology and B i o i p g e n i ria A.C. 1996. Mexico, D.F. Gutiérrez AA: Chapter 65"Regulation of Ce 11 Division in Highe - Eukaryotes". In Sperela is N (Ed): The Cell Physiology Sourcebook. -2nd. edition .Academic Press; New York, NY, USA .1998, 1003-1020. Rocha,., Br iones, M., Ackermann, R.F., Antón, B.Maidment, N.T., Evans, C.J. and Engel, J.Jr. (1996). Pentylenetetrazol-induced - - - Kindling; early Involvement of excitatory and inhibitory systems.

Epilepsy Res. 26: 105-113. Rocha, L., Evans, C. . and Maidment.NT (1997). Amygda 1 a kindling - -modifies extracellular opioid peptide content in hippocampus: a -microdialysis study.J.Neurochem 68: 616-624. Rocha L.and Ka? Fman.D. (1998) In vivo admi n i s tr a t on on c-fos antisense accelerating oligonucleotides to ygdala kindling.Li e --Sci., 241: 111-114. Rocha, L., Maidment, N.T. Evans, C. ., Ackermann, R.F.and Engel, J.- Jr. '1994) Microdialysis reveals changes in extracellular opioid-peptide levéis in the amygdala induced by amygdaloid Kindling stimulation. Exp.Neurol. , 126: 277-283. Roche, L., Ondarza, R. and Mai dment, N. T. Gabapenti n modifies extra-cellular opioid peptide content in amygdala: a icrodialysis study Epilepsy Res. (In press). Rocha, L., Ondarza, R. and Kaufman, D.L.Antisense oligonucleotides on c-Fos reduces postictal seizure suscepti bi 1 i and of fully - --kindled seizures.Neurosci. Lett. (in press). Several popular articles on gene therapy in cancer, - nervous system, AIDS and cloning were published in the - - Se i en ti fie American, June 1997. pp 96-123.

Claims (12)

1. CLAIMS Having described the invention, the contents of the following clauses are claimed as my property: 1. - The in vi tro administration may be carried out in any type of tissue that is removed from the body of the subject of experimentation or in treatment. This tissue can be kept in vitro culture or not, for periods of minutes to days, in order to place multichannel perfusion tubes inside it - which will allow genetic transfer.
2. 2.- The administration in vivo with the multichannel tubes can be carried out directly on the organ or tissue that requires modi fi cation without needing to be removed from the organism.
3. 3.- Genetic vectors include: (1) any plasmid that is denuded or associated with any chemical element, whether organic (2) -plasmids that contain multiple genes to be expressed under the direction of the same promoter or of several promoters; (3) Viruses of the herpes virus, adenovirus, adeno-associated, retro-virus, lentivirus, or other groups that appear in the future to carry out the genetic transfection and that have undergone or not any genetic coding (4 ) combination of viral vectors and non-vira-1 is.
4. 4.- The gene (s) to be transfected may contain: a) complete genetic sequences of the gene that we wish to express or b) sequences modified to the original.
5. 5.- Any of the vectors may contain any type of promoter to direct the transcription of the gene or the genes that they wish to express.
6. 6.- The use of a cropping system coupled to microtubes-hi poal and with multiple channels for the free diffusion of the vector along the path of the microtube.
7. 7.- The microtubes described here will be hypoallergenic and inert-to avoid maximum or their contamination and rejection by the receptor tissue.
8. 8.- The microtubes can be implanted by means of mi-croe and roar techniques and stereotactic surgery if required, using guides specifically designed for it.
9. 9.- The microtubes may be of radiopaque material or not, depending on the need to evaluate their position within the tissue manipulated by means of radiology.
10. 10.- The microtubes may be used for association with other drugs or chemical, physical or organic elements that exert a synergistic or additive action to the genetic transfection.
11. 11.- The microtubes used for genetic transfection may also contain electrical resistances to improve the efficiency of trans ection.
12. 12.- Any surgical technique may be used to obtain the tissue that is desired to be transfected in vitro or in vivo.