WO2001064956A2 - Identification et utilisation de molecules effectrices et allosteriques pour alterer l'expression genique - Google Patents

Identification et utilisation de molecules effectrices et allosteriques pour alterer l'expression genique Download PDF

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WO2001064956A2
WO2001064956A2 PCT/US2001/006615 US0106615W WO0164956A2 WO 2001064956 A2 WO2001064956 A2 WO 2001064956A2 US 0106615 W US0106615 W US 0106615W WO 0164956 A2 WO0164956 A2 WO 0164956A2
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effector
control module
allosteric control
rna
gene
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PCT/US2001/006615
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WO2001064956A3 (fr
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William S. Marshall
Anastasia Khvorova
Sumedha Jayasena
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Amgen Inc.
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Priority to US10/220,403 priority Critical patent/US20050239061A1/en
Priority to JP2001563643A priority patent/JP2004504806A/ja
Priority to AU2001241900A priority patent/AU2001241900A1/en
Priority to CA002401654A priority patent/CA2401654A1/fr
Priority to MXPA02008470A priority patent/MXPA02008470A/es
Priority to EP01913217A priority patent/EP1290154A2/fr
Publication of WO2001064956A2 publication Critical patent/WO2001064956A2/fr
Publication of WO2001064956A3 publication Critical patent/WO2001064956A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead

Definitions

  • the enzymatic nucleic acid first recognizes and then binds a target through complementary base-pairing, and once bound to the correct site, acts enzymatically upon the target.
  • the enzymatic activity may involve a cleavage reaction. Strategic cleavage of a target RNA, for example, will destroy that RNA's ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its target, it is released from that molecule to search for another target and can repeatedly bind and cleave new targets.
  • RNA molecules can perform besides the cleavage of other RNA molecules. See for example, Robertson and Joyce, Nature 344:467, 1990; Ellington and Szostak, Nature 346:818, 1990; Piccirilli, et al, Science 256:1420, 1992; Noller, et al, Science 256:1416, 1992; Ellington and Szostak, Nature 355:850, 1992; Bock, et al, Nature 355:564, 1992; Beaudry and Joyce, Science 257:635, 1992; and Oliphent, et al, Mol. Cell. Biol 9:2944, 1989.
  • RNA molecules with a given function can be selected from a complex mixture of random molecules in what has been referred to as "in vitro genetics” (Szostak, TIBS 19:89, 1992) or "in vitro evolution".
  • in vitro genetics Szostak, TIBS 19:89, 1992
  • in vitro evolution a large pool of RNA molecules bearing random and defined sequences is synthesized and that complex mixture, for example, approximately 10 individual sequences, is subjected to a selection and enrichment process.
  • control or regulation of gene expression is a highly desired objective in the fields of protein production, diagnostics, transgenics, cell therapy and gene therapy.
  • a variety of expression control systems have been described as means to transcriptionally control the expression of a transgene in a recipient host cell.
  • Control means or gene switches include, but are not limited to, the following systems.
  • Rapalogs may be used to dimerize chimeric proteins which contain a small molecule-binding domain and a domain capable of initiating a biological process, such as a DNA-binding protein or transcriptional activation protein (as described in WO 9641865 (PCT/US96/099486); WO 9731898 (PCT US97/03137) and WO 9731899 (PCT/US95/03157)).
  • the dimerization of the proteins can be used to initiate transcription of the transgene.
  • An alternative regulation technology uses a method of storing proteins, expressed from the gene of interest, inside the cell as an aggregate or cluster.
  • the gene of interest is expressed as a fusion protein that includes a conditional aggregation domain which results in the retention of the aggregated protein in the endoplasmic reticulum.
  • the stored proteins are stable and inactive inside the cell.
  • the proteins can be released, however, by administering a drug (e.g., small molecule ligand) that removes the conditional aggregation domain and thereby specifically breaks apart the aggregates or clusters so that the proteins may be secreted from the cell. See Science 287:816-817 and 826-830, 2000.
  • Mifepri stone (RU486) is used as a progesterone antagonist.
  • the binding of a modified progesterone receptor ligand-binding domain to the progesterone antagonist activates transcription by forming a dimer of two transcription factors which then pass into the nucleus to bind DNA.
  • the ligand binding domain is modified to eliminate the ability of the receptor to bind to the natural ligand.
  • the modified steroid hormone receptor system is further described in U.S. Patent No. 5,364,791; WO 9640911 and WO 9710337.
  • ecdysone a fruit fly steroid hormone
  • cytoplasmic receptor a ecdysone receptor
  • the receptor then translocates to the nucleus to bind a specific DNA response element (promoter from ecdysone-responsive gene).
  • the ecdysone receptor includes a transactivation domain/DNA-binding domain/ligand-binding domain to initiate transcription.
  • the ecdysone system is further described in U.S. Patent No. 5,514,578; WO 9738117; WO 9637609 and WO 9303162.
  • Another control means uses a positive tetracycline -controllable transactivator.
  • This system involves a mutated tet repressor protein DNA-binding domain (mutated tet R - 4 amino acid changes which resulted in a reverse tetracycline-regulated transactivator protein, i.e., it binds to a tet operator in the presence of tetracycline) linked to a polypeptide which activates transcription.
  • mutated tet repressor protein DNA-binding domain mutated tet R - 4 amino acid changes which resulted in a reverse tetracycline-regulated transactivator protein, i.e., it binds to a tet operator in the presence of tetracycline
  • Knockout and transgenic animals are well known to those skilled in the art. Swanson et al., Annu. Rep. Med.
  • a knockout animal has been genetically altered to disrupt the expression of a targeted gene, resulting in the elimination of the target gene product. Knockout animals are widely used to demonstrate the function of a protein of interest. In particular, the elimination of the expression of the targeted gene in the knockout animal can indicate the effect of inhibiting the protein product of the gene.
  • targeted gene disruption can cause developmental defects which, although not indicative of the effect of target gene inhibition in an adult animal, result in embryonic lethality. Thus, certain disruptions of gene function cannot be studied in a viable animal.
  • Another limitation of current knockout technology is the effect of developmental compensation on targeted gene disruption. In the course of development, other related gene products may compensate for the lost function of the disrupted gene and, thus, obscure its function in the adult animal.
  • These well known limitations of the technology can significantly restrict the utility of targeted gene disruption in knockout animals.
  • transgenic animals an additional copy of the gene of interest is introduced into the organism and results in the over expression of the gene product. Protein over expression during development can cause defects or lead to compensation for or inhibition of over expression. These problems can obscure the effects of transgene over expression and limit the ability to interpret the biological effects of target gene over expression. The ability to create a conditional knockout animal is particularly important and relevant to overcome these limitations.
  • Figure 1 is a diagrammatic representation of the present invention involving an allosteric control module containing a self-cleaving RNA domain, the activity of which is inhibited by interacting with an effector, thereby resulting in the translation of the mRNA. It will be appreciated by those skilled in the art that the allosteric control module may be placed 5 ' or 3 ' of the gene of interest.
  • Figure 3 is a diagrammatic representation involving an allosteric control module containing a self-splicing RNA domain, the activity of which is initiated by interacting with an effector.
  • the presence of effector provides for the activation of the allosteric control module such that splicing of the precursor mRNA occurs to result in the formation of a translatable mRNA.
  • Figure 5 is a diagrammatic representation of the present invention involving an allosteric control module containing a self-splicing RNA domain.
  • the activity of the alloste ⁇ c control module is inhibited by interacting with an effector.
  • the means to identify a useful effector molecule is desc ⁇ bed as is the use of the identified effector to evolve a cognate aptamer.
  • the construction of an alloste ⁇ c control module is desc ⁇ bed in which a catalytic RNA forms a part of or is linked to the effector-bindmg RNA domain or aptamer, thereby placing the activity of the catalytic RNA under the control of the effector and reqm ⁇ ng the presence of the effector for activation or inactivation.
  • RNA molecules are constructed in which at least one portion is capable of binding an effector and another portion is a catalytic RNA.
  • the present invention involves both the evolution of RNA sequences which bind the effector and a selection process in which the alloste ⁇ c control modules are identified by their catalytic function in the presence and absence of the effector.
  • regulatable catalytic RNAs may be selected for use in cleaving, splicing or ligating a target RNA in the presence of an effector, or in cleaving, splicing or ligating a target RNA in the absence of an effector.
  • allosteric control modules are useful in altering the expression of a target RNA molecule in a controlled fashion. It is particularly useful when the target RNA molecule is formed in or delivered to the cell in combination with the allosteric control module.
  • Disadvantages associated with previously known control constructs and their uses include potential toxicities in in vivo use, including the transcriptional activation or repression of endogenous genes, the activation or inhibition of proteins or cellular processes that the small molecule entity normally regulates, or the induction of an immune response towards the foreign proteinaceous gene products of the control system.
  • the development of a means of controlling gene expression wherein that means may also be varied by the individual components used would contribute significantly to any strategy of gene therapy as well as to the production of therapeutic proteins.
  • the present invention uniquely solves these problems.
  • the expression of a specific gene can be altered at any step in the process of producing an active protein.
  • the modulation of total protein activity may occur via transcriptional, transcript-processing, translational or post-translational mechanisms. Transcription may be modulated by altering the rate of transcriptional initiation or the progression of RNA polymerase. Transcript- processing may be influenced by circumstances such as the pattern of RNA splicing, the rate of mRNA transport to the cytoplasm, or mRNA stability.
  • the present invention primarily concerns the identification and use of effector molecules and the creation of allosteric control module molecules which act together to alter the expression of a target gene (for example, altering the in vivo concentration of a target protein by altering RNA processing.)
  • the present invention provides an identification process for suitable effectors for use in the control of gene expression, which process has not been previously described.
  • the combination of the aptamer and catalytic RNA domain is selected such that the conformational change may either preclude formation of an active catalytic domain or induce the formation of an active catalytic domain.
  • the effector-induced conformational change may be selected to cause the inhibition of or a reduction in the activity of the catalytic domain due to steric interference between the aptamer and the catalytic domain tertiary structures.
  • the effector-induced conformational change may be selected to cause the initiation of or an increase in the activity of the catalytic domain. Therefore, the term "activation" or “activated” is used herein to refer to either the initiation of or an increase in or enhancement of catalytic activity.
  • the domains of the allosteric control module may be non-overlapping or partially overlapping such that one or more domains are encoded in part by the same polynucleotide. Thus, the domains are primarily distinguished by their function rather than by their sequence.
  • the domains may be separately prepared and then joined to form the allosteric control module, or the allosteric control module may be prepared as a single polynucleotide having both aptamer and catalytic domains.
  • the allosteric control module of the present invention does not have true "enzymatic" activity.
  • aptamers The in vitro evolution of aptamers begins with a pool of RNA molecules created by chemical and/or enzymatic synthesis.
  • the desired aptamer is selected based upon its ability to interact (e.g., recognize and bind) with an effector.
  • the effector is a predetermined molecule which is used to select and further evolve a suitable aptamer.
  • the aptamer may be constructed and libraries of molecules screened to identify and select a suitable effector.
  • the aptamer is not an isolated and purified chemical entity. Instead, the aptamer is encoded by a DNA which is delivered to a cell, and the aptamer becomes a portion of the mRNA transcribed from that DNA in the host cell.
  • the effectors of the present invention do not modify a biological activity of an aptamer because the aptamers of the present invention have no inherent physiological activity in the recipient cell.
  • the aptamer will contain 20 to 300 nucleotides, selected as described herein for binding to a specific effector.
  • the small size of the molecule typically 200 nucleotides or less, preferably between 20-30 nucleotides in length
  • the RNA is random only as originally used in the evolution process.
  • the product of the evolution process i.e., the aptamer(s) is not a random sequence. It is a specific sequence which binds with a high degree of affinity and specificity to a defined effector.
  • aptamers of the allosteric control modules described herein are not limiting in the invention. Those skilled in the art will recognize that all that is important in an aptamer of the present invention is that it selectively and specifically interact with a suitable effector, and that it have the ability to alter the catalytic activity of the allosteric control module when the aptamer has interacted with the effector. Multiple aptamers (as well as multiple allosteric control modules) may also be used so that multiple effectors and even multiple different effectors may be used to react with allosteric control modules and thereby alter gene expression by altering the precursor mRNA.
  • domain refers to a polynucleotide which provides a selected activity or function to the allosteric control modules of the present invention.
  • effector refers to a molecule which interacts with an aptamer. Binding may be the result of the interaction and may include, but is not limited to, hydrogen binding, hydrophobic interactions, intercollations, etc.
  • Suitable molecules for use as effectors of the present invention include, but are not limited to, organic or inorganic molecules, peptides, polypeptides, proteins, oligonucleotides, polynucleotides, nucleic acids, naturally occurring metabolites and biological effectors, lipids, carbohydrates (polysaccharides, sugar), fatty acids, and polymers.
  • the preferred effector molecules of the present invention are distinguished from those described in the art in that the present effectors have either no pharmalogical effect or are used at concentrations wherein a pharmalogical effect either is not observed or is negligible.
  • "Suitability" of the effector for use with the allosteric control module may include the following characteristics. (1) The effector has little or no pharmalogical effect in the dosage range used in altering gene expression or has a negligible pharmalogical effect.
  • the effector may be used as a pharmaceutical agent that has an effect on a structure or process other than the allosteric control module, then that effect does not cause any harm to the patient not in need of treatment by that pharmaceutical agent.
  • the effector will undergo biodistribution to that cell or tissue which will contain the allosteric control module.
  • the effector has the ability to pass to subcellular structures, i.e., to the allosteric control module in the nucleus of cells transformed for regulated gene expression. This may be by means of intracellular diffusion or transport, and most preferably, by intranuclear diffusion or transport.
  • the effector has the ability to interact with an aptamer wherein the interaction occurs with high specificity and a high affinity.
  • Additional considerations for the identification of a suitable effector may further include the following. (1) If the effector is a molecule which has a dose- effect relationship, then the molecule typically is used at a daily maximum dose which is less than the usual daily minimum dose of the molecule when used for an approved indication. Preferably such an effector is used at a dosage level which is below 25% of the effective dose (ED25) of the molecule when used as a pharmaceutical agent. It will be appreciated by those skilled in the art, however, that such a preference is based on a case-by-case evaluation of the agent. For example, in the case of an effector which is an antiviral agent, the effector could be used at any dosing range in the absence of the virus.
  • the dosage level is below the ED 10 for the molecule.
  • the effector is a molecule which has a dose-effect relationship
  • the molecule is used as an effector at a dose which is below the lowest effective dose (threshold dose) of the molecule when used as a pharmaceutical agent.
  • the use of a pharmaceutical agent related to the effector is not contraindicated in patients having the condition to be treated by the regulated gene.
  • non-significant effects would include, for example, events such as headache, dizziness, lightheadedness, sedation, nausea, vomiting, rash, constipation, diarrhea, abdominal pain, euphoria, dysphoria, fatigue, arthralgia which can be controlled by dose adjustment or other common intervention or which occurs in less than five percent of the population receiving the effector.
  • the effector is a small molecule.
  • the nature of the effector can be chosen to be exogenously supplied, such as some non-toxic molecule or drug which readily enters at least the cells containing the targeted RNA.
  • an entirely endogenous system in which the controlling effector is some endogenous metabolite or macromolecule which is directly or indirectly related to the pathology to be corrected or the gene to be expressed or the molecule to be produced.
  • a protein encoded by the target RNA could be the effector.
  • the construct may be designed such that the activity of the allosteric control module is dependent on binding to the expressed protein and as the level of protein increases the activity of the module increases to cause a decrease in expression. As the level of target RNA falls due to alteration (e.g., cleavage) by the allosteric control module, the concentration of the protein (as ligand or effector) also falls.
  • the "pharmalogical activity" of a molecule is used to refer to the activity of that molecule as a drug or medication.
  • a “database” as used herein refers to any compilation of information on potential effectors, such as small molecules, containing information concerning the suitability of the effector for use in controlling gene expression.
  • the "catalytic activity" of the allosteric control module refers to the activity of the "catalytic domain" or "catalytic sequence” which is a nucleic acid which acts on a target nucleic acid in a desirable manner. Examples of possible actions include, but are not limited to binding of the target, reacting with the target in a way which modifies/alters the target as by cleavage, splicing or ligation or the functional activity of the target, or facilitating the reaction between the target and another molecule.
  • the catalytic activity is a self-cleaving activity, a ligase activity, or a splicing activity. Such activities are often associated with ribozymes.
  • Ribozymes including ribozyme-like molecules and portions of such molecules, may be used to form the catalytic domain of the allosteric control module of the present invention. It will be appreciated by those skilled in the art that it is primarily the catalytically active portion of the naturally occurring ribozyme or non-naturally occurring ribozyme- like molecules that is used in the allosteric control modules of the present invention, but that additional domains also may be used. For example, if the allosteric control module is self-cleaving, then in addition to an aptamer and a catalytic domain, the module will further include a substrate domain.
  • the "catalytic activity" of the allosteric control module merely refers to the alteration or modification of an interaction with a target RNA.
  • the catalytic domain may be designed such that it may or may not be consumed in the process, and therefore, the domain is not required to be a true "catalyst”.
  • the RNA ligase may have complementarity in a substrate binding region to a specified target polynucleotide, and also has a catalytic activity to specifically join RNA in that target.
  • complementarity is meant a nucleic acid that can base pair (e.g., form hydrogen bond(s)) with other RNA by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions. This complementarity functions to provide sufficient hybridization of the ligase domain to the target RNA to allow the reaction to occur. Complementarity of 100% is preferred, but complementarity as low as 50-75% may also be useful.
  • the nucleic acids may be modified at the base, sugar, and/or phosphate groups.
  • non-natural polynucleotide refers to a polynucleotide sequence or construct that does not occur in nature.
  • the preferred allosteric polynucleotides of the present invention do not occur in nature.
  • isolated polynucleotide refers to a nucleic acid molecule of the invention that is free from at least one contaminating nucleic acid molecule with which it is naturally associated, and preferably substantially free from any other contaminating mammalian nucleic acid molecules which would interfere with its use in protein production or its therapeutic or diagnostic use.
  • splice recognition region refers to a sequence in the precursor RNA which serves as a splice donor, splice acceptor or spliceosome binding site.
  • the splice donor is typically a site at the 3' end of the exon (located at the 5' end of the intron to be removed), and the splice acceptor refers to a site at the 5' end of the adjacent exon to be ligated (the 3' end of the intron to be removed).
  • “Spliceosome” refers to a large, multicomponent complex of cellular protein and RNA that binds to and directs the processing of the pre -RNA into mRNA by cleavage, the removal of introns, and the ligation of exons.
  • the 5'-UTR signals the beginning of RNA transcription and contains sequences that direct the mRNA to the ribosomes and cause the mRNA to be bound by ribosomes so that protein synthesis can occur.
  • the second region is known as an open reading frame and contains the information that can be translated into the amino acid sequence of the protein or function as a bioactive RNA.
  • the third region located at the 3' end, contains the signals for the termination of translation and for the addition of a polyadenylation tail (poly(A)) and is not translated into protein (i.e., the 3'-UTR).
  • poly(A) polyadenylation tail
  • the 3'-UTR can provide mRNA stability.
  • the intron/exon boundary will be that portion in a particular gene where an intron section connects to an exon section.
  • the terms "TATA box” and "CAP site” are used as they are recognized in the art.
  • vector is used to refer to any molecule (e.g., nucleic acid, plasmid, virus, small molecule, liposome, carrier molecule, etc.) used to transfer coding information to a host cell.
  • molecule e.g., nucleic acid, plasmid, virus, small molecule, liposome, carrier molecule, etc.
  • control sequences and “control elements” are used to refer collectively to non-coding regulatory sequences including, but not limited to, promoters, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, enhancers, and the like, which are operably linked to the DNA encoding a gene of interest to provide for the transcription and translation of the coding sequence in a recipient cell. Not all of these control elements need always be present so long as the sequence encoding the gene of interest is capable of being transcribed and translated in an appropriate host cell in accordance with the expression regulatory means of the present invention.
  • a “promoter” is used to refer to a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis.
  • An "origin of replication” is a sequence on a vector or host cell chromosome that renders extragenomic elements (e.g., viruses or plasmids) capable of replicating independently of the host cell genome.
  • Enhancers are cis-acting elements of DNA that stimulate or inhibit transcription of adjacent genes. An enhancer that inhibits transcription is also termed a “silencer”.
  • control sequence need not be contiguous with the coding sequence, so long as it functions correctly.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
  • induction means that the level of translation of a target mRNA is increased above that observed in the absence of the regulation means of the present invention.
  • induction of expression may range from the initiation of the translation of the target mRNA to an increase in the translation of the target mRNA.
  • phrase "specifically controlling the expression of the gene of interest” as used herein means altering the expression of the gene of interest without altering the expression of other genes in the cell in a way which would cause an adverse effect on (a) an organism containing the cell in the case where the cell is within the organism or (b) the growth or the culturing of the cell, in the case where the cell is being grown or cultured to make a product where the amount of product produced is associated with expression of the gene of interest.
  • the production of a recombinant protein or peptide can be affected by the efficiency with which DNA (or an episomal nucleic acid) is transcribed into mRNA.
  • Conventional control systems seek to affect the transcription event.
  • the production of a recombinant protein or peptide can also be affected by the efficiency with which precursor mRNA is modified to form the mRNA which is translated into protein.
  • the novel constructs and methods of the present invention advantageously provide for the regulated expression of a gene of interest, such as a therapeutic protein, by altering the process of pre-mRNA processing.
  • the allosteric control modules of the present invention contain catalytic and effector-binding domains that are specifically selected such that the interaction of the effector-binding domain or aptamer with an effector alters the activity of the allosteric control modules.
  • the interaction of the effector and aptamer may result in a conformational change in the allosteric control module.
  • the conformational change can result in either an increase or a decrease in the activity of the catalytic domain of the module. This in turn affects whether or not the precursor mRNA is appropriately modified to form a mRNA capable of being translated into a protein of interest.
  • a conformational change may be caused by the binding energy derived from the effector-aptamer interaction which is used to shift the thermodynamic balance between two possible confirmations of the allosteric control module.
  • the allosteric control module is either activated or inactivated by the effector.
  • the present invention includes an expression regulation format involving an allosterically activated self-cleaving RNA as the catalytic domain of the allosteric control module.
  • the allosteric control module may be encoded in a mRNA with the gene of interest.
  • the DNA sequence is designed to encode a self-cleaving site that separates the cap and message sequences.
  • the aptamer and catalytic domain may be selected such that in the absence of an effector, the catalytic domain is active and that activity results in the cleavage of the pre- mRNA or mRNA.
  • the gene of interest is not expressed because the mRNA can not be translated.
  • a preferred embodiment of the present invention involves constructs which provide gene expression in the presence of the effector.
  • the aptamer and catalytic domain may be selected such that in the presence of an effector, the catalytic domain is active and that activity results in the cleavage of the mRNA.
  • the gene of interest is not expressed because the mRNA can not be translated.
  • the catalytic domain is inactive or unable to act upon the RNA substrate.
  • the mRNA is not cleaved and the gene of interest is expressed.
  • the gene is expressed.
  • the catalytic domain may include an engineered intron which contains a self-cleaving site.
  • the aptamer and effector are selected such that the domain does not cleave if effector is absent.
  • the mRNA is not recognized as an active molecule and is not translated.
  • the presence of the el in the pre-mRNA can result in the inhibition of gene expression by a variety of mechanisms that can act individually or jointly.
  • Such mechanisms include, but are not limited to 1) the el may be designed to contain several stop codons which would arrest translation; 2) the el could code for nonsense amino acids resulting in a protein with multiple inactivating mutations; and 3) the presence of the el could activate the mRNA surveillance system native to cells that would sequester or destroy the altered pre-mRNA.
  • the format may involve an engineered intron designed such that in the presence of effector the allosteric control module is inactive and in the absence of the effector the module is active.
  • the present invention further includes an expression regulation format involving an inhibitable allosteric self-splicing intron.
  • the allosteric control module is inserted into an intron in a region necessary for spliceosome assembly such that the action of the self-splicing intron results in the removal of a nucleotide sequence necessary for normal splicing.
  • This embodiment is depicted in Figure 6.
  • the allosteric control module is active, thus leading to the removal of the vital splice recognition site.
  • the removal of the splice recognition site results in an altered open reading frame that leads to the inhibition or reduction of protein expression.
  • the allosteric control module is inactive and, as a result, normal splicing occurs at the splice recognition sites, the correct open reading frame is formed, and the protein is expressed.
  • Pharmacokinetic evaluations may include analyses of an effector's absorption, distribution, metabolism and excretion profiles in in vitro cell systems, animals, animal disease models, normal humans and patients.
  • the absorption profile includes the rate of absorption, the maximum plasma concentration achieved, the effect of formulation modifications, the effect of different salt and crystal forms, the effect of food and other medications on absorption and the like.
  • the distribution profile includes the determination of the location and concentration of the effector in the various tissues and fluids of the body, protein binding and the like.
  • Effector physical properties of interest include solubility, chemical stability, such as the effect of temperature, moisture and light, crystal form (solid), salt form (solid or in solution) and the like, solution stability, effect on solution pH, crystal vs. amorphous solid vs. oil vs. liquid, crystal density, etc.
  • effector formulation properties are evaluated. These properties include, but are not limited to, stability, effect of the crystal form and size on absorption, effect of the salt form on absorption, solid compressibility and malleability, solid flowability, uniformity of crystal size, compatibility with other formulation ingredients, packing density, blend and content uniformity of each formulation.
  • Toxicology involves determining the toxic and other side effect profile of the effector, its metabolites and its formulation, generally initially in animals and later in humans, to ascertain the potential risks involved in administering the effector.
  • Analyses may include an evaluation of undesirable effects on the central nervous system, cardiovascular system, pulmonary system, gastrointestinal system, renal system, hepatic system, genitourinary system, hematopoietic system, immunologic system and dermal system.
  • the analyses may include determining toxic dose, maximum tolerable dose, agonistic or antagonistic activity against other enzymes, receptors, binding proteins and the like, carcinogenicity, immunogenicity, and the like. Means and methods of toxicological analysis are well known in the art.
  • the final selection of a suitable effector will include testing similar to that performed for any new molecular entity (NME) proposed for human use.
  • NME new molecular entity
  • a variety of screening strategies may be used.
  • NME new molecular entity
  • recent advances in the understanding of the molecular biology and functional specificity of metabolic enzymes and absorption and transport mechanisms have provided a mechanistic basis for gathering absorption and metabolism data utilizing "humanized” in vitro systems.
  • the development and availability of these humanized in vitro systems coupled with advances in analytical instrumentation are speeding the development process. It is increasingly possible to conduct high- throughput pharmacokinetic screening of new molecules.
  • the following description will highlight the in vitro and in vivo methods and techniques that are being applied.
  • NMEs New higher throughput methods are also being developed to screen NMEs for physicochemical properties such as solubility that could influence NME absorption (Tarbit et al, Curr. Opin. Chem. Biol. 2:411-416, 1998.) Others are developing methods to determine if NMEs are substrates of various intestinal transporters (e.g., p-glycoprotein). Permeability data, coupled with physiochemical and transport data, should enhance the ability to predict absorption in the future and will lead to faster selection of lead candidates with desirable absorption characteristics.
  • PK parameters e.g., clearance, volume of distribution, elimination half-life, and oral bioavailability
  • the process may involve the following steps. 1) A candidate mixture of nucleic acids of differing sequence is prepared.
  • the candidate mixture typically includes nucleic acids having regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences.
  • the fixed sequence regions may be used for a variety of reasons, including: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the effector, or (c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture.
  • the randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
  • the candidate mixture is contacted with the selected effector under conditions favorable for an interaction between the effector and nucleic acids of the candidate mixture.
  • the interaction between the effector and the nucleic acids can be considered as forming aptamer-effector pairs between the effector and those nucleic acids having the strongest affinity for the effector.
  • the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the effector will generally increase. With repeated cycles, the process yields a candidate mixture containing one or more unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the effector.
  • the aptamer will typically contain about 20 to about 50 nucleotides, or more preferably about 20 to about 30 nucleotides, evolved for binding to a specific effector.
  • the small size of the molecule is advantageous for cell delivery as compared with conventional expression regulation constructs which are much larger in size.
  • in vitro evolution techniques may be used to create and identify separate aptamers which may then be used as modular units with catalytic structures for the construction of a variety of different allosteric control modules.
  • the allosteric control modules may be evolved as a single unit with separate regions or domains including an aptamer or effector-binding domain, a catalytic domain, and in some embodiments additional domains including, but not limited to, a target RNA recognition domain and a substrate domain.
  • aptamer binding e.g., large combinatorial libraries of effectors (e.g., organic compounds, peptides, small molecules, etc.) produced by chemical synthesis can be evaluated for aptamer binding.
  • Phage display libraries may also be screened for the presence of an effector to bind a selected aptamer.
  • the aptamer may be identified and the effector selected through a screening process.
  • the other main component of the allosteric control module is the catalytic domain.
  • in vitro evolution may also be used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage, ligation and splicing.
  • the catalytic domain is a highly specific construct, with the specificity of activity depending not only on the base- pairing mechanism of binding, but also on the mechanism by which the molecule affects the expression of the RNA to which it binds. For example, if inhibition is caused by cleavage of the RNA target, specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA.
  • the present invention contemplates, in one embodiment, the provision of a desired gene that encodes a protein that is defective or missing from a target cell genome in a patient.
  • the present invention also contemplates a method of treating a patient suffering from a disease state by providing the patient with human cells genetically engineered to encode a required protein.
  • Yet another embodiment is the delivery of a gene to correct a genetic defect.
  • the gene of interest is delivered to the recipient cell with an allosteric control module which has been designed to alter the expression of the gene in response to the presence or absence of an identified effector.
  • Exemplary uses of constructs of the present invention include gene therapy for hereditary diseases. These diseases include, but are not limited to: familial hypercholesterolemia or type II hyperlipidemias (LDL receptor), familial lipoprotein lipase deficiency or type I hyperlipidemias (lipoprotein lipase), phenylketonuria (phenylalanine hydroxylase), urea cycle deficiency (ornithine transcarbamylase), von Gierke's disease (e.g., glycogen storage disease, type I; glucose-6-phosphotases), alpha 1-antitrypsin deficiency (alpha 1-antitrypsin), cystic fibrosis (cystic fibrosis transmembrane conductant regulator), von
  • Willebrand's disease and hemophilia A (Factor VIII), hemophilia B (Factor IX), sickle cell anemia (beta globin), beta thalassemias (beta globin), alpha thalassemias (alpha globin), hereditary sperocytosis (spectrin), severe combined immune deficiency (adenosine deaminase), Duchenne muscular dystrophy (dystrophin minigene), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyl transferase), Gaucher's disease (beta-glucocerebrosidase), Nieman-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase), and maple syrup urine disease (branched-chain keto acid dehydrogenase).
  • Suitable transgenes for use in the present invention further include, but are not limited to, those encoding proteins such as: nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophins 3 and 4/5 (NT-3 and 4/5), glial cell derived neurotrophic factor (GDNF), transforming growth factors (TGF), and acidic and basic fibroblast growth factor (aFGF and bFGF); sequences encoding tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC); sequences encoding superoxide dismutase (SOD 1 or 2), catalase and glutathione peroxidase; sequences encoding interferons, lymphokines, cytokines and antagonists thereof such as tumor necrosis factor (TNF), CD4 specific antibodies, and TNF or CD4 receptors; sequences encoding GABA receptor isoforms, the GABA synthesizing enzyme glutamic acid decarboxy
  • the vaccine vectors may be used to generate intracellular immunity if the gene product is cytoplasmic (e.g., if the gene product prevents integration or replication of a virus). Alternatively, extracellular/systemic immunity may be generated if the gene product is expressed on the surface of the cell or is secreted.
  • a host especially a human host
  • Immunization of a human host with a vector of the invention typically involves administration by inoculation of an immunity-inducing dose of the virus by the parenteral route (e.g., by intravenous, intramuscular or subcutaneous injection), by surface scarification or by inoculation into a body cavity.
  • parenteral route e.g., by intravenous, intramuscular or subcutaneous injection
  • surface scarification or by inoculation into a body cavity e.g., by intravenous, intramuscular or subcutaneous injection
  • inoculation typically involves administration by inoculation of an immunity-inducing dose of the virus by the parenteral route (e.g., by intravenous, intramuscular or subcutaneous injection), by surface scarification or by inoculation into a body cavity.
  • inoculations typically between about 1000 and about 10,000,000 infectious units each, as measured in susceptible human or nonhuman primate cell lines, are sufficient to effect immunization of a human host. Additional uses of the materials and methods described herein include
  • Monoclonal antibodies could be produced by growing the hybridoma in tissue culture or in vivo; 2. increasing plant derived products as used in fragrances and perfumes, flavoring compounds or sweeteners e.g. the basic proteins from Thaumatococcus danielli, insecticides, anti-fungal compounds or pesticides;
  • biodegradation is effected by the conversion of petroleum products to emulsified fatty acids.
  • Bacteria useful in this invention include, but are not restricted to, Archromobacter, Arthrobacter, Flavobacterium, Nocardia, Pseudomonas (e.g. Pseudomonas oleovorans) and Cytophaga.
  • Yeast useful in this invention include, but are not restricted to, Candida (e.g., Candida tropicalis), Rhodotorula, and Trichosporon;
  • the rate limiting step in gluconic acid production by e.g., Pseudomonas sp., Gluconobacter sp., and Acetobacter sp , or the rate limiting step in the production of ammo acids by bacte ⁇ a or fungi;
  • Streptocucus sp Other examples include anionic polysacha ⁇ des from Arthrobacter viscosus, bacte ⁇ al alginates from Azotobacter vmelandn, and xanthan from Xanthomonas campest ⁇ s;
  • a further use of the allosteric control modules of the present invention is in the creation of conditional knockout and transgenic animals.
  • conditional knockouts the targeted gene product is expressed normally in the genetically altered animal and expression is inhibited only in the presence of an effector.
  • effectors described herein and the allosteric control modules evolved by the methods described herein to conditional knockouts one desires to evolve allosteric self-cleaving RNAs that are activated by a small molecule effector identified by the inventive methods.
  • the altered gene construct could be introduced to adult animals via viral or naked DNA transfer methods, akin to those being contemplated for gene therapy applications.
  • over expression of the gene of interest would normally be inhibited due the insertion of the desired allosteric control modules as described herein.
  • the subsequent dosing of the animal with the effector would then result in the over expression of the gene product for assessment of functional outcomes.
  • vectors may be delivered through implanting into patients certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides, fragments, variants, or derivatives.
  • Such cells may be animal or human cells, and may be derived from the patient's own tissue (autologous) or from another source, either human (allogeneic) or non-human (xenogeneic).
  • the cells may be immortalized.
  • the cells may be encapsulated to avoid the infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow release of the protein product(s) but prevent destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
  • the D ⁇ A constructs described herein can be incorporated into a variety of vectors for introduction into cells.
  • Suitable vectors include, but are not restricted to, naked D ⁇ A, plasmid D ⁇ A vectors, viral D ⁇ A vectors (such as adenovirus or adeno-associated virus vectors), viral R ⁇ A vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra) and non-viral vectors (such as DNA complexed with cationic lipids or packaged within liposomes).
  • an expression vector will also include a) a transcription initiation region; b) a transcription termination region, and c) expression control sequences.
  • the constructs of the present invention may be used with any vector system, a preferred system involving the use of rAAV particles is described as an exemplary system for the following descriptions of pharmaceutical compositions.
  • compositions may comprise a therapeutically effective amount of an rAAV particle product in admixture with a pharmaceutically acceptable agent such as a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable agent such as a pharmaceutically acceptable carrier.
  • An exemplary carrier material may be water for injection, preferably supplemented with other materials common in solutions for administration to mammals.
  • an rAAV particle therapeutic compound will be administered in the form of a composition comprising particles in conjunction with one or more physiologically acceptable agents.
  • Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers.
  • Other standard pharmaceutically acceptable agents may be included as desired.
  • other compositions may comprise a buffer or preservative.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which the rAVV particle composition is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation may involve the formulation of rAVV particles with an agent, such as injectable microspheres, bio-erodible particles or beads, or liposomes, that provides for the controlled or sustained release of the product which may then be delivered as a depot injection.
  • the preparations of the present invention may include other components, for example parenterally acceptable preservatives, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, antioxidants and surfactants, as are well known in the art.
  • a method for identifying an effector for use in the evolution of aptamers and the alteration of gene expression involves the following steps.
  • the first step is the selection a set of desired characteristics for an effector, wherein the desired characteristics include one or more of the following: a) at least 1% bioavailability; b) biodistribution to tissue containing an allosteric control module; c) the ability to pass to the nucleus of the cell; d) either no drug interactions or manageable drug interactions; e) either no toxicity or acceptable toxicity at the dosage range used; f) either no side effects or acceptable side effects at the dosage range used; g) either no pharmacological effect at the dosage range used in regulating transgene expression or a negligible pharmacological effect; and h) physical properties suitable for the in vitro evolution of an aptamer.
  • the preferred effectors include the nonnucleoside reverse transcriptase inhibitors. These compounds have good oral bioavailability, are nontoxic and have no known activity other than against the viral target.
  • nucleic acids are converted into complementary DNA (if the starting pool of nucleic acids is RNA).
  • Nucleic acids with desired trait may be separated or partitioned from the rest of the population of nucleic acids by a variety of methods. For example, a filter-binding assay can be used to separate the fraction that binds the desired effector from those that do not.
  • the fraction of the population that is bound by the effector (for example) may be the population that is desired (active pool).
  • the binding of the effector and the RNA is assessed by applying the RNA pool or mixture to an affinity matrix containing the effector with which the aptamer will specifically bind or react.
  • the non-binding RNA species are removed or washed away, and the specifically-binding species are eluted from the effector for further use in the evolution process.
  • a new piece of DNA (containing new oligonucleotide primer binding sites for PCR and restriction sites for cloning) may be introduced to the termini of molecules in the active pool (to reduce the chances of contamination from previous cycles of selection) to facilitate PCR amplification and subsequent cycles (if necessary) of evolution.
  • Amplification is preferably performed by means of reverse transcription of the eluted species into DNA followed by polymerase chain reaction. The result of the amplification process is the production of a large number of the selected RNA-encoding DNA molecules.
  • the final pool of nucleic acids with the desired trait may be cloned into a plasmid vector and transformed into bacterial hosts. Recombinant plasmids can then be isolated from transformed bacteria, and the identity of clones can be determined using DNA sequencing techniques.
  • the cycles are typically repeated five or more times.
  • the evolutionary steps of selection and amplification may be continued until no significant improvement in binding strength of RNA to effector is achieved upon repetition of a cycle.
  • the selected population is passed over an affinity column containing the negative selection target and those nucleic acids which bind to the negative selection target are removed from the selected pool.
  • the selected population may be passed over an affinity column containing the desired effector, and the pool is then challenged by the addition of a negative selection target.
  • this process would also involve the performance of two to three negative selections using the negative selection target and a late-round, highly evolved pool that was evolved using the effector.
  • the binding of certain sequences to the negative selection target would be used to subtract those sequences from the evolved pool. This method allows one to quickly eliminate from several hundred to several thousand nucleic acid sequences that demonstrate a high affinity for both the effector and molecules having similar structural characteristics.
  • the amplification process may be any process or combination of process steps that increases the amount or number of copies of a molecule or class of molecules.
  • amplification occurs after members of the test mixture have been partitioned, and it is the facilitating nucleic acid associated with a desirable product that is amplified.
  • the amplification of RNA molecules can be carried out by a sequence of three reactions: the use of reverse transcription to make cDNA copies of selected RNAs, the use of the polymerase chain reaction to increase the copy number of each cDNA, and the transcription of the cDNA copies to obtain RNA molecules having the same sequences as the selected RNAs.
  • Ribozymes ribozyme-like molecules and portions of such molecules are used to form the catalytic domains of the present invention.
  • Ribozymes which are useful in the present invention include, but are not limited to, molecules in the classes of hammerhead, axehead, hairpin, hepatitis delta virus, neurospora, self- splicing introns (including group I and group II), newt satellite ribozymes, Tetrahymena ribozymes, ligases, peptide ligases, phosphatases and polymerases.
  • the nucleic acids of these molecules may be used or the molecules may be used as the starting point for the production of ribozyme-like, synthetic, non-naturally occurring sequences.
  • the allosteric control module may include additional components or domains including a substrate domain (for example, in the case of self-cleaving catalytic domains) or a recognition domain (to aid in the recognition of the site at which catalytic activity is to be directed).
  • the allosteric control module may also be designed such that the catalytic domain and aptamer are joined by a structural bridge, wherein the interaction of the aptamer and effector results in an alteration of the bridge which in turn results in an alteration of catalytic activity of the catalytic domain (see Soukup and Breaker, Proc. Nat. Acad. Sci. USA, 96:3584- 3589, 1999).
  • the allosteric control module is then tested in vitro and/or in vivo.
  • the optimal constructs in an effector-activated expression control system will provide the maximum inhibition of expression in the absence of effector and the maximum enhancement of expression in the presence of effector.
  • the optimal constructs will provide the maximum inhibition of expression in the presence of effector and the maximum enhancement of expression in the absence of effector.
  • the catalytic domain may be synthesized by procedures for normal chemical synthesis of RNA as described in Usman et al, J. Am. Chem. Soc. 109:7845, 1987; Scaringe et al, Nucleic Acids Res. 18:5433, 1990; and Wincott et al, Nucleic Acids Res. 23:2677-2684, 1995. The details will not be repeated here, but such procedures may involve the use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 -end, and phosphoramidites at the 3 '-end.
  • the catalytic activity of the molecules can be optimized as described by Draper et al, PCT W093/23569, and Sullivan et al, PCT W094/02595; Ohkawa et al. , Nucleic Acids Symp. Ser. 27: 15-6, 1992; Taira et al. , Nucleic Acids Res. 19:5125-30, 1991 ; Ventura et al. , Nucleic Acids Res. 21:3249-55, 1993; and Chowrira et al , J. Biol. Chem. 269:25856, 1994.
  • One embodiment of the present invention involves effector identification in accordance with Example 1 together with an evolution and selection of an aptamer.
  • the aptamer evolution first involves preparing a pool or mixture of random sequence single-stranded RNA (ssRNA) with constant regions that are necessary for reverse transcription and PCR amplifications. Typically the individual ssRNAs contain at least 20 nucleotides, but fewer nucleotides may be used.
  • the mixture of ssRNA is then contacted with an effector.
  • the RNAs which bind to the effector are separated from the remainder of RNAs in the pool which do not bind to the effector.
  • RNAs are amplified to form DNA, and the amplified DNA is used to form an enriched mixture of RNAs which bind to the effector.
  • the effector-recognition, partitioning and amplification steps are performed for one or more cycles as needed to identify one or more of the RNAs as an aptamer(s) which best bind the effector.
  • the evolved aptamer or aptamers are then used in an allosteric control module.
  • the selection of the aptamer for use in an allosteric control module is performed by first linking the aptamer to a catalytic RNA to form an allosteric control module; and then identifying those allosteric control modules in which the interaction of the effector and aptamer alters the activity of the catalytic RNA. This analysis may be performed in vivo or in vitro.
  • This example describes the evolution of an allosteric regulatable hammerhead ribozyme comprising an aptamer specific for theophylline.
  • Theophylline was chosen as an effector for the reason, among others, that it has been approved for use by the FDA since 1940 and its safety and toxicity profiles are acceptable and well-known.
  • Jenison et al. previously characterized a theophylline-binding aptamer, Science 263:1425-1429, 1994, to which Zimmerman et al., assigned its secondary structure. Nature Struct. Biol 4:644- 649, 1997.
  • the two RNA strands that connect the hammerhead catalytic domain to the theophylline aptamer domain contains only six nucleotides and was completely randomized. The complexity of this library is 1.7 x 10 6 individual molecules. Selection was initiated with 2 nmoles of synthetic single-stranded DNA
  • Template 1 (SEQ ID NO: 1; Fig. 1) which was made into its double stranded form by the polymerase chain reaction (PCR).
  • DNA template in the post-transcription mixture was removed by a brief DNAse I treatment (20 U/500 ⁇ L) at 37 °C for 15 min.
  • the ribozyme library containing the random sequence region in the Stem-II was isolated by running transcribed RNA on an 8% polyacrylamide gel under denaturing conditions (denaturing PAGE).
  • Ribozymes that did not cleave in response to Theophylline were removed by incubating the RNA in a ribozyme cleavage buffer (RZCL buffer: 50 mM Tris- HCl (pH 7.5 at 25 °C) and 10 mM MgCl 2 ) at 25 °C, punctuated at 30 min intervals by incubation at 85 °C for 30 sec. This negative selection was carried out for 5 cycles of thermal punctuation. The RNA population resistant to ligand- independent self-cleavage was isolated by denaturing PAGE.
  • RZCL buffer 50 mM Tris- HCl (pH 7.5 at 25 °C) and 10 mM MgCl 2 )
  • RNA population A positive selection of the ligand-independent cleavage resistant RNA population was carried out by incubating this RNA in RZCL buffer containing 200 ⁇ M Theophylline at 25 °C for 5 min to facilitate Theophylline-dependent cleavage of ribozymes. The resulting population of 5' fragments upon self-cleavage was isolated by denaturing PAGE.
  • the 5 '-fragment RNA population was subsequently used as the template for reverse transcription in 30 ⁇ L reaction volume containing 50 mM KC1, 50 mM Tris-HCl (pH 8.0 at 25 °C), 5 mM MgCl 2 , 5 mM DTT, 1 mM dNTP each, 10 U of avian myeloblastosis virus reverse transcriptase, and 500 pmoles of Reverse T7-11 primer at 42 °C for 30 min.
  • the resulting cDNA was used as the template for PCR to obtain the template to generate RNA for the next round of in vitro selection.
  • PCR products (0.1 pmoles) were cloned into pT7Blue-3 Vector (Novagen , Madison, WI) in EcoRV site with Perfectly Blunt Cloning Kit (Novagen) according to the protocol supplied by the manufacturer.
  • the ligation mixture was transformed into NovaBlue Singles Competent Cells (Novagen) using standard protocols. Plasmids in the transformants were isolated and sequenced with U 19 vector-based primer (SEQ ID NO: 4) (5' GTTTTCCCAGTCACGACGT 3') and subjected to further analysis.
  • SEQ ID NO: 4 5' GTTTTCCCAGTCACGACGT 3'
  • ribozyme TA-50 The self-cleaving ability of ribozyme TA-50 is strongly dependent on the presence of theophylline (Fig. 9). The highest cleavage activity of TA-50 ribozyme was observed with theophylline concentration between 10-50 ⁇ M. Since this concentration of theophylline is within the therapeutically achievable range, it is possible to envision an in vivo application of TA-50 or another ribozyme with similar performance characteristics to modulate gene expression in response to theophylline intake.
  • Effector identification is performed in accordance with Example 1 followed by the evolution and selection of an aptamer which involves the steps of: a) preparing a pool of random sequence ssRNA wherein each ssRNA comprises an aptamer, a proposed catalytic domain and one or more constant regions suitable for reverse transcription and PCR amplification; b) identifying those RNAs which have catalytic activity; c) amplifying the catalytically active RNAs to form coding DNA molecules; d) transcribing the amplified DNA to form an enriched mixture of catalytically active RNA; e) contacting the mixture with an effector; f) selecting those RNAs which bind to the effector but which do not retain catalytic activity upon binding the effector; g) amplifying the selected RNAs to form coding DNA molecules; h) transcribing the amplified DNA to
  • the evolution of the allosteric control module involves the steps of: a) preparing a pool of random sequence ssRNA wherein each ssRNA comprises an aptamer, a proposed catalytic domain and one or more constant regions suitable for reverse transcription and PCR amplification; b) contacting the mixture with an effector; c) selecting those RNAs which bind to the effector but which do not demonstrate catalytic activity upon binding the effector; d) amplifying the selected RNAs to form DNA molecules; e) transcribing the amplified DNA to form a RNA mixture; f) selecting those RNA as one or more allosteric control modules which demonstrate catalytic activity in the absence of the effector; g) amplifying the selected RNAs; h) transcribing the amplified RNA to form an enriched mixture of allosteric control modules having a catalytic activity which is inactivated or inhibited in the presence of the effector; and i) performing steps b) through
  • the "catalytic activity” is a self-cleaving activity and the self-cleaving allosteric control module is used for the inhibition or reduction the expression of a gene of interest in the absence of the effector.
  • the catalytic activity involves ligase activity or splicing activity.
  • Effector identification is performed in accordance with Example 1 followed by the evolution and selection of an aptamer which involves the steps of: a) preparing a mixture of random sequence ssRNA wherein each ssRNA comprises an aptamer, a proposed catalytic domain and one or more constant regions suitable for reverse transcription and PCR amplification; b) identifying those RNAs which do not demonstrate catalytic activity in the absence of effector; c) amplifying the identified RNAs to form coding DNA molecules; d) transcribing the amplified DNA to form an enriched mixture of RNA; e) contacting the mixture with an effector; f) identifying those RNAs which bind to the effector and demonstrate catalytic activity upon binding the effector; g) amplifying the identified RNAs to form coding DNA molecules; h) transcribing the amplified DNA to form an enriched mixture of allo
  • the evolution of the allosteric control module involves the steps of: a) preparing a pool of random sequence ssRNA wherein each ssRNA comprises an aptamer, a proposed catalytic domain and one or more constant regions suitable for reverse transcription and PCR amplification; b) contacting the pool with an effector; c) identifying those RNAs which bind to the effector and demonstrate catalytic activity while bound to the effector; d) amplifying the identified RNAs to form coding DNA molecules; e) transcribing the amplified DNA to form an enriched mixture of RNA having a catalytic activity in the presence of effector; f) selecting those RNA which are catalytically inactive in the absence of effector; g) amplifying the selected RNAs to form coding DNA molecules; h) transcribing the amplified DNA to form an enriched mixture of allosteric control modules having a catalytic activity which is activated in the presence of effector; and i)
  • the "catalytic activity” is a self-cleaving activity and the self-cleaving of the allosteric control module results in the formation of a functional mRNA encoding a gene of interest.
  • the catalytic activity involves ligase activity or splicing activity.
  • An alternate method of selecting an effector involves the steps of: a) providing an allosteric control module suitable for use in the modulation of gene expression; b) contacting the allosteric control module with one or more effectors; and c) determining whether or not the interaction of the allosteric control module and an effector results in an alteration of the catalytic activity of the allosteric control module.
  • Another method of determining whether a molecule not previously known to be an effector may be used in combination with an allosteric control module to specifically alter the expression of a gene of interest involves the steps of: (a) contacting a sample which contains a predefined number of eucaryotic cells with the molecule to be tested, each cell comprising a DNA construct encoding, i) an allosteric control module, and ii) a reporter gene that produces a detectable signal, coupled to, and under the control of, a promoter, under conditions wherein the molecule if capable of acting as a modulator of the gene of interest, causes a detectable signal to be produced by the reporter gene; (b) quantitatively determining the amount of the signal produced in (a); (c) comparing the amount of signal determined in (b) with the amount of signal produced and detected in the absence of any molecule being tested or with the amount of signal produced and detected upon contacting the sample in (a) with other molecules, thereby identifying the test molecule as an effector which causes a
  • the Figures are schematics of several specific embodiments of this technology to regulate expression of genes.
  • the system's utility can be extended beyond direct medical applications into more basic research applications, diagnostic applications and environmental testing applications.
  • the allosteric control module involves a self- cleaving catalytic domain
  • the interaction of the effector and allosteric control module results in the expression of the gene of interest.
  • the mRNA is untranslatable.
  • the allosteric control module should return to the active conformation and the expression levels decrease due to the presence of untranslatable mRNA.
  • the allosteric control module involves a self-cleaving catalytic domain
  • the interaction of the effector and allosteric control module may be designed to result in the inactivation of the mRNA and the non-expression of the gene of interest in the presence of effector. In the absence of the effector, the mRNA is translatable.
  • the allosteric control module involves a self- splicing catalytic domain, the interaction of the effector and allosteric control module results in the expression of the gene of interest. In the absence of the effector, the mRNA is untranslatable.
  • the interaction of the effector and allosteric control module may be designed to result in the inactivation of the mRNA and the non-expression of the gene of interest. In the absence of the effector, the mRNA is translatable.
  • the DNA constructs may include a regulatable allosteric control module (e.g., self-cleaving format) placed under the control of a constitutive promoter.
  • the same construct may include the coding region for the gene of interest, a stop codon and a poly(A) tail.
  • the introduction of the vector into a cell will result in the production of mRNA encoding both the allosteric control module and gene. If the allosteric control module is active in the absence of effector, then the mRNA will be cleaved by the catalytic domain to yield pieces available for exonucleolytic attack; that is, mRNA will be inactivated. When the effector is administered and enters the cells, the allosteric control module becomes inactive and the mRNA will be translated normally. Hence, gene expression will be under the inducible control of the allosteric control module.
  • a regulatable allosteric control module e.g., self-cleaving format

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Abstract

La présente invention concerne la mise au points d'un module de régulation allostérique dans lequel l'ARN catalytique forme une partie/est lié à un domaine d'ARN de liaison d'effecteur ou à un aptamère. Ces produits de synthèse placent l'activité de l'ARN catalytique sous le contrôle de l'effecteur et exige la présence d'un effecteur approprié pour l'activation ou l'inactivation. En outre, cette invention concerne un dispositif d'identification de molécules effectrices utiles, ainsi que leur utilisation pour élaborer des aptamères correspondants. Cette invention implique à la fois l'élaboration de séquence d'ARN qui se lient à l'effecteur, et un procédé de sélection dans lequel les modules de régulation allostérique sont identifiés par leur fonction catalytique en présence et en l'absence de l'effecteur. Les ARN catalytiques régulables obtenus peuvent être utilisés pour modifier l'expression d'une molécule d'ARN cible de manière contrôlée.
PCT/US2001/006615 2000-03-01 2001-03-01 Identification et utilisation de molecules effectrices et allosteriques pour alterer l'expression genique WO2001064956A2 (fr)

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US10/220,403 US20050239061A1 (en) 2000-03-01 2001-03-01 Identification and use of effectors and allosteric molecules for the alteration of gene expression
JP2001563643A JP2004504806A (ja) 2000-03-01 2001-03-01 遺伝子発現を変化させるためのエフェクターおよびアロステリック分子の特定と使用
AU2001241900A AU2001241900A1 (en) 2000-03-01 2001-03-01 The identification and use of effectors and allosteric molecules for the alteration of gene expression
CA002401654A CA2401654A1 (fr) 2000-03-01 2001-03-01 Identification et utilisation de molecules effectrices et allosteriques pour alterer l'expression genique
MXPA02008470A MXPA02008470A (es) 2000-03-01 2001-03-01 Identificacion y uso de efectores y moleculas alostericas para la alteracion de la expresion de genes.
EP01913217A EP1290154A2 (fr) 2000-03-01 2001-03-01 Identification et utilisation de molecules effectrices et allosteriques pour alterer l'expression genique

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US6506887B1 (en) 1999-07-29 2003-01-14 Somalogic, Incorporated Conditional-selex
EP1410021A2 (fr) * 2000-10-20 2004-04-21 Canji, Inc. Regulation de l'expression genique au moyen d'aptameres
EP1555874A2 (fr) * 2002-10-10 2005-07-27 Oxford Biomedica (UK) Limited Regulation des genes au moyen d'apatameres et de complexes modulateurs a des fins de therapie genique
WO2016166303A1 (fr) * 2015-04-16 2016-10-20 Wageningen Universiteit Criblage commandé par riborégulateur et sélection des biocatalyseurs souhaités
WO2016166310A1 (fr) * 2015-04-16 2016-10-20 Wageningen Universiteit Expression génique inductible par riborégulateur
WO2021195214A1 (fr) 2020-03-24 2021-09-30 Generation Bio Co. Vecteurs d'adn non viraux et leurs utilisations pour exprimer des agents thérapeutiques du facteur ix
WO2021195218A1 (fr) 2020-03-24 2021-09-30 Generation Bio Co. Vecteurs d'adn non viraux et leurs utilisations pour exprimer des agents thérapeutiques de la maladie de gaucher
WO2024040222A1 (fr) 2022-08-19 2024-02-22 Generation Bio Co. Adn à extrémités fermées clivable (adnce) et ses procédés d'utilisation

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JP5299750B2 (ja) * 2008-03-31 2013-09-25 長崎県 粘土鉱物系抗微生物材料、その製造方法及び用途
WO2010117464A1 (fr) * 2009-04-09 2010-10-14 Sangamo Biosciences, Inc. Intégration ciblée dans des cellules souches
US9721061B2 (en) * 2014-02-21 2017-08-01 President And Fellows Of Harvard College De novo design of allosteric proteins

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6506887B1 (en) 1999-07-29 2003-01-14 Somalogic, Incorporated Conditional-selex
US6706482B2 (en) 1999-07-29 2004-03-16 Somalogic, Inc. Conditional-selex
EP1410021A2 (fr) * 2000-10-20 2004-04-21 Canji, Inc. Regulation de l'expression genique au moyen d'aptameres
EP1410021A4 (fr) * 2000-10-20 2005-02-16 Canji Inc Regulation de l'expression genique au moyen d'aptameres
US6949379B2 (en) 2000-10-20 2005-09-27 Canji, Inc. Aptamer-mediated regulation of gene expression
EP1555874A2 (fr) * 2002-10-10 2005-07-27 Oxford Biomedica (UK) Limited Regulation des genes au moyen d'apatameres et de complexes modulateurs a des fins de therapie genique
EP1555874A4 (fr) * 2002-10-10 2006-10-04 Oxford Biomedica Ltd Regulation des genes au moyen d'apatameres et de complexes modulateurs a des fins de therapie genique
WO2016166303A1 (fr) * 2015-04-16 2016-10-20 Wageningen Universiteit Criblage commandé par riborégulateur et sélection des biocatalyseurs souhaités
WO2016166310A1 (fr) * 2015-04-16 2016-10-20 Wageningen Universiteit Expression génique inductible par riborégulateur
WO2021195214A1 (fr) 2020-03-24 2021-09-30 Generation Bio Co. Vecteurs d'adn non viraux et leurs utilisations pour exprimer des agents thérapeutiques du facteur ix
WO2021195218A1 (fr) 2020-03-24 2021-09-30 Generation Bio Co. Vecteurs d'adn non viraux et leurs utilisations pour exprimer des agents thérapeutiques de la maladie de gaucher
WO2024040222A1 (fr) 2022-08-19 2024-02-22 Generation Bio Co. Adn à extrémités fermées clivable (adnce) et ses procédés d'utilisation

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