Source: http://www.google.com/patents/US7923207?ie=ISO-8859-1&dq=7,339,580
Timestamp: 2015-03-02 05:56:19
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Matched Legal Cases: ['Application No. 05852038', 'Application No. 05852038', 'Application No. 05852038', 'Application No. 05852038', 'Application No. 05852038', 'Application No. 05852038', 'Application No. 555248', 'Application No. 555248', 'Application No. 555248', 'Application No. 555248', 'Application No. 555248', 'Application No. 200703649', 'Application No. 05852038', 'Application No. 200703649', 'Application No. 200580047054', 'Application No. 05852038', 'Application No. 05852038']

Patent US7923207 - Apparatus and system having dry gene silencing pools - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsA reverse transfection apparatus can be used for introducing siRNA into a cell to effect gene silencing. Such an apparatus can include a well plate having a well configured for transfecting cells. The well can include a substantially dry gene silencing composition that has at least two siRNAs which silences...http://www.google.com/patents/US7923207?utm_source=gb-gplus-sharePatent US7923207 - Apparatus and system having dry gene silencing poolsAdvanced Patent SearchPublication numberUS7923207 B2Publication typeGrantApplication numberUS 11/283,482Publication dateApr 12, 2011Filing dateNov 18, 2005Priority dateNov 22, 2004Fee statusPaidAlso published asUS20060110829, WO2006058046A2, WO2006058046A3Publication number11283482, 283482, US 7923207 B2, US 7923207B2, US-B2-7923207, US7923207 B2, US7923207B2InventorsBarbara Robertson, Devin Leake, Kathryn Robinson, William S. Marshall, Anastasia KhvorovaOriginal AssigneeDharmacon, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (100), Non-Patent Citations (169), Classifications (10), Legal Events (4) External Links: USPTO, USPTO Assignment, EspacenetApparatus and system having dry gene silencing pools
The well plates, systems, kits, and methods of the present invention can be configured for use in high content screening (�HCS�) applications and high throughput screening (�HTS�) applications with or without the use of laboratory automation equipment. Also, the well plates, systems, kits, and methods can also be used with automated systems, such as robotic systems. However, the well plates, systems, kits, and methods can also be used in RTF protocols without the aid of automated delivery systems, or robotics, and thus can provide an efficient alternative to costly robotic delivery systems for laboratories using manual processing. Thus, the well plates, systems, kits, and methods provide versatility in choice such that high throughput screening can be done in a cost effective manner.
As used herein, the term �2′ modification� is meant to refer to a chemical modification of a nucleotide that occurs at the second position atom. As such, the 2′ modification can include the conjugation of a chemical modification group to the 2′ carbon of the ribose ring of a nucleotide, or a nucleotide within an oligonucleotide or polynucleotide. Thus, a 2′ modification occurs at the 2′ position atom of a nucleotide. Examples of a 2′ modification can include a 2′-O-aliphatic, 2′-O-alkyl, 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-isopropyl, 2′-O-butyl, 2′-O-isobutyl, 2′-O-ethyl-O-methyl (i.e., �CH2CH2OCH3), 2′-O-ethyl-OH (i.e., �OCH2CH2OH), 2′-orthoester, 2′-ACE group orthoester, 2′-halogen, and the like.
As used herein, the term �antisense strand� is meant to refer to a polynucleotide or region of a polynucleotide that is at least substantially (e.g., 80% or more) or 100% complementary to a target nucleic acid of interest. Also, the antisense strand of a dsRNA is complementary to its sense strand. An antisense strand may be comprised of a polynucleotide region that is RNA, DNA, or chimeric RNA/DNA. Additionally, any nucleotide within an antisense strand can be modified by including substituents coupled thereto, such as in a 2′ modification. The antisense strand can be modified with a diverse group of small molecules and/or conjugates. For example, an antisense strand may be complementary, in whole or in part, to a molecule of messenger RNA (�mRNA�), an RNA sequence that is not mRNA including non-coding RNA (e.g., tRNA, rRNA, and the like), or a sequence of DNA that is either coding or non-coding. The antisense strand includes the antisense region of polynucleotides that are formed from two separate strands, as well as unimolecular siRNAs that are capable of forming hairpin structures with complementary base pairs. The terms �antisense strand� and �antisense region� are intended to be equivalent and are used interchangeably.
As used herein, the terms �complementary� and �complementarity� are meant to refer to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in anti-parallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenosine.
Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can hydrogen bond with a nucleotide unit of an anti-parallel polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 90% complementarity. �Substantial complementarity� refers to polynucleotide strands exhibiting 79% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected so as to be non-complementary. Accordingly, complementarity does not consider overhangs that are selected so as not to be similar or complementary to the nucleotides on the anti-parallel strand.
As used herein, the term �conjugate� is meant to refer to a molecule, large molecule, or macromolecular structure that is coupled with either the sense strand or antisense strand of an siRNA. That is, the moiety coupled to the siRNA is considered the conjugate. For clarity purposes, the siRNA can include a conjugate that is coupled thereto by a covalent bond, ionic interaction, and like couplings. Usually, a conjugate is coupled with an siRNA in order to impart a functionality other than increasing the stabilization or targeting specificity. For examples, some conjugates, such as cholesterol, can be used to enhance the ability of the siRNA to enter a cell. Other conjugates can be labels that can be used to detect transfection or the presence of the siRNA in the cell. Usually, the conjugate is coupled to the siRNA through a linker group.
As used herein, the terms �dried� or �dry� as used in connection with gene silencing compositions is meant to refer to a composition that is not fluidic and does not flow. However, this does not exclude small amounts of water or other solvents, and includes amounts of water remaining in an RNA preparation that has equilibrated at standard or ambient conditions, for example, at one atmosphere of pressure, room temperature, and ambient humidity, such that the preparation is not in a substantially liquid form but instead is �dried� in the well. For example, an siRNA preparation is �dried� or substantially �dry� if, at about one atmosphere pressure, at about 20 to 40� C., and at about 50 to about 95% humidity, the preparation is equilibrated and, when the well plate is inverted or tilted to, for example, 90� from horizontal, the RNA preparation does not displace or flow within the well. This is in comparison to a liquid preparation which would flow or run when tilted. In various embodiments, methods for using the dry gene silencing composition in order to perform a transfection can include solubilizing or suspending the dried preparation in a suitable aqueous medium to form a mixture. Additionally, the suitable aqueous medium can include a polynucleotide carrier capable of facilitating introduction of the siRNA into a cell, and exposing the mixture to one or more cells to achieve transfection.
As used herein, the term �duplex region� is meant to refer to the region in two complementary or substantially complementary polynucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between the polynucleotide strands. For example, a polynucleotide strand having 21 nucleotide units can base pair with another polynucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary such that the �duplex region� has 19 base pairs. The remaining bases may, for example, exist as 5′ and/or 3′ overhangs. Further, within the duplex region, 100% complementarity is not required, and substantial complementarity is allowable within a duplex region. Substantial complementarity refers to 79% or greater complementarity and can result from mismatches and/or bulges. For example, a single mismatch in a duplex region consisting of 19 base pairs results in 94.7% complementarity, rendering the duplex region substantially complementary.
As used herein, the term �functionality� is meant to refer to the level of gene specific silencing induced by an siRNA. In general, functionality is expressed in terms of percentages of gene silencing. Thus, 90% silencing of a gene (e.g., F90) refers to situations in which only 10% of the normal levels of gene expression are observed. Similarly, 80% silencing of a gene (e.g., F80) refers to situations in which only 20% of the normal levels of gene expression are observed.
As used herein, the term �gene silencing� is meant to refer to a process by which the expression of a specific gene product is inhibited by being lessened, attenuated, and/or terminated. Gene silencing can take place by a variety of pathways. In one instance, gene silencing can refer to a decrease in gene product expression that results from the RNAi pathway, wherein an siRNA acts in concert with host proteins (e.g., RISC) to degrade mRNA in a sequence-dependent manner. Alternatively, gene silencing can refer to a decrease in gene product expression that results from siRNA mediated translation inhibition. In still another alternative, gene silencing can refer to a decrease in gene product expression that results from siRNA mediated transcription inhibition. The level of gene silencing can be measured by a variety of methods, which can include measurement of transcript levels by Northern Blot Analysis, B-DNA techniques, transcription-sensitive reporter constructs, expression profiling (e.g., DNA chips), and related technologies and assays. Alternatively, the level of gene silencing can be measured by assessing the level of the protein encoded by a specific gene that is translated from the corresponding mRNA. This can be accomplished by performing a number of studies including Western Blot analysis, measuring the levels of expression of a reporter protein, such as calorimetric or fluorescent properties (e.g., GFP), enzymatic activity (e.g., alkaline phosphatases), or other well known analytical procedures.
As used herein, the term �mismatch� includes a situation in which Watson-Crick base pairing does not take place between a nucleotide of a sense strand and a nucleotide of an antisense strand, where the non-base paired nucleotides are flanked by a duplex comprising base pairs in the 5′ direction beginning directly after (e.g., in the 5′ direction) the non-base paired nucleotides and in the 3′ direction beginning directly after (e.g., in the 3′ direction) the non-base paired nucleotides. An example of a mismatch would be an A across from a G, a C across from an A, a U across from a C, an A across from an A, a G across from a G, a C across from a C, and the like. Mismatches are also meant to include an abasic residue across from a nucleotide or modified nucleotide, an acyclic residue across from a nucleotide or modified nucleotide, a gap, or an unpaired loop. In its broadest sense, a mismatch as used herein includes any alteration at a given position that decreases the thermodynamic stability at or in the vicinity of the position where the alteration appears, such that the thermodynamic stability of the duplex at the particular position is less than the thermodynamic stability of a Watson-Crick base pair at that position.
As used herein, the term �nucleotide� is meant to refer to a ribonucleotide, a deoxyribonucleotide, or modified form thereof, as well as an analog thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs. Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5′-position pyrimidine modifications, 8′-position purine modifications, modifications at cytosine exocyclic amines, and 2′-position sugar modifications (e.g., 2′ modifications). Such modifications include sugar-modified ribonucleotides in which the 2′-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl or aliphatic moiety. Nucleotides are well known in the art. Also, reference to a first nucleotide or nucleotide at a first position refers to the nucleotide at the 5′-most position of a duplex region, and the second nucleotide is the next nucleotide toward the 3′ end. In instances the duplex region extends to the end of the siRNA, the 5′ terminal nucleotide can be the first nucleotide.
As used herein, the terms �off-target� and �off-target effects� are meant to refer to any instance where an siRNA, such as a synthetic siRNA or shRNA, is directed against a given target mRNA, but causes an unintended effect by interacting either directly or indirectly with another mRNA, a DNA, a cellular protein, or other moiety in a manner that reduces non-target protein expression. Often, this can happen when an siRNA interacts with non-target mRNA that has the same or similar polynucleotide sequence as the siRNA. For example, an �off-target effect� may occur when there is a simultaneous degradation of other non-target mRNA due to partial homology or complementarity between that non-target mRNA and the sense and/or antisense strand of the siRNA.
As used herein, the term �on-target� is meant to refer to a set of modifications of an siRNA that increase the likelihood that the siRNA will preferentially target and interact with a target mRNA or DNA so as to inhibit production of the polypeptide encoded thereby. This increases the specificity of the siRNA for silencing the target gene. For example, an on-target modification can include a siRNA where the first and second nucleotide of the sense region each has a 2′-O-methyl moiety, and the antisense strand is phosphorylated at its 5′ end, wherein such an on-target modification also refers to a proprietary modification coined On-Target� (Dharmacon, Inc.). In any event, on-target modifications can be used to help reduce off-target effects. Also, an siRNA can have a sense region that has complementarity to the antisense region of the siRNA, and wherein the antisense region is the region that has complementarity to a target mRNA.
As used herein, the terms �rational design� and �rationally designed� are meant to refer to the selection or design of one or more siRNA(s) for use in a gene silencing application based upon one or more criteria that are independent of the target sequence. As such, rationally designed siRNA are selected to specifically interact with and inhibit polypeptide translation from a selected mRNA. Thus, for any one target mRNA there may be hundreds of potential siRNA having 18 to 31 base pairs that are 100% complementary to the target mRNA. In part, this is because a single mRNA may have multiple sequences that can be specifically targeted by the siRNA. However, it is likely that not all of the siRNA will have equal functionality. Through empirical studies, a number of other factors including the presence or absence of certain nitrogenous bases at certain positions, the relative GC content, and the like, can affect the functionality of particular siRNA. Additional information regarding rationally designed siRNA can be found in commonly owned U.S. patent application Ser. No. 10/714,333, filed on Nov. 14, 2003, related PCT application PCT/US03/36787, published on Jun. 3, 2004 as WO 2004/045543 A2, U.S. patent application Ser. No. 10/940,892, filed on Sep. 14, 2004, published as U.S. Patent Application Publication 2005/0255487, related PCT application PCT/US 04/14885, filed on May 12, 2004, and U.S. Patent Application Publication 2005/0246794, which are all incorporated herein by reference.
As used herein, the term �reverse transfection� and abbreviation �RTF� are each meant to refer to a process for introducing nucleic acid, such as an siRNA, into a cell. Such an introduction of an siRNA into a cell can be accomplished by combining the nucleic acid and cell in a well, wherein the cell has not yet been previously adhered or maintained on the growth surface. The reverse transfection proceeds by contacting the nucleic acid onto a cellular surface in a manner such that the nucleic acid can enter into the cell. Usually, the siRNA is complexed with a lipid or other polynucleotide carrier prior to being contacted to the cells. Reverse transfection differs from forward transfection because the cells have not been seeded and maintained on the cellular growth surface of a well or other container before addition of the siRNA.
As used herein, the term �sense strand� is meant to refer to a polynucleotide or region that has the same nucleotide sequence, in whole or in part, as a target nucleic acid such as a messenger RNA or a sequence of DNA. The term �sense strand� includes the sense region of a polynucleotide that forms a duplex with an antisense region of another polynucleotide. Also, a sense strand can be a first polynucleotide sequence that forms a duplex with a second polynucleotide sequence on the same unimolecular polynucleotide that includes both the first and second polynucleotide sequences. As such, a sense strand can include one portion of a unimolecular siRNA that is capable of forming hairpin structure, such as an shRNA. When a sequence is provided, by convention, unless otherwise indicated, it is the sense strand or region, and the presence of the complementary antisense strand or region is implicit. The phrases �sense strand� and �sense region� are intended to be equivalent and are used interchangeably.
As used herein, the terms �siRNA library� or �RTF siRNA library� is meant to refer to an array of siRNAs for use in analyzing a particular biological pathway or gene target. An siRNA library comprises various siRNA pool reagents for analyzing a particular pathway or gene target. A pool typically comprises two or more non-identical siRNA directed against a single target gene. Usually, a pool includes four or more non-identical siRNA that are rationally designed. An exemplary list of siRNA libraries is provided in Table 1 below. Sequences used in certain siRNA libraries, including pool reagents, are provided in Table I and Table II of U.S. Provisional Application Ser. No. 60/678,165.
As used herein, the terms �siRNA pool,� �pool,� �pool of siRNAs,� and �pool reagents� are meant to refer to two or more siRNA, typically four siRNA, directed against a single target gene, mRNA, and/or translation of a protein. The siRNA of the pool reagent can be rationally designed by being selected according to non-target specific criteria as described herein and in the incorporated references. For example, two nanomoles of each pool reagent can be sufficient for transfecting cells in about 200 wells of multiple 96-well plates, using 100 nM siRNA concentration. Pool reagents can be plated as a pool (i.e., the two or more siRNA of Dharmacon's SMARTpool� Reagent in a single transfection well). The individual siRNAs that comprise the SMARTpool� Reagent can also be plated individually on the same plate as the SMARTpool� Reagent.
As used herein, the term �transfection� is meant to refer to a process by which nucleic acids are introduced into a cell. The list of nucleic acids that can be transfected is large and includes, but is not limited to, siRNA, shRNA, sense and/or anti-sense sequences, DNA, RNA, and the like. There are multiple modes for transfecting nucleic acids into a cell including, but not limited to, electroporation, calcium phosphate delivery, DEAE-dextran delivery, lipid delivery, polymer delivery, molecular conjugate delivery (e.g., polylysine-DNA or -RNA conjugates, antibody-polypeptide conjugates, antibody-polymer conjugates, or peptide conjugates), microinjection, laser- or light-assisted microinjection, optoporation or photoporation with visible and/or nonvisible wavelengths of electromagnetic radiation, and the like. Transfections can be �forward transfections� whereby cells are first plated in wells and then treated with a nucleic acid or they can be �reverse transfections� (RTF) whereby the nucleic acid is combined with the cells before or during being plated and/or attached to the bottom of the well. Any mode of transfecting cells, such as those described above, can be used with the present invention by inducing the nucleic acid to be introduced into a cell after the siRNA is solubilized or suspended in the aqueous medium to implement reverse transfection. Details regarding a mode of reverse transfection are described in more detail below
As used herein, the term �well plate� is meant to refer to a substrate that is divided into distinct regions that prevent migration from one distinct region to another distinct region, wherein the distinct regions are wells. For example, each well of a multi-well well plate may contain a horizontal well floor that may be curved or flat, as well as have sidewalls. Additionally, well plates are well known in the art.
In one embodiment, the results of the RTF protocol to induce gene silencing can be detected or monitored using systems for performing high content screening (�HCS�) or high throughput screening (�HTS�). An HCS analysis can be used to measure specific translocation and morphology changes, receptor trafficking, cytotoxicity, cell mobility, cell spreading, and the like. HCS studies can be performed on an ArrayScan� HCS Reader, or a KineticScan� HCS Reader (Cellomics, Inc.) Additional information on HCS can be found in U.S. Pat. Nos. 6,902,883, 6,875,578, 6,759,206, 6,716,588, 6,671,624, 6,620,591, 6,573,039, 6,416,959, 5,989,835, wherein each is incorporated herein by reference. HTS analyses can be performed using a variety of available readers, typically of the fluorescence from each well as a single measurement.
Due to the unique and highly sensitive nature of the RNAi pathway, methodologies particularly useful for introducing pools of siRNAs into cells have been developed. Accordingly, new RTF methodologies were developed for use with pools of siRNAs. As such, recently developed protocols for implementing siRNA RTF were modified by augmenting such protocols with recently developed siRNA technologies based on rationale design, siRNA stabilization, siRNA targeting specificity, and pooling siRNAs. Thus, improved methods for implementing gene silencing with pools of siRNAs can be performed with RTF protocols (�siRNA pool RTF�).
In one embodiment, the present invention includes the use of gene silencing solutions dried in the bottom of a well in a well plate. The well plates used in connection with the present invention are preferably formatted and distinct well arrays (e.g., a 48, 96, 384, or 1536-well plate) that can be purchased from any number of commercial sources of cell culture plates and other cell culture surface-containing devices, including products such as NUNC�, NUNCLON�, MICROWELL� and FLUORONUNC� plates (e.g., each of which may be obtained from Nalge Nunc International of Rochester, N.Y., and Nunc A/S of Denmark), COSTAR�, COSTAR THERMOWELL� and CORNING� plates (e.g., each of which is available from Corning), BD FALCON� and OPTILUX� plates (e.g., available from Becton, Dickinson and Company) and GREINER�, CELL COAT� and CELLSTAR� plates (e.g., available from Greiner Bio-One).
In one embodiment, the well plate can be characterized by being configured to be suitable for cell growth and propagation. A well plate can be made of glass, polystyrene, other polymeric material or any equivalent materials, and can have rounded and/or flat well floors. However, certain analytical equipment can have enhanced functionality when using flat bottom surfaces. Additionally, wells having substantially flat floors can provide uniform cell spacing and monolayer formation. Thus, it can be preferable for the well floor to have a substantially flat bottom surface. The well floor can have a physical or chemical treatment, such as irradiation, corona discharge, plasma discharge, or microwave plasma discharge of polystyrene. Such treatments can be conventional in tissue culture surfaces upon which adherent eukaryotic cells may adhere and grow. Additionally, the wells may not be modified by any chemical coating, or they can be coated with poly-L-lysine (�PLL�), laminin, collagen, or equivalent substances that improve the adherence of cells.
Additionally, it can be preferable for each plate to have between 6 and 2000 wells, and more preferably having 1536 wells, 384 wells, or 96 wells. Also, it can be preferable for the wells to have a volume that varies between about 5 to about 2000 microliters (�uL�), and the total culture area, which is represented by the well bottom surface or cell floor, to range between about 0.02 cm2 to about 4.2 cm2, and about 0.3 cm2 to about 0.35 cm2 for a 96-well plate.
In one embodiment, the total amount of siRNA in the gene silencing composition can be present in an amount for transfecting cells in only the well in which it is contained. As such, the total concentration of siRNA can be less than about 100 nM when solubilized or suspended in the aqueous medium during RTF. More preferably, the total concentration of siRNA can be less than about 50 nM when solubilized or suspended in the aqueous medium during RTF. Even more preferably the total concentration of siRNA can be less than about 25 nM when solubilized or suspended in the aqueous medium during RTF. In an additional preference, the total concentration of siRNA can be less than about 10 nM when solubilized or suspended in the aqueous medium during RTF. Most preferably, the total concentration of siRNA can be less than about 1 nM when solubilized or suspended in the aqueous medium during RTF. Additionally, the siRNA of each pool can be present in equal amounts. Alternatively, the siRNA of each pool can be present at different amounts depending on functionality, where a more functional siRNA may be present at a low concentration than a less functional siRNA. Moreover, the amount of siRNA can vary in each well. For example, the amount of siRNA in a 96-well plate can be from 0.1 picomoles (�pm�) to about 100 pm, more preferably about 1 pm to about 75 pm, and most preferably about 10 pm to about 62.5 pm per well, where corresponding amounts of siRNA can be calculated for plates having other numbers of wells.
In one embodiment, it can be preferably to select the siRNAs from a list that have been identified from being rationally designed. As such, each of the siRNAs can be selected from Table I of incorporated U.S. Provisional Application having Ser. No. 60/678,165. Table I is entitled �siGENOME Sequences for Human siRNA,� and consists of columns �Gene Name,� �Accession No.,� �Sequence,� and �SEQ. ID NO.� Table I lists about 92,448 19-mer siRNA sense strand sequences, where antisense strand sequences were omitted for clarity. The siRNA sequences listed in Table I of the includes SEQ. ID NOS. 1 to about 92,448, wherein each preferably can also include a 3′ UU overhang on the sense strand and/or on the antisense strand. Each of the about 92,448 sequences of Table I can also comprise a 5′ phosphate on the antisense strand. Of the about 92,448 sequences listed in Table I of the incorporated provisional application, about 19,559 have an on-targeting set of modifications. A list of sequences, identified by SEQ. ID NO., that have on-target modifications is presented in Table II, entitled �List of Table I Sequences Having On-Target Modifications Identified by SEQ. ID NO.� On-target modifications are on SEQ. ID NOS. 1-22,300. The siRNA in the gene silencing compositions may be used as part of a pool.
Briefly, in order to identify whether a given lipid is acceptable for siRNA pool RTF, two or more well characterized siRNAs can be tested under a variety of lipid, media, and siRNA concentrations using the optimizing RTF protocols described herein. Subsequently, the level of silencing of the targeted gene and the level of cell death are quantified using art-accepted techniques. Suitable lipids for siRNA pool RTF include OLIGOFECTAMINE�, TransIT-TKO�, or TBIO Lipid 6�, LIPOFECTAMINE� 2000, lipids DharmaFECT� 1, DharmaFECT� 2, DharmaFECT� 3, and DharmaFECT� 4 (Dharmacon, Inc.). The term �DharmaFECT�� (followed by any of the numerals 1, 2, 3, or 4) or the phrase �DharmaECT� transfection reagent,� refers to one or more lipid-based transfection reagents that have been optimized to transfect siRNA rather than larger nucleic acids (e.g., plasmids). Additional information on lipids can be obtained in U.S. Pat. Nos. 5,674,108, 5,834,439, 6,110,916, 6,399,663, and 6,716,582, and international publications WO 00/12454 and WO 97/42819.
The formation of a functional siRNA-lipid complex can be prepared by combining a pool of siRNAs and the lipid. As such, an appropriate volume of lipid at a selected concentration can be combined with a volume of media and/or buffer to form a lipid-media or lipid-buffer having a suitable concentration of lipid. For example, a volume of lipid media ranging from about 5-50 microliters (�uL�) can include about 0.03-2 micrograms (�ug�) of lipid to be introduced into each well of a 96-well plate, and the amount of lipid can be changed to correspond with other well sizes. The choice of media and/or buffer for siRNA pool RTF can improve the efficiency of the RTF protocol. Some media contain one or more additives that induce cell toxicity and/or non-specific gene modulation during RTF. Examples of preferred media or buffers include Opti-MEM� (GIBCO, Cat. # 31985-070), HyQ-MEM-RS� (HyClone, Cat.# SH30564.01), Hanks Balanced Salt Solution�, or equivalent media. A suitable media can be identified by employing the optimization protocol described herein.
The lipid-media or lipid-buffer can be introduced into a well by a variety of methods including hand-held single and multi-channel pipettes, or more advanced and automated delivery systems that can inject measured volumes of the lipid solution into a well. The lipid solution can be incubated in the well that contains the dried gene silencing composition for a period of time that is sufficient to solubilize or suspend the siRNAs, and to form siRNA-lipid complexes (e.g., lipoplexes). In general, the process of siRNA solubilization and lipoplex formation can require about 20 minutes, but usually not more than 120 minutes. The complex formation process is generally performed at room temperature, but can be performed at temperatures ranging from 4-37� C. In some instances, the lipid and siRNAs can be mixed by agitating the plate (e.g., swirl, vortex, sonicate) for brief periods (e.g., seconds�minutes) to enhance the rate of siRNA solubilization and complex formation.
FIGS. 1A and 1B illustrate embodiments of plate arrangements similar with the foregoing concentrations arrangements. While the wells are shown to be square, it should be recognized that they can be any shape. Also, the well plate can include any number of wells, and the number of wells depicted is merely for example. In the figures the wells are defined as follows: �Tc� indicates a transfection control well, wherein the increasing corresponding numbers identify different transfection controls; blank wells indicate wells devoid or substantially devoid of any siRNA; �+� indicates a positive control; �−� indicates negative controls; �P1� through �P1N� indicate a first pool which silences a first gene at a concentration gradient; �P2� through �P2N� indicate a second pool which silences a second gene at a concentration gradient; �1A� through �1N� indicate a first individual siRNA of the first pool at a concentration gradient; �2A� through �2N� indicate a second individual siRNA of the first pool at a concentration gradient; �3A� through �3N� indicate a third individual siRNA of the first pool at a concentration gradient; �4A� through �4N� indicate a first individual siRNA of the second pool at a concentration gradient; �5A� through �5N� indicate a second individual siRNA of the second pool at a concentration gradient; and �6A� through �6N� indicate a third individual siRNA of the second pool at a concentration gradient. Thus, FIG. 1A illustrates a well plate assaying a single pool, and FIG. 1B illustrates a well plate assaying multiple pools. Additionally, a well plate can include more than two pools. Also, the pools and single siRNA can be rationally designed, and/or have modifications or conjugates.
FIG. 1C illustrates another embodiment of a plate arrangement similar with the foregoing concentrations arrangements. The wells are defined as follows: �Tc� indicates a transfection control well, wherein the increasing corresponding numbers identify different transfection controls; blank wells indicate wells devoid or substantially devoid of any siRNA; �+� indicates a positive control; �−� indicates negative controls; �P1� indicates a first pool in triplicate which silences a first gene at a standard concentration; �P1A� through �P1N� indicates the first pool at a concentration gradient A-N, each in triplicate; �P2� indicates a second pool in triplicate which silences a second gene at a standard concentration; and �P2A� through �P2N� indicate the second pool at a concentration gradient A-N, each in triplicate. Accordingly, multiple wells can be used to test each gene silencing composition and/or condition.
FIG. 1D illustrates an embodiment of a plate arrangement similar with the foregoing concentrations arrangements. The wells are defined as follows: �Tc� indicates a transfection control well, wherein the increasing corresponding numbers identify different transfection controls; blank wells indicate wells devoid or substantially devoid of any siRNA; �+� indicates a positive control; �−� indicates negative controls; �P1� through �PN� indicate a first pool which silences a first gene through an Nth pool which silence an Nth gene at a standard concentration; and �P1A� through �P1N� indicate the first pool at a concentration gradient A-N, wherein second pool (e.g., �P2A�-�P2N�) through Nth pool (e.g., �PNA�-�PNN�) each have a similar concentration gradient. Thus, multiple pools can be studied at different concentrations, wherein the multiple pools can be related or different.
FIG. 1E illustrates an embodiment of a plate arrangement similar with the foregoing concentrations arrangements. The wells are defined as follows: �Tc� indicates a transfection control well, wherein the increasing corresponding numbers identify different transfection controls; blank wells indicate wells devoid or substantially devoid of any siRNA; �+� indicates a positive control; �−� indicates negative controls; �P1A� is a first pool that silences a first gene; �P1B� is a second pool that silences the first gene; �P1C� is a third pool that silences the first gene; �P1D� is a fourth pool that silences the first gene; �P1N� is an Nth pool that silences the first gene; �P2A� through �PNA� indicate a second through Nth pools that silence related second through Nth genes; and the corresponding wells in each of the �P2A� through �PNA� rows are A-N pools which silences the gene of the row. Thus, plates can be arranged with multiple pools targeting multiple related genes for silencing.
In one embodiment, each pool of siRNAs (e.g., SMARTpool� reagent) can designed with Dharmacon, Inc.'s multi-component, proprietary SMARTselection� algorithm. The reagents can be deposited and dried in well of a well plate in triplicate and can be used for a single transfection into the cell type of interest for rapid screening of the siRNA library.
For example, at least one well plate can include an RTF siRNA library, wherein each plate has a maximum of 80 wells having rationally designed siRNA, such as SMARTpool� siRNA reagents. Each gene silencing composition can include the rationally designed siRNAs at about 6.25 pmol of total siRNA per about 0.3 cm2 to about 0.35 cm2 cell culture area or well floor. As such, the amount of each individual siRNA in each pool is about 1.56 pmol about 0.3 cm2 to about 0.35 cm2 per cell culture area or well floor. The gene silencing compositions are presented in a matrix format, wherein each SMARTpool� siRNA reagent contains four different siRNA duplexes targeting a single gene. Such a well plate can be prepared as a kit that includes a DharmaFECT� transfection reagent supplied as a liquid at a concentration of 1 ug/uL. The kit preferably also comprises DharmaFECT� Cell Culture Reagent supplied as a liquid.
A series of studies were conducted to asses the ability of certain siRNA sequences to have toxic effects, to assess whether off-targeting causes toxicity, and whether off-targeting can be minimized using pools of siRNA. Accordingly, a population of randomly selected siRNAs derived from a walk targeting DBI (e.g., NM�020548, position 202-291) was assessed for the ability of certain siRNA sequences to induce toxicity. The collection of siRNAs consisted of 90 individual (e.g., 19 bp) duplexes, and covered the respective regions in single base steps. Duplexes were forward transfected into HeLa cells using LIPOFECTAMINE� 2000, and a threshold of 75% cell viability was used as the cutoff to distinguish toxic from nontoxic sequences.
The ability of rationally designed pools of siRNA to silence four separate genes simultaneously was studied with individual and pools of siRNAs targeting G6PD, GAPDH, PLK, and UQC. Pools of siRNA (e.g., 4 siRNA per gene) were forward transfected into cells at a total siRNA concentration of 100 nM, 6.25 nM per siRNA, using LIPOFECTAMINE� 2000, and assayed twenty-four hours later by B-DNA. FIG. 3 is a graphical representation of the results which demonstrated that pools of rationally designed molecules are capable of simultaneously silencing four different genes. The ability to target multiple genes in an RTF format will significantly simplify the ability to use RTF for screening large (e.g., genome-sized) collections of siRNA.
The genes involved in the kinase pathway are studied by siRNA RTF to determine the genes responsible for cell viability. Rationally designed pools of siRNAs targeting the 779 members of the kinase family are solubilized in RNase-free water and dried in individual wells of PLL coated 96-well plates. The amount of each pool of siRNA is a total of approximately 25 nM for 125 uL of total solution. A lipid solution having 0.1 ug of DharmaFECT� 1 lipid in 25 uL total volume of Hanks Balanced Saline Buffer is added to each well and incubated for 20-40 minutes to solubilize and complex the siRNA before 10,000 HeLa cells in media are added for a final volume of 125 uL. The plates are maintained between 24 and 72 hours and assayed for cell viability. A comparison between the cell viability of cultures that were treated with lipid alone (i.e., control wells) and cultures treated with individual members of the Kinase siRNA array allows the identification of genes that are essential for HeLa cell viability.
The genes involved in the cytokine receptor family are studied by siRNA RTF to determine the genes responsible for cell viability. Rationally designed pools of siRNAs targeting the 166 members of the cytokine receptor family are solubilized in RNase-free water and dried in individual wells of PLL coated 96-well plates. The amount of each pool of siRNA is a total of approximately 25 nM for 125 μL of total solution. A lipid solution having 0.1 ug of DharmaFECT� 1 lipid in 25 uL total volume of Hanks Balanced Saline Buffer is added to each well and incubated for 20-40 minutes to solubilize and complex the siRNA before 10,000 HT-29 cells in media are added for a final volume of 125 uL. The plates are maintained between 24 and 72 hours and assayed for cell viability. A comparison between the cell viability of cultures that were treated with lipid alone (i.e., control wells) and cultures treated with individual members of the cytokine receptor siRNA array allows the identification of genes that are essential for HT-29 cell viability.
The ability of a pool of siRNA to be directed against a selected gene was studied in an RTF protocol. To assess the effectiveness of pools of siRNA directed against a single target individual siRNAs and pools of three or four siRNAs directed against GAPDH, MAP2K1, or MAP2K2 were reverse transfected into 10,000 HeLa cells using DharmaFECT� 1. At 48 hours after addition of cells the cultures were assessed for cell viability and target silencing knockdown (e.g., B-DNA assay). In this study, the siRNA are designated as follows: GAPDH siRNA duplex 1, duplex 2, duplex 3, and duplex 4; MAP2K1 siRNA duplex 1, duplex 2, duplex 4, and duplex 5; MAP2K2 siRNA duplex 1, duplex 2, duplex 4, and duplex 7.
The ability of a pool of siRNA to be directed against a selected gene was studied in an RTF protocol. To assess the effectiveness of pools of siRNA directed against a single target combinations of individual siRNAs directed to multiple targets. The siRNA directed to GAPDH, MAP2K1, or MAP2K2 were reverse transfected into 10,000 HeLa cells using DharmaFECT� 1 using a RTF procedure substantially similar to that described in Example 5.
In one example, a multi-well RTF plate or series of plates can be designed in order to optimize RTF with siRNA. Accordingly, the plates can be configured to include any of the following variables: (1) the concentration of pools of siRNA can be between 0.01-250 nM, more preferably between 0.05 and 100 nM, even more preferably between 0.1 and 50 nM, still even more preferably between 0.5 and 25 nM, and most preferably between 0.75 and 10 nM or about 1 nM; (3) the types of polynucleotide carrier can be a lipid such as DharmaFECT� 1, DharmaFECT� 2, DharmaFECT� 3, or DharmaFECT� 4; (3) the concentration of the lipid polynucleotide carrier can be at concentrations of 0.05-1 ug per 100 uL of solution, more preferably at concentrations of 0.05-0.5 ug of lipid per 100 uL of solution, even more preferably still at concentrations of 0.05-0.25 ug of lipid per 100 uL of solution, and most preferably at concentrations of 0.05-0.1 ug per 100 uL of solution; (4) the types of media and/or buffer used to complex the lipid can be preferably Opti-MEM�, more preferably HyQ-MEM�, and most preferably buffered salt solutions such as Hanks Buffered salt solution or equivalent mixtures; and (5) the types and amounts of cells having densities of 1,000 to 35,000 cells per about 0.3 cm2 to about 0.35 cm2 preferred densities of 2,000-30,000 cells, more preferably 2,000-20,000 cells, even more preferably 2,000-15,000 cells, and most preferably cell densities of 2,000-10,000 cells per about 0.3 cm2 to about 0.35 cm2. The siRNA can be used to study the silencing of selected target genes, or control siRNA can be used to silence known genes in a reproducible manner.
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