Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-22
2001-04-10
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S022100, C536S023100, C536S024310
Reexamination Certificate
active
06214556
ABSTRACT:
1. AREA OF THE INVENTION
The method to be patented here provides a new possibility for the differential diagnosis of cancer diseases. It leads to a deeper understanding of carcinogenesis and of the pathogenesis of polygenic inherited diseases. The method furthermore concerns the identification of all the genes participating in the development of diseases. As in the past, cell differentiation and the differentiation of higher organisms remains essentially not understood. Here too, the method promises to considerably increase knowledge.
The levels of observation that have been well studied by the methodological developments of recent years in molecular biology include the gene itself, the translation of genes in RNA, and the resulting proteins. When, during the course of the development of an individual, a gene is switched on, and how the activation and inhibition of certain genes in certain cells and tissues is controlled, can be correlated with a high degree of probability with the extent and the character of the methylation of the gene or the genome. In this regard, it is reasonable to assume that pathogenic conditions are expressed in a modified methylation pattern of individual genes or of the genome.
The state of the art is a method which allows the study of the methylation pattern of individual genes. More recent additional developments of this method also allow the analysis of minute quantities of starting material, where, however, the total number of measurement points remains at most a two-digit number, in theoretical range of values of at least 10
7
measurement points. Using the method to be patented, it is now possible, for the first time, to examine any desired sections of the genome with any desired number of measurement points. Thus, the method allows the identification of causes for genetic diseases of all types that could not be determined by any other means, and it allows the development of new treatment strategies and the identification of target proteins for new drugs.
2. STATE OF THE ART
2.1 State of the Art of Molecular Analysis of Cell Phenotypes
The study of gene expression can be at the RNA level or at the protein level. Both levels in principle reflect important phenotypic parameters. Protein assays using two-dimensional gels (McFarrel method) have been known for approximately 15 years. Using these assays, it is possible to elaborate the analysis of the chromatographic positions of several thousand proteins. Very early on, such electropherograms were already processed or evaluated with data processing means. In principle, the validity of the method is high, however, it is inferior to the modern methods of gene expression based on RNA analysis in two regards.
In particular, the detection of proteins that are of regulatory importance, from small quantities of cells, fails because of the fact that the sensitivity of the methods used is much too low. Indeed, in contrast to nucleic acids, proteins cannot be amplified. In addition, the method is very complex, not amenable to automation, and very expensive. In contrast, RNA analysis presents considerable advantages, and due to of the use of PCR it is more sensitive. Above all, each RNA species recognized to be important can be identified immediately by its sequence.
Overexpression or underexpression of individual RNAs with a known sequence can usually be easily detected; however, in connection with the applications discussed here, they are only valid in exceptional cases.
The method of “differential displays” at best allows a semiquantitative study of expression. Expression products amplified by PCR are separated by gel electrophoresis. The validity is limited as a result of the resolution of the gel electrophoresis. In addition, the method is insufficiently sensitive and robust for use in routine diagnosis (Liang, P. and Pardee, A. B., Science 257, 967-971).
Genes with high overexpression or underexpression are frequently identified by subtractive techniques. Here, cDNA clones of a cell or tissue species to be examined are plated. Against the clones, CDNA is hybridized as comparison material. Expression patterns cannot be reliably prepared using this technique.
One activity of the American “human genome project” is the systematic sequencing of expressed genes. The data obtained from this can be used to build expression chips, which allow the study of practically all expressed sequences of a cell or tissue type in a single experiment.
2.2 State of the Art in the Analysis of Cancer Diseases
Mutations in genes always trigger cancer diseases [sic], that is, cell degeneration. The causes of these mutations can be exogenous influences, or events in the cell. In a few exceptional cases, an individual mutation, which frequently affects larger regions of the genome (translocations, deletions), results in the degeneration of the cell; but in most cases a chain of mutations on different genes is involved, and it is only their combined effect that results in the malignant disease. These results on the DNA level are also reflected on the RNA and protein levels. In this context, it is highly probable that a multiplication occurs, because it is certain that in many cases the quantity and type of one RNA influences the extent of the synthesis of several other RNA species. This leads to a change in the synthesis rates of the corresponding proteins, which, in turn, can result in deregulating metabolism, and thus initiate the mechanism of regulation and counter regulation. The result is a gene expression pattern of the cells in question, that has been modified in a very specific (but largely nondeterminable) manner
the specificity is for a certain carcinoma, for the stage of the carcinoma, and the degree of malignancy of the carcinoma. So far, such phenomena have been outside the realm of study of natural sciences. Indeed, it has been impossible to examine the gene expression or the metabolism of a cell in its totality. Chip technology for the first time provided such a possibility (Schena, M. et al., Science 270, 467-470).
If one wishes to solve the diagnostic problem of early diagnosis of tumors on the molecular level, then one is confronted, today, with an insurmountable difficulty, with very few exceptions: Because, for most tumors, the knowledge of the molecular events, that is, the different mutations, is only fragmentary; researchers do not know what to look for in medical examination material. This means it is absolutely impossible to apply the remarkable sensitivity and specificity of the polymerase chain reaction. Examples are certain intestinal tumors, Ewing's sarcoma, and certain forms of leukemia, which are in fact each defined by a single, precisely described mutation. In those cases, it is possible to identify the degenerated cell among millions of normal cells. However, even within these apparently unambiguously defined tumor groups, there are such differences in the behavior that the conclusion must be drawn that additional unknown genetic parameters (such as, for example, the genetic background of the individual) play an important role. Immunological tumor markers are helpful auxiliary parameters, but they continue to make only a modest contribution, in addition to the other conventional diagnostic parameters. However, they can be used for the purpose of preselecting suspect cells.
Histology plays an important and indispensable role in the identification of degenerated tissues, but not precisely in early diagnosis.
Thus, because most tumors are not sufficiently characterized for diagnostic purposes on the molecular level, as a rule, no possibilities exist to proceed to a subdivision into stages or even a subdivision by degrees of risk. Such a subdivision, however, is an absolute prerequisite for an improved selection of treatments and, above all, for the development of effective new drugs and of gene therapy.
2.3 State of the Art in Research on the Number, Type and Properties of the Possible Stable States of Cells of Higher Organisms
In recent times, there has been an increase in the number o
Olek Alexander
Olek Sven Stefan
Walter Jörn
Brusca John S.
Epigenomics AG
Kriegsman & Kriegsman
Siu Stephen
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