Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-24
2001-06-26
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091210, C435S091400, C435S091510, C536S023100, C536S023200, C536S023500, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06251590
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the fields of biotechnology, medicine, biology and biochemistry. Applications thereof are aimed at human health, animal and plant care. More particularly, the invention makes it possible to identify nucleic acid sequences whereby both novel screening tools for identifying molecules of therapeutic interest and novel gene therapy means can be developed. The invention also provides information on molecular toxicity as well as pharmacogenomic data.
The present invention primarily describes a set of original methods for identifying nucleic acid sequences which rely on demonstrating qualitative differences between RNAs derived from two distinct physiological states being compared, in particular those derived from a diseased tissue or organ and healthy equivalents thereof. More precisely, these methods are intended to specifically clone differentially spliced alternative introns and exons with respect to a pathological condition and a healthy state or with respect to two physiological conditions one wishes to compare.
DISCUSSION OF THE PRIOR ART
The characterization of gene expression alterations which underlie, or are linked to a given disorder raises substantial hope regarding the discovery of novel therapeutic targets and of original diagnostic tools. However, identifying a genomic or complementary DNA sequence, whether through positional cloning or quantitative differential screening techniques, yields only little information if at all therefor the function or functional domains involved in regulation defects related to the disease under study.
DETAILED DESCRIPTION OF THE INVENTION
The instant invention describes a set of original methods aimed at identifying differences in splicing occurring between two distinct pathophysiological conditions. Identifying such differences provides information on qualitative but not on quantitative differences as has been the case for techniques described so far. The techniques disclosed in the present invention are hence all encompassed under the term of “qualitative differential screening”. The methods of the invention may be used to identify novel targets or therapeutic products, to devise genetic research and/or diagnostic tools, to construct nucleic acid libraries, and to develop methods for determining the toxicological profile or potency of a compound for example.
One particular object of the invention is first to provide a method for identifying alternatively (differentially) spliced nucleic acid regions occurring between two physiological conditions, comprising hybridizing RNAs derived from the test condition with cDNAs originating from a standard condition and identifying nucleic acids which correspond to alternative forms of splicing.
Another object of the invention is to provide a method for cloning differentially spliced nucleic acids occurring between two physiological states, comprising hybridizing RNAs derived from the test condition with cDNAs originating from the standard condition and cloning nucleic acids representative of alternative splicing patterns
In a particular embodiment, the method of nucleic acid identification and/or cloning according to the invention comprises running two hybridizations in parallel consisting of:
(a) hybridizing RNAs derived from the test condition with cDNAs derived from the standard condition;
(b) hybridizing RNAs derived from the standard condition with cDNAs derived from the test condition; and
(c) identifying and/or cloning those nucleic acids corresponding to alternative forms of splicing.
The present invention is equally directed to the preparation of nucleic acid libraries, to the nucleic acids and libraries thus prepared, as well as to uses of such materials in all fields of biology/biotechnology, as illustrated hereinafter.
As indicated hereinabove, the present invention relates in particular to methods for identifying and cloning nucleic acids representative of a physiological state. In addition, the nucleic acids identified and/or cloned represent the qualitative characteristics of a given physiological state in that these nucleic acids are generally involved to a great extent in the physiological state being observed. Thus, the qualitative methods of the invention afford direct exploration of genetic elements and protein products thereof, playing a functional role in the development of a pathophysiological state.
The methods of the invention are partly based on an original step consisting of cross hybridization between RNAs and cDNAs belonging to distinct physiological states. This or these cross hybridization procedures allow one to demonstrate in a convenient manner unpaired regions, i.e. regions present in RNAs in one physiological condition and not in RNAs from another physiological condition. Such regions essentially correspond to alternative forms of splicing, typical of a given physiological state, and thus form genetic elements or markers of particular use in the fields of therapeutics and diagnostics as set forth below.
The invention first deals with a method for identifying nucleic acids of interest comprising hybridizing RNAs of a test sample with cDNAs of a standard sample. This hybridization procedure makes it possible to identify the differences in splicing between the conditions under study, and in particular the splicings which are characteristic of the test condition.
According to one variant of the invention, the method allows therefore one to generate a nucleic acid population characteristic of splicing events that occur in a physiological test condition as compared to the standard (reference) condition (FIG.
1
A). As indicated hereinafter, this population can be used for the cloning and characterization of nucleic acids, including their use in diagnostics, screening, therapeutics and antibody production or synthesis of whole proteins or protein fragments. This population can also be used to generate libraries that may be used in different application fields as shown hereinafter (FIG.
1
C).
According to a further variant of the invention, the method comprises a first hybridization as described hereinbefore and a second hybridization, conducted in parallel, between RNAs derived from a standard condition and cDNAs derived from the test condition. This variant has great advantage since it allows one to generate two nucleic acid populations, one representing the characteristics (qualities) of the test condition with respect to the standard condition, while the other representing the characteristics of the standard condition in relation to the test condition (FIG.
1
B). These two populations can be utilized as nucleic acid sources, or as libraries which serve as finger prints of a particular physiological condition, as will be more fully described in the following (FIG.
1
C).
The present invention may be applied to all types of biological materials. In particular, the biological material can be any cell, organ, tissue, sample, biopsy material, etc. containing nucleic acids. In case of an organ, tissue or biopsy material, the samples can be cultured so as to facilitate access to the constituent cells. Of interest are samples derived from mammals (especially human beings), plants, bacteria and lower eucaryotic cells (yeasts, fungal cells, etc.). Relevant materials are exemplified in particular by a cancer biopsy, neurodegenerative plaque or cerebral zone biopsy displaying neurodegenerative signs, a skin sample, a blood cell sample obtained by collecting blood, a colorectal biopsy, a bronchial washing biopsy, etc. Examples of cells include muscular cells, hepatic cells, fibroblasts, nervous cells, epidermal and dermal cells, blood cells such as B-, T-lymphocytes, mastocytes, monocytes, granulocytes and macrophages.
As indicated above, the qualitative differential screening according to the invention allows the identification of nucleic acids characteristic of a particular physiological condition (condition B) in relation to a standard (reference) physiological condition (condition A), that are to be cloned or used f
Bracco Laurent
Schweighoffer Fabien
Tocque Bruno
Bieker-Brady Kristina
Clark & Elbing LLP
ExonHit Therapeutics S.A.
Horlick Kenneth R.
Siew Jeffrey
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