Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-04-22
2002-10-01
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S348000, C435S320100, C536S023100, C536S024100
Reexamination Certificate
active
06458559
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to monovalent and multivalent RNA aptamers, constructed DNA molecules and engineered genes which encode the RNA aptamers of the present invention, as well as expression systems, host cells, and transgenic organisms which express the RNA aptamers of the present invention.
BACKGROUND OF THE INVENTION
Cells and organisms are complex adaptive systems in which numerous biological processes are driven by sophisticated macromolecular machinery and regulated by elaborate signal transduction networks, both usually composed of multiple proteins. To better understand and control such processes, new technologies are needed to intervene in protein functions in the real time and space of the living cells. In many cases, such in vivo destructive approaches are needed to expand and extend results obtained from in vitro reconstruction studies. On the other hand, many diseases are known to be caused by either overexpression of certain endogenous genes (such as oncogenes in cancer) or expression of exogenous genes (as in the case of a virus infection), and “anti-gene” therapies are called for to avert or ameliorate the morbidity and mortality caused by these gene products. To inactivate a specific gene or gene product, different techniques are directed at three distinct types of targets: DNA, RNA, and protein. For example, a gene can be altered by homologous recombination, the expression of the genetic code can be blocked at the RNA level by antisense oligonucleotides or ribozymes, and the protein function can be altered or inhibited by antibodies or drugs.
A particularly useful tool resulting from the change of the protein coding function of genes is a conditional allele which displays its mutant phenotype only under certain non-permissive conditions, making it possible to obtain viable cells or organisms when a critical protein is under investigation. More importantly, with a conditional allele it is also possible to target and change specific genes in specific stages of development so that the details of a wrongly assembled protein machine can be identified. Recently there have been many new refinements of this technique. Notably, Struhl and colleagues developed a two-pronged approach to create yeast strains with conditional alleles in which the addition of copper ion leads to the simultaneous cessation of MRNA synthesis and destruction of the target protein in the cell (Moqtaderi et al., “TBP-Associated Factors Are Not Generally Required for Transcriptional Activation in Yeast,”
Nature
383:188-191 (1996)). However, the generation of conditional mutants in higher (i.e., multicellular) eukaryotes is quite difficult. In addition, it is often impossible to assay individual domains or discrete functional surfaces of a protein, since the function of the whole protein is abolished.
Small molecular mass drugs and drug derivatives that directly target proteins have been used not only clinically to rectify disease phenotype, but also in basic research that yielded ample information in mechanistic studies both in vitro and in vivo. These are usually cell-permeable, low molecular weight organic molecules identified from natural sources or designed and synthesized in the laboratory. Usually they are specific ligands of proteins, affecting protein functions upon binding. In many cases they are mimetics of the natural ligands of their targets (or receptors, as they are called in pharmacodynamics). In vivo experiments can be conducted easily with drugs at the cellular level since the administration may be simple diffusion governed by Fick's law. But systemic drug delivery to the organism is usually complicated by many pharmacokinetic factors, making it difficult to institute dosage regimens and assess drug effects at high temporal-resolution. The biggest limitation of using small molecular protein ligands is their availability. It is usually not easy to find such a ligand for a predetermined protein target, either from natural sources or by design. Recently, a general procedure for manipulating protein in vivo at the cellular level was developed, in which a gain of function results from the use of synthetic “dimerizers” derived from an immunosuppressive drug (Ho et al., “Dimeric Ligands Define a Role for Transcriptional Activation Domains in Reinitiation,”
Nature
382:822-826 (1996)). Although this “three-part invention” (Crabtree and Schreiber, “Three-Part Inventions: Intracellular Signaling and Induced Proximity,”
TIBS
21:418-422 1996)) may overcome the difficulty to a certain extent, a ligand-binding domain has to be appended to the target proteins.
As specific protein binding ligands, antibodies can be custom-made for virtually any given protein, due to the clonal selection and maturation function of the immune system. Antibodies raised against specific proteins have made possible many technological advances in the field of molecular biology, including modern immunochemistry (Harlow and Lane,
Antibodies: A Laboratory Manual
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)). But the in vivo utility of protein reagents like antibodies is severely limited by difficulties in their delivery and their own immunogenicity.
RNA has distinct advantages over proteins and small organic molecules when considering its use to inactivate protein function in vivo. An RNAencoding sequence can be linked to a promoter and this artificial gene introduced into cells or organisms. Depending on the regulatory sequence included, this provides a unique way of constructing a time and/or tissue specific suppresser gene. Such RINA expressing genes are usually smaller than protein-coding genes and can be inserted easily into gene therapy vectors. Unlike a foreign or altered protein, RNA is less likely to evoke an immune response. Antisense molecules and ribozymes have been developed as “code blockers” to inactivate gene function, with their promise of rational drug design and exquisite specificity (Altman, “RNase P in Research and Therapy,”
Bio/Technology
13:327-329 (1995); Matteucci and Wagner, “In Pursuit of Antisense,”
Nature
384 Suppl. (6604):20-22 (1996)). Mechanistically, both antisense oligodeoxynucleotides (“ODNs”) and bioengineered ribozymes are expected to achieve specific binding in the first step of their action by forming a stable duplex (or triplex in some cases of the ODNs) with a target nucleotide sequence based on Watson-Crick or Hoogsteen base pairing. However, this mechanism and their ability to disrupt the function of a single gene has never been proven. Furthermore, a wide variety of unexpected non-antisense effects have come to light, especially with the chemically modified compounds. Although some of these side effects may have clinical value, the use of antisense compounds as research reagents is severely limited (Branch, “A Good Antisense Molecule is Hard to Find,”
TIBS
23:45-50 (1998)).
Recently, RNA aptamers have also been explored as research and therapeutic reagents for their ability directly to disrupt protein function. Selection of aptamers in vitro allows rapid isolation of extremely rare RNAs that have high specificity and affinity for specific proteins. Exemplary RNA aptamers are described in U.S. Pat. No. 5,270,163 to Gold et al., Ellington and Szostak, “In vitro Selection of RNA Molecules That Bind Specific Ligands,”
Nature
346:818-822 (1990), and Tuerk and Gold, “Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase,”
Science
249:505-510 (1990). Unlike antisense compounds, whose targets are one dimensional lattices, RNA aptamers can bind to the three dimensional surfaces of a protein. Moreover, RNA aptamers can frequently discriminate finely among discrete functional sites of a protein (Gold et al., “Diversity of Oligonucleotide Functions,”
Annu. Rev. Biochem
. 64:763-797 (1995)). As research and therapeutic reagents, aptamers not only have the combined advantages of antibodies and small molecular mass drugs, but in vivo production of RNA aptamers a
Lis John T.
Shi Hua
Brusca John S.
Cornell Research Foundation Inc.
Nixon & Peabody LLP
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