WO2012005898A2

WO2012005898A2 - Chinese hamster ovary (cho) cell transcriptome, corresponding sirnas and uses thereof

Info

Publication number: WO2012005898A2
Application number: PCT/US2011/040427
Authority: WO
Inventors: Anthony Rossomando; Stuart Pollard; John M. Maraganore; Greg Hinkle; Brian Bettencourt
Original assignee: Alnylam Pharmaceuticals Inc
Current assignee: Alnylam Pharmaceuticals Inc
Priority date: 2010-06-15
Filing date: 2011-06-15
Publication date: 2012-01-12
Anticipated expiration: 2012-12-15
Also published as: WO2012005898A3

Abstract

The present invention provides a transcriptome of a Chinese hamster ovary cell comprising the genes expressed by the CHO cells and a set of siRNAs targeting these transcripts.

Description

CHINESE HAMSTER OVARY (CHO) CELL TRANSCRIPTOME, CORRESPONDING

SIRNAS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Patent Cooperation Treaty International Application which claims the benefit of priority of US Provisional Application No. 61/354,932 filed June 15, 2010, the contents of which are each incorporated herein by reference in its entirety.

LENGTHY TABLE

[0002] The specification includes a lengthy Table 1. Lengthy Table 1 is provided herein in an electronic format on a CD, as file "CHO.15 Jun2010.patent.masterTable.txt (Table I), and is incorporated herein by reference in their entirety. Please refer to the end of the specification for access instructions.

SEQUENCE LISTING

[0003] The instant application contains a "lengthy" Sequence Listing which has been submitted via CD-R in lieu of a paper copy, and is hereby incorporated in its entirety. Said CD-R, recorded on May 26, 2011, are labeled CRF, "Copy 1,", "Copy 2," and "Copy 3," respectively, and each contains only one single self-extracting file named 200211PC.EXE (50,249,728 bytes), which subsequently contains one uncompressed ASCII text file named 200211PC.TXT (752,307,648 bytes).

BACKGROUND

Field of the Invention

[0004] The invention relates generally to transcriptomes, organized transcriptomes and sy tems and methods using the transcriptomes for designing targeted modulation of biomolecule production in cells. The invention further related to engineering cells and cell lines for more effective and efficient production of biomolecules.

Background of the Invention

[0005] Cell culture techniques are used to manufacture a wide range of biological products, including biopharmaceuticals, biofuels, metabolites, vitamins and nutraceuticals. A number of strategies have been developed to enhance productivity, yield, efficiency, and other aspects of cell culture bioprocesses in order to facilitate industrial scale production and meet applicable standards for product quality and consistency. Traditional strategies for optimizing cell culture bioprocesses involve adjusting physical and biochemical parameters, such as culture media (e.g., pH or nutrients) and conditions (e.g., temperature or duration), and selecting host cells having desirable phenotypes. More recently, genetic approaches have been developed for optimizing cell culture bioprocesses by introducing recombinant DNA into host cells, where the DNA encodes an exogenous protein that influences production of a biological product or regulates expression of an endogenous protein that influences production of the biological product.

However, such methods require costly and time-consuming laboratory manipulations and can be incompatible with certain genes, proteins, host cells, and biological products.

[0006] Chinese hamster ovary (CHO) cells have been widely used in various bioprocesses. However, little is known about expression of genes in these cells, and thus, targeted and intelligent modulation of bioprocesses of these cells cannot readily be done or designed.

[0007] Accordingly, there is a need in the art for new genetic approaches for optimizing cell culture bioprocesses involving a wide range of host cells, such a CHO cells, and biological products produced in these cells.

SUMMARY OF THE INVENTION

[0008] The present invention provides a transcriptome of a CHO cell comprising the genes expressed by the CHO cells and a set of siRNAs targeting these transcripts.

[0009] The invention also includes systems configured for using the CHO transcriptome data and an organized compilation of the CHO transcriptome data outlining at least one functional aspect of each of the transcript, and the corresponding siRNAs to allow design and selection of appropriate targets and effector RNA molecules for optimization of biological processes, particularly in the CHO cells.

[0010] Accordingly, the invention provides a system for selecting a sequence of at least one RNA effector molecule suitable for modulating protein expression in a cell, the system comprising: a computer system, having a one or more processors and associated memory, and a database comprising at least one cell transcriptome information, the information comprising, a sequence for each transcript of the transcriptome, and optionally, a name of the transcript, and a pathway the transcript plays a role; and at least one RNA effector molecule information, the information comprising at least the sequence of the RNA effector molecule and optionally target specificity of the RNA effector molecule, wherein each RNA effector molecule is designed to match at least one or more sequences in the at least one cell transcriptome; a program on the computer system adapted and configured to receive from a user, input parameters, comprising at least one of, a cell type selection, a target organism selection, a cellular pathway selection, a cross-reactivity selection, an amount of transcript selection a target gene name and/or sequence selection, and optionally a method of delivery selection comprising either in vivo or in vitro delivery options; and further optionally user address information; a first module configured to check the parameters against the sequences in the database for a matching combination of the parameters and transcriptome transcript sequences; and a second module to display a selected sequence of at least one RNA effector molecule suitable for modulating protein expression in the cell. The system can also include a module for executing one or more data processing algorithms for determining appropriate RNA effector molecules as a function the targets identified.

[0011] In some embodiments, the system further comprises a storage module for storing the at least one RNA effector molecule in a container, wherein if there are two or more RNA effector molecules, each RNA effector molecule is stored in a separate container, and a robotic handling module, which upon selection of the matching combination, selects a matching container, and optionally adds to the container additives based on a user selection for in vivo or in vitro delivery, and optionally further packages the container comprising the matching RNA effector molecule to be sent to the user address.

[0012] In some embodiments, the at least one cell transcriptome sequence information consists essentially of SEQ ID NOs: 1-9771.

[0013] In some embodiments the RNA effector molecule is selected from the group consisting of an siRNA, a formulated siRNA, an siRNA mixture, and any combination thereof.

[0014] In some embodiments the at least one RNA effector molecule sequence information consists essentially of an RNA effector molecule comprising, consisting essentially of or consisting of an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18 or at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772-3152399.

[0015] In some embodiments, the invention provides a method for selecting an RNA effector molecule for modulating protein expression in a cell using the system of any one of the described systems.

[0016] In some embodiments, the system further comprises genome information of the cell, wherein by a user selection, the RNA effector molecules can be matched to target genomic sequences, comprising promoters, enhancers, introns and exons present in the genome.

[0017] In some embodiments, the invention provides a Chinese hamster ovary (CHO) cell transcriptome comprising a selection or a compilation of transcripts having SEQ ID NOs: 1- 9771. [0018] In some embodiments, the invention provides the CHO cell transcriptome, wherein the CHO cell transcriptome sequences are a part of a database.

[0019] In some embodiments, the invention provides at least one siRNA directed to any one of the CHO cell transcriptome transcript set forth in Table 1. In some embodiments, the siRNA is selected from the group of siRNAs set forth in Table 1, i.e., wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18 or at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772-3152399.

[0020] In some embodiments, the siRNA sequences are part of a database.

[0021] The invention further provides, a method for improving a cell line, the method comprising modulating at least one protein translated from a transcript selected from Table 1.

[0022] The invention also provides a method for improving a cell line, the method comprising modulating at least two transcripts using an effector RNA molecule, wherein a first transcript affects a first cell culture phenotype and a second transcript affects a second, different cell culture phenotype, wherein the cell culture phenotypes are selected from the group consisting of a cell growth rate, a cellular productivity, a peak cell density, a sustained cell viability, a rate of ammonia production or consumption, or a rate of lactate production or consumption, and wherein the first and second transcripts are selected from the group consisting of SEQ ID NOs: 1-9771. In some embodiments, the method further comprises modulating a third transcript affecting a third cell culture phenotype different from the first and second cell culture phenotypes.

[0023] In some embodiments, the siRNA is selected from the group of siRNAs set forth in Table 1, i.e., wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18 or at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772-3152399.

[0024] In some embodiments, the cell line is a CHO cell line.

[0025] The invention also provides an engineered cell line with an improved cellular productivity, improved cell growth rate, or improved cell viability, comprising a population of engineered cells, each of which comprising an engineered construct modulating one or more transcripts selected from Table 1. In some embodiments, the siRNA is selected from the group of siRNAs set forth in Table 1, i.e., wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18 or at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772-3152399. In some embodiments, the engineered construct comprises an siRNA selected from the group consisting of SEQ ID NOs: 9772-3152399. [0026] The invention further provides compositions and methods for producing a biological product from a host cell, particularly from CHO cell. The methods comprising contacting the cell with an RNA effector molecule, such as one or more siRNA molecules targeting the CHO transcriptome transcripts, directed to, a portion of which is complementary to a target transcript, maintaining the cell in a bioreactor for a time sufficient to modulate expression of the target gene, wherein the modulation enhances production of the biological product from the cell, and isolating the biological product from the cell.

[0027] In various embodiments, the biological product is a polypeptide, a metabolite, a nutraceutical, a chemical intermediate, a biofuel, a food additive, or an antibiotic.

[0028] In one embodiment, the cell is contacted with a plurality of different RNA effector molecules to modulate expression of multiple target genes. In one embodiment, the RNA effector molecules is added to the cell culture medium used to maintain the cells under conditions that permit production of a biological product. The RNA effector molecules can be added at different times or simultaneously. In one embodiment, one or more of the different RNA effector molecules is added by continuous infusion into the cell culture medium, for example, to maintain a continuous average percent inhibition or RNA effector molecule concentration.. In one embodiment, each of the different RNA effector molecules is added at the same frequency or different frequencies. In another embodiment, each of the different RNA effector molecules is added at the same concentration or at different concentrations. In still another embodiment, the RNA effector molecule is added at a given starting concentration of each of the different RNA effector molecules (e.g., at 1 nM each), and further supplemented with continuous infusion of the RNA effector molecule.

[0029] As used herein, the term "RNA effector molecule" refers to an RNA agent capable of modulating the expression of a target gene, as defined herein, within a host cell, or a

polynucleotide agent capable of forming such an RNA agent within a host cell. RNA effector molecules described herein are substantially complementary to at least a portion of the target gene such as the coding region, the promoter region and the 3' untranslated region (3'-UTR) of the target gene or completmentary to at least a portion of the target gene and modulate expression of target genes by one or more of a variety of mechanisms, including but not limited to, Argonaute-mediated post-transcriptional cleavage of target gene mRNA transcripts

(sometimes referred to in the art as RNAi) and/or other pre-transcriptional and pre-translational mechanisms.

[0030] In various embodiments, the RNA effector molecule can comprise siRNA, miRNA, dsRNA, saRNA, shRNA, piRNA, tkRNAi, eiRNA, pdRNA, a gapmer, an antagomir, or a ribozyme. A skilled artisan will be able to design these molecules based on the CHO

transcriptome target sequences provided herein.

[0031] In one embodiment, the RNA effector molecule can activate a target gene. In another embodiment, the RNA effector can inhibit a target gene.

[0032] In some embodiments, the maintaining step further comprises monitoring at least one measurable parameter selected from the group consisting of cell density, medium pH, oxygen levels, glucose levels, lactic acid levels, temperature, and protein production.

[0033] In further embodiments, the methods further comprise contacting the cell with a second agent. The second agent can be selected from the group consisting of: an antibody; a growth factor; an apoptosis inhibitor; a kinase inhibitor, such as a MAP kinase inhibitor, a CDK inhibitor, and K252a; a phosphatase inhibitor, such as sodium vanadate and okadaic acid; a protease inhibitor; and a histone demethylating agent, such as 5-azacytidine.

[0034] In some embodiments, the host cell, such as a CHO cell, further comprises a genetic construct encoding the biological product or a virus receptor.

[0035] In some embodiments, the biological product is a polypeptide and the target gene encodes a protein that affects post-translational modification in the host cell. In various embodiments, the post-translational modification can be protein glycosylation, protein deamidation, protein disulfide bond formation, methionine oxidation, protein pyroglutamation, protein folding, or protein secretion.

[0036] In additional embodiments, the target gene encodes a protein that affects a physiological process of the host cell, such as a CHO cell. In various embodiments, the physiological process is apoptosis, cell cycle progression, carbon metabolism or transport, lactate formation, or RNAi uptake and/or efficacy.

[0037] In further embodiments, the target gene encodes a pro-oxidant enzyme, a protein that affects cellular pH, or a viral protein.

[0038] In one embodiment, the invention provides a cultured eukaryotic cell, such as a CHO cell, containing at least one RNA effector molecule provided herein.

[0039] In another embodiment, the invention provides a composition for enhancing production of a biological product in cell culture by modulating the expression of a target gene in a host cell, such as a CHO cell. The composition typically includes one or more RNA effector molecules described herein and a suitable carrier or delivery vehicle.

[0040] In another embodiment, the composition is formulated for administration to cells according to a dosage regimen described herein, e.g., at a frequency of 6h, 12h, 24h, 36h, 48h, 72h, 84h, 96h, 108h or more. In another embodiment, the administration of the composition can be maintained during one or more cell growth phases e.g., lag phase, early log phase, mid-log phase, late-log phase, stationary phase and/or death phase.

[0041] In another embodiment, a composition containing two or more RNA effector molecules directed against separate target genes is used to enhance production of a biological product in cell culture by modulating expression of a first target gene and at least a second target gene in the cultured cells.

[0042] In another embodiment, a first RNA effector molecule is administered to a cultured cell, such as a CHO cell, and then a second RNA effector molecule is administered to the cell (or vice versa). In a further embodiment, the first and second RNA effector molecules are administered to a cultured cell substantially simultaneously.

[0043] In another embodiment, a composition containing a RNA effector molecule described herein, e.g., a dsRNA directed against a host cell target gene, is administered to a cultured cell with a non-RNA agent useful for enhancing the production of a biological product by the cell.

[0044] In one embodiment, a vector is provided for inhibiting the expression of a target gene in a cultured cell, such as a CHO cell, where the target gene encodes a protein that affects production of a biological product by the cell. In one embodiment, the vector includes at least one regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of an RNA effector molecule described herein.

[0045] In another embodiment, the invention provides a cell containing a vector for inhibiting the expression of a target gene in a cell. The vector includes a regulatory sequence operably linked to a polynucleotide encoding at least one strand of an RNA effector molecule described herein.

[0046] Still another aspect of the invention encompasses kits for enhancing production of a biological product by a cultured cell, such as a CHO cell. In one embodiment, the kits comprise an RNA effector molecule which modulates expression of a target gene encoding a protein that affects production of the biological product. In another embodiment, the kits comprise a modified cell line, such as a modified CHO cell line, which expresses an RNA effector molecule which modulates expression of a protein that affects production of the biological product. The kits can also comprise instructions for carrying out methods provided herein.

[0047] In one embodiment, the kits further comprise suitable culture media for growing host cells and/or constructs (e.g., plasmid, viral, etc.) for introducing a nucleic acid sequence encoding an RNA effector molecule into host cells. In still another embodiment, the kits can further comprise reagents for detecting and/or purifying the biological product. Non-limiting examples of suitable reagents include PCR primers, polyclonal antibodies, monoclonal antibodies, affinity chromatography media, and the like. [0048] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 shows a diagrammatic view of a computer system according to one embodiment of the invention.

[0050] Figure 2 shows a diagrammatic view of a computer system according to an alternative embodiment of the invention.

[0051] Figure 3 shows a diagram of the data structures according to one embodiment of the invention.

[0052] Figure 4 shows a flow diagram of a method according to one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The invention provides a set of transcripts that are expressed in Chinese hamster ovary (CHO) cells, also called "the CHO cell transcriptome". The invention further provides siRNA molecules designed to target any one of the transcripts of the CHO cell transcriptome. Uses of the transcriptome in a form of an organized CHO cell transcript sequence database for selecting and designing CHO cell modulating effector RNAs are also provided in the form of methods and systems. The invention further provides a selection of siRNAs targeted against each of the transcripts in the CHO transcriptome, and uses thereof for engineering or modifying CHO cells, for example, for improved production of biomolecules. Accordingly, the invention also provides modified CHO cells.

[0054] The invention is based upon discovery of a set of transcripts that were identified in CHO cells pooled under different conditions, including early-, mid-, and late-log phase cells, that were grown in standard conditions under chemically defined media at 37°C. The transcripts are set forth in Table 1, Tables 2-8, and in SEQ ID file 200211PC.TXT.

[0055] The discovery of the CHO transcriptome is useful for specifically modifying one or more cellular processes in the CHO cell, for example, for the production of biomolecules in such cells. For example, based on the known expressed transcripts, one can modulate apoptosis regulating genes, cell cycle genes, DNA amplification (DHFR) regulating genes, virus gene production regulating genes, e.g., in the case of viral promoters that are used to drive biomolecule production in the cells, glycosylation-associated genes, carbon metabolism regulating genes, prooxidant enzyme encoding genes. By modulating the known expressed genes or transcripts one can further modulate protein folding, methionine oxidation, protein pyroglutamation, disulfide bond formation, protein secretion, cell viability, specific productivity of cell, nutrient requirements, and internal cell pH.

[0056] The invention therefore also provides methods for modulating production of a biological product in a host cell, particularly in a CHO cell, the methods comprising the steps of contacting the cell with an RNA effector molecule, a portion of which is complementary to at least a portion of a target gene, maintaining the cell in a bioreactor for a time sufficient to modulate expression of the target gene, wherein the modulation enhances production of the biological product and recovering the biological product from the cell.

[0057] The following detailed description discloses the nucleic acid sequences of the transcripts of the CHO transcriptome, the proteins the transcripts are translated into, and at least some of the pathways the transcribed proteins play a role in. The description also sets forth a compilation of siRNA molecules as RNA effector molecules, which are designed to target the sequences of the transcriptome. Systems, including computer assisted systems and methods, for selecting appropriate RNA effector molecules to modulate gene expression in a cell, particularly in a CHO cell based on the known transcriptome transcript sequences are also described.

CHO cell transcriptome

[0058] We have discovered a defined set of transcripts expressed in a CHO cell. The defined set of transcripts in referred to herein as a "transcriptome". The transcript name, at least one pathway the transcript plays a role in, an associated SEQ ID NOs and corresponding exemplary siRNA molecule SEQ ID NOs are set forth as a list in Table 1.

[0059] The sequences of the transcrips in the CHO cell transcriptome are set forth in the associated text file 20021 IPC. TXT incorporated herein by reference.

[0060] The transcriptome can be further divided into sub-parts based on the function of each of the transcripts. The list of Table 1 also sets forth at least one pathway or function, a protein translated from the transcript plays a role in. Tables 2-8 give exemplary pathways the transcripts of the CHO transcriptome play a role in.

[0061] Discovery of the entire CHO transcriptome allows designing engineered cells wherein particular cellular pathways have been modified. Such modification can be done, for example using effector RNA molecules, such as siRNAs.

[0062] In some embodiments, the at least one cell transcriptome sequence information consists essentially of SEQ ID NOs: 1-9771. In some embodiments, the transcriptome information comprises, and in some embodiments it consists of SEQ ID NOs: 1-9771.

[0063] In some embodiments the RNA effector molecule is selected from the group consisting of an siRNA, a formulated siRNA, an siRNA mixture, and any combination thereof.

[0064] In some embodiments, the siRNA is selected from the group of siRNAs set forth in Table 1 or text file 20021 IPC. TXT incorporated herein by reference, i.e., wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772-3152399. In some embodiments the antisense strand comprises 16-19 contiguous nucleotides. In some embodiments the at least one RNA effector molecule sequence information comprises, consists essentially of, or consists of SEQ ID NOs: 9772- 3152399 set forth in text file 200211PC.TXT.

[0065] In some embodiments, the invention provides a method for selecting an RNA effector molecule for modulating protein expression in a cell using the system of any one of the described systems.

[0066] Tables 2-8 included at the end of the specification set forth additional targets that can be modulated for improved quality/quantity of expression.

Systems and methods for selecting RNA effector molecules [0067] Based on the known CHO transcriptome, we have developed methods and systems for selecting RNA effector molecules to affect the cells through manipulating cellular processes, for example, to improve production of biomolecules in the cells.

[0068] Accordingly, the invention provides databases and systems comprising and using the CHO transcriptome sequences and optionally also an organized compilation of the CHO transcriptome outlining at least one functional aspect of each of the transcript, such as the transcripts role in a particular cellular process or pathway, and the corresponding siRNAs to allow design and selection of targets and effector RNA molecules for optimization of biological processes, particularly in the CHO cells.

[0069] Functional aspects of transcripts relate to their role in, for example apoptosis, cell cycle, DNA amplification (DHFR), virus gene production, e.g., in the case of viral promoters that are used to drive biomolecule production in the cells, glycosylation, carbon metabolism, prooxidant enzymes, protein folding, methionine oxidation, protein pyroglutamation, disulfide bond formation, protein secretion, cell viability, specific productivity of cell, nutrient requirements, internal cell pH. Other cellular processes are known to a skilled artisan, and can be found, for example, at Gene Ontology database available through World Wide Web at geneontology.org.

[0070] Accordingly as shown in Figure 1, the invention provides a system 100 for selecting a sequence of at least one RNA effector molecule suitable for modulating protein expression in a cell, the system comprising: a computing device 110, having a processor 112 and associated memory 114, and a database 120 comprising at least one cell transcriptome information, the information comprising, a sequence for each transcript of the transcriptome, and optionally, a name of the transcript, and a pathway the transcript plays a role in; and at least one RNA effector molecule information, the information comprising at least the sequence of the RNA effector molecule and optionally target specificity of the RNA effector molecule, wherein each RNA effector molecule is designed to match at least one or more sequences in the at least one cell transcriptome; a computer program, stored in associated memory 114, executed by the computing device 110 and configured to receive from a user via a user input device 118, parameters comprising a cell type selection, a target organism selection, a cellular pathway selection, a cross-reactivity selection, a target gene name and/or sequence selection, and optionally a method of delivery selection comprising either in vivo or in vitro delivery options; and further optionally user address information; a first module configured to check the parameters against the sequences in the database for a matching combination of the parameters and transcriptome transcript sequences; and a second module to display a selected sequence of at least one RNA effector molecule suitable for modulating protein expression in the cell. [0071] The computing device 110 and associated programs stored in the associated memory 114 can be adapted and configured to provide a user interface, such as a graphical user interface which allows the user to input search target parameters, for example, using one or more drop down menus or structured or free form text input, and selects the appropriate parameters for finding an appropriate target in the desired cell. For example, if a user wishes to find a target for modulating carbon metabolism in a CHO cell, the user identifies the target cell as "CHO", and pathway as "carbon metabolism", and the server performs a search through the database that would identify, e.g., transcripts for Gluts, PTEN and LDH genes and matches them with the appropriate siRNA molecules from the siRNA database part. This output information can be presented to the user on a computer display 116 or other output device, such as a printer (not shown).

[0072] The system can be a stand alone system or an internet-based system, wherein the computations and selection of effector RNA molecules is performed in same or different locations. As shown in Figure 1, the transcriptome information can be stored in a database 120 and accessed by the computing device 110. As used herein, the term database includes any organization of data regardless of whether it is structured or unstructured that allows retrieval of the information requested. The database can be a fiat file or set of flat files stored in the associated memory, one or more tables stored in the associated memory, a set of discrete data elements stored in the associated memory. The database can also include any well known database program that allows a user to directly or indirectly (through another program) access the data. Examples of these include Microsoft^® Access^®, Oracle^® database, and MySQL™.

[0073] In an alternative embodiment of the invention shown in Figure 2, the system 200 can be a network based system. The system 200 can include a server system 210 and one or more client systems 240 and 250 connected to a network 230, such as a private user network or the Internet. The server system 210 and client systems 240 and 250 can be computing devices as described herein. Server system 210 can include one or more processors 212, a computer display 216, associated memory 214 and one or more computer programs or software adapted and configured to control the operations and functions of the server system 210. The transcriptome information can be stored in a database 220 and accessed by the server system 210. The server system 210 can include one or more network interfaces for connecting via wire or wirelessly to the network 230. Examples of server systems include computer servers based on Intel^® and AMD^® microprocessor architectures available from Hewlett Packard^®, Dell^® and Apple^®. Client systems 240 and 250 can include one or more processors 242 and 252, computer displays 246 and 256, user input devices 248 and 258, associated memory 244 and 254 and one or more computer programs or software adapted and configured to control the operations and functions of the client systems 240 and 250. The client systems 240 and 250 can include one or more network interfaces for connecting via wire or wirelessly to the network 230. Examples of client systems include desktop and portable computers based on Intel^® and AMD^® microprocessor architectures available from Hewlett Packard^®, Dell^® and Apple^® and smaller network enabled, handheld devices such as PDA and smart phones available from Research In Motion

(Blackberry^®), Apple^® (iPod^®, iPad^® and iPhone^® devices) and HTC^®.

[0074] In accordance with one embodiment, the server system 210 is a web server, for example based in Microsoft^® IIS or .Net products or Apache, and uses a web based application accessed by a remote client system via the Internet to search the database of transcriptome information to identify RNA effector molecules that may be suitable for modulating protein expression in a cell. The system can include or be connected to a fulfillment system that allows a user to select and purchase desired quantities of the identified RNA effector molecules to be delivered to the user.

[0075] One can also provide a system by selling software to be run by a computer, wherein the databases and algorithms matching the parameters with sequence information and other information are provided to the user. The user may then either synthesize the effector RNA molecules or separately order them from a third party provider.

[0076] In some embodiments, the system further comprises a storage module for storing the at least one RNA effector molecule in a container, wherein if there are two or more RNA effector molecules, each RNA effector molecule is stored in a separate container, and a robotic handling module, which upon selection of the matching combination, selects a matching container, and optionally adds to the container additives based on a user selection for in vivo or in vitro delivery, and optionally further packages the container comprising the matching RNA effector molecule to be sent to the user address. Exemplary additives that can be added to the siRNA or a mixture of siRNAs are set forth below, under heading "Compositions containing RNA effector molecule."

[0077] The storage module can be a refrigerated module linked to the system components.

[0078] The system may also be linked to a nucleic acid or other biomolecule synthesizer.

[0079] The robotic handling module can be any system that can retrieve, and optionally mix components from the storage module, and or the biomolecule synthesizer, and optionally package the container(s). The robotic handling module can comprise one or more parts functioning based upon the commands from the system. The robotic handling module may be in the same or different location as where the computations are performed.

[0080] In some embodiments, the system further comprises genome information of the cell, wherein by a user selection, the RNA effector molecules can be matched to target genomic sequences, comprising promoters, enhancers, introns and exons present in the genome. [0081] The terms "system," "computing device" and "computer-based system" refer to the computer hardware, associated software, and data storage devices used to analyze the information of the present invention. In one embodiment, the computer-based systems of the present invention comprises one or more central processing units (e.g., CPU, PAL, PLA, PGA), computer input means (e.g., keyboard, cursor control device, touch screen, mouse), output means (e.g., computer display, printer) and data storage devices (e.g., RAM, ROM, volatile and nonvolatile memory devices, hard disk drives, network attached storage, optical storage devices, magnetic storage devices, solid state storage devices). As such, any convenient computer-based system can be employed in the present invention. Further, the computing device can include an embedded system based on a combination of computing hardware and associated software or firmware.

[0082] A "processor" includes any hardware and/or software combination which can perform the functions under program control. For example, any processor herein may be a programmable digital microprocessor such as available in the form of an embedded system, a programmable controller, mainframe, server or personal computer (desktop or portable). Where the processor is selectively programmable, suitable programs, software or firmware can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based). For example, a magnetic medium or optical disk can store the program or operating instructions and can be read and transferred to each processor at its corresponding station.

[0083] "Computer readable medium" as used herein refers to any storage or transmission medium that participates in providing instructions and/or data to a computer for execution and/or processing. Examples of storage media include floppy disks, magnetic media (tape, disk), UBS, optical media (CD-ROM, DVD, Blu-Ray), solid state media, a hard disk drive, a RAM, a ROM or integrated circuit, a magneto-optical disk, or a computer readable card such as a PCMCIA card and the like, whether or not such devices are internal or external to the computer. A file containing information can be "stored" on computer readable medium, where "storing" means recording information such that it is accessible and retrievable at a later date by a computer.

[0084] With respect to computer readable media, "permanent memory" or "non-volatile memory" refers to memory that is permanently stored on a data storage medium. Permanent memory is not erased by termination of the electrical supply to a computer or processor. A computer hard-drive, ROM, CD-ROM, floppy disk and DVD are all examples of permanent memory. Random Access Memory (RAM) is an example of non-permanent or volatile memory. [0085] To "record" or "store" data, programming or other information on a computer readable medium refers to a process for storing information, using any convenient method. Any convenient data storage structure can be chosen, based on the means used to access the stored information.

[0086] An associated "memory" or "memory unit" refers to any device which can store information for subsequent retrieval by a processor, and may include magnetic or optical devices (such as a hard disk, floppy disk, CD, or DVD), or solid state memory devices (such as volatile or non- volatile RAM). An associated memory or memory unit may have more than one physical memory device of the same or different types (for example, an associated memory may have multiple memory devices such as multiple hard drives or multiple solid state memory devices or some combination of hard drives and solid state memory devices).

[0087] In some embodiments of the invention, the system can include hardware components or systems of hardware components and software components that carry out specific tasks (such as managing input and output of information, processing information, etc.) of the system and can be carried out by the execution of software applications on and across the one or more computing devices that make up the system. The present inventions can include any convenient type of computing device, e.g., such as a server, main-frame computer, a work station, etc. Where more than one computing device is present, each device can be connected via any convenient type of communications interconnect, herein referred to as a network, using well know interconnection technologies including, for example, Ethernet (wired or wireless - "WiFi"), Bluetooth^®, ZigBee^®, 3G, 4G. Where more than one computing device is used, the devices can be co-located or they may be physically separated. Various operating systems may be employed on any of the computing devices, where representative operating systems include Microsoft Windows^®, Mac OS, Sun Solaris, Linux, Unix, OS/400, Android, Chrome OS, and others. The functional elements of system may also be implemented in accordance with a variety of software facilitators, platforms, or other convenient method.

[0088] Items of data can be "linked" to one another in a memory when the same data input (for example, filename or directory name or search term) retrieves the linked items (in a same file or not) or an input of one or more of the linked items retrieves one or more of the others.

[0089] Figure 3 shows a diagrammatic view of the data structure 300 according to one embodiment of the invention. In one embodiment, in a first layer of searching 310 a user can input field terms which can be linked to target RNA, such as by their associated sequence ID in the database and in accordance with the invention, to execute a software module to search for one or more of the input field terms to return one or more sequence IDs of the target. In a second layer of searching 312 , each target RNA can be linked to one or more RNA effector molecules, such as by their associated sequence ID and in accordance with the invention, thus for each target identified, a software module can be executed to perform a subsequent search for some or all of targets identified and can return one or more sequence IDs for desired RNA effector molecules and return a listing of the RNA effector molecules and their sequence IDs.

[0090] Alternatively, for each target identified, a software module can be executed that implements one or more well known algorithms for determining the desired RNA effector molecules and return a listing of the RNA effector molecules and their sequence IDs.

[0091] Figure 4 shows a flow chart of the method for identifying RNA effector molecules according to one embodiment of the invention. The method 400 includes presenting the user with an input screen 402 that allows the user to input the desired parameters for finding the target in the desired cell. The input can be free form text or one or more drop-down boxes allowing the user to select predefined terms. At 404, the user selects the appropriate user interface element, for example a "search" button and the system searches the database for targets associated with the input parameters. At 406, the user can be presented with a list of targets, each associated with a check box and the user can select or unselect the check box associated with each target to further refine their search. At 408, the user selects the appropriate user interface element, for example a "search" button and the system can search the database for RNA effector molecules associated with the input targets and/or use well know algorithms to determine RNA effector molecules associated with the input targets. The system can, for example, search for RNA effector molecules and if, none are found, use the well known algorithms to determine appropriate RNA effector molecules. Subsequently, the determined molecules can be added to the database and appear in subsequent searches. Alternatively, even where RNA effector molecules are found, the system can, in addition, use the well known algorithms to determine additional appropriate RNA effector molecules. At 410, the results of the user interface element selection are presented. At 412 the user can be provided with optional functions such as ordering the reported RNA effector molecule from information found in the database. For example, online procurement can be provided as described in U.S. Published Patent Application No. US 2005-0240352 which is hereby incorporated by reference in its entirety.

[0092] In one example of the system and using the system, a person, such as a customer, is experiencing problems in protein production using a cell line.

[0093] The problem may include, but is not limited to, in post translational modification of the protein, such as in glycosylation, in too much fucosylation, and /or another process, such as too much lactic acid buildup or too low yield.

[0094] The system on the invention allows the user to input parameters, such as the problem or multiple problems they are experiencing (too low cell growth rate or too much fucosylation) and/or a target gene, or transcript or multiple target genes or transcripts that they wish to modulate, such as FUT8, GMDS, and/or TSTA3, into the user interface.

[0095] The system takes the parameters and matches them with sequence data and RNA effector molecule data and delivers suggested RNA effector molecule(s) to the customer. For example, the system can match the problem to a cellular pathway, such as glycosylation, with transcripts known to play a role in glycosylation, and then matches the RNA effector molecules targeting these sequences and delivers, e.g. a list of siRNA sequences that the customer may experiment with.

[0096] If the customer wishes to receive one or more of the sequences, the customer can order or instruct the system to synthesize and/or send the appropriate nucleic acids to the customer- defined location.

[0097] The system may also send instructions to a nucleotide synthesis system to make the sequences. The synthesizer may be in the same location or in a remote location from the other system parts. The system may also select ready-made sequences from a storage location and provide packaging information so that the appropriate molecules can be sent to the customer- defined location. If the customer wishes to obtain different mixtures of the RNA effector molecules, such can be defined prior to submitting the final order and then the system will instruct the robotic component to mix the appropriate RNA effector molecules, such as siRNA duplexes, e.g comprising an antisense and sense strand, in one vial or tube or other container.

RNA effector molecules

[0098] We have further discovered a set of siRNA molecules that target at least one of the transcripts in the CHO cell transcriptome. Table 1 also sets forth a set of siRNA molecules that target the transcrips in the CHO cell transcriptome.

[0099] Such siRNA molecules are particularly useful as RNA effector molecules.

[0100] As used herein, the term "portion," when used in reference to an RNA effector molecule refers to a nucleic acid sequence of at least 3 nucleotides up to and including nucleic acid sequences one nucleotide shorter than the entire RNA effector molecule. Thus, the term portion refers to a region of an RNA effector molecule having a desired length to affect complementary binding to a region of a target gene. One of skill in the art can vary the length of the portion that is complementary to the target gene, such that an RNA effector molecule having desired characteristics (e.g., inhibition of a target gene) is produced. While not wishing to be bound by theory, RNA effector molecules provided herein can modulate expression of target genes by one or more of a variety of mechanisms, including but not limited to, Argonaute- mediated post-transcriptional cleavage of target gene mR A transcripts (sometimes referred to in the art as RNAi) and/or other pre-transcriptional and/or pre-translational mechanisms.

[0101] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0102] Complementary sequences within an RNA effector molecule, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter

oligonucleotide, may yet be referred to as fully complementary for the purposes described herein.

[0103] Complementary sequences, as used herein, can also include, or be formed entirely from, non- Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non- Watson-Crick base pairs includes, but are not limited to, G U Wobble or Hoogstein base pairing. [0104] The terms complementary, fully complementary and substantially complementary herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an RNA effector molecule agent and a target sequence, as will be understood from the context of their use.

[0105] As used herein, a polynucleotide that is "substantially complementary to at least part of a target gene refers to a polynucleotide that is substantially complementary to a contiguous portion of a target gene of interest (e.g., an mRNA encoded by a target gene, the target gene's promoter region or 3' UTR). For example, a polynucleotide is complementary to at least a part of a target mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoded by a target gene.

[0106] RNA effector molecules can comprise a single strand or more than one strand, and can include, e.g., double stranded RNA (dsRNA), microRNA (miRNA), antisense RNA, promoter- directed RNA (pdRNA), Piwi-interacting RNA (piRNA), expressed interfering RNA (eiRNA), short hairpin RNA (shRNA), antagomirs, decoy RNA, DNA, plasmids and aptamers.

[0107] The term "double-stranded RNA" or "dsRNA," as used herein, refers to an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti- parallel and substantially complementary nucleic acid strands, which will be referred to as having sense and antisense orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length.

Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therebetween, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecules, the molecules can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a "hairpin loop") between the 3 '-end of one strand and the 5 '-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a "linker." The term "sRNA effector molecule" is also used herein to refer to a dsRNA as described above.

[0108] In some embodiments, the RNA effector molecule is a promoter-directed RNA (pdRNA) which is substantially complementary to at least a portion of a noncoding region of an mRNA transcript of a target gene. In one embodiment, the pdRNA is substantially

complementary to at least a portion of the promoter region of a target gene mRNA at a site located upstream from the transcription start site, e.g., more than 100, more than 200, or more than 1,000 bases upstream from the transcription start site. In another embodiment, the pdRNA is substantially complementary to at least a portion of the 3'-UTR of a target gene mRNA transcript. In one embodiment, the pdRNA comprises dsRNA of 18-28 bases optionally having 3 ' di- or tri-nucleotide overhangs on each strand. The dsRNA is substantially complementary to at least a portion of the promoter region or the 3'-UTR region of a target gene mRNA transcript. In another embodiment, the pdRNA comprises a gapmer consisting of a single stranded polynucleotide comprising a DNA sequence which is substantially complementary to at least a portion of the promoter or the 3 '-UTR of a target gene mRNA transcript, and flanking the polynucleotide sequences (e.g., comprising the 5 terminal bases at each of the 5' and 3' ends of the gapmer) comprising one or more modified nucleotides, such as 2' MOE, 2'OMe, or Locked Nucleic Acid bases (LNA), which protect the gapmer from cellular nucleases.

[0109] pdRNA can be used to selectively increase, decrease, or otherwise modulate expression of a target gene. Without being limited to a particular theory, it is believed that pdRNAs modulate expression of target genes by binding to endogenous antisense RNA transcripts which overlap with noncoding regions of a target gene mRNA transcript, and recruiting Argonaute proteins (in the case of dsRNA) or host cell nucleases (e.g., RNase H) (in the case of gapmers) to selectively degrade the endogenous antisense RNAs. In some embodiments, the endogenous antisense RNA negatively regulates expression of the target gene and the pdRNA effector molecule activates expression of the target gene. Thus, in some embodiments, pdRNAs can be used to selectively activate the expression of a target gene by inhibiting the negative regulation of target gene expression by endogenous antisense RNA. Methods for identifying antisense transcripts encoded by promoter sequences of target genes and for making and using promoter-directed RNAs are described, e.g., in International Publication No. WO 2009/046397, herein incorporated by reference in its entirety.

[0110] Expressed interfering RNA (eiRNA) can be used to selectively increase, decrease, or otherwise modulate expression of a target gene. Typically with eiRNA, the dsRNA is expressed in the first transfected cell from an expression vector. In such a vector, the sense strand and the antisense strand of the dsRNA may be transcribed from the same nucleic acid sequence using, for example, two convergent promoters at either end of the nucleic acid sequence or separate promoters transcribing either a sense or antisense sequence. Alternatively, two plasmids can be cotransfected, with one of the plasmids designed to transcribe one strand of the dsRNA while the other is designed to transcribe the other strand. Methods for making and using eiRNA effector molecules are described, for example, in International Publication No. WO 2006/033756, and in U.S. Pat. Pub. Nos. 2005/0239728 and 2006/0035344, which are incorporated by reference in their entirety.

[0111] In some embodiments, the RNA effector molecule comprises a small single-stranded Piwi-interacting RNA (piRNA effector molecule) which is substantially complementary to at least a portion of a target gene, as defined herein, and which selectively binds to proteins of the Piwi or Aubergine subclasses of Argonaute proteins. Without being limited to a particular theory, it is believed that piRNA effector molecules interact with RNA transcripts of target genes and recruit Piwi and/or Aubergine proteins to form a ribonucleoprotein (RNP) complex that induces transcriptional and/or post-transcriptional gene silencing of target genes. A piRNA effector molecule can be about 25-50 nucleotides in length, about 25-39 nucleotides in length, or about 26-31 nucleotides in length. Methods for making and using piRNA effector molecules are described, e.g., in U.S. Pat. Pub. No. 2009/0062228, herein incorporated by reference in its entirety.

[0112] In some embodiments, the RNA effector molecule is an siRNA or shRNA effector molecule introduced into an animal host cell by contacting the cell with an invasive bacterium containing one or more siRNA or shRNA effector molecules or DNA encoding one or more siRNA or shRNA effector molecules (a process sometimes referred to as transkingdom RNAi (tkRNAi)). The invasive bacterium can be an attenuated strain of a bacterium selected from the group consisting of Listeria, Shigella, Salmonella, E. coli, and Bifidobacteriae, or a non-invasive bacterium that has been genetically modified to increase its invasive properties, e.g., by introducing one or more genes that enable invasive bacteria to access the cytoplasm of host cells. Examples of such cytoplasm-targeting genes include listeriolysin O of Listeria and the invasin protein of Yersinia pseudotuberculosis. Methods for delivering RNA effector molecules to animal cells to induce transkingdom RNAi (tkR Ai) are described, e.g., in U.S. Pat. Pub. Nos. 20080311081 to Fruehauf et al. and 20090123426 to Li et al., both of which are herein incorporated by reference in their entirety. In one embodiment, the RNA effector molecule is an siRNA molecule. In one embodiment, the RNA effector molecule is not an shRNA molecule.

[0113] In some embodiments, the RNA effector molecule comprises a microRNA (miRNA). miRNAs are small noncoding RNA molecules that are capable of causing post-transcriptional silencing of specific target genes, e.g., by inhibiting translation or initiating degradation of the targeted miRNA. In some embodiments, the miRNA is completely complementary with the target nucleic acid. In other embodiments, the miRNA has a region of noncomplementarity with the target nucleic acid, resulting in a "bulge" at the region of non-complementarity. In some embodiments, the region of noncomplementarity (the bulge) is flanked by regions of sufficient complementarity, e.g., complete complementarity, to allow duplex formation. Preferably, the regions of complementarity are at least 8 to 10 nucleotides long (e.g., 8, 9, or 10 nucleotides long). miRNA can inhibit gene expression by, e.g., repressing translation, such as when the miRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, when the miRNA binds its target with perfect or a high degree of complementarity.

[0114] In further embodiments, the RNA effector molecule may comprise an oligonucleotide agent which targets an endogenous miRNA or pre-miRNA. For example, the RNA effector may target an endogenous miRNA which negatively regulates expression of a target gene, such that the RNA effector alleviates miRNA-based inhibition of the target gene.

[0115] The oligonucleotide agent can include naturally occurring nucleobases, sugars, and covalent internucleotide (backbone) linkages and/or oligonucleotides having one or more non- naturally-occurring features that confer desirable properties, such as enhanced cellular uptake, enhanced affinity for the endogenous miRNA target, and/or increased stability in the presence of nucleases. In some embodiments, an oligonucleotide agent designed to bind to a specific endogenous miRNA has substantial complementarity, e.g., at least 70, 80, 90, or 100% complementary, with at least 10, 20, 25 or more bases of the target miRNA.

[0116] miRNA or pre-miRNA can be 16-100 nucleotides in length, and more preferably from 16-80 nucleotides in length. Mature miRNAs can have a length of 16-30 nucleotides, preferably 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. miRNA precursors can have a length of 70-100 nucleotides and can have a hairpin conformation. In some embodiments, miRNAs are generated in vivo from pre-miRNAs by the enzymes Dicer and Drosha. miRNAs or pre-miRNAs can be synthesized in vivo by a cell-based system or can be chemically synthesized. miRNAs can comprise modifications which impart one or more desired properties, such as improved stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, and/or cell permeability, e.g., by an endocytosis-dependent or - independent mechanism. Modifications can also increase sequence specificity, and consequently decrease off-site targeting.

[0117] In some embodiments, the RNA effector molecule comprises a single-stranded R A molecule that interacts with and directs the cleavage of RNA transcripts of a target gene. It is particularly preferred that single stranded RNA effector molecules comprise a 5 ' modification including one or more phosphate groups or analogs thereof to protect the effector molecule from muclease degradation.

[0118] In some embodiments, the RNA effector molecule comprises an antagomir.

Antagomirs are single stranded, double stranded, partially double stranded or hairpin structures that target a microRNA. An antagomir consisting essentially of or comprises at least 12 or more contiguous nucleotides substantially complementary to an endogenous miRNA and more particularly a target sequence of an miRNA or pre -miRNA nucleotide sequence. Antagomirs preferably have a nucleotide sequence sufficiently complementary to a miRNA target sequence of about 12 to 25 nucleotides, preferably about 15 to 23 nucleotides, to allow the antagomir to hybridize to the target sequence. More preferably, the target sequence differs by no more than 1, 2, or 3 nucleotides from the sequence of the antagomir. In some embodiments, the antagomir includes a non-nucleotide moiety, e.g., a cholesterol moiety, which can be attached, e.g., to the 3' or 5' end of the oligonucleotide agent.

[0119] In some embodiments, antagomirs are stabilized against nucleolytic degradation by the incorporation of a modification, e.g., a nucleotide modification. For example, in some embodiments, antagomirs contain a phosphorothioate comprising at least the first, second, and/or third internucleotide linkages at the 5' or 3' end of the nucleotide sequence. In further embodiments, antagomirs include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0- dimethylaminoethyl (2*-0-DMAOE), 2*-0-dimethylaminopropyl (2*-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0~N-methylacetamido (2'-0-NMA). In some preferred embodiments, antagomirs include at least one 2'-0-methyl-modified nucleotide.

[0120] In some embodiments, the RNA effector molecule comprises an aptamer which binds to a non-nucleic acid ligand, such as a small organic molecule or protein, e.g., a transcription or translation factor, and subsequently modifies (e.g., inhibits) activity. An aptamer can fold into a specific structure that directs the recognition of a targeted binding site on the non-nucleic acid ligand. Aptamers can contain any of the modifications described herein. [0121] In some embodiments, the RNA effector molecule is a single-stranded antisense nucleic acid having a nucleotide sequence that is complementary to at least a portion of a sense nucleic acid of a target gene, e.g., the coding strand of a double-stranded cDNA molecule or an RNA sequence, e.g., a pre-mRNA, mRNA, miRNA, or pre-miRNA. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid target. In an alternative embodiment, the RNA effector molecule comprises a duplex region of at least 9 nucleotides in length.

[0122] Given a coding strand sequence (e.g., the sequence of a sense strand of a cDNA molecule), antisense nucleic acids can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid can be complementary to a portion of the coding or noncoding region of an RNA, e.g., the region surrounding the translation start site of a pre- mRNA or mRNA, e.g., the 5' UTR. An antisense oligonucleotide can be, for example, about 10 to 25 nucleotides in length (e.g., 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length). In some embodiments, the antisense oligonucleotide comprises one or more modified nucleotides, e.g., phosphorothioate derivatives and/or acridine substituted nucleotides, designed to increase its biological stability of the molecule and/or the physical stability of the duplexes formed between the antisense and target nucleic acids. Antisense oligonucleotides can comprise ribonucleotides only, deoxyribonucleotides only (e.g., ohgodeoxynucleotides), or both deoxyribonucleotides and ribonucleotides. For example, an antisense agent consisting only of ribonucleotides can hybridize to a complementary RNA and prevent access of the translation machinery to the target RNA transcript, thereby preventing protein synthesis. An antisense molecule including only deoxyribonucleotides, or deoxyribonucleotides and ribonucleotides, can hybridize to a complementary RNA and the RNA target can be subsequently cleaved by an enzyme, e.g., RNAse H, to prevent translation. The flanking RNA sequences can include 2'-0- methylated nucleotides, and phosphorothioate linkages, and the internal DNA sequence can include phosphorothioate internucleotide linkages. The internal DNA sequence is preferably at least five nucleotides in length when targeting by RNAse H activity is desired.

[0123] In some embodiments, a plurality of RNA effector molecules are used to modulate expression of one or more target genes. As used herein, the term "plurality" refers to at least 2 or more RNA effector molecules e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 80, 100 RNA effector molecules or more. The term "plurality" can also refer to at least 2 or more target genes, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 target genes or more.

[0124] A skilled artisan will recognize that the term "RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a "nbonucleoside" includes a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside with one, two or three phosphate moieties. However, the terms ribonucleoside and ribonucleotide can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below.

However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. An RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2'-0-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesterol derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino-modified nucleoside, 2'-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more modified ribonucleosides, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.

[0125] In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an RNA effector molecule agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term "RNA effector molecule."

[0126] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an RNA effector molecule, e.g., a dsRNA. For example, when a 3 '-end of one strand of a dsRNA extends beyond the 5 '-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a

deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5' end, 3' end or both ends of either an antisense or sense strand of a dsRNA.

[0127] dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture media, blood, and serum. Generally, the single- stranded overhang is located at the 3 '-terminal end of an antisense strand or, alternatively, at the 3 '-terminal end of a sense strand. The dsRNA having an overhang on only one end will also have one blunt end, generally located at the 5 'end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. In one embodiment, the antisense strand of a dsRNA has a 1 - 10 nucleotide overhang at the 3 ' end and/or the 5 ' end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3' end and/or the 5' end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

[0128] The terms "blunt" or "blunt ended" as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a blunt ended dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

[0129] The term "antisense strand" refers to the strand of an R A effector molecule, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus. The antisense strand can form one strand of duplex siRNA.

[0130] The term "sense strand," as used herein, refers to the strand of an RNA effector molecule that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

[0131] As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an RNA effector molecule or a plasmid from which an RNA effector molecule is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and USSN 61/045,228, filed on April 15, 2008. These applications are hereby incorporated by reference in their entirety.

Delivery of RNA effector molecule

[0132] The delivery of an RNA effector molecule to cells according to methods provided herein can be achieved in a number of different ways. Delivery can be performed directly by administering a composition comprising an RNA effector molecule, e.g. a dsRNA, to the cell culture media. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the RNA effector molecule. These alternatives are discussed further below.

Direct delivery

[0133] RNA effector molecules can be modified to prevent rapid degradation of the dsRNA by endo- and exo-nucleases and avoid undesirable off-target effects. For example, RNA effector molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. In an alternative embodiment, RNA effector molecules can be delivered using a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an RNA effector molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient cellular uptake. Cationic lipids, dendrimers, or polymers can either be bound to RNA effector molecules, or induced to form a vesicle or micelle (see e.g., Kim SH., et al (2008) Journal of Controlled Release

129(2): 107-116, which is incorporated herein by reference in its entirety) that encases the RNA effector molecule. Methods for making and using cationic- RNA effector molecule complexes are well within the abilities of those skilled in the art (see e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25: 197-205, which are incorporated herein by reference in their entirety). Separate and Temporal Administration

[0134] Where the RNA effector molecule is a double-stranded molecule, such as a small interfering RNA (siRNA), comprising a sense strand and an antisense strand, the sense strand and antisense strand can be separately and temporally exposed to a cell, cell lysate or cell culture. The phrase "separately and temporally" refers to the introduction of each strand of a double- stranded RNA effector molecule to a cell, cell lysate or cell culture in a single-stranded form, e.g., in the form of a non-annealed mixture of both strands or as separate, i.e., unmixed, preparations of each strand. In some embodiments, there is a time interval between the introduction of each strand which can range from seconds to several minutes to about an hour or more, e.g., 12, 24, 48, 72, 84, 96, 108 hours or more. Separate and temporal administration can be performed with canonical or non-canonical RNA effector molecules.

[0135] It is also contemplated herein that a plurality of RNA effector molecules are administered in a separate and temporal manner. Thus, each of a plurality of RNA effector molecules can be administered at a separate time or at a different frequency interval to achieve the desired average percent inhibition for the target gene. For example, RNA effector molecules targeting Bak can be administered more frequently than RNA effector molecule targeting LDH or Bax, as the expression of Bak recovers faster following treatment with a Bak RNA effector molecule. In one embodiment, the RNA effector molecules are added at a concentration from approximately O.OlnM to 200nM. In another embodiment, the RNA effector molecules are added at an amount of approximately 50 molecules per cell up to and including 500,000 molecules per cell. In another embodiment, the RNA effector molecules are added at a concentration from about 0.1 fmol/106 cells to about 1 pmol/106 cells.

Vector encoded dsRNAs

[0136] In another aspect, an RNA effector molecule for modulating expression of a target gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al, International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299 which are incorporated herein by reference in their entirety). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extra chromosomal plasmid (see Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292 which is incorporated herein by reference in its entirety).

[0137] The individual strand or strands of an RNA effector molecule can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

[0138] RNA effector molecule expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an RNA effector molecule as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. RNA effector molecule expressing vectors can be delivered directly to target cells using standard transfection and transduction methods.

[0139] RNA effector molecule expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-T O^® by Minis Bio LLC, Madison, WI). Multiple lipid transfections for RNA effector molecule-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0140] Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper- dependent or gutless adenovirus. Replication-defective viruses can also be advantageous.

Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of an RNA effector molecule will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the RNA effector molecule in target cells. Other aspects to consider for vectors and constructs are further described below.

[0141] Vectors useful for the delivery of an RNA effector molecule will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the RNA effector molecule in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

[0142] Expression of the RNA effector molecule can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., glucose levels (see Docherty et al., 1994, FASEB J. 8:20-24, which is incorporated herein by reference in its entirety). Such inducible expression systems, suitable for the control of dsR A expression in cells include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D 1 -thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the RNA effector molecule transgene.

[0143] In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an RNA effector molecule can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993), which is incorporated herein by reference in its entirety). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an RNA effector molecule are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al, Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy, which is herein incorporated by reference in its entirety. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644- 651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.

3: 110-114 (1993), each of which is herein incorporated by reference in its entirety. Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, each of which is herein incorporated by reference in its entirety.

[0144] Adenoviruses are also contemplated for use in delivery of RNA effector molecules. A suitable AV vector for expressing an RNA effector molecule featured in an embodiment of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, is described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010, herein encorporated by reference in its entirety.

[0145] Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146, herein incorporated by reference in its entirety). In one embodiment, the RNA effector molecule can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61 : 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

[0146] Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

[0147] The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

[0148] The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Compositions containing RNA effector molecule

[0149] In one embodiment, the invention provides compositions containing an RNA effector molecule, as described herein, and an acceptable carrier. The composition containing the RNA effector molecule is useful for enhancing the production of a biological product by a cultured eukaryotic cell by modulating the expression or activity of a target gene in the cell. Such pharmaceutical compositions are formulated based on the mode of delivery. Provided herein are exemplary RNA effector molecules useful in improving the production of a biological product. In one embodiment, the RNA effector molecule is an siRNA. In another embodiment, the RNA effector molecule is not an shRNA.

[0150] In another embodiment, a composition is provided herein comprising a plurality of RNA effector molecules that permit inhibition of expression of Bax (for example, wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 3023234-3023515), Bak (SEQ ID NOs: 2259855-2260161), and LDH (for example, wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1297283-1297604). This composition can optionally be combined (or administered) with at least one additional RNA effector molecule targeting a cellular process including, but not limited to: carbon metabolism and transport, apoptosis, RNAi uptake and/or efficiency, reactive oxygen species production, cell cycle control, protein folding, pyroglutamation protein modification, deamidase, glycosylation, disulfide bond formation, protein secretion, gene amplification, viral replication, viral infection, viral particle release, control of pH, and protein production.

[0151] In one embodiment, the compositions described herein comprise a plurality of RNA effector molecules. In one embodiment of this aspect, each of the plurality of RNA effector molecules is provided at a different concentration. In another embodiment, each of the plurality of RNA effector molecules is provided at the same concentration. In another embodiment, at least two of the plurality of RNA effector molecules are provided at the same concentration, while at least one other RNA effector molecule in the plurality is provided at a different concentration. It is appreciated one of skill in the art that a variety of combinations of RNA effector molecules and concentrations can be provided to a cell in culture to produce the desired effects described herein.

[0152] The compositions featured herein are administered in amounts sufficient to inhibit expression of target genes. In general, a suitable dose of RNA effector molecule will be in the range of 0.001 to 200.0 milligrams per unit volume or cell density per day. In another embodiment, the RNA effector molecule is provided in the range of 0.00 InM to 200mM per day, generally in the range of 0. InM to 500 nM. For example, the dsRNA can be administered at O.OlnM, 0.05nM, O.lnM, 0.5 nM, 0.75nM, 1 nM, 1.5 nM, 2 nM, 3 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, ΙΟΟηΜ, 200nM, 400nM, or 500nM per single dose.

[0153] The composition can be administered once daily, or the RNA effector molecule can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the RNA effector molecule contained in each sub dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation, which provides sustained release of the RNA effector molecule over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents to a particular site, such as could be used with the agents of the present invention. In one embodiment, an RNA effector molecule is contacted with the cells in culture at a final concentration of InM. It should be noted that when administering a plurality of RNA effector molecules that one should consider that the total dose of RNA effector molecules will be higher than when each is administered alone. For example, administration of three RNA effector molecules each at InM (e.g., for effective inhibition of target gene expression) will necessarily result in a total dose of 3nM to the cell culture. One of skill in the art can modify the necessary amount of each RNA effector molecule to produce effective inhibition of each target gene while preventing any unwanted toxic effects to the cell culture resulting from high concentrations of either the RNA effector molecules or delivery agent.

[0154] The effect of a single dose on target gene transcript levels can be long-lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

[0155] It is also noted that, in certain embodiments, it can be beneficial to contact the cells in culture with an RNA effector molecule such that a constant number (or at least a minimum number) of RNA effector molecules per each cell is maintained. Maintaining the levels of the RNA effector molecule as such can ensure that modulation of target gene expression is maintained even at high cell densities.

[0156] Alternatively, the amount of an RNA effector molecule can be administered according to the cell density. In such embodiments, the RNA effector molecule(s) is added at a

concentration of at least 0.01 fmol/106 cells, at least 0.1 fmol/106 cells, at least 0.5 fmol/106 cells, at least 0.75 fmol/106 cells, at least 1 fmol 106 cells, at least 2 fmol/106 cells, at least 5 fmol/106 cells, at least 10 fmol/106 cells, at least 20 fmol/106 cells, at least 30 fmol/106 cells, at least 40 fmol/106 cells, at least 50 fmol/106 cells, at least 60 fmol/106 cells, at least 100 fmol/106 cells, at least 200 fmol/106 cells, at least 300 fmol/106 cells, at least 400 fmol/106 cells, at least 500 fmol/106 cells, at least 700 fmol/106 cells, at least 800 fmol/106 cells, at least 900 fmol/106 cells, or at least 1 pmol/106 cells, or more.

[0157] In an alternate embodiment, the RNA effector molecule is administered at a dose of at least 10 molecules per cell, at least 20 molecules per cell, at least 30 molecules per cell, at least 40 molecules per cell, at least 50 molecules per cell, at least 60 molecules per cell, at least 70 molecules per cell, at least 80 molecules per cell, at least 90 molecules per cell at least 100 molecules per cell, at least 200 molecules per cell, at least 300 molecules per cell, at least 400 molecules per cell, at least 500 molecules per cell, at least 600 molecules per cell, at least 700 molecules per cell, at least 800 molecules per cell, at least 900 molecules per cell, at least 1000 molecules per cell, at least 2000 molecules per cell, at least 5000 molecules per cell or more. In some embodiments, the RNA effector molecule is administered at a dose within the range of 10- 100 molecules/cell, 10-90 molecules/cell, 10-80 molecules/cell, 10-70 molecules/cell, 10-60 molecules/cell, 10-50 molecules/cell, 10-40 molecules/cell, 10-30 molecules/cell, 10-20 molecules/cell, 90-100 molecules/cell, 80-100 molecules/cell, 70-100 molecules/cell, 60-100 molecules/cell, 50-100 molecules/cell, 40-100 molecules/cell, 30-100 molecules/cell, 20-100 molecules/cell, 30-60 molecules/cell, 30-50 molecules/cell, 40-50 molecules/cell, 40-60 molecules/cell, or any range therebetween.

[0158] In one embodiment of the methods described herein, the RNA effector molecule is provided to the cells in a continuous infusion. The continuous infusion can be initiated at day zero (e.g., the first day of cell culture or day of inoculation with an RNA effector molecule) or can be initiated at any time period during the biological production process. Similarly, the continuous infusion can be stopped at any time point during the biological production process. Thus, the infusion of an RNA effector molecule or composition can be provided and/or removed at a particular phase of cell growth, a window of time in the production process, or at any other desired time point. The continuous infusion can also be provided to achieve a "desired average percent inhibition" for a target gene, as that term is used herein. In one embodiment, a continuous infusion can be used following an initial bolus administration of an RNA effector molecule to a cell culture. In this embodiment, the continuous infusion maintains the concentration of RNA effector molecule above a minimum level over a desired period of time. The continuous infusion can be delivered at a rate of 0.03 - 3 pmol/liter of culture/h, for example, at 0.03 pmol/l/h, 0.05 pmol/l/h, 0.08 pmol/l/h, 0.1 pmol/l/h, 0.2 pmol/l/h, 0.3 pmol/l/h, 0.5 pmol/l/h, 1.0 pmol/l/h, 2 pmol/l/h, or 3 pmol/l/h, or any value therebetween. In one embodiment, the RNA effector molecule is administered as a sterile aqueous solution. In another embodiment, the RNA effector molecule is formulated in a cationic or non-cationic lipid formulation. In still another embodiment, the RNA effector molecule is formulated in a cell medium suitable for culturing a host cell (e.g., a serum- free medium). In one embodiment, an initial concentration of RNA effector molecule(s) is supplemented with a continuous infusion of the RNA effector molecule to maintain modulation of expression of a target gene. In another embodiment, the RNA effector molecule is applied to cells in culture at a particular stage of cell growth (e.g., early log phase) in a bolus dosage to achieve a certain concentration (e.g., 1 nM), and provided with a continuous infusion of the RNA effector molecule.

[0159] The RNA effector molecule(s) can be administered once daily, or the RNA effector molecule treatment can be repeated (e.g., two, three, or more doses) by adding the composition to the culture medium at appropriate intervals/frequencies throughout the production of the biological product. As used herein the term "frequency" refers to the interval at which transfection of the cell culture occurs and can be optimized by one of skill in the art to maintain the desired level of inhibition for each target gene. In one embodiment, RNA effector molecules are contacted with cells in culture at a frequency of every 48 hours. In other embodiments, the RNA effector molecules are administered at a frequency of e.g., every 4h, every 6h, every 12h, every 18h, every 24h, every 36h, every 72h, every 84h, every 96h, every 5 days, every 7 days, every 10 days, every 14 days, every 3 weeks, or more during the production of the biological product. The frequency can also vary, such that the interval between each dose is different (e.g., first interval 36h, second interval 48h, third interval 72h etc).

[0160] The term "frequency" can be similarly applied to nutrient feeding of a cell culture during the production of a biological product. The frequency of treatment with RNA effector molecule(s) and nutrient feeding need not be the same. To be clear, nutrients can be added at the time of RNA effector treatment or at an alternate time. The frequency of nutrient feeding can be a shorter interval or a longer interval than the RNA effector molecule treatment. As but one example, the dose of RNA effector molecule can be applied at a 48h interval while nutrient feeding may be applied at a 24h interval. During the entire length of the interval for producing the biological product (e.g., 3 weeks) there can be more doses of nutrients than RNA effector molecules or less doses of nutrients than RNA effector molecules. Alternatively, the amount of treatments with RNA effector molecule(s) is equal to that of nutrient feedings.

[0161] The frequency of RNA effector molecule treatment can be optimized to maintain a desired average percent inhibition of a particular target gene. As used herein, the term "desired average percent inhibition" refers to the average degree of inhibition of target gene expression over time that is necessary to produce the desired effect and which is below the degree of inhibition that produces any unwanted or negative effects. For example, the desired inhibition of Bax/ Bak is typically >80% inhibition to effect a decrease in apoptosis, while the desired average inhibition of LDH may be less (e.g., 70%) as high degrees of LDH average inhibition (e.g., 90%) decrease cell viability. In some embodiments, the desired average percent inhibition is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or even 100% (i.e., absent). One of skill in the art can use routine cell death assays to determine the upper limit for desired percent inhibition (e.g., level of inhibition that produces unwanted effects). One of skill in the art can also use methods to detect target gene expression (e.g., RT-PCR) to determine an amount of an RNA effector molecule that produces gene modulation. The percent inhibition is described herein as an average value over time, since the amount of inhibition is dynamic and can fluctuate slightly between doses of the RNA effector molecule.

[0162] In one embodiment of the methods described herein, the RNA effector molecule is added to the culture medium of the cells in culture. The methods described herein can be applied to any size of cell culture flask and/or bioreactor. For example, the methods can be applied in bioreactors or cell cultures of 1L, 3L, 5L, 10L, 15L, 40L, 100L, 500L, 1000L, 2000L, 3000L, 4000L, 5000L or larger. In some embodiments, the cell culture size can range from 0.01L to 5000L, from 0.1L to 5000L, from 1L to 5000L, from 5L to 5000L, from 40L to 5000L, from 100L to 5000L, from 500L to 5000L, from 1000 to 5000L, from 2000 to 5000L, from 3000 to 5000L, from 4000 to 5000L, from 4500 to 5000L, from 0.01L to 1000L, from 0.01 to 500L, from 0.01 to 100L, from 0.01 to 40L, from 15 to 2000L, from 40 to 1000L, from 100 to 500L, from 200 to 400L, or any integer therebetween.

[0163] The RNA effector molecule(s) can be added during any phase of cell growth including, but not limited to, lag phase, stationary phase, early log phase, mid-log phase, late-log phase, exponential phase, or death phase. It is preferred that the cells are contacted with the RNA effector molecules prior to their entry into the death phase. In some embodiments, such as when targeting an apoptotic pathway, it may be desired to contact the cell in an earlier growth phase such as the lag phase, early log phase, mid-log phase or late-log phase (e.g., Bax/Bak inhibition). In other embodiments, it may be desired or acceptable to inhibit target gene expression at a later phase in the cell growth cycle (e.g., late-log phase or stationary phase), for example when growth-limiting products such as lactate are formed (e.g., LDH inhibition).

[0164] RNA effector molecules featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, RNA effector molecules can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1- dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a CI -20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or acceptable salt thereof.

[0165] In one embodiment, a RNA effector molecules featured in the invention are fully encapsulated in the lipid formulation (e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle). As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SPLPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683, herein incorporated by reference. The particles in this embodiment typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids, when present in the nucleic acid-lipid particles of the present invention, are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964, all of which are incorporated herein by reference.

[0166] In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1 :1 to about 50:1, from about 1 :1 to about 25:1, from about 3:1 to about 15: 1, from about 4:1 to about 10: 1, from about 5:1 to about 9: 1, or about 6: 1 to about 9: 1.

[0167] The cationic lipid of the formulation preferably comprises at least one protonatable group having a pKa of from 4 to 15. The cationic lipid can be, for example, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I - (2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane

(DLinDMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1 ,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1 ,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin- MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), 1 -Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol

(DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l ,3]- dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 70 mol % or about 40 mol % to about 60 mol % of the total lipid present in the particle. In one embodiment, cationic lipid can be further conjugated to a ligand.

[0168] The non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),

dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl- phosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE),

dimyristoylphosphoethanolamme (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-0- monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidyethanolamme (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

[0169] The lipid that inhibits aggregation of particles may be, for example, a

polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG- dimyristyloxypropyl (CI 4), a PEG-dipalmityloxypropyl (CI 6), or a PEG- distearyloxypropyl (C 18). The lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle. In one embodiment, PEG lipid can be further conjugated to a ligand.

[0170] In some embodiments, the nucleic acid-lipid particle further includes a steroid such as, cholesterol at about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0171] In one embodiment, the lipid particle comprises a steroid, a PEG lipid and a cationic lipid of formula (I):

formula (I)

wherein

each X^a and X^b, for each occurrence, is independently a C 1 -6 alkylene;

n is 0, 1, 2, 3, 4, or 5;

h Pv is independently

m is 0, 1, 2, 3 or 4;

Y is absent, O, NR², or S;

Pv¹ is alkyl alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; and

R² is H, alkyl alkenyl or alkynyl; each of which is optionally substituted each of which is optionally substituted with one or more substituents. In one example, the lipidoid ND98-4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid RNA effector molecule nanoparticles (e.g., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide CI 6, 100 mg/mL. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous RNA effector molecule (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid RNA effector molecule nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7 (e.g., about pH 6.9, about pH 7.0, about pH 7.1 , about pH 7.2, about pH 7.3, or about pH 7.4).

[0172] LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

[0173] Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total sRNA effector molecule concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated RNA effector molecule can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton- XI 00. The total RNA effector molecule in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" RNA effector molecule content (as measured by the signal in the absence of surfactant) from the total RNA effector molecule content. Percent entrapped RNA effector molecule is typically >85%. For lipid nanoparticle formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

[0174] In some embodiments, RNA effector molecules featured in the invention are formulated in conjunction with one or more penetration enhancers, surfactants and/or chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and

ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, l-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.

[0175] The compositions of the present invention may be formulated into any of many possible dosage forms, including but not limited to, tablets, capsules, gel capsules, liquid syrups, and soft gels. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium

carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. Emulsions

[0176] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding Ο.ΐμιη in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al, in Remington's Pharmaceutical Sciences, Mack

Publishing Co., Easton, Pa., 1985, p. 301, all of which are incorporated herein by reference). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain components in addition to the dispersed phases and the active drug, which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in- water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not provide. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0177] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion- style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199, all of which are herein incorporated by reference). [0178] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199, all of which are herein incorporated by reference). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285, all of which are herein incorporated by reference).

[0179] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0180] Large varieties of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and

antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199, all of which are incorporated herein by reference).

[0181] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These gums and synthetic polymers disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0182] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0183] In one embodiment, the compositions of NA effector molecules and nucleic acids are formulated as microemulsions. A "microemulsion" may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245, all of which are herein incorporated by reference). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol, to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215, herein incorporated by reference). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271, herein incorporated by reference). [0184] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate micro emulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume I, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335, all of which are herein incorporated by reference). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0185] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),

hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1 -propanol, and 1 -butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film resulting from the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems that are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0186] Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, and decreased toxicity (see e.g., U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143, all of which are herein incorporated by reference). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or RNA effector molecules. [0187] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the R A effector molecules and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories— surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92, herein incorporated by reference). Each of these classes has been discussed above.

Liposomes

[0188] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0189] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous interior contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0190] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0191] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; and liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (see e.g., Wang, B et al., Drug delivery:

principles and applications, 2005, John Wiley and Sons, Hoboken, NJ; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245, all of which are herein incorporated by reference). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0192] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0193] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration liposomes present several advantages over other formulations. Such advantages include reduced side- effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0194] Several reports have detailed the ability of liposomes to deliver agents including high- molecular weight DNA into the skin. Agents including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications of these agents resulted in the targeting of the upper epidermis.

[0195] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged polynucleotide molecules to form a stable complex. The positively charged polynucleotide/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985, herein incorporated by reference).

[0196] Liposomes which are pH-sensitive or negatively-charged entrap the polynucleotide rather than form a complex with it. Since both the polynucleotide and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some polynucleotides are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture.

Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274, herein incorporated by reference).

[0197] One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. [0198] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al, Journal of Drug Targeting, 1992, 2, 405-410, herein incorporated by reference). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265, herein incorporated by reference).

[0199] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl

dilaurate/cholesterol/po- lyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.

S.T.P.Pharma. Sci., 1994, 4, 6, 466 herein incorporated by reference).

[0200] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as

monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al, FEBS Letters, 1987, 223, 42; Wu et al, Cancer Research, 1993, 53, 3765, both of which are herein incorporated by reference).

[0201] Various liposomes comprising one or more glycolipids are known in the art.

Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64, herein incorporated by reference) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and

phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949, herein incorporated by reference). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester, both of which are herein incorporated by reference. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn- dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al), which is herein incorporated by reference.

[0202] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778), herein incorporated by reference, described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79), herein incorporated by reference, noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899, both of which are herein incorporated by reference). In addition, antibodies can be conjugated to a polyakylene derivatized liposome (see e.g., PCT Application US 2008/0014255, herein incorporated by reference). Klibanov et al. (FEBS Lett., 1990, 268, 235), herein incorporated by reference, described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91), herein incorporated by reference, extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of

distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 Bl and WO 90/04384 to Fisher, both of which are herein incorporated by reference. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 Bl), all of which are herein incorporated by reference in their entirety. Liposomes comprising a number of other lipid- polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.), all of which are herein incorporated by reference. Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.), herein incorporated by reference. U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al), both of which are herein incorporated by reference, describes PEG- containing liposomes that can be further derivatized with functional moieties on their surfaces. Methods and compositions relating to liposomes comprising PEG can be found in, US Patent Nos. 6,049,094; 6,224,903; 6,270,806; 6,471,326; and 6,958,241, all of which are herein incorporated by reference.

[0203] As noted above, liposomes may optionally be prepared to contain surface groups, such as antibodies or antibody fragments, small effector molecules for interacting with cell-surface receptors, antigens, and other like compounds, and these groups can facilitate delivery of liposomes and their contents to specific cell populations. Such ligands can be included in the liposomes by including in the liposomal lipids a lipid derivatized with the targeting molecule, or a lipid having a polar-head chemical group that can be derivatized with the targeting molecule in preformed liposomes. Alternatively, a targeting moiety can be inserted into preformed liposomes by incubating the preformed liposomes with a ligand-polymer-lipid conjugate.

[0204] Lipids can be derivatized using a variety of targeting moieties, such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies by covalently attaching the ligand to the free distal end of a hydrophilic polymer chain, which is attached at its proximal end to a vesicle-forming lipid. There are a wide variety of techniques for attaching a selected hydrophilic polymer to a selected lipid and activating the free, unattached end of the polymer for reaction with a selected ligand, and as noted above, the hydrophilic polymer polyethyleneglycol (PEG) has been widely studied (Allen, T. M., et al, Biochemicia et Biophysica Acta 1237:99-108 (1995); Zalipsky, S., Bioconjugate Chem., 4(4):296-299 (1993); Zalipsky, S., et al., FEBS Lett. 353:71-74 (1994); Zalipsky, S., et al., Bioconjugate Chemistry, 705-708 (1995); Zalipsky, S., in STEALTH LIPOSOMES (D. Lasic and F. Martin, Eds.) Chapter 9, CRC Press, Boca Raton, Fla. (1995), all of which are herein incorporated by reference).

[0205] A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al, herein incorporated by reference, discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al., herein incorporated by reference, discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes and is herein incorporated by reference. WO 97/04787 to Love et al, herein incorporated by reference, discloses liposomes comprising dsRNAs targeted to the raf gene. In addition, methods for preparing a liposome composition comprising a nucleic acid can be found in, U.S. Patent Nos. 6,011,020; 6,074,667; 6,110,490; 6,147,204; 6, 271, 206; 6,312,956; 6,465,188; 6,506,564; 6,750,016; and 7,112,337, all of which are herein incorporated by reference in their entirety.

[0206] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the

environment in which they are used, e.g., they are self-optimizing, self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition.

[0207] Surfactants have a wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285, both of which are herein incorporated by reference).

[0208] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant.

Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0209] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0210] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0211] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides. [0212] The use of surfactants in drug products, formulations and in emulsions has been reviewed (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285, both of which are herein incorporated by reference).

Penetration Enhancers

[0213] In one embodiment, the present invention employs various penetration enhancers to affect the efficient delivery of nucleic acids, particularly RNA effector molecules, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non- lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0214] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92, both of which are herein incorporated by reference). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0215] Surfactants: In connection with the present invention, "surfactants" (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of RNA effector molecules through cellular membranes and other biological barriers is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92, both of which are herein incorporated by reference); and perfluorochemical emulsions, such as FC-43 (see Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252, herein incorporated by reference).

[0216] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1- monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1 -monocaprate, 1- dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, CI -20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol, 1992, 44, 651-654, all of which are herein incorporated by reference).

[0217] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw- Hill, New York, 1996, pp. 934-935, both are herein incorporated by reference). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro- fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al, J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583, all of which are herein incorporated by reference).

[0218] Chelating Agents: "Chelating agents," as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of RNA effector molecules through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339, herein incorporated by reference). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and -amino acyl derivatives of beta-diketones (enamines) (see e.g., Katdare, A. et al., Excipient development for pharmaceutical,

biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51, all of which are herein incorporated by reference).

[0219] Non-chelating non-surfactants: As used herein, "non-chelating non-surfactant" penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of RNA effector molecules through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33, herein incorporated by reference). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1- alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92, herein incorporated by reference); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626, herein incorporated by reference).

[0220] Agents that enhance uptake of RNA effector molecules at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188, herein incorporated by reference), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731, herein incorporated by reference), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™

(Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX

(Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA),

Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA),

Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X- tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse,

Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^a D 1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, CA, USA), PerFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, CA, USA ), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B- Bridge International, Mountain View, CA, USA), among others.

[0221] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

[0222] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal.

Other Components

[0223] The compositions of the present invention may additionally contain other adjunct components so long as such materials, when added, do not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0224] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0225] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 0% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

[0226] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in the instant methods. The dosage of compositions featured in the invention lies generally within a range of concentrations that includes the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

Definitions

[0227] For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[0228] "G," "C," "A," "T" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without

substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

[0229] A "biological product" can include any substance capable of being produced by a cultured host cell and recovered in useful quantities, including but not limited to, polypeptides (e.g., glycoproteins, antibodies, peptide-based growth factors), carbohydrates, lipids, fatty acids, metabolites (e.g., polyketides, macro lides), and chemical intermediates. The biological products can be used for a wide range of applications, including as biotherapeutic agents, vaccines, research or diagnostic reagents, fermented foods, food additives, nutraceuticals, biofuels, industrial enzymes (e.g., glucoamylase, lipase), industrial chemicals (e.g., lactate, fumarate, glycerol, ethanol), and the like. [0230] In some embodiments, the biological product is a polypeptide. The polypeptide can be a recombinant polypeptide or a polypeptide endogenous to the host cell. In some embodiments, the polypeptide is a glycoprotein and the host cell is a mammalian cell. Non-limiting examples of polypeptides that can be produced according to methods provided herein include receptors, membrane proteins, cytokines, chemokines, hormones, enzymes, growth factors, growth factor receptors, antibodies, antibody derivatives and other immune effectors, interleukins, interferons, erythropoietin, integrins, soluble major histocompatibility complex antigens, binding proteins, transcription factors, translation factors, oncoproteins or proto-oncoproteins, muscle proteins, myeloproteins, neuroactive proteins, tumor growth suppressors, structural proteins, and blood proteins (e.g., thrombin, serum albumin, Factor VII, Factor VIII, Factor IX, Factor X, Protein C, and von Willebrand factor, etc.). As used herein, a polypeptide encompasses glycoproteins or other polypeptides which has undergone post-translational modification, such as deamidation, glycation, and the like.

[0231] An exemplary process for the industrial-scale production of a heterologous polypeptide in cell culture (e.g., mammalian cell culture) includes the following steps:

(i)inoculating mammalian host cells containing a transgene encoding the heterologous protein into a seed culture vessel containing cell culture medium and propagating the cells to reach a minimum threshold cross-seeding density; (ii) transferring the propagated seed culture cells, or a portion thereof, to a large-scale bioreactor; (iii) propagating the large-scale culture under conditions allowing for rapid growth and cell division until the cells reach a predetermined density; and (iv) maintaining the culture under conditions that disfavor continued cell growth and/or cell division and facilitate expression of the heterologous protein.

[0232] The cells can be cultured in a stirred tank bioreactor system in a fed batch culture process in which the host cells and culture medium are supplied to the bioreactor initially and additional culture nutrients are fed, continuously or in discrete increments, throughout the cell culture process. The fed batch culture process can be semi-continuous, wherein periodically whole culture (including cells and medium) is removed and replaced by fresh medium.

Alternatively, a simple batch culture process can be used in which all components for cell culturing (including the cells and culture medium) are supplied to the culturing vessel at the start of the process. A continuous perfusion process can also be used, in which the cells are immobilized in the culture, e.g., by filtration, encapsulation, anchoring to microcarriers, or the like, and the supernatant is continuously removed from the culturing vessel and replaced with fresh medium during the process.

[0233] Steps (i) - (iii) of the above method generally comprise a "growth" phase, whereas step (iv) generally comprises a "production" phase. In some embodiments, fed batch culture or continuous cell culture conditions are tailored to enhance growth and division of the cultured cells in the growth phase and to disfavor cell growth and/or division and facilitate expression of the heterologous protein during the production phase. For example, in some embodiments, a heterologous protein is expressed at levels of about 1 mg/L, or about 2.5 mg/L, or about 5 mg/L or higher. The rate of cell growth and/or division can be modulated by varying culture conditions, such as temperature, pH, dissolved oxygen (d0₂) and the like. For example, suitable conditions for the growth phase can include a pH of between about 6.5 and about 7.5, a temperature between about 30° C to about 38° C, and a d0₂ between about 5-90% saturation. In some embodiments, the expression of a heterologous protein can be enhanced in the production phase by inducing a temperature shift to a lower culture temperature (e.g., from about 37° C to about 30° C), increasing the concentration of solutes in the cell culture medium, or adding a toxin (e.g., sodium butyrate) to the cell culture medium. A variety of additional protocols and conditions for enhancing growth during the growth phase and/or protein expression during the production phase are known in the art.

[0234] In one embodiment, after the production phase the heterologous protein is recovered from the cell culture medium using various methods known in the art. Recovering a secreted heterologous protein typically involves removal of host cells and debris from the medium, for example, by centrifugation or filtration. In some cases, particularly if the protein is not secreted, protein recovery can also be performed by lysing the cultured host cells, e.g., by mechanical shear, osmotic shock, or enzymatic treatment, to release the contents of the cells into the homogenate. The protein can then be separated from subcellular fragments, insoluble materials, and the like by differential centrifugation, filtration, affinity chromatography, hydrophobic interaction chromatography, ion-exchange chromatography, size exclusion chromatography, electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), ammonium sulfate precipitation, and the like. Procedures for recovering and purifying particular types of proteins are known in the art.

[0235] In some embodiments, the production of a heterologous protein is enhanced by contacting cultured cells with an RNA effector molecule provided herein during the growth phase to modulate expression of a target gene encoding a protein that affects cell growth, cell division, cell viability, apoptosis, nutrient handling, and/or other properties related to cell growth and/or division. In further embodiments, the production of a heterologous protein is enhanced by contacting cultured cells with an RNA effector molecule which transiently inhibits expression of the heterologous protein during the growth phase.

[0236] In yet further embodiments, the modulation of expression (e.g., inhibition) of a target gene by an RNA effector molecule can be alleviated by contacting the cell with second RNA effector molecule, wherein at least a portion of the second RNA effector molecule is

complementary to a target gene encoding a protein that mediates RNAi in the host cell. For example, the modulation of expression of a target gene can be alleviated by contacting the cell with an RNA effector molecule that inhibits expression of an argonaute protein (e.g., argonaute- 2) or other component of the RNAi pathway of the cell. In one embodiment, the biological product is a recombinant protein and expression of the product is transiently inhibited by contacting the cell with a first RNA effector molecule targeted to the transgene encoding the product. The inhibition of expression of the product is then alleviated by contacting the cell with a second RNA effector molecule targeted against a gene encoding a protein of the RNAi pathway of the cell.

[0237] In additional embodiments, the production of a heterologous protein is enhanced by contacting cultured cells with an RNA effector molecule, provided herein, during the production phase to modulate expression of a target gene encoding a protein that affects protein expression, post-translational modification, folding, secretion, and/or other processes related to production and/or recovery of the heterologous protein. In further embodiments, the production of a heterologous protein is enhanced by contacting cultured cells with an RNA effector molecule which inhibits cell growth and/or cell division during the production phase.

[0238] In some embodiments, the production of a biological product in a cultured host cell is enhanced by contacting the cell with an RNA effector molecule which modulates expression of a protein of a contaminating virus such that the infectivity and/or load of the virus in the host cell is reduced. In additional embodiments, production of a biological product in a cultured host cell is enhanced by contacting the cell with an RNA effector molecule which modulates expression of a host cell protein involved in viral infection or reproduction such that the infectivity and/or load of contaminating viruses in the host cell is reduced.

[0239] In some embodiments, the enhancement of production of a biological product upon modulation of a target gene is detected by monitoring one or more measurable bioprocess parameters, such as a parameter selected from the group consisting of: cell density, medium pH, oxygen levels, glucose levels, lactic acid levels, temperature, and protein production. Protein production can be measured as specific productivity (SP) (the concentration of a product, such as a heterologously expressed polypeptide, in solution) and can be expressed as mg/L or g/L; in the alternative, specific productivity can be expressed as pg/cell/day. An increase in SP can refer to an absolute or relative increase in the concentration of a product produced under two defined set of conditions.

[0240] In some embodiments, the enhancement of production of a biological product is achieved by improving viability of the cells in culture. As used herein, the term "improving cell viability" refers to an increase in cell density (e.g., as assessed by a Trypan Blue exclusion assay) or a decrease in apoptosis (e.g., as assessed using a TU EL assay) of at least 10% in the presence of an RNA effector molecule(s) compared to the cell density or apoptosis levels in the absence of such a treatment. In some embodiments, the increase in cell density or decrease in apoptosis in response to treatment with an RNA effector molecule(s) is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even 100% compared to untreated cells. In some embodiments, the increase in cell density in response to treatment with an RNA effector molecule(s) is at least 2-fold, at least 5- fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or higher than the cell density in the absence of the RNA effector molecule(s).

[0241] A "host cell," as used herein, is any cell capable of being grown and maintained in cell culture under conditions allowing for production and recovery of useful quantities of a biological product, as defined herein. Host cells can be unmodified cells or cell lines, or cell lines which have been genetically modified (e.g., to facilitate production of a biological product). In some embodiments, the host cell is a cell line that has been modified to allow for growth under desired conditions, such as in serum-free media, in cell suspension culture, or in adherent cell culture.

[0242] A mammalian host cell may be preferred where the biological product is a

recombinant polypeptide, particularly if the polypeptide is a biotherapeutic agent or is otherwise intended for administration to or consumption by humans. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell, which is a predominant cell line used for the expression of many recombinant proteins. Additional mammalian cell lines commonly used for the expression of recombinant proteins include, but are not limited to, 293HEK cells, HeLa cells, COS cells, N1H/3T3 cells, Jurkat Cells, NSO cells and HUVEC cells.

[0243] In some embodiments, the host cell is a CHO cell derivative that has been genetically modified to facilitate production of recombinant proteins or other biological products. For example, various CHO cell strains have been developed which permit stable insertion of recombinant DNA into a specific gene or expression region of the cells, amplification of the inserted DNA, and selection of cells exhibiting high level expression of the recombinant protein. Examples of CHO cell derivatives useful in methods provided herein include, but are not limited to, CHO-K1 cells, CHO-DUKX, CHO-DUKX Bl, CHO-DG44 cells, CHO-ICAM-1 cells, and CHO-hlFNy cells. Methods for expressing recombinant proteins in CHO cells are known in the art and are described, in U.S. Pat. Nos. 4,816,567 and 5,981,214, herein incorporated by reference in their entirety.

[0244] As used herein the term "contacting a cell" refers to the treatment of a cell in culture with an agent e.g., at least one RNA effector molecule, often prepared in a composition comprising a delivery agent (e.g., Lipofectamine). The step of contacting a cell with an RNA effector molecule(s) can be repeated more than once (e.g., twice, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, l lx, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx or more). In one embodiment, the cell is contacted such that the target gene is modulated only transiently, e.g., by addition of an RNA effector molecule composition to the cell culture medium used for the production of the biological product where the presence of the RNA effector molecules dissipates over time, i.e., the RNA effector molecule is not constitutively expressed in the cell.

[0245] "Introducing into a cell," when referring to an RNA effector molecule, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an RNA effector molecule can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0246] The term "modulates expression of," and the like, in so far as it refers to a target gene, herein refer to the modulation of expression of a target gene, as manifested by a change (e.g., an increase or a decrease) in the amount of target gene mRNA which can be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of a target gene is modulated, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

[0247] Alternatively, the degree of modulation can be given in terms of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene, or the number of cells displaying a certain phenotype, e.g., stabilization of microtubules. In principle, target gene modulation can be determined in any host cell expressing the target gene, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given RNA effector molecule modulates the expression of a target gene and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.

[0248] For example, in certain instances, expression of a target gene is inhibited. In one example, expression of a target gene is inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an RNA effector molecule provided herein. In some embodiments, a target gene is inhibited by at least about 60%, 70%, or 80% by

administration of an RNA effector molecule. In some embodiments, a target gene is inhibited by at least about 85%, 90%, or 95% or more by administration of an RNA effector molecule as described herein. In other instances, expression of a target gene is activated by at least about 10%, 20%, 25%, 50%, 100%, 200%, 400% or more by administration of an RNA effector molecule provided herein.

[0249] A "bioreactor," as used herein, refers generally to any reaction vessel suitable for growing and maintaining cells such that the cells produce a biological product, and for recovering such biological product. Bioreactors described herein include cell culture systems of varying sizes, such as small culture flasks, Nunc multilayer cell factories, small high yield bioreactors (e.g., MiniPerm, INTEGRA-CELLine), spinner flasks, hollow fiber-WAVE bags (Wave Biotech, Tagelswangen, Switzerland), and industrial scale bioreactors. In some embodiments, the biological product is produced in a bioreactor having a capacity suitable for pharmaceutical or industrial scale production of biological products (e.g., a volume of at least 2 liters, at least 5 liters, at least 10 liters, at least 25 liters, at least 50 liters, at least 100 liters, or more) and means of monitoring pH, glucose, lactate, temperature, and/or other bioprocess parameters.

[0250] As used herein, a "target gene" refers to a gene which encodes a protein that affects one or more aspects of the production of a biological product by a host cell, such that modulating expression of the gene enhances production of the biological product. Target genes can be derived from the host cell, endogenous to the host cell (present in the host cell genome), transgenes (gene constructs inserted at ectopic sites in the host cell genome), or derived from a pathogen (e.g., a virus, fungus or bacterium) which is capable of infecting the host cell or the subject who will use the biological product or derivatives thereof (e.g., humans).

[0251] In some embodiments, the target gene is an endogenous gene of the host cell. For example, the target gene can encode the biological product or a portion thereof when the biological product is a polypeptide. The target gene can also encode a host cell protein that directly or indirectly affects one or more aspects of the production of the biological product. Examples of target genes that affect the production of polypeptides include genes encoding proteins involved in the secretion, folding or post-translational modification of polypeptides (e.g., glycosylation, deamidation, disulfide bond formation, methionine oxidation, or

pyroglutamation); genes encoding proteins that influence a property or phenotype of the host cell (e.g., growth, viability, cellular pH, cell cycle progression, apoptosis, carbon metabolism or transport, lactate formation, susceptibility to viral infection or RNAi uptake, activity or efficacy); and genes encoding proteins that impair the production of a biological product by the host cell (e.g., a protein that binds or co-purifies with the biological product).

[0252] In some embodiments, production of a biological product is enhanced by reducing the expression of a protein that binds to the product. For example, in producing a growth factor, a hormone, or a cell signaling protein, it may be advantageous to reduce or inhibit expression of its receptor/ligand so that its production in the cell does not elicit a biological response. It will be known to a skilled artisan that a receptor can be a cell surface receptor or an internal (e.g., nuclear) receptor. Therefore, in one example, production of a biological product such as an interferon (e.g., interferon-beta) can be enhanced by modulating (e.g., reducing) the level of the receptor present in the cell (e.g., IFNAR1 (SEQ ID NOs: (2436536-2436863)) or IFNAR2 receptor). The expression of the binding partner can be modulated by contacting the host cell with an RNA effector molecule directed at the receptor gene according to methods described herein.

[0253] In some embodiments, the target gene encodes a host cell protein that indirectly affects the production of the biological product such that inhibiting expression of the target gene enhances production of the biological product. For example, the target gene can encode an abundantly expressed host cell protein which does not directly influence production of the biological product but indirectly decreases its production, for example by utilizing cellular resources that could otherwise enhance production of the biological product.

[0254] Proteins produced in a cell culture on an industrial-scale are typically secreted by cultured cells and recovered and purified from the surrounding cell culture media. In general, the rate of protein production and the yield of recovered protein is directly related to the rate of protein folding and secretion by the host cells. For example, an accumulation of misfolded proteins in the endoplasmic reticulum (ER) of host cells can slow or stop secretion via the unfolded protein response (UPR) pathway. The UPR is triggered by stress-sensing proteins in the ER membrane which detect excess unfolded proteins. UPR activation leads to the upregulation of chaperone proteins (e.g., Bip) which bind to misfolded proteins and facilitate proper folding. UPR activation also upregulates the transcription factors XBP-1 (SEQ ID NOs: 187955-188152) and CHOP (SEQ ID NOs: 2813622-2813956). CHOP generally functions as a negative regulator of cell growth, differentiation and survival, and its upregulation via the UPR causes cell cycle arrest and increases the rate of protein folding and secretion to clear excess unfolded proteins from the cell. An increase the rate of protein secretion by the host cells can be measured by, e.g., monitoring the amount of protein present in the culture media over time.

[0255] The present invention provides methods for enhancing the production of a secreted polypeptide in cultured eukaryotic host cells by modulating expression of a target gene which encodes a protein that affects protein secretion by the host cells. In some embodiments, the target gene encodes a protein of the UPR pathway, such as IRE1, PERK, ATF4 (SEQ ID NOs: 1552067-1552460), ATF6 (SEQ ID NOs: 570138-570498), eIF2alpha (SEQ ID NOs: 1828122- 1828492), GRP78 (SEQ ID NOs: 292590-292837), GRP94 (SEQ ID NOs: 180574-180954), calreticulin (SEQ ID NOs: 895691-896051) or a variant thereof, or a protein that regulates the UPR pathway, such as a transcriptional control element (e.g., the cis-acting UPR element (UPRE)).

[0256] In some embodiments, the protein that affects protein secretion is a molecular chaperone selected from the group consisting of: (SEQ ID NOs: 677203-677558), HSP47 (SEQ ID NOs: 777036-777317), HSP60 (SEQ ID NOs: 494743-495086), Hsp70 (SEQ ID NOs:

3147029-3147080), HSP90, HSP100, protein disulfide isomerase (SEQ ID NOs: 72748-72996, , peptidyl prolyl isomerase (SEQ ID NOs: 38781-39067, 1074139-1074475, 1127061-1127426, 1649170-1649515, 2197146-2197532, 2253978-2254373, 2261765-2262058, 2275330-2275633, 2579547-2579908, and 3115010-3115199), calnexin (SEQ ID NOs: 61559-61785), Erp57 (SEQ ID NOs: 774355-774677), and BAG-1.

[0257] In some embodiments, the protein that affects protein secretion is selected from the group consisting of: gamma-secretase, pi 15 (SEQ ID NOs: 89340-89737), a signal recognition particle (SRP) protein, secretin, and a kinase (e.g., MEK).

[0258] In some embodiments, the production of a biological product is enhanced by inducing a stress response in the host cells which causes growth arrest and increased productivity. A stress response can be induced, e.g., by limiting nutrient availability, increasing solute concentrations, or low temperature or pH shift, and oxidative stress. Along with increased productivity, stress responses can also have adverse effects on protein folding and secretion. In some embodiments, such adverse effects are ameliorated by modulating the expression of a target gene encoding a stress response protein, such as a protein that affects protein folding and/or secretion described herein.

[0259] The production of biological products in cell culture can be negatively affected by proteins which have an affinity for the biological product or a molecule or factor that binds specifically to the biological product. For example, a number of heterologous proteins have been shown to bind the glycoproteins heparin and heparan sulfate at host cell surfaces. This can lead to the co-purification of heparin, heparan sulfate, and/or heparin/heparan sulfate -binding proteins with recombinant protein products, decreasing yield and reducing homogeneity, stability, biological activity, and/or other properties of the recovered proteins. Examples of heterologous proteins which have been shown to bind heparin and/or heparan sulfate include BMP3 (bone morphogenetic protein 3 or osteogenin); TNF-alpha; GDNF; TGF-beta family members; and HGF. Therefore, in one embodiment, the production of a heterologous protein, such as BMP3, TNF-alpha, GDNF, a TGF-beta family members, or HGF, or another biological product in cultured host cells is enhanced by contacting the cells with an RNA effector molecule which modulates (e.g., inhibits) expression and/or production of heparin and/or heparan sulfate. In one embodiment, the level of heparin and/or heparan sulfate is reduced by modulating expression of a host cell enzyme involved in the production of heparin and/or heparan sulfate, such as a host cell xylotransferase (SEQ ID NOs: 1554774-1555054).

[0260] Proteins expressed by cultured eukaryotic host cells can undergo several potential post-translational modifications that can impair protein production and/or the structure, biological activity, stability, homogeneity, and/or other properties of protein products. Many of these modifications occur spontaneously during cell culture and polypeptide expression and can occur at several sites, including the peptide backbone, the amino acid side-chains, and the amino and/or carboxyl termini of a given polypeptide. In addition, a given polypeptide can comprise several different types of modifications. For example, proteins expressed in mammalian cells can be subject to acetylation, acylation, ADP-ribosylation, amidation, ubiquitination, methionine oxidation, disulfide bond formation, methylation, demethylation, sulfation, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, hydroxylation, iodination, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, gluconoylation, sequence mutations, N-terminal glutamine cyclization and deamidation, and asparagine deamidation. N-terminal asparagines deamidation can be reduced by contacting the cell with an RNA effector molecule targeting the N-terminal Asn amidase (encoded, for example by SEQ ID NO: 5950), wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1999410-1999756.

[0261] Post-translational modifications can require additional bioprocess steps to separate modified and unmodified polypeptides, increasing costs and reducing efficiency of protein production. Accordingly, in some embodiments, production of a polypeptide in a cultured host cell is enhanced by modulating the expression of a target gene encoding a protein that affects post-translational modification. In additional embodiments, protein production is enhanced by modulating the expression of a first target gene encoding a protein that affects a first post- translational modification and modulating the expression of a second target gene encoding a protein that affects a second post-translational modification.

[0262] In some embodiments, protein production is enhanced by modulating expression of a target gene which encodes a protein involved in protein deamidation. Proteins can be deamidated via several pathways, including the cyclization and deamidation of N-terminal glutamine and deamidation of asparagine. Thus, in one embodiment, the protein involved in protein deamidation is N-terminal asparagine amidohydrolase. Protein deamidation can lead to altered structural properties, reduced potency, reduced biological activity, reduced efficacy, increased immunogenicity, and/or other undesirable properties and can be measured by several methods, including but not limited to, separations of proteins based on charge by, e.g., ion exchange chromatography, HPLC, isoelectric focusing, capillary electrophoresis, native gel electrophoresis, reversed-phase chromatography, hydrophobic interaction chromatography, affinity chromatography, mass spectrometry, or the use of L-isoaspartyl methyltransf erase.

[0263] In some embodiments, the biological product is a glycoprotein and protein production is enhanced by modulating expression of a target gene which encodes a protein involved in protein glycosylation. Glycosylation patterns are important determinants of the structure and function of mammalian glycoproteins and can influence the solubility, thermal stability, protease resistance, antigenicity, immunogenicity, serum half-life, stability, and biological activity of glycoproteins.

[0264] In various embodiments, the protein that affects glycosylation is selected from the group consisting of: dolichyl-diphosphooligosaccharide-protein glycosyltransferase (SEQ ID NOs: 2742894-2743239), UDP glycosyltransferase, UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase (SEQ ID NOs: 851115-851489, 1552461-1552728, 1562813-1563108, and 1635173-1635561), UDP-galactose-ceramide galactosyltransferase, fucosyltransferase (209841- 210227), protein O-fucosyltransferase (916726-917035), N-acetylgalactosaminytransferase (57147-57422, 65737-65999, 1013002-1013376, 1363583-1363970, 1546609-1546999, 1965217-1965613, 2876241-2876595), O-GlcNAc transferase (607012-607348), oligosaccharyl transferase (89738-90024, 262368-262621), O-linked N-acetylglucosamine transferase, and alpha-galactosidase (SEQ ID NOs: 1600968-1601288) and beta-galactosidase (690601-690989).

[0265] In some embodiments, the biological product is iduronate 2-sulfatase (IDS). IDS is an exosulfatase that hydrolyzes sulfate esters in human lysosomes. A deficiency in active IDS in humans leads to Hunter syndrome (mucopolysaccharidosis type II), which is characterized by the accumulation of heparan sulfate and dermatan sulfate fragments in lysosomes. Hunter syndrome can be treated by administration of exogenous IDS, such as a wild-type recombinant human IDS.

[0266] Human IDS is a glycoprotein and its activity can be enhanced by modulating the degree of glycosylation. Thus, in one embodiment, methods are provided herein for enhancing production of a recombinant human IDS in a host cell by contacting cultured host cells with an RNA effector molecule capable of modulating expression of a host cell gene involved in the glycosylation of recombinant IDS. Exemplary target genes include, e.g., the glycosylation enzymes described herein. Recombinant IDS is preferably produced in mammalian cells, such as CHO cells. Examples of preferred cell lines include CHO-KI cells and CHO-Lecl cells. The recombinant IDS preferably has the same glycosylation pattern but an enhanced degree of glycosylation compared to wild-type IDS (e.g., IDS isolated from human liver). The enhanced glycosylation of highly glycosylated forms of IDS produced by methods provided herein preferably results in the IDS having a molecular weight that is at least 5 kDa greater than wild- type IDS, or more preferably at least 10 kDa greater than wild-type IDS, or even more preferably at least 15 kDa, 20 kDa, or 25 kDa or more greater than wild-type IDS. Highly glycosylated forms of recombinant IDS produced by methods provided herein preferably exhibit enhanced enzymatic activity relative to the wild-type enzyme (e.g., IDS having an average degree of glycosylation).

[0267] The enzymatic activity of recombinant and wild-type IDS can be assayed using methods known in the art, including, e.g., the methods described in Bielicki et al., et al., Biochem, J., 271 : 75-86 (1990) using the radiolabeled disaccharide substrate IdoA2S-anM6S, herein incorporated by reference.

[0268] In another embodiment, the biological product is arylsulfatase A. A deficiency of arylsulfatase A in humans leads to the accumulation of sulfatides, particularly in the cells of the nervous system, resulting in progressive damage to the nervous system. Like iduronate 2- sulfatase, arylsulfatase A is a glycoprotein which requires glycosylation for optimal enzymatic activity. Thus, in one embodiment, methods are provided herein for enhancing production of a recombinant human IDS in a host cell by contacting cultured host cells with an RNA effector molecule capable of modulating expression of a host cell gene involved in the glycosylation of recombinant IDS. Recombinant IDS is preferably produced in mammalian cells, such as CHO cells.

[0269] The use of bioprocesses for the manufacture of biological products such as polypeptides at an industrial scale is often confounded by the presence of pathogens, such as active viral particles, and other adventitious agents (e.g., prions), often necessitating the use of expensive and time consuming steps for their detection, removal (e.g., viral filtration) and/or inactivation (e.g., heat treatment) to conform to regulatory procedures. Such problems can be exacerbated due to the difficulty in detecting and monitoring the presence of such viruses.

Accordingly, in some embodiments, methods are provided for enhancing production of a biological product by modulating expression of a target gene affecting the susceptibility of a host cell to pathogenic infection. For example, in some embodiments, the target gene is a host cell protein which mediates viral infectivity, such as the transmembrane proteins XPR1 (SEQ ID NOs: 62021-62362), RDR, Fiver, CCR5, CXCR4, CD4, Pitl and Pit2 (SEQ ID NOs: 3068222- 3068455).

[0270] In further embodiments, methods are provided for enhancing production of a biological product by modulating expression of a target gene affecting the infectivity of a pathogen. In various embodiments, the target gene is a gene of a virus that has been known to contaminate mammalian cell cultures, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein Barr virus (EBV), human T-lymphotropic virus (HTLV), human herpesvirus 8, reovirus type 3 (Reo3), Sendai virus, simian virus 40, feline sarcoma virus, human papillomavirus, hantavirus, herpes B virus, Marburg virus, lactic dehydrogenase virus (DHM), lymphocytic choriomeningitis Virus (LCM), minute virus of mice (MVM), mouse adenovirus (MAV), mouse cytomegalovirus (MCMV), mouse rotavirus (EDIM), pneumonia virus of mice (PVM), pseudorabies virus (PRV), murine leukemia virus (MuLV), Semliki Forest virus (SFV), simian virus 5 (sv5), vesivirus, Kilham rat virus (KRV), Toolan virus (HI), bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus, parainfluenza virus type 3, simian virus 40 (SV40), and simian virus 20 (SV20).

[0271] In further embodiments, methods are provided wherein host cells are contacted with one or more RNA effector molecules against target genes derived from a pathogen (e.g., a virus, fungus or bacterium) or a plurality of pathogens capable of infecting the host cell or the subject who will use the biological product or derivatives thereof (e.g., humans). Therefore, in another embodiment, RNA effector compositions are provided comprising one or more RNA effector molecules that are complementary to at least a portion of a target gene of a pathogen selected from the group consisting of: human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, hepatitis E virus, hepatitis F virus, Epstein Barr virus (EBV), human T-lymphotropic virus (HTLV), human herpesvirus 8, reovirus type 3 (Reo3), Sendai virus, simian virus 40, feline sarcoma virus, human papillomavirus, hantavirus, cytomegalovirus, herpes simplex virus 1, herpes simplex virus 2, varicella zoster virus, roseolovirus, herpes B virus, influenze A virus, influenza B virus, influenza C virus, parvovirus, Marburg virus, lactic dehydrogenase virus (DHM), lymphocytic choriomeningitis Virus (LCM), minute virus of mice (MVM), mouse adenovirus (MAV), mouse cytomegalovirus (MCMV), mouse rotavirus (EDIM), pneumonia virus of mice (PVM), pseudorabies virus (PRV), murine leukemia virus (MuLV), Semliki Forest virus (SFV), simian virus 5 (sv5), vesivirus, Kilham rat virus (KRV), Toolan virus (HI), bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus, parainfluenza virus type 3, simian virus 40 (SV40), and simian virus 20 (SV20). The RNA effector composition is contacted with a host cell such that the replication, lysis and/or release of virus particles by the cell is inhibited.

[0272] In one embodiment, the biological product is an antibody (e.g., a monoclonal antibody). Monoclonal antibodies produced in mammalian host cells contain an N-linked glycosylation site on each heavy chain. The heavy chain glycans are typically complex structures with high levels of core fucosylation. The fucose residues attached via an a1,6 linkage to the innermost N-acetylglucosamine (GlacNAc) residues of the Fc region N-linked oligosaccharides are the most important carbohydrate structures for antibody activity. For example, non- fucosylated antibodies are associated with dramatically increased Antibody-Dependent Cellular Cytotoxicity (ADCC) activity. Other enzymes contributing to fucosylation of polypeptides include GDP mannose dehydratase (GMDS), which catalyzes the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose, the GDP-4-keto-6-deoxy-D-mannose epimerase-reductase (also known as TSTA3, or FX), catalyzing the the two-step epimerase and the reductase reactions in GDP-D-mannose metabolism, converting GDP-4-keto-6-D-deoxymannose to GDP- L-fucose, which is the substrate of several fucosyltransferases.

[0273] Thus, in one embodiment, the production of an antibody, such as a monoclonal or polyclonal antibody, is enhanced by modulating expression of a target gene encoding a fucosyltransferase, such as FUT8 (SEQ ID NOs: 209841-210227), a target gene encoding a GDP mannose dehydratase, or a target gene encoding GDP-4-keto-6-deoxy-D-mannose epimerase- reductase. In another embodiment, expression of a combination of these genes is modulated. ADCC activity can be assessed using an in vitro ADCC assay, such as those described in US Patent Nos. 5,500,362, 5,821,337, and 6,737,056, herein incorporated by reference in their entirety, and peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells as effector cells. ADCC activity can also be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998), herein incorporated by reference in its entirety.

[0274] In one embodiment, methods are provided herein for enhancing production of a recombinant antibody by or a fragment or derivative thereof by contacting a cell, such as a CHO cell with one or more RNA effector molecules of Table 7, wherein the contacting modulates expression of the fucosyltransferase (FUT8). In another embodiment, a cell is contacted with one or more RNA effector molecules wherein the contacting modulates expression of a GDP mannose dehydratase (GMDS) (encoded, for example, by SEQ ID NO: 5069). An RNA effector molecule targeting GMDS can comprise an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1688202-1688519. In another embodiment, a cell is contacted with one or more RNA effector molecules wherein the contacting modulates expression of a gene encoding GDP-4-keto-6-deoxy-D-mannose epimerase-reductase (encoded by TSTA3), (encoded, for example, by SEQ ID NO: 5505). An RNA effector molecule targeting TSTA3 can comprise an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1839578-1839937. In still another embodiment, a cell is contacted with a plurality of RNA effector molecules targeting the expression of more than one of FUT8, GMDS, and TSTA3.

[0275] Reduced sialic content of antibodies is believed to further increase ADCC. Therefore, in still another embodiment, a cell is contacted with one or more RNA effector molecules wherein the contacting modulates expression of a sialyltransferase. The sialyltransferase activity in a cell can be modulated by contacting the cell with an RNA effector molecule targeting at least one sialyltransferase gene. Listed below are some sialyltransferases that can be modulated, as well as the RNA effector molecules targeting sialyltransferases:

[0276] SEQ ID NO: 2088 ST3 beta-galactoside alpha-2,3-sialyltransferase 1

[0277] RNA effectors: SEQ ID NO:681105-681454

[0278] SEQ ID NO: 4319 ST3 beta-galactoside alpha-2,3-sialyltransferase 2

[0279] RNA effectors: SEQ ID NO: 1435989-1436317.

[0280] SEQ ID NO: 3411 ST3 beta-galactoside alpha-2,3-sialyltransferase 3

[0281] RNA effectors: SEQ ID NO: 1131123-1131445.

[0282] SEQ ID NO: 2167 ST3 beta-galactoside alpha-2,3-sialyltransferase 4

[0283] RNA effectors: SEQ ID NO: 707535-707870

[0284] SEQ ID NO: 3484 ST3 beta-galactoside alpha-2,3-sialyltransferase 5

[0285] RNA effectors: SEQ ID NO: 1155324-1155711

[0286] SEQ ID NO: 4186 ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N- acetylgalactosaminide alpha-2,6-sialyltransferase 6

[0287] RNA effectors: SEQ ID NO: 1391079-1391449

[0288] The RNA effector molecules targeting the sialyltransferases comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence of the SEQ ID NOs presented above (i.e., SEQ ID NOs: 681105-681454, 707535-707870, 1131123-1131445, 1155324-1155711, 1391079-1391449, 1435989-1436317).

[0289] In still another embodiment, a cell is contacted with at least one RNA effector molecule targeting one of FUT8, GMDS, and TSTA3, and another RNA effector molecule targeting one sialyltransferase. In a particular embodiment, a cell is contacted with RNA effector molecules targeting FUT8 and ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N- acetylgalactosaminide alpha-2,6-sialyltransferase 6.

[0290] In additional embodiments, the biological product is an antibody derivative, such as a humanized antibody, a chimeric antibody, a single chain antibody, a bispecific antibody, a Fab fragment, a F(ab')2 fragment, an anti-idiotypic (anti-Id) antibody, or an epitope binding fragment. Methods for the production of antibodies and antibody fragments are known in the art and are described, e.g., in U.S. Pat. No. 4,816,397 to Boss et al, U.S. Pat. No. 4,376,110 to Kohler and Milstein, U.S. Pat. No. 4,946,778 to Bird, U.S. Pat. No. 4,816,567 to Cabilly et al., U.S. Pat. No. 5,816,397 to Boss et al., U.S. Pat. No. 5,585,089 to Queen, U.S. Pat. No. 5,225,539 to Winter, Kohler and Milstein, Nature, 256:495-497 (1975), Kozbor et al., Immunology Today, 4:72 (1983), and Cole et al, Proc. Natl. Acad. Sci. USA, 80:2026-2030 (1983), all of which are herein incorporated by reference in their entirety.

[0291] In further embodiments, production of a glycoprotein is enhanced by modulating expression of a sialidase or a sialytransferase enzyme. Terminal sialic acid residues of glycoproteins are particularly important determinants of glycoprotein solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity. For example, when terminal sialic acid is removed from serum glycoproteins, the desialylated proteins have significantly decreased biological activity and lower circulatory half-lives relative to sialylated counterparts. The amount of sialic acid in a glycoprotein is the result of two opposing processes, i.e., the intracellular addition of sialic acid by sialytransferases and the removal of sialic acid by sialidases. Thus, in some embodiments, production of a glycoprotein is enhanced by inhibiting expression of a sialidase and/or activating expression of a sialytransferase.

[0292] In some embodiments, protein production is enhanced by modulating expression of a glutaminyl cyclase which catalyzes the intramolecular cyclization of N-terminal glutamine residues into pyroglutamic acid, liberating ammonia (pyroglutamation). Glutaminyl cyclase modulation can be accomplished by contacting the cell with an RNA effector molecule targeting the glutaminyl cyclase gene (for example, encoded by SEQ ID NO: 5486), wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1832626-1832993.

[0293] In some embodiments, production of proteins containing disulfide bonds is enhanced by modulating expression of a protein that affects disulfide bond oxidation, reduction, and/or isomerization, such as protein disulfide isomerase or sulfhydryl oxidase. Disulfide bond formation can be particularly problematic for the production of multi-subunit proteins or peptides in eukaryotic cell culture. Examples of multi-subunit proteins or peptides include receptors, extracellular matrix proteins, immunomodulators, such as MHC proteins, full chain antibodies and antibody fragments, enzymes and membrane proteins.

[0294] In some embodiments, protein production is enhanced by modulating expression of a protein that affects methionine oxidation. Reactive oxygen species (ROS) can oxidize methionine (Met) to methionine sulfoxide (MetO), resulting in increased degradation and product heterogeneity, and reduced biological activity and stability. In some embodiments, the target gene encodes a methionine sulfoxide reductase, which catalyzes the reduction of MetO residues back to methionine. For example, wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 2044387-2044676, SEQ ID NOs: 2557492-2557809, and SEQ ID NOs: 3076104-3076309.

[0295] For optimal production of biological products in cell culture bioprocesses described herein, it is desirable to grow host cells at high density and to maximize cell viability.

Accordingly, in one embodiment, production of a biological product is enhanced by modulating expression of a protein that affects apoptosis or cell viability, such as a protein selected from the group consisting of: BI , BAD (SEQ ID NOs: 3049436-3049721), BID (SEQ ID NOs:

2582517-2582823), BIM, HRK, BCLG, HR, NOXA, PUMA (SEQ ID NOs: 1712045-1712425), BOK, BOO, BCLB, CASP2 (SEQ ID NOs: 2718675-2719039), CASP3 (SEQ ID NOs:

1924836-192 195), CASP6 (SEQ ID NOs: 2408466-2408843), CASP7 (SEQ ID NOs: 2301618- 2301960), CASP8 (SEQ ID NOs: 2995593-2995870), CASP9 (SEQ ID NOs: 1412589- 1412860), CASP10, BAX, BAK, BCL2, p53 (SEQ ID NOs: 1283506-1283867), APAFI, and HSP70 (SEQ ID NOs: 3147029-3147080).

[0296] In one embodiment, a plurality of different RNA effector molecules are contacted with the cell culture and permit modulation of one or more target genes. In one embodiment, the RNA effector molecules are contacted with the cell culture during production of the biological product.

[0297] In another embodiment, a plurality of different RNA effector molecules is contacted with the cells in culture to permit modulation of Bax, Bak and LDH expression. In another embodiment, RNA effector molecules targeting Bax and Bak are co-administered to a cell culture during production of the biological product and can optionally contain at least one additional RNA effector molecule or agent.

[0298] In one embodiment, when a plurality of different RNA effector molecules are used to modulate expression of one or more target genes the plurality of RNA effector molecules are contacted with the culture simultaneously or separately. In addition, each RNA effector molecule can have its own dosage regime. For example, in one embodiment one may prepare a

composition comprising a plurality of RNA effector molecules are contacted with a cell.

[0299] Alternatively, one may administer one RNA effector molecule at a time to the cell culture. In this manner, one can easily tailor the average percent inhibition desired for each target gene by altering the frequency of administration of a particular RNA effector molecule. For example, full inhibition (i.e., >80%) of lactate dehydrogenase (LDH) is not always necessary to significantly improve production of a biological product and under some conditions may even be detrimental to cell viability. Thus, one may desire to contact a cell with an RNA effector molecule targeting LDH at a lower frequency (e.g., less often) than the frequency of contacting with the other RNA effector molecules (e.g., Bax/ Bak). Alternatively, the cell can be contacted with an RNA effector molecule targeting LDH at a lower dosage (e.g., lower multiples over the IC50) than the dosage for other RNA effector molecules (e.g., Bax/Bak). Contacting a cell with each RNA effector molecule separately can also prevent interactions between RNA effector molecules that can reduce efficiency of target gene modulation. For ease of use and to prevent potential contamination it may be preferred to administer a cocktail of different RNA effector molecules, thereby reducing the number of doses required and minimizing the chance of introducing a contaminant to the cell culture.

[0300] Reactive oxygen species (ROS) are toxic to host cells and can mediate non-specific oxidation, degradation and/or cleavage and other structural modifications of the biological product which lead to increased heterogeneity, decreased biological activity, lower recoveries, and/or other impairments to of biological products produced by methods provided herein.

Accordingly, in one embodiment, production of a biological product is enhanced by modulating expression of a pro-oxidant enzyme, such as a protein selected from the group consisting of: NAD(p)H oxidase, peroxidase, such as a glutathione peroxidase (for example, glutathione peroxidase 1, glutathione peroxidase 4, glutathione peroxidase 8 (putative), glutathione peroxidase 3, encoded by SEQ ID NOs: 7213, 7582, 8011, 9756, respectively (RNA effectors: SEQ ID NOs: 2439217-2439612, 2560559-2560895, 2703865-2704225, 3151589-3151685), myeloperoxidase, constitutive neuronal nitric oxide synthase (cnNOS), xanthine oxidase (XO) (SEQ ID NOs: 374846-375216) and myeloperoxidase (MPO), 15 -lipoxygenase- 1 (2480018- 2480362), NADPH cytochrome c reductase, NAPH cytochrome c reductase, NADH cytochrome b5 reductase (SEQ ID NOs: 569460-569777, 1261910-1262218, 2195311-2195681, 3146048- 3146071, 259827-260060), and cytochrome P4502E1.

[0301] In some embodiments, protein production is enhanced by modulating expression of a protein that affects the cell cycle of host cells, such as a cyclin (e.g.,cyclin M4, cyclin J, cyclin T2, cyclin-dependent kinase inhibitor 1A (P21), cyclin-dependent kinase inhibitor IB, cyclin M3, cyclin-dependent kinase inhibitor 2B (pl5, inhibits CDK4), cyclin E2, S100 calcium binding protein A6 (calcyclin), cyclin-dependent kinase 5, regulatory subunit 1 (p35), cyclin Tl, inhibitor of CDK, cyclin Al interacting protein 1 by use of corresponding RNA effectors comprising an an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 2447340-2447632, SEQ ID NOs: 2463782-2464073, SEQ ID NOs: 2466004- 2466274, SEQ ID NOs: 2659502-2659871, SEQ ID NOs: 2731076-2731440, SEQ ID NOs: 2748583-2748914, SEQ ID NOs: 2895015-2895359, SEQ ID NOs: 2904183-2904530, SEQ ID NOs: 2966362-2966657, SEQ ID NOs: 3088848-3089061, SEQ ID NOs: 3107706-3107919, SEQ ID NOs: 3122589-3122734, respectively), or a cyclin dependent kinase (CDK). In some embodiments, the cyclin dependent kinase is selected from the group consisting of: CDK2 (SEQ ID NOs: 1193336-1193684), CD 4 (SEQ ID NOs: 1609522-1609852), P10 (SEQ ID NOs: 3013998-3014274), P21 (SEQ ID NOs: 2659502-2659871), P27 (SEQ ID NOs: 2731076- 2731440), p53, P57, pl6INK4a, P14A F, and CDK4 (SEQ ID NOs: 1609522-1609852). For example, in various embodiments, the expression of one or more proteins that affect cell cycle progression can be transiently modulated during the growth and/or production phases of heterologous protein production in order to enhance expression and recovery of heterologous proteins.

[0302] Production of lactic acid in CHO cell cultures inhibits cell growth and influences metabolic pathways involved in glycolysis and glutaminolysis (Lao and Toth, Biotechnol Prog., 13(5): 688-91 (1997), herein incorporated by reference in its entirety). The accumulation of lactate in cell cultures is caused mainly by the incomplete oxidation of glucose to C02 and H20, in which most of the glucose is oxidized to pyruvate and finally converted to lactate by lactate dehydrogenase (LDH). The accumulation of lactic acid in cell culture is detrimental to achieving high cell density and viability. Accordingly, in one embodiment, protein production is enhanced by modulating expression of a protein that affects lactate formation, such as lactate

dehydrogenase A.

[0303] In one embodiment, a cell culture is treated as described herein with RNA effector molecules that permit modulation of Bax, Bak, and LDH expression. In another embodiment, the RNA effector molecules targeting Bax, Bak, and LDH may be administered in combination with one or more additional RNA effector molecules and/or agents. Provided herein is a cocktail of RNA effector molecules targeting Bax, Bak, and LDH expression, which can optionally be combined with additional RNA effector molecules or other bioactive agents as described herein.

[0304] In some embodiments, production of a biological product is enhanced by modulating expression of a protein that affects cellular pH, such as lactate dehydrogenase or lysosomal V- type ATPase.

[0305] In some embodiments, production of a biological product is enhanced by modulating expression of a protein that affects carbon metabolism or transport, such as a protein selected from the group consisting of: GLUT1 (for example, by contacting the cell with an RNA effector molecule wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 438155-438490), GLUT2, GLUT3, GLUT4, PTEN (with an RNA effector molecule wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 69091-69404), and lactate dehydrogenase (with an RNA effector molecule wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1297283-1297604).

[0306] In some embodiments, production of a biological product is enhanced by modulating expression of cofilin (for example a muscle cofilin 2, or non-muscle cofilin-1). In one embodiment, a cell with an RNA effector molecule wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 435213-435610, targeting the CHO muscle cofilin 2 (SEQ ID NO: 1366). In another embodiment, a cell with an RNA effector molecule wherein the RNA effector molecule comprises an antisense strand comprising at least 16 contiguous nucleotides (e.g., at least 17, at least 18, at least 19 nucleotides) of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1914036-1914356, targeting the CHO non-muscle cofilin 1 (SEQ ID NO: 5716).

[0307] In some embodiments, production of a biological product is enhanced by modulating expression of a viral gene, such as a gene encoding a protein selected from the group consisting of: a MuLV protein, MVM protein, Reo-3 protein, PRV protein, or a vesivirus protein.

[0308] In some embodiments, production of a biological product is enhanced by modulating expression of a protein that affects uptake or efficacy of an RNA effector molecule in host cells, such as a protein selected from the group consisting of: ApoE, Mannose/GalNAc-receptor, and Eril . In various embodiments, the expression of one or more proteins that affects RNAi uptake or efficacy in host cells is modulated according to a method provided herein concurrently with modulation of one or more additional target genes, such as a target gene described herein, in order to enhance the degree and/or extent of modulation of the one or more additional target genes.

[0309] As used herein, an "RNA effector composition" comprises an effective amount of an RNA effector molecule and an acceptable carrier. As used herein, "effective amount" refers to that amount of an RNA effector molecule effective to produce an intended modulatory effect on a bioprocess for the production of a biological product.

[0310] The term "acceptable carrier" refers to a carrier for administration of an RNA effector molecule to cultured eukaryotic host cells. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. [0311] In some embodiments, RNA effector compositions comprise two or more RNA effector molecules, e.g., comprise two, three, four or more RNA effector molecules. In various embodiments, the two or more RNA effector molecules are capable of modulating expression of the same target gene and/or one or more additional target genes. Advantageously, certain compositions comprising multiple RNA effector molecules are more effective in enhancing production of a biological product, or one or more aspects of such production, than separate compositions comprising the individual RNA effector molecules.

[0312] In some embodiments, a composition can comprise two or more RNA effector molecules capable of modulating expression of multiple genes relating to a common biological process or property of the host cell. In one embodiment, an RNA effector composition comprises two or more RNA effector molecules which modulate expression of proteins of two or more contaminating viruses such that the combination of RNA effector molecules inhibits infectivity and/or decreases viral load with respect to a broad spectrum of viruses. For example, an RNA effector composition can comprise two or more RNA effector molecules capable of modulating expression of two or more genes selected from the group consisting of: MuLV, Reo- 3, MMLV, PRV and vesivirus. In another embodiment, an RNA effector composition comprises two or more RNA effector molecules which modulate expression of two or more proteins involved in two or more post-translational modification pathways, e.g., as described herein. In one embodiment, an RNA effector composition can comprise RNA effector molecules targeting a plurality of viruses.

[0313] As used herein the term "comprising " or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0314] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0315] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0316] The phrase "genome information" as used herein and throughout the claims and specification is meant to refer to sequence information from partial or entire genome of an organism, including protein coding and non-coding regions. These sequences are present every cell originating from the same organisms. As opposed to the transcriptome sequence information, genome information comprises not only coding regions, but also, for example, intronic sequences, promoter sequences, silencer sequences and enhancer sequences. Thus, the "genome information" can refer to, for example a human genome, a mouse genome, a rat genome. One can use complete genome information or partial genome information to add an additional dimension to the database sequences to increase the potential targets to modify with an R A effector molecule.

[0317] The phrase "play a role" as used herein and throughout the claims and specification is meant to refer to any activity of a transcript or a protein in a molecular pathway known to a skilled artisan or identified elsewhere in this specification. Such pathways in cellular activities include, but are not limited to apoptosis, cell division, glycosylation, growth rate, a cellular productivity, a peak cell density, a sustained cell viability, a rate of ammonia production or consumption, or a rate of lactate production.

[0318] Tables 2-8 set forth below identify targets based on their function or role that they play in a cell:

LENGTHY TABLE ACCESS INSTRUCTIONS

[0319] The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site. An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Claims

CLAIMS We claim:

1. A system for selecting a sequence of at least one RNA effector molecule suitable for modulating protein expression in a cell, the system comprising:

a. a computer system including at least one processor and associated memory, the memory storing at least one computer program for controlling the operation of the computer system.

b. a database, connected to the computer system, comprising at least one cell

transcriptome information, the information comprising, a sequence for each transcript of the transcriptome, and optionally, a name of the transcript, and a pathway the transcript plays a role; and at least one RNA effector molecule information, the information comprising at least the sequence of the RNA effector molecule and optionally target specificity of the RNA effector molecule, wherein each RNA effector molecule is designed to match at least one or more sequences in the at least one cell transcriptome;

c. a user interface program module executed by the computer system and configured to receive user parameters comprising at least one of, a cell type selection, a target organism selection, a cellular pathway selection, a cross-reactivity selection, an amount of transcript selection, a target gene name and/or sequence selection, and optionally a method of delivery selection comprising either in vivo or in vitro delivery options; and further optionally user address information; d. a first module executed by the computer system and configured to check the

parameters against the sequences in the database for a matching combination of the parameters and transcriptome transcript sequences; and

e. a second module executed by the computer system and configured to display a selected sequence of at least one RNA effector molecule suitable for modulating protein expression in the cell.

2. The system of claim 1 further comprising a storage module for storing the at least one RNA effector molecule in a container, wherein if there are two or more RNA effector molecules, each RNA effector molecule is stored in a separate container, and a robotic handling module, which upon selection of the matching combination, selects a matching container, and optionally adds to the container additives based on a user selection for in vivo or in vitro delivery, and optionally further packages the container comprising the matching RNA effector molecule to be sent to the user address.

3. The system of any of the preceding claims, wherein the at least one cell transcriptome sequence information consists essentially of SEQ ID NOs: 1-9771.

4. The system of any of the preceding claims, wherein the RNA effector molecule is selected from the group consisting of siRNA, miRNA, dsRNA, saRNA, shRNA, piRNA, tkRNAi, eiRNA, pdRNA, a gapmer, an antagomir, a ribozyme and any combination thereof.

5. The system of any of the preceding claims, wherein the RNA effector molecule is selected from the group consisting of an siRNA, a formulated siRNA, an siRNA mixture, and any combination thereof.

6. The system of any of the preceding claims, wherein the RNA effector molecule

comprises an antisense strand comprising at least 16 contiguous nucleotides of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772- 3152399.

7. The system of any of the preceding claims, wherein the RNA effector molecule

comprises an antisense strand comprising 16-19 contiguous nucleotides of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772- 3152399.

8. The system of any of the preceding claims, wherein the sequence of the at least one RNA effector molecule consists essentially of SEQ ID NOs: 9772-3152399.

9. The system of any of the preceding claims, wherein a plurality of RNA effector molecules are selected that match at least one or more sequences in at least one transcriptome.

10. A method for selecting an RNA effector molecule for modulating protein expression in a cell using the system of any one of the preceding claims.

11. The system of any of the preceding claims further comprising genome information of the cell, wherein by a user selection, the RNA effector molecules can be matched to target genomic sequences, comprising promoters, enhancers, introns and exons present in the genome.

12. A Chinese hamster ovary (CHO) cell transcriptome comprising a selection or a

compilation of transcripts having SEQ ID NOs: 1-9771.

13. The CHO cell transcriptome of claim 12, wherein the CHO cell transcriptome

sequences are a part of a database.

14. An siRNA directed to any one of the CHO cell transcriptome transcript of claim 11.

15. The siRNA of claim 14, wherein the siRNA comprises an antisense strand comprising at least 16 contiguous nucleotides of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772-3152399.

16. The siRNA of claim 14 and 15, wherein the siRNA comprises an antisense strand comprising 16-19 contiguous nucleotides of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772-3152399.

17. The siRNA of any one of claims 14-16, wherein the siRNA is selected from the group consisting of SEQ ID NOs: 9772-3152399.

18. The siRNA of any of the preceding claims, wherein the siRNA sequences or an

antisense sequence thereof are part of a database.

19. A method for improving a cell line, the method comprising modulating at least one protein translated from a transcript selected from Table 1.

20. A method for improving a cell line, the method comprising modulating at least two transcripts using an effector RNA molecule, wherein a first transcript affects a first cell culture phenotype and a second transcript affects a second, different cell culture phenotype, wherein the cell culture phenotypes are selected from the group consisting of a cell growth rate, a cellular productivity, a peak cell density, a sustained cell viability, a rate of ammonia production or consumption, or a rate of lactate production or consumption, and wherein the first and second transcripts are selected from the group consisting of SEQ ID NOs 1-9771.

21. The method of claim 20, further comprising modulating a third transcript affecting a third cell culture phenotype different from the first and second cell culture phenotypes.

22. The method of any one of claims 20-21 , wherein the RNA effector molecule is

selected from the group consisting of siRNA, miRNA, dsRNA, saRNA, shRNA, piRNA, tkRNAi, eiRNA, pdRNA, a gapmer, an antagomir, or a ribozyme.

23. The method of any one of claims 20-22, wherein the RNA effector molecule

24. The method of any one of claims 20-23, wherein the RNA effector molecule

25. The method of any one of claims 20-24, wherein the effector RNA molecule is selected from the group consisting of SEQ ID NOs 9772-3152399.

26. The method of any one of claims 20-25, wherein the cell line is a CHO cell line.

27. An engineered cell line with an improved cellular productivity, improved cell growth rate, or improved cell viability, comprising a population of engineered cells, each of which comprising an engineered construct modulating one or more transcripts selected from Table 1.

28. The engineered cell line of claim 27, wherein the engineered construct modulating one or more transcripts comprises an RNA effector molecule selected from the group consisting of siRNA, miRNA, dsRNA, saRNA, shRNA, piRNA, tkRNAi, eiRNA, pdRNA, a gapmer, an antagomir, a ribozyme and any combination thereof.

29. The engineered cell line of claim 27, wherein the RNA effector molecule comprises an antisense strand comprising 16-19 contiguous nucleotides of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 9772-3152399

30. The engineered cell line of any one of claims 27-29, wherein the engineered construct comprises an siRNA selected from the group consisting of SEQ ID NOs 9772-

3152399 or an antisense molecule thereof.