Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
  • C12M1/26—Inoculator or sampler
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
  • C12M1/16—Apparatus for enzymology or microbiology containing, or adapted to contain, solid media
  • C12M1/18—Multiple fields or compartments
• • 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
  • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

• the present invention relates to a system and method for studying a biological agent. More particularly, the present invention relates to a system and method that uses gene silencing for determining a cellular response to the biological agent.
• RNA interference RNA interference
• siRNA synthetic dsRNA
• siRNA having specific sequences can be produced to target complementary DNA and/or mRNA encoding a specific protein.
• the siRNA can interact with the natural RNAi pathway to silence a target gene and inhibit production of the encoded polypeptide.
• the ability to silence a specific gene and inhibit production of the encoded protein has been used for basic research of gene function and cellular pathway analysis.
• lipid-nucleic acid e.g., lipoplex
• RTF reverse transfection
• screening techniques have been used to study the interaction between biological agents and cells. These screening techniques can be used to identify which biological agents, such as drugs, have an effect on a particular cell, or interact with a polypeptide or protein produced by the cell. Additionally, screening techniques can be used with infectious biological agents to identify cell types that may or may not be susceptible to an infection, as well as identify drugs that can treat or prevent such infections. Thus, screening techniques can provide a wide array of valuable information that can be used for treating and/or preventing diseases. However, new screening techniques for studying biological agents continue to be developed in order to test various physiological responses.
• embodiments of the present invention include well plates, kits, systems, and methods of using the same for determining the effect of a biological agent on cells, which can include the biological agent being introduced into cells in the presence of gene silencing.
• the well plates, kits, and systems include siRNAs that are configured to be implemented in an RTF format with biological agents in order to determine whether or not a silenced target gene is involved in a cellular response to the biological agent.
• the method can include adding an aqueous medium to a first test well in a well plate that has a substantially dry gene silencing composition.
• the gene silencing composition can have at least a first siRNA which silences at least a first target gene.
• the gene silencing composition can be configured such that the first siRNA is capable of being solubilized or suspended in the aqueous medium in an amount sufficient for transfecting cells in the first test well.
• cells are added to the first test well under conditions that permit transfection. Additionally, cells are added to a first control well. Any control well can include control siRNA or it can be devoid or substantially devoid of having siRNA. Also, the cells can be added to the wells under conditions that permit the siRNA to be introduced into the cell. The cells can be added in an amount of about 1x10 3 to about 3.5xlO 4 per about 0.3 cm 2 to about 0.35 cm 2 of cell growth surface area. Any mode of transfection can be used to introduce the siRNA into the cells. Subsequently, the cells can be maintained in the test well and the control well for any appropriate duration before a biological agent is added to the well. This can include maintaining the test well and test control well under conditions so that cell growth, cell division, transfection and/or gene silencing occurs.
• a biological agent is added to the test and control wells after the cells are plated and/or after any gene silencing has occurred.
• the biological agent can be added to a well before the cells are added.
• the cells in the test well and control well can be maintained or incubated with the biological agent for a period of time sufficient for the biological agent to have an effect on normal cells in normal conditions. That is, the duration the biological agent is incubated with the cell cultures is a time sufficient for the effect of the biological agent to be observed in cells that have not been otherwise treated with siRNA.
• a determination can be made as to whether or not a first response to the biological agent by the cells in the first well is different from a first control response to the biological agent by the cells in the first control well.
• the siRNA may silence a gene that is involved in a cellular response to the biological agent.
• the test response is different from the first control response it can be an indication that the target gene that was silenced is involved in the response by the cell to the biological agent.
• the test response is substantially the same as the first control response, the first target gene may not be involved in a response by the cell to the biological agent.
• the method of studying a response by a cell to a biological agent can include adding cells to a second control well.
• the second control well can be a well that does not receive the biological agent, and can include control siRNA or it can be devoid or substantially devoid of having any siRNA.
• the second control well can be cultured and maintained in a manner similar to the test well and the first control well so that meaningful comparative data can be obtained. After being maintained in a manner substantially similar with the test well and/or first control well a determination can be made as to whether the first response to the biological agent by the cells in the first test well is different from a second control response to the absence of the biological agent by the cells in the second control well.
• the target gene may be involved in the response by the cell to the biological agent.
• the first test response is substantially the same as the second control response the target gene may not be involved in the response by the cell to the biological agent.
• the first control response in the first control well can be compared to the second control response in the second control well, which can provide data regarding the efficacy of the testing procedure.
• a control well that does not receive a biological agent, such as the second control well can be used in a comparison with the test well in place of the first control well.
• Figures 1A-1E are schematic diagrams that illustrate different embodiments of siRNA arrangements on a multi-well plate that can be used to study a biological agent.
• FIGS 2A-2D are schematic diagrams that illustrate different embodiments of control plate arrangements that can be used to optimize studies of biological agents in the presence of gene silencing.
• the present invention is related to an apparatus and system for use in effecting gene silencing in cells in the presence of a biological agent.
• the apparatus includes plates with wells that have dry gene silencing compositions comprised of at least a first siRNA, which can be solubilized or suspended in an aqueous medium for use in RTF protocols.
• the siRNA can be configured for silencing cellular genomic or transgenes.
• the biological agent is a microorganism the genomic or transgenes within the microorganism can be silenced in order to assess any changes in the cellular response.
• the systems which can be provided as kits, can include the plates, biological agent, polynucleotide carriers, polynucleotide carrier solutions, an aqueous medium, and the like.
• the well plates, systems, kits, and methods of the present invention can be configured for use in high content screening ("HCS") or high throughput screening ("HTS”) of biological agents with or without the use of laboratory automation equipment.
• HCS high content screening
• HTS high throughput screening
• the well plates, systems, kits, and methods can also be used with automated systems, such as robotic systems.
• 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.
• the well plates, systems, kits, and methods provide versatility in choice such that high throughput screening of biological agents can be done in a cost effective manner.
• the present invention can be used to screen for the contribution of a large number of genes to a cellular response to a biological agent. This can be used to screen the entire genome to identify the gene or combination of genes that encode for products that are involved in the cellular response or cellular event that is induced by the biological agent.
• the genes of a microorganism can be silenced to assess the effect on infection, replication, and release from a cell.
• 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.
• 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.
• 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.
• 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 such as non-coding RNA (e.g., tRNA, rRNA, and the like), or a sequence of DNA that is either coding or non-coding.
• mRNA messenger RNA
• non-coding RNA e.g., tRNA, rRNA, and the like
• 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.
• active agent is meant to refer to a molecule or combination of molecules that have some activity or cause an effect on a cell or cellular process. Such activities or effects can be either beneficial or detrimental to the viability of a cell.
• active agents include chemicals, toxic chemicals, known drugs, potential drugs, nutraceuticals, vitamins, polypeptides, proteins, hormones, cytokines, enzymes, and the like.
• biological agent is meant to refer to a chemical or pathogen that can be introduced into a cell.
• the biological agent can have a property that induces a physiological response in a subject such as a plant or an animal (e.g., humans, mice, rats, and the like) or a plant.
• the biological agent can induce a cellular response when introduced into a cell.
• Biological agents can be molecules or combinations of molecules that can be produced by natural processes or chemical reactions. Additionally, biological agents can be microorganisms that are capable of being produced by replication.
• the terms “dried” or “dry” as used in connection with gene silencing compositions are 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.
• 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.
• 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.
• 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.
• 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.
• host proteins e.g., RISC
• gene silencing can refer to a decrease in gene product expression that results from siRNA mediated translation inhibition.
• 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.
• 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.
• a reporter protein such as colorimetric or fluorescent properties (e.g., GFP), enzymatic activity (e.g., alkaline phosphatases), or other well known analytical procedures.
• microorganism is meant to refer to a microscopic organism that can be characterized as being alive or capable of replication. Accordingly, microorganisms can be either benign or pathogenic. Examples of pathologic microorganisms include viruses, bacteria, fungi, protozoa, and the like.
• polynucleotide is meant to refer to polymers of nucleotides linked together through internucleotide linkages.
• a polynucleotide includes DNA, RNA, DNA/RNA, hybrids including polynucleotide chains of regularly and/or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an -OH, then and -H, then an -OH, then an -H, and so on at the 2' position of a sugar moiety), and modifications of these kinds of polynucleotides.
• polynucleotides include nucleotides with various modifications or having attachments of various entities or moieties to the nucleotide units at any position.
• 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.
• rationally designed siRNA are selected to specifically interact with and inhibit polypeptide translation from a selected mRNA.
• siRNA for any one target mRNA there may be hundreds of potential siRNA of 18 - 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.
• reverse transfection and abbreviation "RTF” are each meant to refer to a process for introducing nucleic acid, such as an siRNA, into a cell.
• nucleic acid such as an siRNA
• RTF abbreviation
• 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.
• the siRNA is complexed with a lipid or other polynucleotide carrier prior to being contacted to the cells; however, other modes of transfection can be used to introduce the siRNA into the cell.
• 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.
• 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.
• 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.
• 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.
• a sense strand can include one portion of a unimolecular siRNA that is capable of forming hairpin structure, such as an shRNA.
• 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.
• siRNA is meant to refer to a small inhibitory RNA duplex that induces gene silencing by operating within the RNA interference ("RNAi") pathway.
• RNAi RNA interference
• siRNA are dsRNA that can vary in length, and can contain varying degrees of complementarity between the antisense and sense strands, and between the antisense strand and the target sequence.
• Each siRNA can include between 17 and 31 base pairs, more preferably between 18 and 26 base pairs, and most preferably 19 and 21 base pairs.
• Some, but not all, siRNA have unpaired overhanging nucleotides on the 5' and/or 3' end of the sense strand and/or the antisense strand.
• siRNA includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region, which may be referred to as short hairpin RNA ("shRNA").
• shRNA short hairpin RNA
• 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.
• a pool includes four or more non-identical siRNA that are rationally designed.
• Table 1 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 the incorporated provisional application.
• siRNA pool 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, rnRNA, 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. 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).
• siRNAs that comprise the SMARTpool ® Reagent can also be plated individually on the same plate as the iSM4i?rpoolTM Reagent.
• target is used in a variety of different forms throughout this document and is defined by the context in which it is used.
• target gene is meant to refer to the gene that encodes the protein to be silenced by the siRNA, and encodes for the production of the target mRNA.
• target rnRNA is meant to refer to an mRNA against which a given siRNA is direct to silence the transcription of the polypeptide product.
• target sequence and “target site” are meant to refer to a sequence within the mRNA, miRNA, or DNA coding or promoter region to which the sense strand of an siRNA exhibits varying degrees of homology and the antisense strand exhibits varying degrees of complementarity.
• target polypeptide or "target protein” is meant to refer to the gene product encoded by the target gene, target mRNA, and/or target sequence.
• siRNA target can refer to the gene, mRNA, or protein against which the siRNA is directed to for silencing.
• target silencing can refer to the state of silencing a gene, or the corresponding mRNA or protein.
• 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.
• 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, cholesterol conjugates, or peptide conjugates), microinjection, laser- or light-assisted microinjection such as, for example, optoporation or photoporation with visible and/or nonvisible wavelengths of electromagnetic radiation, and the like.
• electroporation calcium phosphate delivery
• DEAE-dextran delivery lipid delivery
• polymer delivery e.g., polylysine-DNA or -RNA conjugates, antibody- polypeptide conjugates, antibody-polymer conjugates, cholesterol conjugates, or peptide conjugates
• microinjection e.g., laser- or light-assisted microinjection such as, for example, optoporation or photop
• 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.
• RTF reverse transfections
• 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.
• the present invention provides well plates, systems, kits, and methods for implementing reverse transfection ("RTF") of siRNAs.
• RTF reverse transfection
• the RTF protocol can be implemented to silence a selected gene or combination of genes to study the ability of a gene product to interact with or be associated with a cellular response to a biological agent, wherein the biological agent can be an active agent or a microorganism.
• the RTF protocol can be used to assess genes involved in an infectious pathway. This can be done by using gene silencing to study genes in the cell that respond to the pathogenic infection or silencing genes in the pathogen.
• the present invention includes an RTF protocol for introducing siRNA into a cell to study the effect of gene silencing on a biological agent that is also added to the cell.
• a method can include providing a well plate that includes a first test well having a substantially dry gene silencing composition.
• the gene silencing composition can include at least a first siRNA which silences at least a first target gene so that the production of the corresponding gene product is inhibited or stopped.
• the gene silencing composition can include a pool of siRNAs which silences the target gene.
• the siRNA is present in the first test well as part of the dry gene silencing composition so that the plates can be prepared, sealed, stored, and/or shipped long before an RTF protocol is performed.
• the dry gene silencing composition can stably retain the siRNA in a functional condition within the well, and be suspended or solubilized with an aqueous medium during the RTF protocol.
• a well plate having the gene silencing composition can be manufactured and hermetically sealed in an inert environment within a sterile container, wherein the plate can include different wells with predefined types of siRNA for specific gene targets, which can include an individual siRNA and pools of siRNAs. Such types of siRNA and intended gene targets for silencing are described in more detail below.
• An aqueous medium can be added to the test well that contains the gene silencing composition to suspend or solubilize the siRNA into the aqueous medium.
• the aqueous medium is allowed to solubilize or suspend the siRNA for a sufficient duration so that most or all of the siRNA is in the aqueous medium.
• cells are added to the test well under conditions that permit the siRNA to be introduced into the cells. The cells are usually added in an amount of about IxIO 3 to about 3.5xlO 4 cells per about 0.3 cm 2 to about 0.35 cm 2 of cell growth surface area.
• the conditions that promote an siRNA entering a cell can be described by typical cell culture techniques used for plating cells that are well known in the art. That is, the cells can be added to the well that contains the siRNA in a manner similar to ordinary plating. Additionally, any mode of transfection can be used to introduce the siRNA into the cells.
• the test well containing the siRNA and cells can be incubated for a sufficient duration for gene silencing to occur, which is typically less than 72 hours, more preferably less than 48 hours, and most preferably about 24 hours or less.
• the aqueous medium that comprises the cells can be used to solubilize or suspend the siRNA with the cells.
• the RTF protocol can include adding a polynucleotide carrier to the test well to form an siRNA-carrier complex, wherein the siRNA-carrier complex is suspended or solubilized in the aqueous medium. After the cells are added, the siRNA-carrier complex can contact the cell surface to induce endocytosis of the complex.
• the polynucleotide carrier can be added as part of the aqueous medium or in addition thereto. Thus, the polynucleotide carrier can be presented in an aqueous medium as either solubilized or suspended therein.
• the polynucleotide carrier can be a cationic lipid, polymer, lipopolymer, and the like.
• the siRNA can include a conjugate, such as a cholesterol or polypeptide, which can enhance passive delivery of the siRNA into the cells, and thus eliminate the need for polynucleotide carrier; however, such passive delivery conjugates can be advantageously used with polynucleotide carriers.
• a conjugate such as a cholesterol or polypeptide
• the well plate can be maintained under conditions so that cell growth, cell division, transfection, and/or gene silencing occurs.
• the cells are maintained in the presence of the siRNA for about 6 to about 72 hours to effect gene silencing, more preferably about 12 to about 48 hours, even more preferably from about 18 hours to about 36 hours, and most preferably about 24.
• the cells are incubated with the siRNA for a time period sufficient for silencing a gene so that the amount of corresponding gene product decreases.
• the production of a target polypeptide can be silenced by at least 50%, more preferably by at least 70%, even more preferably by at least 80%, and most preferably by at least 90%.
• cells can be added to a first control well.
• the first control well can include control siRNA or it can be devoid or substantially devoid of having any siRNA.
• the cells can be maintained in the control well for any appropriate duration before a biological agent is added to the well. This can include maintaining the control well under conditions so that cell growth, cell division, transfection, and/or gene silencing occurs.
• a biological agent is added to the cells in the test well and the cells in the first control well, hi different studies, it can be appropriate for the biological agent to be added to the wells before, during, or after the cells are added to the wells. Additionally, the biological agent can be added to the wells before or after gene silencing occurs in the test well.
• the biological agent is typically added from about 0.25 to about 72 hours after the cells are added to the wells, more preferably from about 1 hours to about 48 hours, even more preferably from about 6 hours to about 36 hours, and most preferably from about 12 to about 24 hours after the cells are added to the wells.
• the cells in the test well and first control well can be maintained or incubated with the biological agent for a period of time sufficient for the biological agent to have an effect on normal cells in normal conditions. That is, the duration the biological agent is incubated with the cell cultures is a time sufficient for the effect of the biological agent to be observed in cells that have not been otherwise treated with any test reagent such as siRNA.
• the cells in the test well and first control well can be assayed for a response to the biological agent.
• the response to the biological agent can be detected by any protocol that measures a cellular response to the biological agent, or directly measures the presence of the biological agent in the cells.
• the cellular response can be detected by methods described herein or well-known methods in the art, which can include toxicity studies and protein analysis. This can also include photometric analyses that direct light through the wells, wherein it can be favorable for the test well and first control well to each have a substantially flat well floor comprised of a translucent or transparent material such as polystyrene.
• a determination can be made as to whether or not a first response to the biological agent by the cells in the test well is different from a first control response to the biological agent by the cells in the first control well. In the instance the first response is different from the first control response the first siRNA may silence a gene that is involved in a cellular response to the biological agent.
• the first response being different from the first control response may be linked to a biological pathway related to the biological agent being compromised.
• the first response when the first response is different from the first control response it can be an indication that the first target gene is involved in the response by the cell to the biological agent.
• the first target gene when the first response is substantially the same as the first control response, the first target gene may not be involved in a response by the cell to the biological agent.
• the method of studying a response by a cell to a biological agent can include adding cells to a second control well.
• the second control well can be a well that does not receive the biological agent, and can be used in addition to or in place of the first control well that does receive the biological agent.
• the second control well can be cultured and maintained as the test well and the first control well so that meaningful comparative data can be obtained.
• the second control well can include control siRNA or it can be devoid or substantially devoid of having any siRNA.
• the first response is different from the second control response the first target gene may be involved in the response by the cell to the biological agent.
• the first response is substantially the same as the second control response the first target gene may not be involved in the response of the cell to the biological agent.
• the first control response can be compared to the second control response when both are used, which can provide data regarding the efficacy of the testing procedure.
• the cells in the test well and first control well can contain the target gene.
• the target gene can be a genomic gene in the cells.
• the target gene can be a plasmid that has been transfected into the cells.
• the target gene may be contained in the cells of the first well and first control well by being within a virus, fungi, bacteria, or other microbe that has entered the cell. This can include the target gene being a microbial genomic gene or a transgene.
• the present invention includes a method of determining a cellular response to a biological agent. This can include identifying a gene involved in a response by a cell to a biological agent.
• the study can be performed by adding an aqueous medium to a plurality of wells in a well plate.
• the plate can include at least a first set of wells that contain substantially dry gene silencing compositions.
• the gene silencing compositions can have at least a first siRNA which silences a first target gene.
• the gene silencing composition can be configured such that the first siRNA is capable of being solubilized or suspended in the aqueous medium in an amount sufficient for transfecting cells in the well.
• cells can be added to the first set of wells under conditions that permit transfection.
• Cells can also be added to a first set of control wells, wherein a well in the first set of control wells corresponds to a well in the first set of wells that contains a gene silencing composition.
• the cells, first siRNA, and first set of control wells can be selected to provide a biological comparison between the results obtained from the study.
• the cells in the first set of wells and in the first set of control well can be maintained for any duration before the biological agent is added to the first set of wells.
• the biological agent may or may not be added to the first set of control wells depending on the study.
• the cells can be incubated with the biological agent before a cellular response thereto is detected and/or measured.
• the cells in the test well transfected with the siRNA in the RTF format can be assessed for cell viability, gene silencing, target protein production, and the like.
• the cells in either the first control well or the second control well can be assessed for cell viability, gene silencing, protein production, and the like.
• the cell viability studies can be performed in the well plate in accordance with well known procedures.
• the gene silencing and/or activity of the biological agent can also be assessed with the contents in the well by various techniques well known in the art to assess the presence or absence of target proteins.
• the amount of gene silencing and/or activity of the biological agent can be assessed by removing the contents from the well by well known assays.
• the well is designed to be compatible with optical detection systems such as, for example, UV, luminescence, fluorescence, or light scattering detection systems.
• optical detection systems such as, for example, UV, luminescence, fluorescence, or light scattering detection systems.
• the walls of the well can be made opaque, or rendered such that light scattering that can interfere with optical detection is reduced or minimized.
• the cellular response to the biological agent in the presence of gene silencing can be detected or monitored using systems for performing high content screening ("HCS") or high throughput screening ("HTS").
• HCS high content screening
• HTS high throughput screening
• 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. Patent Nos.
• HTS analyses can be performed using a variety of available readers, typically of the fluorescence from each well as a single measurement.
• the invention includes a well plate configured for having the contents of a well transferred to a location, device, or system wherein detection of the results of an siRNA RTF protocol is carried out.
• wet transfer detection systems can be employed that include systems wherein cells are transferred from wells to a substrate such as nitrocellulose. Following the transfer of the well contents to the substrate a detection protocol can be implemented.
• An example of such a well plate transfer system can include nitrocellulose, wherein the well contents can be treated such that cell membranes are permeabilized or disrupted so as to gain access to intracellular contents.
• the transfer of the well contents to the nitrocellulose can be achieved by any suitable method including gravity or use of a vacuum manifold.
• the nitrocellulose containing the well contents can then be further subjected to a detection protocol that uses antibody-based detection systems and the like to detect the presence or level of one or more contents of the cells that comprise a particular well.
• the number of cells in a test well or control well can be varied to optimize gene silencing and/or activity of the biological agent. It has been found that siRNA RTF protocols can have more favorable results with lower cell densities compared to RTF protocols using DNA.
• 96-well plates can include cell densities of about 1,000-35,000 cells per well, more preferably about 2,000-30,000 cells per well, even more preferred are cell densities of about 2,500- 20,000 cells per well, still more preferably about 3,000-15,000 cells per well, and most preferable are cell densities of about 3,500-10,000 cells per well. These cell densities can be favorable for studying biological agents in the presence of gene silencing.
• the number of cells per well can be extrapolated to wells having different cell culture areas.
• One possible equation for calculating the appropriate number of cells that are placed in a given well is based on a 96-well plate having a cell culture area of about 0.3 cm 2 to about 0.35 cm 2 , wherein well # 2 is the 96-well plate, and is described as follows:
• the present invention is directed to studying the effect of gene silencing on a cellular response to a biological agent.
• the study protocols can include any of the following: (1) selecting the type of plate; (2) selecting an appropriate solution to deliver and dry the siRNAs in the wells; (3) selecting an individual siRNA or pool of siRNAs to silence specific genes; (4) identifying any modifications or conjugates that can be applied to the individual siRNA in order to enhance siRNA stability and/or specificity; (5) applying and drying the siRNAs on well floor so that it can be solubilized or suspended in an appropriate aqueous medium; (6) selecting an appropriate mode of transfection to introduce the siRNA into the cells; (7) solubilizing or suspending the siRNAs; (8) selecting an appropriate polynucleotide carrier and complexing the siRNA with the polynucleotide carrier to form an siRNA-carrier complex; (9) combining the siRNA-carrier complex with the cell type or types of choice; (10) selecting a biological agent to test; (11)
• 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 NUNCTM, NUNCLONTM, MICROWELLTM and FLUORONUNCTM plates (e.g., each of which may be obtained from Nalge Nunc International of Rochester, NY, and Nunc A/S of Denmark), COSTARTM, COSTAR THERMO WELLTM and CORNINGTM plates (e.g., each of which is available from Coming), BD FALCONTM and OPTILUXTM plates (e.g., available from Becton, Dickinson and Company) and GREINERTM, CELL COATTM and CELLSTARTM plates (e.g., available from Greiner Bio-
• 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.
• certain analytical equipment can have enhanced functionality when using flat well floors.
• wells having substantially flat floors can provide uniform cell spacing and monolayer formation.
• 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.
• 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.
• PLL poly-L-lysine
• laminin laminin
• collagen or equivalent substances that improve the adherence of cells.
• each plate can have between 6 and 2000 wells, and more preferably having 1536 wells, 384 wells, or 96 wells.
• the wells 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 cm to about 4.2 cm , and about 0.3 cm to about 0.35 cm for a 96-well plate.
• the wells are not coated with materials such as MATRIGELTM (Beckinson Dickerson), or are not
• a well plate in accordance with the foregoing can be configured to be a gene silencing plate.
• the well plate can include a gene silencing composition in one or more wells.
• the well plates can be gene silencing plates by having an siRNA-containing solution applied to at least one well, which is then dried in a manner that removes the solution and leaves a dried gene silencing composition.
• the gene silencing composition can have at least one siRNA that silences at least a first target gene or a pool of siRNAs that silence the target gene.
• the gene silencing composition can have a single siRNA directed against a family of related genes.
• the well plate can have a well having multiple siRNAs targeting a single gene, or multiple siRNAs targeting multiple genes.
• the gene silencing plates can be prepared as described in the incorporated reference having Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. as inventors
• the siRNA delivered to a test well is present in a total amount that can provide a desired final concentration after being solubilized or suspended in the aqueous medium. It can be preferable that the final concentration of siRNA to effect gene silencing can be less than or equal to about 250 nM, more preferably less than or equal to 100 nM, even more preferably less than or equal to about 50 nM, still even more preferably less than or equal to about 25 nM, and most preferably less than or equal to about 10 nM. In some instances it the siRNA may be present at less than or equal to 1 nM.
• the amount of siRNA delivered to a well in a 96-well plate can be from 0.1 picomoles ("pm") to 100 pg, 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.
• the total amount of siRNA added to each well can be sufficient for use in a single RTF protocol within that well. That is, the siRNA in the gene silencing composition can be present in an amount to only be used with the cells added to the well. As such, the total amount of siRNA dried in the well can be insufficient for performing two RTF protocols in two different wells.
• the siRNA solution can be transferred to the well floor by any method of delivering liquids into wells of a well plate, which can be manual or automated. Various methods can be used to dry the siRNA solution into a gene silencing composition. In one embodiment, the plates are allowed to dry at room temperature in a sterile setting which allows the siRNA solution to evaporate leaving behind the pool of siRNAs.
• the dried plates can be sterile so that cells are not contaminated during the RTF protocol.
• Dried plates are preferably vacuum-sealed or sealed in sterile packages in the presence of inert gases, and stored at temperatures ranging from -8O 0 C to 37 0 C for extended periods of time without loss of silencing functionality.
• the plates having the dry gene silencing compositions in at least one well can be stored at room temperature and shipped via traditional routes and still maintain the integrity and functionality of the siRNA.
• the well plate can have various wells that can be used for control and calibration functions.
• the well plate can have at least one well devoid or substantially devoid of siRNA.
• the well plate can have at least one well that includes at least a first control siRNA.
• the control siRNA can include at least one of the following: (a) an siRNA that is capable of silencing a known gene; (b) transfection control siRNA; (c) an siRNA having a fluorescent marker; (d) siRNA having at least one toxic motif; (e) a non-functional siRNA; or (f) a siRNA that inhibits being taken in and processed by RISC.
• the foregoing dry gene silencing compositions include at least a first siRNA which silences at least a first target gene, and can include a pool of siRNAs.
• the gene silencing composition is configured such that the siRNA is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the well.
• the total amount of siRNAs in the well is sufficient for implementing reverse transfection only for that well.
• the siRNA it is optional for the siRNA to have at least one of a hairpin structure, modification or a conjugate.
• the siRNA can " be rationally designed to target the gene.
• the siRNA is selected to optimize functionality in silencing the target gene.
• the siRNA has between 50% and 100% gene silencing functionality, more preferably between 70% and 100%, even more preferably between 80% and 100%, and most preferably a gene silencing functionality between 90% and 100%.
• the design of a functional siRNA can be based on providing modifications that increase target specificity, increase stability, are rationally designed for particular mRNA targets, and combinations thereof.
• siRNA can target a promoter region, the open reading frame, the 5' UTR, the 3' UTR, or other regions of DNA.
• siRNA can be preferably to select siRNA from a list that have been identified from being rationally designed.
• the siRNA can be selected from Table I and Table II of U.S. Provisional Application Serial 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 92,448 19-mer siRNA sense strand sequences, where antisense strand sequences were omitted for clarity.
• the siRNA sequences listed in Table I includes SEQ. ID NOs.
• each preferably can also include a 3' UU overhang on the sense strand and/or on the antisense strand.
• Each of the 92,448 sequences of Table I when used in an siRNA, can also comprise a 5' phosphate on the antisense strand.
• 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.
• an siRNA can be configured as an shRNA.
• an shRNA is a form of siRNA that includes a hairpin structure having a loop region connecting a sense region with an antisense region.
• the shRNA can have a substantially similar functionality compared to other types of siRNA.
• an shRNA is not considered a modified siRNA unless the nucleotides include modifications as described in more detail below.
• the siRNA is presented as a hairpin shRNA, the size and orientation of the strands can vary. Additional information regarding shRNA can be found in the incorporated reference having Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. as inventors.
• the present invention includes siRNA having a modification that increases specificity for gene silencing. Accordingly, specificity modifications can be incorporated into any siRNA in order to decrease off-targeting. Such specificity modifications can be an aspect of on-targeting. A more complete description of specificity modifications can be found in the incorporated reference having Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. as inventors.
• the present invention includes siRNA having stability enhancing modifications.
• the stability modifications can be used with or without specificity modifications, and vice versa.
• siRNA having stability modifications can be advantageous because they can prevent degradation by nucleases.
• the stability modifications can increase the potential shelf life of siRNA, and increase the ability to manufacture and store plates having dry gene silencing compositions for extended periods of time.
• a more complete description of stabilizing modifications can be found in the incorporated reference having Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. as inventors.
• the siRNA can include a conjugate, such as a label, coupled to the sense and/or antisense strands.
• the conjugate can perform a variety of functions or provide additional benefits to the siRNA.
• a cholesterol conjugate can increase the penetration of the siRNA through a cell membrane with or without being complexed with a carrier.
• the conjugates can be labels that can be monitored or identified in order to determine whether or not a labeled siRNA entered a cell.
• a complete description of conjugates that can be coupled to siRNA can be found in the incorporated reference having Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. as inventors. V. Polynucleotide Carriers
• the present invention includes polynucleotide carriers that can interact with siRNA, and transport the siRNA across a cell membrane.
• modes of transfection can be implemented without carriers, such as by electrophoresis, precipitation, particle bombardment, optoporation, and microinjection.
• polynucleotide carriers include a positive charge that interacts with the negatively charged phosphates on the polynucleotide backbone.
• Polynucleotide carriers that are well-known in the art of cellular nucleic acid deliver.
• Preferred polynucleotide carriers include cationic polymers, lipids, lipopolymers, lipid-peptide mixtures, and the like that are capable of complexing with an siRNA and delivering the siRNA into a cell in a manner that retains the gene silencing functionality without being overly toxic.
• lipids or lipid-peptide mixtures are preferable for introducing an siRNA into a target cell.
• the lipid is a cationic lipid.
• Cationic lipids that can be used to introduce siRNA into cells can be characterized by having little or no toxicity (e.g., defined as less than 15-20% toxicity), which can be measured by AlamarBlue or equivalent cell viability assays.
• Such lipids can facilitate delivery of sufficient amounts of siRNA into cells in order to induce gene silencing.
• Peptides that have affinity to one or more proteins, lipids, lipid-polysaccharide, or other components of the cell membrane can be conjugated to the siRNA and used independent of lipids or advantageously combined with one or more lipids to form a polynucleotide carrier.
• Such lipid-peptide mixtures can enhance RTF of siRNA.
• Cholesterol conjugates can be similarly coupled to the siRNA and be used independent of polynucleotide carriers or advantageously combined therewith.
• suitable lipids for siRNA pool RTF include OLIGOFECTAMINETM, TransIT-TKOTM, or TBIO Lipid 6TM, LIPOFECTAMINETM 2000, lipids DharmaFECTTM 1, DharmaFECJTM 2, DharmaFECTTM 3, and DharmaFECTTM 4 (Dharmacon, Inc.).
• lipids can be prepared by combining siRNA and the lipid.
• 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.
• 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 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- MEMTM (GIBCO, Cat. # 31985-070), HyQ-MEM-RSTM (HyClone, Cat.# SH30564.01), Hanks Balanced Salt SolutionTM, or equivalent media.
• 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).
• 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 typically is performed at temperatures ranging from 4-37 0 C.
• the lipid and siRNAs can be mixed by agitating the plate (e.g., swirl, vortex, sonicate) for brief periods (e.g., seconds to minutes) to enhance the rate of siRNA solubilization and complex formation.
• agitating the plate e.g., swirl, vortex, sonicate
• brief periods e.g., seconds to minutes
• the present invention includes biological agents that can be studied for their effect on cells that have had a particular gene or combination of genes silenced with siRNA. Accordingly, a biological agent can be introduced to a cell or to the cell culture medium to determine whether or not the silencing of a particular gene alters the effect of the biological agent on the cell compared to a cell that does not have a gene silenced. Also, the effects of silencing multiple genes in a single cell or different genes in different cells can be compared so that a broad spectrum analysis of the biological agent can be obtained. Moreover, the entire genome of any organism can be screened for being associated with a response to a biological agent.
• the biological agents in accordance with the present invention can be substances that are physiologically active agents, such as molecules or combinations of molecules, or microorganisms that are benign or pathologic in nature.
• the biological agent is an active agent, which includes substances known or suspected of having biological or physiological activity. That is, the substance can interact with proteins, cells, lipids, polypeptides, and the like within an organism and cause a change in the organism.
• Active agents include a wide variety of molecules and combinations of molecules including small molecules that can be used as drugs, chemicals that can have adverse or beneficial physiological interactions, nucleic acids like plasmid DNA, and polypeptides or proteins like enzymes, cytokines, and hormones. Additionally, macromolecular substances or structures, such as polymers, can be used. For example, a cellular response to latex can be studied in the presence of gene silencing. Accordingly, any active agent can be used in the present invention in order to be studied in the presence of gene silencing. This allows the selective silencing of a specific gene to identify genes that maybe responsible for proteins or pathways that respond to the presence of the active agent.
• the active agent is a drug, which includes known substances that are used as drugs as well as substances suspected of being usable as drugs.
• the drag can be studied by being introduced to cells that have a selected gene or combination of genes silenced wherein the drug can interact with external or internal components of a cell. This can allow for the activity of the drag to be assessed in the absence of selected gene products.
• a disease model can be created by the selective silencing of a single gene or family of genes, and the drag can be administered to the cell so that its ability to function as a therapeutic agent can be monitored.
• a gene product suspected of adversely interacting with a drag can be silenced to determine the effect of the drag on the cell in the absence of the gene product.
• a single drag can be studied with different genes being silenced so that phenotypic changes can be related to drag activity. Thus, a wide array of studies can be performed on drag activity in the presence of a silenced gene.
• the active agent is a chemical other then a drag.
• the chemical can be known or suspected to have biological or physiological activity that is beneficial or adverse to an organism.
• the chemical can be studied in the presence of a silenced gene or combination of genes so that the effect of the chemical on a cell can be determined.
• a chemical suspected of having a toxic or detrimental effect on an organism can be tested on selected cell types from that organism, wherein various genes in the cells can be silenced so that the toxicity or cellular function can he monitored in the presence of the suspected toxin. This can be beneficial for identifying genes involved in a response to a toxic chemical.
• the same procedure could be used to identify biological pathways that adversely react to certain chemicals that may otherwise have a beneficial use.
• the active agent is a polypeptide or protein such as a hormone, enzyme, cytokine, or the like.
• the active agent can be any naturally occurring or synthetic polypeptide that is known or suspected of having biological or physiological activity.
• Polypeptides can be studied in the present invention by being introduced to cells having a selected gene or combinations of genes that have been silenced. This can allow for a broad array of screenings of polypeptides and activities in various cellular conditions and cell phenotypes.
• a polypeptide having a suspected therapeutic use can be studied with cells having selected genes silenced to determine which genes or family of genes may interact with the polypeptide
• a disease model can be created by the selective silencing of a gene or genes in a particular pathway so that the activity or function of a polypeptide can be studied in the model. This can enable the activity of a polypeptide in response to a disease to be studied or identify polypeptides that can be used to treat the disease.
• the importance of one or more genes in the response of cells to a specific biological agent can be tested. Also, the importance of one or more genes in a particular phenotype (e.g., cell growth, cell recognition, cell differentiation) can be studied in the presence of a biological agent. For example, various genes in a cell can be silenced, such as genes of the human genome, a particular family of genes (e.g., kinase genes), or genes of a particular pathway.
• the ability of the cells to respond to a particular substance or compound e.g., a drug or toxic chemical
• a particular substance or compound e.g., a drug or toxic chemical
• untreated cells i.e., cells not transfected with an siRNA
• the method can identify a gene that plays a role in the cellular response to the substance or compound.
• a cellular response to an active agent can be compared between a test well having a test siRNA and a control well either devoid of any siRNA or having a control siRNA.
• a cellular response to the active agent can be measured or identified by comparing at least one of retention, excretion, metabolism, activity, or absorption of the active agent by the cells in the test well compared to the cells in the control well.
• any change in cell function and/or morphology of the cells in the test well compared to the cells in the control well can be monitored and detected. Monitoring and/or detection can be done visually by the experimenter. Alternatively, monitoring and/or detection can be automated using technologies designed for HCS analysis.
• gene silencing can be used in a method for assessing the contribution of a gene to cellular interaction, retention, excretion, metabolism, or absorption with respect to the active agent.
• cell cultures having a gene or combination of genes that have been silenced can be treated with a particular compound (e.g., a potential therapeutic compound or toxic compound).
• a particular compound e.g., a potential therapeutic compound or toxic compound.
• the ability of cells to retain, excrete, metabolize, absorb, or adsorb that compound can be measured and compared with cells that are not administered the compound or cells with no gene silencing.
• any active agent can be a part of a system or kit that includes a well plate having a gene silencing composition that silences genes that may interact with the active agent.
• gene silencing in cells can be used to identify particular phenotypes that are generated by an active agent. Accordingly, the effect of the active agent on the cell can be determined when a specific gene or combination of genes has been silenced. This can be especially beneficial in the instance the silenced gene or combination of genes creates a disease model. As such, the effect of the drag on a cell can be monitored and identified instead of the effect of the cell on the drug (i.e., absorption, excretion, metabolism, and the like is not studied).
• Phenotypic changes in a cell that are generated by an active agent in the presence of gene silencing can be used to simulate the effect of a drags in the absence of a gene product, and thereby can provide information related to whether or not certain active agents are suitable for a specific therapeutic regimen. Additionally, similar studies can be performed by administering multiple active agents in the presence of gene
• Microorganisms that interact with an organism or cell can be studied in accordance with the present invention by being introduced to cell cultures having a selected gene or combinations of genes that have been silenced. This can allow for a cellular response to such a microorganism to be studied when the production of a selected cellular polypeptide, such as a protein, has been silenced. Similarly, this protocol can enable one to identify the contribution of various host genes to a pathogenic infection. Also, this can allow for selected microorganism genes to be studied because the siRNA in the gene silencing composition can be used to silence a microorganism genomic gene. As such, a cell that has not had any host genomic genes silenced can be used to study microorganisms having silenced genes.
• the biological agent is a pathogen.
• the pathogen can be a microbe that can cause an infection associated with phenotypic changes, cellular toxicity, and death.
• pathogens include viruses, bacteria, yeasts, protozoa, and the like. Accordingly, a cellular response or contribution to an infection can be studied when a specific gene or combination of genes in the cell have been silenced. This can include silencing genomic genes of the cell or transgenes that have been stably or transiently transfected into the cell.
• the gene being silenced can be genomic genes of the pathogen. This can allow for an infectious pathway to be studied when cellular or pathogenic genes have been silenced.
• the pathogen can be used in an amount that is toxic to the cells, which can cause the cells to have inhibited cellular functions or cause cell death.
• the viability or function of a cell in a test well can be compared to a cell in a control well in response to gene silencing and the presence of a microorganism such as a pathogen.
• the production or non-production of a target polypeptide in response to the pathogen can be detected in cells that have a target gene that is silenced and/or not silenced.
• various aspects of a pathogenic infection can be identified by administering the pathogen to cells that have had a specific gene or combination of genes silenced.
• the pathogen is a virus it can be modified to contain a reporter vector which does not include viral genes that aid in the infection. This can allow for a selected host genomic gene or combinations of genes to be silenced so that the effect on viral infection can be determined because the virus will not have any infectious genes to use for inducing the infection.
• the cellular mechanisms that contribute to the virus infecting the cell can be identified, which can allow for designing preventive therapies to inhibit the onset of such infections.
• gene silencing can be used to study various other aspects of interactions that occur between a cell and a microorganism.
• the present invention includes a method for determining a cellular response to a biological agent in the presence of gene silencing when the cell has been sensitized to the biological agent.
• Sensitization usually occurs after repeated exposure to a biological agent, which can be either an active agent or a microorganism. Sensitization can be detrimental when beneficial substances, such as drugs, begin to cause serious and unwanted physiological complications. Also, sensitization has been linked to chemical dependencies and addictions to various drugs. Moreover, sensitization by a microorganism can cause inflammation or other allergic reactions. Sensitization to a biological agent is typically manifested by an enhanced response following repeated administration of the same dose the biological agent. Accordingly, the same dose begins to cause increased cellular responses over time.
• the mechanisms for such sensitizations can be identified and studied by silencing specific genes or combinations of genes in a cell.
• the method of studying sensitization with gene silencing can be performed in substantially the same manner as the foregoing RTF protocols; however, the cells have been previously sensitized to the biological -agent.
• the cells can be sensitized to the biological agent by repeated dosings that can be constant or varied from dose-to-dose. This can include sensitizing the cell to an active agent or a microorganism.
• Such sensitization studies are particularly well suited for HCS and HTS assays, and the information can be used in producing bioinformatics indicative of the genes associated with sensitization to the biological agent.
• a sensitization study can be performed in the presence of two biological agents.
• the cell can be sensitized to one biological agent and the other biological agent can be a drug or potential drug to reduce the sanitization.
• a potential therapeutic agent can be screened with the sensitizing biological agent so that the sensitization can be studied.
• the genes involved in the sensitized cellular response can be studied in the presence of the potential therapeutic agent.
• gene silencing can be used to identify the genes involved in the cellular response to the potential therapeutic agent in the presence of the sensitizing biological agent.
• a neurological cell can be sensitized to cocaine.
• Cocaine is a well characterized psychostimulant that is highly addictive, and functions by blocking the re-uptake of dopamine in neurons, which contributes to its rewarding properties.
• Neurological cells can be sensitized with cocaine and monitored for increasing levels of dopamine in the culture.
• Genes encoding for polypeptides, such as kinases, adapter proteins, transcription factors, and the like, can be silenced before cocaine is administered. After cocaine is administered, an altered response can be indicative of genes that are responsible for the sensitization.
• the present invention provides a method of screening a library of siRNAs with a biological agent sensitized background.
• sensitized background studies are performed with drugs; however, they can also be performed with other types of biological agents.
• sensitized background screens can allow for a drug or potential drug that is toxic at therapeutic levels to be studied at levels below its traditional toxicity level.
• a drug may have a particular detrimental effect on a cancer cell at a specific concentration, but the drug induces systemic toxicity when dosed at that concentration.
• the functional concentration of the drug is too high to use as an anti-cancer therapeutic because of systemic toxicity.
• a sensitized background study can be performed by reducing the concentration of the drug (i.e., so there is no toxicity), while screening a library, such as an siRNA library and/or a drug library, to look for secondary targets that generate a particular phenotype.
• This can include silencing a library of genes in different cell cultures with a corresponding library of siRNA and then introducing the drug into the different cell cultures at various sub-toxic concentrations.
• Such sensitized background studies can allow for a lower concentration of the drug to be tested in the presence of gene silencing to generate a particular phenotype such as toxicity, change in morphology, or change in cell function. Additionally, various lower concentrations that are not overly toxic can be screened together in different cell cultures with a particular library of siRNAs.
• siRNA library screen performed in a drug sensitized background can be beneficial for identifying combinations of drug targets that are particularly effective from a therapeutic perspective. Also, the sensitized background siRNA library screens can be used to identify genes that are in the same pathway, in redundant pathways, or in mutually dependent pathways. VIII. Synthetic Lethal Combinations
• the present invention includes determining a synthetic lethal combination of a silenced gene and a biological agent.
• Synthetic lethal combinations typically refer to two different stimuli that are benign or non-functional alone, but are lethal when combined.
• a biological agent may not induce cellular toxicity, but is toxic when a gene has been silenced.
• the siRNA may silence a gene that encodes for a gene product that is vital for cell viability in the presence of the biological agent.
• Synthetic lethal combinations can include a silenced gene produce and a biological agent, which can be either an active agent or a microorganism
• the mechanisms for such synthetic lethal combinations can be identified and studied by silencing specific genes or combinations of genes in a cell in the presence of a biological agent.
• the method of studying of identifying lethal combinations with gene silencing can be performed in substantially the same manner as the foregoing RTF protocols.
• the combination of an active agent and a silenced gene may result in a lethal combination, wherein suitable cell viability is obtained in the absence of the active agent or the gene silencing.
• microorganisms such as viruses, can be studied to detect silenced genes that form a synthetic lethal with the microorganism. This may be used to detect gene products that are vital to be produced in the presence of the microorganism.
• synthetic lethal combinations can be studied by combinations that results in a particular phenotype. Typically, the particular phenotype that results is cell death; however, decreased viability or compromised morphologies may be representative phenotypes. Accordingly, screening for synthetic lethal combinations can include introducing a biological agent into different cells that have a different gene or combination of genes that have been silenced. The synthetic lethal combinations can be identified by determining the combinations that generate a particular phenotype. As such, the genes identified with these phenotypic screens may or may not be related to the compound itself or how a normal cell would respond to the compound (e.g., excretion, metabolism, adsorption, absorption, and the like).
• a cell can be treated with paclitaxel and then administered siRNA to silence a gene or combination of genes.
• This can be used to identify synthetic lethals that are synergistic with paclitaxel.
• the identification of synthetic lethals that are synergistic with paclitaxel can be used to tailor therapies that use paclitaxel or avoid generation of synthetic lethals in patients that are administered paclitaxel.
• the information can be used to generally improve the use of paclitaxel.
• the present invention includes generating libraries of active agents or microorganisms that have synthetic lethals or potential synthetic lethals with silenced genes. These libraries can be generated by employing the screening studies described herein that include silencing a gene or combination of genes in a cell and then administering a first biological agent to the cells. Subsequently, the same gene or combination of genes can be silenced in the same cell type, which is then administered a second biological agent. This process can continue until an entire library of biological agents, or microorganisms have been screened against the same silenced gene or combination of genes. This can be used to identify biological agents that have synergistic activities when combined with a silenced gene.
• This information can be implemented into bioinformatic software so that patterns of synthetic lethal combinations can be identified and monitored. Also, this can be used to test combinations of biological agents, such as a multi-drug combination, a toxin and drag combination, a polypeptide and drag combination, a polypeptide and toxin combination, a drag and pathogen combination, a polypeptide and pathogen combination, a combination of pathogens, and the like.
• biological agents such as a multi-drug combination, a toxin and drag combination, a polypeptide and drag combination, a polypeptide and toxin combination, a drag and pathogen combination, a polypeptide and pathogen combination, a combination of pathogens, and the like.
• a synthetic lethal combination can be identified and studied with a biological agent that can inhibit a gene function in combination with siRNA induced gene silencing. Accordingly, such a lethal combination can be identified and studied by employing an RTF protocol as described herein. Additionally, the biological agent, which can be an active agent or a microorganism, can be administered to the cells before, during, or after the siRNA induces gene silencing. Various combinations of inhibited gene functions and silenced genes can be screened for synthetic lethal combinations. While the synthetic lethal combinations can be assessed by cell viability, HCS and HTS applications can also be used.
• RTF plates that include multiple wells having different dry gene silencing compositions can have the wells organized into predefined arrangements for testing a biological agent in the presence of gene silencing.
• Such arrangements can correspond to the type of assay being employed with the RTF plate and the number of variables being studied. For example, when a family of genes is being studied in the presence of an active agent or pathogen, the siRNA that targets a single gene can be organized in one column or row while pools of siRNAs targeting the gene can be organized in a different column or row. This format can be continued until siRNA targeting all the genes in a family for silencing are present, which may include multiple plates.
• the wells of a single plate or multiple plates can be organized into a pre-selected arrangement so that particular siRNAs are in a pre-selected pattern to show the activity of the biological agent when different genes have been silenced.
• the pre-selected pattern can include control wells, such as those that include one or more negative and/or positive siRNA controls, and transfection controls.
• the pre-selected pattern can include wells that are empty or substantially devoid of siRNA, which can be used as controls and for calibrations.
• This can allow for RTF plates to have gene silencing compositions at standardized positions and amounts of siRNAs, which is beneficial for using standardized well plates in multiple experiments that can be conducted over time without introducing variability between the plates.
• the use of standardized plate arrangements can provide a series of plates that can be used over time and provide data that can be analyzed together. Also, it provides for the ability to use different biological agents in different plates so that the cellular responses of different biological agents in the presence of the same silenced gene can be identified.
• a plate comprising a plurality of columns of wells can include a transfection control in the first column, positive controls for RNAi in the second column, negative controls for RNAi in a third column, a pool of siRNA directed against a single target in a fourth column, and individual members of the siRNA pool that comprise the fourth column are in subsequent columns, such as the fifth through twelfth columns.
• the fifth through twelfth columns can comprise different concentrations of each siRNA in a pool, with the amount of siRNA increasing from well to well or decreasing from well to well.
• the array configuration may be used to easily identify which genes are responsible for cellular interactions with the biological agent or identifying which siRNA targeting a selected gene are more effective in regulating the cellular response to the biological agent.
• the well plate can be arranged so that the concentration of siRNA is constant through multiple wells in a column or row. This can allow for the biological agent to be studied at various concentrations in the presence of constant gene silencing. This . can be beneficial for monitoring cellular responses to various concentrations or amounts of the biological agent when a specific gene or combination of genes has been silenced. Also, other similar variations of plate arrangements can be employed to study biological agents. [0110] Figures IA and IB illustrate embodiments of plate arrangements similar with the foregoing concentrations arrangements for studying a biological agent. As shown, the plate arrangement can be useful for studying the effect of pools of siRNAs and individual siRNAs on a cellular response to a biological agent.
• the wells are shown to be square, it should be recognized that they can be any shape.
• the well plate can include any number of wells, and the number of wells depicted is merely for exemplification 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; “Pl” through “PI N " 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; “IA” through “IN” indicate a first individual siRNA of the first pool at a concentration gradient; “2A” through “2 N “ indicate a second individual siRNA of the first pool at a concentration gradient; “3 A” through “3 N “ indicate a third individual siRNA of the first pool at a concentration gradient; "4A”
• FIG. 1C illustrates another embodiment of a similar plate arrangement with the foregoing concentration arrangements for studying biological agents in the presence of gene silencing.
• 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; “Pl” indicates a first pool in triplicate which silences a first gene at a standard concentration; “PlA” through “PlN” 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 for a response to the biological agent.
• Figure ID illustrates an embodiment of a similar plate arrangement with the foregoing concentration arrangements for studying biological agents in the presence of gene silencing.
• 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; "Pl” through “PN” indicate a first pool which silences a first gene through an N th pool which silence an Nth gene at a standard concentration; and “PlA” through “PlN” indicate the first pool at a concentration gradient A-N, wherein second pool (e.g., "P 2 A” - “P2N”) through N th pool (e.g., "PNA” - “PNN”) each have a similar concentration gradient.
• second pool e.g., "P 2 A” - “P2N
• N th pool e.g., "PNA” - “
• Figure IE illustrates an embodiment of a similar plate arrangement with the foregoing concentration arrangements for studying biological agents in the presence of gene silencing.
• 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; "PlA” is a first pool that silences a first gene; “PlB” is a second pool that silences the first gene; “PlC” is a third pool that silences the first gene; “PlD” is a fourth pool that silences the first gene; “PlN” is an N th pool that silences the first gene; “P2A” through “PNA” indicate a second through N th pools that silence related second through N th genes; and the corresponding wells in each of the "P2A” through “PNA” rows are A - N pools which silences the gene of the row.
• FIG. 2A is a schematic diagram that illustrates an embodiment of a well plate having control siRNA.
• Control siRNA can be useful in optimizing RTF protocols to be used with biological agents.
• wells Al-Hl, G2, and H2 are blank wells devoid of siRNA.
• wells Al-Hl are available for RTF testing controls.
• Well G2 can be used as a mock transfection control, and well H2 can be an untreated cell control.
• Wells A2-F2 contain negative and positive transfection control siRNAs.
• control wells include the following: well A2 includes a non-targeting negative control siRNA ⁇ e.g., siCONTROLTM Non-Targeting siRNA pool from Dharmacon, Inc.); well B2 includes an siRNA that inhibits being taken in and processed by RISC ⁇ e.g., siCONTROLTM RISC-Free from Dharmacon, Inc.); well C2 includes a dual control siRNA that can be used as a negative control and a transfection control ⁇ e.g., siGLOTM RISC-Free from Dharmacon, Inc.); well D2 includes a positive control siRNA targeting GAPDH control gene ⁇ e.g., siCONTROLTM GAPD from Dharmacon, Inc.); well E2 includes a positive control siRNA targeting cyclophilin B control gene ⁇ e.g., siCONTROLTM Cyclophilin B siRNA from Dharmacon, Inc.); and well F2 is a positive control siRNA targeting Lamin A/C control gene
• the wells in columns 3-12 can each contain a test siRNA or test siRNA pool such as any of Dharmacon' s 5!M4i?rpoolTM siRNA reagents.
• well F2 can be a user- defined control siRNA. This plate configuration can be useful for optimizing conditions for studying the effects of gene silencing with a single concentration of a biological agent.
• FIG. 2B is a schematic diagram that illustrates an embodiment of an optimization well plate or an RTF testing plate having control siRNA.
• the use of control siRNA can be beneficial for testing the efficacy of gene silencing experiments conducted to study the interaction of biological agents and cells.
• rows A- C can contain three negative control siRNAs (e.g., siCONTROLTM Non-Targeting siRNA pool, siCONTROLTM RISC-Free siRNA, siGLOTM RISC-Free siRNA, each from Dharmacon, Inc.).
• Rows D-E can contain three positive transfection control siRNAs (e.g., siCONTROLTM GAPD, siCONTROLTM Cyclophilin B siRNA, siCONTROLTM Lamin AJC siRNA, each from Dharmacon, Inc.).
• Row F can be a user defined control siRNA.
• Row G does not contain siRNA and can be used for mock-transfected cells in order to study the effect of the transfection reagent alone on cell viability and/or mRNA expression.
• Row H does not contain siRNA and is can be used for untreated cells, and can serve as a 100% viability control and/or 100% mRNA level control.
• the illustrated control plate arrangement can be beneficial to optimize conditions for studying the biological agent.
• FIG. 2C is a schematic diagram that illustrates an embodiment of an optimization plate or an RTF testing plate having control siRNA that can be used to study a biological agent.
• the testing plate includes an arrangement of wells that can be used in a procedure for optimizing RTF conditions.
• column 1 is devoid of siRNA, and can be used as a blank that does not receive cells. This column can be used for a standard curve or other experimental controls that may be desired for an RTF testing protocol for assessing the efficacy of gene silencing.
• Column 2 is devoid of siRNA, but receives cells. This column can be used as an untreated reference for different volumes of polynucleotide carrier, such as DharmaFECJTM transfection reagent, which is tested in columns 3—12.
• Columns 3 — 7 can contain negative control siRNAs, which serve as negative control samples for each DharmaFECJTM transfection reagent volume used. As shown, up to five volumes of each DharmaFEC-TTM transfection reagent may be tested in one plate. In each row, the DharmaFECJTM transfection reagent volumes in columns 3-7 can be repeated in columns 8-12. This can allow the negative control siRNAs to serve as references for any gene silencing that occurs in the wells of columns 8-12, which contain positive transfection control siRNAs. Additionally, rows A-D can be seeded with a low cell density, and rows E-H can be seeded with a high cell density.
• the plate can include a combination of different RTF conditions, which can be used to determine the optimal amount of DharmaFECTTM transfection reagent, DharmaFECJTM transfection reagent volume, and cell number.
• various controls can be used to test the efficacy of an experiment that studies a biological agent in the presence of gene silencing.
• Figure 2D is a schematic diagram that illustrates an embodiment of an optimization well plate or an RTF testing plate having control siRNA that can be used to study a biological agent.
• the plate is arranged with blanks, negative control siRNA, and positive control siRNA as in Figure 2C.
• this plate can be assayed with different polynucleotide carrier concentrations.
• Rows A-D can be used with low cell densities with DharmaFECTTM transfection reagent at volumes ranging from 0.03 uL/well (e.g., columns 3 and 8) to 0.5 uL/well (e.g., columns 7 and 12).
• Rows E-H can be used with high cell densities with DharmaFECJTM transfection reagent at volumes ranging from 0.06 uL/well (e.g., columns 3 and 8) to 1.0 uL/well (e.g., columns 7 and 12).
• various parameters such as lipid concentration and cell density, can be used to optimize conditions for studying a biological agent in the presence of gene silencing.
• a well plate has a pre-defined arrangement of test and control siRNAs, it enables the contribution of each gene, member of a family of genes, each gene in a pathway, and/or each gene in a genome to be studied with respect to activity or response to a biological agent.
• Gene silencing experiments can be designed for identifying one or more host (e.g., human) genes that contribute to the ability of a virus, such as the HIV virus, to infect human cells.
• the siRNA in the gene silencing composition can be directed to the viral genes so that the viral infectious pathway can be studied.
• studies can be performed in cells that are susceptible to HIV infection (e.g., JC53 cells), wherein the HIV virus is added to the cells before, during, or after gene silencing.
• the cells in each well can be subjected to a lethal titer of the HIV virus.
• the cellular response to the HIV can be studied by detecting the presence and/or amounts of targeted polypeptides.
• Test wells that contain living cells or a substantially larger number of living cells than control wells can be used to identify a host gene that is necessary for viral infection, replication, and/or release.
• a similar study can be performed to study the viral genes involved in the pathogenic infection.
• a plate can have an arrangement of gene silencing compositions containing siRNA directed against a gene or genes whose silencing is known to induce a particular disease state.
• multiple disease models can be created on a single plate by inducing selected gene silencing. This can allow for biological agents to be studied in single or multiple disease models on a single plate. Examples of diseases models that may be included by gene silencing include those associated with cancer, neurological diseases, diabetes, metabolic diseases, diseases of the bone, cartilage, muscle, heart, kidneys, liver, prostate, gastrointestinal tract, and
• the well plate arrangements can be organized in order to study the effects of gene silencing induced by libraries of siRNAs in the presence of biological agents. Examples of such siRNA libraries can be reviewed in Table 1.
• siRNAs comprising the siRNA libraries in Table 1, and more complete descriptions of the use of gene silencing to study the plates/pathways identified in Table 1 are provided in U.S. Provisional Application Serial No. 60/678,165. Moreover additional descriptions of plate arrangements and the types of genes that can be studied using siRNA are provided in U.S. Provisional Application Serial No. 60/678,165, United States Patent Application having Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. as inventors, and United States Patent Application having Attorney Docket No.
• siRNA RTF The genes involved in the kinase pathway are studied by siRNA RTF to determine the genes responsible for cell viability in the presence of a drug.
• 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 DharmaFECTTM 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 drug is added to the plates between 0 and 24 hours after the cells are added. The plates are then maintained between 24 and 72 hours and assayed for cell viability.
• a comparison between the cell viability of different cell cultures that were treated with the drug can be made to identify siRNA that silence genes that are essential for HeLa cell viability in the presence of the drug.
• siRNA RTF The genes involved in the cytokine receptor family are studied by siRNA RTF to determine the genes responsible for cell viability in the presence of a drug.
• 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
• ObarmaFECTTM 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 drug is added between 0 to 24 hours after the cells are added to the wells.
• the plates are maintained between 24 and 72 hours and assayed for cell viability.
• a comparison between the cell viability of different cultures that were treated with the drug in the presence of gene silencing induced by using different members of the cytokine receptor siRNA array can provide the identification of genes that are essential for HT-29 cell viability in the presence of the drag.
• siRNAs targeting the 193 members of the phosphatase family are solubilized in RNase-free water and dried in individual wells of PLL coated 96-well plates. The amount of each siRNA is measured to obtain a final siRNA concentration of approximately 25 nM. About 0.1 ug of DharmaFEC7TM 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 the siRNA(s) and create complexes before about 10,000 3T3 cells are added to obtain a final volume of 125 uL.
• the cells are then cultured for a period between 24 and 72 hours before human growth hormone (hGH) is added to selected cells. After 5-7 days, the cells are then lysed with a buffer containing 0.5% (v/v) Triton X-100, and adipose conversion is quantified by assaying the adipogenic enzyme, glycerophosphate dehydrogenase.
• hGH human growth hormone
• Selected gene products that may be involved in a cellular response to an HIV virus can be studied by inducing silencing of the genes in the presence of the virus. This can be accomplished with rationally designed siRNAs that target the selected genes of the human genome.
• the selected genes are solubilized in RNase- free water and dried in individual wells of PLL coated 96-well plates. The amount of each siRNA added to each well is measured to obtain a final concentration of approximately 25 nM.
• a single plate or multiple plates containing an arrangement of gene silencing compositions can be used to determine whether gene silencing affects cell differentiation.
• gene silencing compositions having siRNA that function as miRNA inhibitors can be used to study cell differentiation. After the siRNA have been solubilized or suspended in an aqueous medium, about 10,000- 25,000 3T3-L1 mouse pre-adipocyte cells can be added to each well. After sufficient gene silencing, the cells can be treated with a cocktail of dexamethasome, insulin, and IBMX. About four days later, the cocktail can be replaced with media inoculated with insulin.
• the cells can be stained with Sudan Ill-Oil Red, and the wells having different gene silencing compositions are scanned to identify cultures in which the cells have not differentiated into adipocytes. This can be useful in studying the effect of the miRNA pathway in cell differentiation.
• a multi-well RTF plate or series of plates can be designed in order to study a biological agent in the presence of gene silencing.
• the plates can be configured to include any of the following variables: (1) the concentration of individual or 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 Dharmar ⁇ CJTM 1, DharmaFECZTM 2, DharmaFECITM 3, or DharmaFECZTM 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 concentration

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