CELLS FOR VIRUS AND PROTEIN PRODUCTION

I. PRIORITY

This application claims priority to U.S. Provisional Application 61/581 ,622, entitled "Cells for Virus and Protein Production," filed on December 29, 2012. This application is hereby incorporated by reference in its entirety.

II. BACKGROUND

Disclosed are general mechanisms and modified cells for high level production of virus or protein. The methods are based on an understanding of the factors that limit virus and protein production obtained from an extensive insertional mutagenesis study. In that study, genes that regulate the stress responses of the cell were identified, compared, and reviewed. In certain embodiments, the disclosed cells can be used in mammalian cell systems, such as Vero and Madin Darby Canine Kidney (MDCK), and can be used for influenza virus production. In other embodiments, particular cell lines can be used to produce blood products, such as factor VIII or butyrylcholinesterase (BChE), a protective measure against volatile nerve agents.

The disclosed systems and methods can be used to produce high yield cell lines from any cell, and can be used to produce any protein or any virus because of the generality of the modifications of the cefls. For example, the systems and methods can be used for Vero, MDCK, C3A, and CHO cells and cell lines.

The availability of these systems will substantially shorten the time necessary for the production of sufficient vaccine in the face of a pandemic threat, such a pandemic flu or nerve agent threats. Disclosed are high yield BChE production systems. BChE is one of the only known agents that can protect against organophosphorus nerve agents, such as Sarin. Since BChE protection against nerve agents requires a substantial dose of the protein, the availability of a cell based system will enable much more rapid production than current systems based on purification from human plasma or from transgenic animals.

III. SUMMARY

Disclosed are methods and compositions related to cells and systems which can be used for virus or protein or peptide production. IV. DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions

1. A, an, the

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

2. About

"About" modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use

formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term "about" also encompasses amounts that differ due to aging of a composition or formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term "about" the claims appended hereto include equivalents to these quantities.

3. Activity

Activity or like terms refers to the actions or states disclosed herein, such as the actions or states of cell proliferation, modulating binding or modulating a signaling pathway, and transactivation and downstream transactivation. 4. Administration

Administration and like terms means applying or bringing into contact.

5· Antibodies

a) Antibodies Generally

The term "antibodies" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibodies" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with the molecules disclosed herein, such as a protein, cell, or nucleic acid disclosed herein such that protein, cell, or nucleic acid can be recognized as bound by the antibody and/or its normal activity is modulated, such as decreased or increased. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl Acad. Sci. USA, 81 :6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedure which produces mono clonal antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4-co-receptor complexes described herein. The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et a!.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

As used herein, the term "antibody" or "antibodies" can also refer to a human antibody and/or a humanized antibody, as well as a chimeric antibody, or any antibody produced using recombinant biotechnology. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

b) Human antibodies

The disclosed human antibodies can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. {Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J Immunol, 147(l):86-95, 1991). Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381, 1991 ; Marks et al, J. Mol. Biol, 222:581, 1991).

The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol, 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H>) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.

c) Humanized antibodies

Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321 :522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol, 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567 (Cabilly et al.), U.S. Patent No. 5,565,332 (Hoogenboom et al.), U.S. Patent No. 5,721,367 (Kay et al.), U.S. Patent No. 5,837,243 (Deo et al.), U.S. Patent No. 5, 939,598 (Kucherlapati et al.), U.S. Patent No. 6, 130,364 (Jakobovits et a!.), and U.S. Patent No. 6, 180,377 (Morgan et al.).

6. Apoptosis

Apoptosis and like terms is the process of programmed cell death (PCD) that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Apoptosis contrasts to necrosis, in that apoptosis produces cell fragments called apoptotic bodies that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage.

7. ATF4

ATF is a gene encoding Activating Transcription Factor 4, and exemplary sequences encoding ATF can be found in Table 5.

8. Basal Levels

Basal levels are normal in vivo levels prior to, or in the absence of, or addition of an agent such as an agonist or antagonist to activity. 9. Bio reactor

A bioreactor or like terms refers to any manufactured or engineered device or system that supports a biologically active environment. In one case, a bioreactor can be a vessel in which a chemical process is carried out involving organisms or biochemically active substances derived from such organisms. This process can either be aerobic or anaerobic. Bioreactors are typically cylindrical, ranging in size from liters to cubic meters, and are often made of stainless steel. In certain embodiments, the bioreactor can grow cells or tissues in the context of cell culture.

Operationally, a bioreactor can be classified as batch, fed batch or continuous (e.g. a continuous stirred-tank reactor model). An example of a continuous bioreactor is the chemostat.

Organisms growing in bioreactors may be suspended or immobilized, A simple method, where cells are immobilized, in typically a small scale is a Petri dish with agar gel. Larger scale (for example, greater than 500 liters) immobilized cell bioreactors typically have moving media, also known as Moving Bed Biofilm Reactors (MBBR).

Bioreactors can scale from 1 liter to 10,000 liters or more.

10. Cell

Cell or like term refers to a small usually microscopic mass of protoplasm bounded externally by a semipermeable membrane, optionally including one or more nuclei and various other organelles, capable alone or interacting with other like masses of performing all the fundamental functions of life, and forming the smallest structural unit of living matter capable of functioning independently including synthetic cell constructs, cell model systems, and like artificial cellular systems.

A cell can include different cell types, such as a cell associated with a specific disease, a type of cell from a specific origin, a type of cell associated with a specific target, or a type of cell associated with a specific physiological function. A cell can also be a native cell, an engineered cell, a transformed cell, an immortalized cell, a primary cell, an embryonic stem cell, an adult stem cell, a cancer stem cell, or a stem cell derived cell.

Human consists of about 210 known distinct cell types. The numbers of types of cells can almost unlimited, considering how the cells are prepared (e.g., engineered, transformed, immortalized, or freshly isolated from a human body) and where the cells are obtained (e.g., human bodies of different ages or different disease stages, etc).

The term "cell" as used herein also refers to individual cells, cell lines, or cultures derived from such cells. A "culture" refers to a composition comprising isolated cells of the same or a different type. The term co-culture is used to designate when more than one type of cell are cultured together in the same dish with either full or partial contact with each other.

The disclosed systems and cells include MDC , Vero, CHO, Per.C6, duck stem cells, HEK293, Hela, Hep3b, EB66, EBx, CAP, and C3A.

11. Cellular Target

A "cellular target" or like terms is a molecule such as a protein or nucleic acid whose activity can be modified by an external stimulus. Cellular targets are most commonly proteins such as enzymes, kinases, ion channels, and receptors or nucleic acids.

12. Component

Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

13. Control

The terms control or "control levels" or "control cells" or like terms are defined as the standard by which a change is measured, for example, the controls are not subjected to the experiment, but are instead subjected to a defined set of parameters, or the controls are based on pre- or post-treatment levels. They can either be run in parallel with or before or after a test run, or they can be a pre-determined standard. For example, a control can refer to the results from an experiment in which the subjects or objects or reagents etc are treated as in a parallel experiment except for omission of the procedure or agent or variable etc under test and which is used as a standard of comparison in judging experimental effects. Thus, the control can be used to determine the effects related to the procedure or agent or variable etc. For example, if the effect of a test molecule on a cell was in question, one could a) simply record the characteristics of the cell in the presence of the molecule, b) perform a and then also record the effects of adding a control molecule with a known activity or lack of activity, or a control composition (e.g., the assay buffer solution (the vehicle)) and then compare effects of the test molecule to the control. In certain circumstances once a control is performed the control can be used as a standard, in which the control experiment does not have to be performed again and in other circumstances the control experiment should be run in parallel each time a comparison will be made.

14. Culturing

"Cell culture" or "culturing" or like terms refers to the process by which either prokaryotic or eukaryotic cells are grown under controlled conditions. "Cell culture" not only refers to the culturing of cells derived from multicellular eukaryotes, such as primary cells and cell lines, especially animal cells, but also the culturing of complex tissues, embryonic material (including avian eggs) and organs.

15. Excipient

An excipient and like terms is generally a pharmacologically inactive substance used as a carrier for the active ingredients of a medication. In many cases, an "active" substance (such as acetyl salicylic acid) may not be easily administered and absorbed by the human body; in such cases the substance in question may be dissolved into or mixed with an excipient. Excipients can also be used to bulk up formulations that contain small amounts of highly active ingredients, to allow for convenient and accurate dosage. In addition to their use in the single-dosage quantity, excipients can be used in the manufacturing process to aid in the handling of the active substance concerned. Depending on the route of administration, and form of medication, different excipients may be used. For oral administration typically tablets and capsules are used. Suppositories are used for rectal administration.

Excipients can be used to stabilize an active ingredient. They can be added excipients, ensuring that the active ingredient stays "active", and stable for a sufficiently long period of time that the she If- life of the product makes it competitive with other products.

Excipients can include Antiadherents, Binders, Coatings, Disintegrants, Fillers and diluents, Flavors, Colors, Lubricants, Glidants, Preservatives, Sorbents, and/or Sweeteners. 16. Endogenous

"Endogenous" or like terms means an action or object or its result coming from within a system. For example, a gene product in a cell would be endogenous if the gene product came from a gene found within the cell naturally. A cell which has been made through recombinant biotechnology (bringing genetic material together that does not naturally exist) means are considered to have at least one exogenous action or object within them, but could also contain many endogenous gene.

17. Exogenous

"Exogenous" or like terms means an action or object or its result coming from outside a system. For example, a gene product in a cell could be exogenous if the gene product came from outside the cell. From outside the cell includes those gene products expressed within a cell, but which are expressed from a nucleic acid, which was itself exogenous. All cells which have been made through recombinant biotechnology (bringing genetic material together that does not naturally exist) means are considered to have at least one exogenous action or object within them.

18. Fluidized bed reactor

A fluidized bed reactor (FBR) is a type of reactor device that can be used to carry out a variety of multiphase chemical reactions. In this type of reactor, a fluid (gas or liquid) is passed through a granular solid material (usually a catalyst possibly shaped as tiny spheres) at high enough velocities to suspend the solid and cause it to behave as though it were a fluid. FBRs can be used for culturing cells, such as human or animal cells.

19. Functional nucleic acid

Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, siRNA, shRNA, miRNA, and external guide sequences. The functional nucleic acid molecules can act as affectors, inhibitors, modulators, can increase or decrease an activity, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the transcription product, such as mRNA of any gene disclosed herein or the genomic DNA of any gene disclosed herein or they can interact with the translation product, polypeptide, of any gene disclosed herein . Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.

a) antisense

Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (k _d )less than or equal to 10 ^"6 , 10 ^~8 , 10 ^"10 , or 10 ^"12 . A representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non-limiting list of United States patents: 5, 135,917, 5,294,533, 5,627, 158, 5,641,754, 5,691,317, 5,780,607, 5,786, 138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025, 198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437. b) aptamers

Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP (United States patent 5,631,146) and theophiiine (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293). Aptamers can bind very tightly with k(js from the target molecule of less than 10 ^~12 M. It is preferred that the aptamers bind the target molecule with a !¾ less than 10 ^"6 , 10 ^~8 , 10 ^"10 , or 10 ^"12 . Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (United States patent 5,543,293). It is preferred that the aptamer have a kj with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the k _d with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide. For example, when determining the specificity of RPL41 aptamers, the background protein could be serum albumin. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424 , 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660 , 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698.

c) Ribozymes

Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (for example, but not limited to the following United States patents: 5,334,711, 5,436,330, 5,616,466, 5,633,133,

5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684,

5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following United States patents: 5,631,115, 5,646,031,

5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, but not limited to the following United States patents: 5,595,873 and

5,652,107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited to the following United States patents: 5,580,967, 5,688,670, 5,807,718, and 5,910,408).

Preferred ribozymes cleave R A or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non- limiting list of United States patents: 5,646,042, 5,693,535, 5,731,295, 5,81 1 ,300, 5,837,855, 5,869,253, 5,877,021 , 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.

d) Triplex

Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a k<j less than 10 ^"6 , 10~ ⁸ , 10 ^"10 , or 10 ¹² . Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834, 185, 5,869,246, 5,874,566, and 5,962,426.

e) EGS

External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells. (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, EMBQ J 14:159-168 (1995), and Carrara et al.. Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)). Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in the following non-limiting list of United States patents: 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877, 162. f) siRNA

Small interfering RNA (siRNA), and like terms, sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, typically between 20-25 nucleotides in length. siRNAs have been shown to have a number of roles in biology, such as its involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to its role in the RNAi pathway, siRNA also acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome.

g) shRNA

shRNA and like terms is a small hairpin RNA or short hairpin RNA (shRNA) and it is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA typically uses a vector introduced into cells and utilizes the U6 or HI promoter to ensure that the shRNA is constitutively expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. It is thought that the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it. If utilizing the appropriate promotes, shRNA is transcribed by RNA polymerase III.

h) miRNA

A microRNA (abbreviated miRNA) is a short ribonucleic acid (RNA) molecule found in eukaryotic cells. A microRNA molecule has relatively few nucleotides (typically an average of 22) compared with other RNAs. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. miRNA can function through the RNAi process.

20. Gene

A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a polypeptide or for an RNA chain that has a function in the organism. It is understood that the designations disclosed herein for biological macromolecules, such as ATF4 and GAS5 represent their native gene, including exons and introns, their transcriptionally expressed product, such as an mRNA, as well as their translated product are all disclosed, throughout this application. 21. Gene construct

A gene construct refers to a vector carrying a region of nucleic acid to be expressed into an expression product, such as an RNA, which can then be processed, hybridize with target nucleic acids, or be translated. When in a cell, a gene construct would be considered exogenous.

22. Gene product

A gene product is either the mRNA, protein, or other product expressed from the gene. A gene product arises through either transcription of the gene or translation of the mRNA encoded by the gene. Included are gene transcription products (transcribed RNA) and gene translation product (proteins).

23. Heterologous

The term "heterologous" or like terms when used with respect to a nucleic acid or polypeptide molecule refers to a nucleic acid or polypeptide from a foreign cell which does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or which is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which they are introduced, but have been obtained from another cell or synthetically or. recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed, similarly exogenous RNA codes for proteins not normally expressed in the cell in which the exogenous RNA is present. Furthermore, it is known that a heterologous protein or polypeptide can be composed of homologous elements arranged in an order and/or orientation not normally found in the host organism, tissue or cell thereof in which it is transferred, i.e. the nucleotide sequence encoding said protein or polypeptide originates from the same species but is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other. 24. Hybridization/selective hybridization

The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene. Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, H, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.

25. Inhibit

By "inhibit" or other forms of inhibit means to hinder, supress, or restrain a particular characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, "inhibits phosphorylation" means hindering or restraining the amount of phosphorylation that takes place relative to a standard or a control.

26. Increase

By "increase" or like terms means raising of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, "increases gene expression or gene product activity" means raising the amount of gene expression or gene product activity that takes place relative to a standard or a control. It is understood that wherever one of these words is used it is also disclosed that it could be, for example, 1%, 5%, 10%, 20%, 50%, 100%, 500%, or 1000% raised from a control.

27. Interferon response

The interferon response and like terms refers to the production of interferons in response to a pathogen, and include the signal pathways down stream of the production of interferons.

28. Isolated

As used herein, the terms" isolated, ""purified," or like terms refer to material which is substantially or essentially free from components that normally accompany it as found in its native state or substantially free from non material components, such as at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% material. 29. Knockout

A knockout or like terms refers to the recombinant interruption of at least one allele of an endogenous gene, such that the gene is not expressed, is reduced in expression, or the product expressed from the gene is non-functional or less functional. A homozygous knockout refers to both alleles being interrupted and a heterozygous knock out refers to a single allele being knocked out.

30. mTO , mTOR related, and mTOR signal pathway

mTOR and like terms refers to the mammalian target of rapamycin (mTOR) also known as mechanistic target of rapamycin or FK506 binding protein 12-rapamycin associated protein 1 (FRAP1) and is a protein which in humans is encoded by the FRAP1 gene. mTOR is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. mTOR belongs to the

phosphatidylinositol 3-kinase-related kinase protein family.

The mTOR related or mTOR signal pathway or like terms refers to the upstream and downstream signaling events that utilize, or include signaling through mTOR.

31. Material

Material is the tangible part of something (chemical, biochemical, biological, or mixed) that goes into the makeup of a physical object.

32. Microbeads

Microbeads are uniform polymer particles, typically 0.5 to 500 micrometres in diameter. Bio-reactive molecules can be adsorbed or coupled to their surface, and used to separate biological materials such as cells, proteins, or nucleic acids.

33. Microcarrier

A microcarrier is a support matrix allowing for the growth of adherent cells in, for example, bioreactors. Microcarriers are typically 125 - 250 micrometre spheres and their density allows them to be maintained in suspension with gentle stirring. Microcarriers can be made from a number of different materials including DEAE-dextran, glass, polystyrene plastic, acrylamide, and collagen, and these microcarrier materials, along with different surface chemistries, can influence cellular behavior, including morphology and proliferation. Surface chemistries can include extracellular matrix proteins, recombinant proteins, peptides, and positively or negatively charged molecules. Microcarriers can be used to grow protein-producing or virus-generating adherent cell populations in the large-scale commercial production of biologies (proteins) and vaccines.

Microcarrier cell culture is typically carried out in spinner flasks, although other vessels such as rotating wall microgravity bioreactors or fluidized bed bioreactors can also support microcarrier-based cultures. Microcarriers can scale and can be used to precisely control cell growth conditions in, for example, computer-controlled bioreactors.

Examples of microcarriers include dextran-based (Cytodex, GE Healthcare), collagen- based (Cultispher, Percell), and polystyrene-based (SoIoHill Engineering) microcarriers.

Microcarriers can differ in their porosity, specific gravity, optical properties, presence of animal components, and surface chemistries.

34. Modulate

To modulate, or forms thereof, means either increasing, decreasing, or maintaining an activity. It is understood that wherever one of these words is used it is also disclosed that it could be 1%, 5%, 10%, 20%, 50%, 100%, 500%, or 1000% increased or decreased from a control.

35. Modulator

A modulator or like terms is a molecule that modulates the activity of a cellular target.

36. Modulated gene

A modulated gene is any gene that has been modulated.

37. Modulated Gene product

A modulated gene product is any gene product that has been modulated.

38. Molecule

As used herein, the terms "molecule" or like terms refers to a biological or biochemical or chemical entity that exists in the form of a chemical molecule or molecule with a definite molecular weight. A molecule or like terms is a chemical, biochemical or biological molecule, regardless of its size.

Many molecules are of the type referred to as organic molecules (molecules containing carbon atoms, among others, connected by covalent bonds), although some molecules do not contain carbon (including simple molecular gases such as molecular oxygen and more complex molecules such as some sulfur-based polymers). The general term "molecule" includes numerous descriptive classes or groups of molecules, such as proteins, nucleic acids, carbohydrates, steroids, organic pharmaceuticals, small molecule, receptors, antibodies, and lipids. When appropriate, one or more of these more descriptive terms (many of which, such as "protein," themselves describe overlapping groups of molecules) will be used herein because of application of the method to a subgroup of molecules, without detracting from the intent to have such molecules be representative of both the general class "molecules" and the named subclass, such as proteins. Unless specifically indicated, the word "molecule" would include the specific molecule and salts thereof, such as pharmaceutically acceptable salts.

39. NFKappaB

NFkappaB (nuclear factor kappa-light-chain-enhancer of activated B cell) is a family of proteins including NFkB l, NFkB2, RelA, RelB and RelC, dimers and heterodimers of which bind to a specific sequence in the promoter of NFkB responsive genes, activating their transcription.

40. Optional

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

41. p53

p53 is a tumor suppressor gene that in humans is encoded by the TP53, and exemplary sequences encoding p53 can be found in Table 5.

42. poly ADP ribosylation

poly ADP ribosyation and ADP-ribosylation and like terms refer to the process of the addition of one or more ADP-ribose moieties to a protein. These reactions are involved in cell signaling and the control of many cell processes, including DNA repair and apoptosis.

43. Polypeptides and variants

"Polypeptide" or like terms as used herein can include peptides, oligopeptides, polypeptides, gene products, expression products, or protein if they are a polymer comprised of at least two consecutive amino acids linked by a peptide bond between the alpha carboxyl group of one amino acid and the amino group of the next amino acid. The term "polypeptide" encompasses naturally occurring or synthetic molecules. Herein, peptides differ from proteins in that a peptide does not have posttranslational modifications and a protein can have such modifications. 2. In addition, as used herein, the term "polypeptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP- ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer- NA mediated addition of amino acids to protein such as arginylation. (See Proteins - Structure and Molecular Properties 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslationai Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983))

a) Protein variants

As discussed herein there are numerous variants of the various proteins that are known and herein contemplated. In addition, to the known functional strain variants there are derivatives of the disclosed proteins which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 1 and 2 and are referred to as conservative substitutions.

TABLE I :Amino Acid Abbreviations

TABLE 2 Amino Acid Substitutions

Original Residue Exemplary Conservative Substitutions, others are known in the art.

Alaser Arglys, gin

Asngln; his

Aspglu

Cysser

Ginasn, lys

Gluasp

Glypro

Hisasn;gln

lleleu; val

Leuile; val

Lysarg; gin;

MetLeu; iie

Phemet; leu; tyr

Serthr

Thrser

Trptyr

Tyrtrp; phe

Valiie; leu

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobic ity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an

electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post- translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology/identity to specific known sequences. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by published algorithms.

Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Bioi. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. it is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular cell from which that protein arises is also known and herein disclosed and described.

It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 1 and Table 2. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way (Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in Biotechnology, 3:348-354 (1992); Ibba,

Biotechnology & Genetic Enginerring Reviews 13: 197-216 (1995), Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech, 12: 158-163 (1994); Ibba and Hennecke,

Bio/technology, 12:678-682 (1994) all of which are herein incorporated by reference at least for material related to amino acid analogs).

Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH ₂ NH-, -CH ₂ S~, -CH ₂ -CH ₂ ~, ~CH=CH— (cis and trans), -COCH ₂ -, - CH(OH)CH ₂ ~, and— CHH ₂ SO— (These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14: 177-185 (1979) (~CH ₂ NH-, CH ₂ CH ₂ ~); Spatola et al. Life Sci 38: 1243-1249 (1986) (-CH H ₂ --S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (— CH--CH--, cis and trans); Almquist et al. J. Med. Chem. 23: 1392-1398 (1980) ( ^» COCH ₂ ~); Jennings- White et al. Tetrahedron Lett 23:2533 (1982) (-COCH ₂ -); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-CH(OH)CH ₂ --); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (~C(OH)CH ₂ ~); and Hruby Life Sci 31 : 189-199 (1982) (~CH ₂ --S~); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is— CH ₂ NH~. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference).

44. Prevent

By "prevent" or like terms or other forms of prevent means to stop a particular characteristic or condition. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce or inhibit. As used herein, something could be reduced but not inhibited or prevented, but something that is reduced could also be inhibited or prevented. It is understood that where reduce, inhibit or prevent are used, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. Thus, if inhibits phosphorylation is disclosed, then reduces and prevents phosphorylation are also disclosed.

45. Primer

"Primers" are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.

46. Probes

"Probes" are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.

47. Nucleic acids

There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode the disclosed proteins, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.

a) Nucleotides and related molecules

A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-l-yl (C), guanin-9-yl (G), uracil- 1-yi (U), and thymin-l-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. An non-limiting example of a nucleotide would be 3 '-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. A modified base includes but is not limited to 5-methyIcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyi and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and

2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uraciI (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifIuoromethyl and other 5-substituted uracils and cytosines, 7-methy I guanine and

7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and

3- deazaguanine and 3-deazaadenine. Additional base modifications can be found for example in U.S. Pat. No. 3,687,808, Englisch et al., Angewandte Chemte, International Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine can increase the stability of duplex formation. Often time base modifications can be combined with for example a sugar modifcation, such as 2'-0-methoxyethyl, to achieve unique properties such as increased duplex stability. There are numerous United States patents such as 4,845,205;

5, 130,302; 5,134,066; 5, 175,273; 5,367,066; 5,432,272; 5,457, 187; 5,459,255; 5,484,908;

5,502,177; 5,525,71 1 ; 5,552,540; 5,587,469; 5,594, 121, 5,596,091 ; 5,614,617; and 5,681,941, which detail and describe a range of base modifications. Each of these patents is herein incorporated by reference.

Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted d to do, alkyl or C ₂ to C _]0 alkenyl and alkynyl. 2' sugar modiifcations also include but are not limited to -0[(CH ₂ ) _n 0] _m CH ₃ , -0(CH ₂ ) _n OCH ₃ , -0(CH ₂ ) _n NH ₂ , -0(CH ₂ ) _n CH ₃ , -0(CH ₂ ) _n -ONH ₂ , and -0(CH ₂ )„ON[(CH ₂ ) _n CH ₃ )] ₂ , where n and m are from 1 to about 10.

Other modifications at the 2' position include but are not limted to: d to do lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, CI, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , S0 ₂ CH ₃ , ON0 ₂ , N0 ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylammo, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 -5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH ₂ and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures such as 4,981 ,957; 5,1 18,800; 5,319,080; 5,359,044;

5,393,878; 5,446, 137; 5,466,786; 5,514,785; 5,519, 134; 5,567,81 1 ; 5,576,427; 5,591,722;

5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotri ester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2 -5' linkage, and the linkage can contain inverted polarity such as 3 -5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, 3,687,808; 4,469,863; 4,476,301 ; 5,023,243; 5, 177, 196; 5, 188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131 ; 5,399,676; 5,405,939;

5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519, 126; 5,536,821 ; 5,541,306; 5,550,111 ;

5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.

It is understood that nucleotide analogs need only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.

Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.

Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones;formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and C¾ component parts. Numerous United States patents disclose how to make and use these types of phosphate replacements and include but are not limited to 5,034,506; 5, 166,315; 5,185,444; 5,214, 134; 5,216, 141 ; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;

5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;

5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage

(aminoethylgiycine) (PNA). United States patents 5,539,082; 5,714,331 ;and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. (See also Nielsen et al., Science, 1991 , 254, 1497-1500).

It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthioI (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiochole sterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1 1 1 1-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al, Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium

l ,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. Numerous United States patents teach the preparation of such conjugates and include, but are not limited to U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541 ,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731 ; 5,580,731 ; 5,591 ,584; 5,109,124; 5, 1 18,802;

5, 138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;

4,667,025; 4,762,779; 4,789,737; 4,824,941 ; 4,835,263; 4,876,335; 4,904,582; 4,958,013;

5,082,830; 5, 1 12,963; 5,214, 136; 5,082,830; 5, 1 12,963; 5,214,136; 5,245,022; 5,254,469;

5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371 ,241, 5,391 ,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481 ;

5,587,371 ; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941 , each of which is herein incorporated by reference.

A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.

48. Ranges

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value " 10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value " 10" is disclosed the "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data are provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular datum point "10" and a particular datum point 1 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 1 1, 12, 13, and 14 are also disclosed.

49. Recombinant polypeptides and proteins

A recombinant protein is any protein that has been made using recombinant methods or from a nucleic acid that was made by recombinant methods. A protein produced from an exogenous nucleic acid in a cell, or any nucleic acid outside of a cell, is a recombinant protein. A partial list of recombinant proteins that can be made in the cells and systems disclosed herein is Human growth hormone (rHGH) (Humatrope from Lilly and Serostim from Serono), Biosynthetic human insulin (BHI) (Humulin from Lilly and Novolin from Novo Nordisk among others), Follicle-stimulating hormone (FSH) as a recombinant gonadotropin preparation, Factor VIII (Kogenate from Bayer replaced blood harvested factor VIII), Erythropoietin (EPO) (Epogen from Amgen), Granulocyte colony-stimulating factor (G-CSF) (filgrastim sold as Neupogen from Amgen; pegfilgrastim sold as Neulasta), alpha-glactosidase A (Fabrazyme by Genzyme), alpha-L-iduronidase (rhIDU; laronidase) (Aidurazyme by BioMarin Pharmaceutical and Genzyme), N-acetyIgalactosamine-4-sulfatase (rhASB; galsulfase) (Naglazyme (TM) by BioMarin Pharmaceutical), Dornase alfa, (a DNase sold under the trade name Pulmozyme by Genentech), Tissue plasminogen activator (TP A) (Activase by Genentech), Glucocerebrosidase (Ceredase by Genzyme), Interferon (IF) (Interferon-beta-la as Avonex from Biogen Idee; Rebif from Serono; Interferon beta- lb as Betaseron from Schering), Insulin-like growth factor 1 (IGF- 1) Animal proteins have recombinant versions as well provided in a partial list as follows: Bovine somatotropin (bST), Porcine somatotropin (pST), and Bovine Chymosin. Viral recombinants include Envelope protein of the hepatitis B virus (marketed as Engerix-B by SmithKline Beecham)

Therapeutic monoclonal antibodies can also be considered a recombinant protein as discussed herein as they are a protein and are produced recombinantly. A partial list includes,

Exam le FDA approved therapeutic monoclonal antibodiesfl]

T.7

Also included can be the expression of virus like particles (VLPs). C3A cells produce a mammalian glycoslation that is unique. Thus, this invention provides proteins shown herein and polypeptides thereof having the glycosylation pattern of C3A cells.

It is understood that the specific recombinant protein discussed herein can be produced using the systems and cells disclosed herein, as well as variants of these as discussed herein, including sequence variants as well as post-translational variants that may arise in production through the systems and cells disclosed herein.

50. Reduce or decrease

By "reduce" or "decrease" or other forms of reduce or decrease or like terms means lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, "reduces gene product" means lowering the amount of gene product that takes place relative to a standard or a control. It is understood that wherever one of these words is used it is also disclosed that it could be 1%, 5%, 10%, 20%, 50%, 100%, 500%, or 1000% lowered from a control.

51. References

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

52. RNA interference (RNAi)

RNAi and like terms is a process within living cells that moderates the activity of the genes within the cells. Co-suppression, post transcriptional gene silencing (PTGS), and quelling are all forms of RNAi. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon.

Two types of small ribonucleic acid (RNA) molecules - microRNA (miRNA) and small interfering RNA (siRNA) - are largely involved in RNA interference. RNA interference has an important role in defending cells against parasitic genes - viruses and transposons - but also in directing development as well as gene expression in general.

The RNAi pathway is found in many eukaryotes including animals and can be initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of -20 nucleotides that are called siRNAs. Each siRNA is unwound into two single- stranded (ss) ssRNAs, namely the passenger strand and the guide strand. The passenger strand will be degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). This can result in post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex.

53. RNA metabolism

RNA metabolism and like terms refers to the process of modifying and degrading RNA, such as mRNA. RNA metabolism includes, for example, RNA splicing, RNA editing, RNA capping, RNA poly A addition, and RNA transport.

54. RNA Editing

RNA editing and like terms describes those molecular processes in which the information content in an RNA molecule is altered through a chemical change in the base makeup.

55. RNA Capping

RNA capping and like terms refers to the alteration of the 5' nucleotide of an RNA transcript, and has a guanine-guanine link via a '-5' triphosphate bond.

56. RNA Poly A addition and poly A tail

RNA A addition and poly A tail and like terms refer to the addition of and presence of a stretch of Adenosines at the 3' end of the RNA.

57. RNA transport

RNA transport and like terms refers to the movement of RNA from the nucleus to the cytoplasm or from the mitochondria to the cytoplasm.

58. RNA splicing

RNA splicing and like terms is the process of removing introns and joining exons of a gene after transcription. This process typically involves the splicesome small nuclear ribonucleoproteins (snRNPs).

59. Sparging

Sparging and like terms refers to agitating a liquid, through the use of gas or air, such as compressed gas or air. 60. Splicing related protein or small nuclear ribonucleoproteins (snRJ Ps) snRNPs and like terms (pronounced "snurps"), or small nuclear ribonucleoproteins, are R A-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs. The action of snRNPs is required to remove introns from pre-mRNA. This is an important aspect of post-transcriptional modification of RNA, occurring only in the nucleus of eukaryotic cells.

The two essential components of snRNPs are protein molecules and RNA. The RNA found within each snRNP particle is known as small nuclear RNA, or snRNA, and is usually about 150 nucleotides in length. The snRNA component of the snRNP gives specificity to individual introns by "recognizing" the sequences of critical splicing signals at the 5' and 3' ends and branch site of introns. The snRNA in snRNPs is similar to ribosomal RNA in that it directly incorporates both an enzymatic and a structural role.

At least five different kinds of snRNPs join the spliceosome to participate in splicing. They can be visualized by gel electrophoresis and are known individually as: Ul, U2, U4, U5, and U6. Their snRNA components are known, respectively, as: U l snRNA, U2 snRNA, U4 snRNA, U5 snRNA, and U6 snRNA.

61. Subject

As used throughout, by a subject or like terms is meant an individual. Thus, the

"subject" can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) and mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In one aspect, the subject is a mammal such as a primate or a human. The subject can be a non-human.

62. Signaling Pathwa (s)

A "signaling pathway" or like terms is a path in a cell from receiving a signal (from within or from without, and propagated from within to without, within to within, or from without to with, for example) (e.g., an exogenous ligand) to a cellular response (e.g., increased expression of a cellular target). In some cases, receptor activation caused by ligand binding to a receptor is directly coupled to the cell's response to the ligand. For example, the neurotransmitter GABA can activate a cell surface receptor that is part of an ion channel. GABA binding to a GABA A receptor on a neuron opens a chloride-selective ion channel that is part of the receptor. GABA A receptor activation allows negatively charged chloride ions to move into the neuron which inhibits the ability of the neuron to produce action potentials. However, for many cell surface receptors, ligand-receptor interactions are not directly linked to the cell's response. The activated receptor must first interact with other proteins inside the cell before the ultimate physiological effect of the ligand on the cell's behavior is produced, such as at a G-Protein Coupled Receptor (GPCR). Often, the behavior of a chain of several interacting cell proteins is altered following receptor activation. The entire set of cell changes induced by receptor activation is called a signal transduction mechanism or pathway. Activation of signaling pathway often results in an activity within the cell, such as apoptosis or transport. The signaling pathway can be either relatively simple or quite complicated.

A signaling pathway occurs when a first molecule interacts with a second molecule which causes some alteration or which allows the second molecule to perform a different activity, such as interacting with a third molecule.

Any signaling pathway with a modifier, such as p53 or apoptosis, refers to a signaling pathway involving that molecule or that process. Thus, a p53 signaling pathway refers to a signaling pathway that comprises p53 as either receiving or transmitting a signal from or to another molecule, as discussed herein. Furthermore, an apoptosis signaling pathway refers to a signaling pathway that comprises apoptosis as the activity occurring because of the signaling between molecules.

A variety of signaling pathways are disclosed herein, such as the interferon response pathway, apoptosis pathway, unfolded protein response pathway, ATF4 pathway, poly ADP ribosylation pathway, p53 pathway, RNA metabolism pathway, NFKappaB pathway, ubiquitin related, transport related pathway, mTOR related pathway, trafficking pathway, and RNA splicing pathway. Furthermore, it is understood that a signaling pathway is disclosed for each gene and gene product, such as miRNA or protein disclosed herein. For example, if a particular protein or miRNA, such as p53 or mir21 is disclosed, the signaling pathway for each is also disclosed.

63. System

A system or like terms as used herein refers to an interdependent group of items forming a unified whole. For example, a computer system are the parts, such as a process, a memory storage device, and other parts which can be used to form a functioning computer. Also for example, a system can be made up of a cell and the necessary reagents for the cell to be passaged. Also by example, the word system can be used as a system, wherein the system comprises a C3A cell and modulated gene product. In this type of system, for example, the system could be a fermenter, having the necessary reagents to culture the cells, or to isolate the cells or to assay the cells. An isolation system, could for example could be a column chromatography system, a batch binding system, a immobilized bead system, a free bead system etc. Those of skill in the art, given the information herein, can create systems around particular pieces of information, once the information is provided, such as information about an interaction between two proteins or a cell with multiple modulated gene products.

64. Selective hybridization

Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25°C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989;

Kunkel et al. Methods Enzymol. 1987: 154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids). A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for. Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art. Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non- limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k _d , or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their kd.

Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules, it is understood that these methods and conditions may provide different percentages of

hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein.

It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein.

65. Sequence similarities It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.

In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by published algorithms.

Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. NatL Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.

66. Substance

A substance or like terms is any physical object A material is a substance. Molecules, Hgands, markers, cells, proteins, and DNA can be considered substances. A machine or an article would be considered to be made of substances, rather than considered a substance themselves.

67. Transformation

The terms "transformation" and "transfection" or like terms mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell possibly including introduction of a nucleic acid to the chromosomal DNA of said cell.

68. Transport related and transport signaling pathway

A transport related, transport signalling pathway, and like terms refers to the processes and signalling within these processes for moving items in, out, within, through, and between cells. Processes from the golgi and to or from endosomes, from the plasma membrane to or from endosomes, and endosomes to or from lysosomes are examples of transport pathways. There are also endocytic and exocytic pathways. The GGAs, AP-1 clathrin, Rabs, such as Rab9, Tip47, caveolin, and ubiquitin are a few proteins and molecules involved in transport pathways.

69. Treating

Treating or treatment or like terms can be used in at least two ways. First, treating or treatment or like terms can refer to administration or action taken towards a subject. Second, treating or treatment or like terms can refer to mixing any two things together, such as any two or more substances together, such as a molecule and a cell. This mixing will bring the at least two substances together such that a contact between them can take place. For instance, "treating a cell to reach confiuency", means to take care or manipulate cells so they reach confluency.

When treating or treatment or like terms is used in the context of a subject with a disease, it does not imply a cure or even a reduction of a symptom for example. When the term therapeutic or like terms is used in conjunction with treating or treatment or like terms, it means that the symptoms of the underlying disease are reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced. It is understood that reduced, as used in this context, means relative to the state of the disease, including the molecular state of the disease, not just the physiological state of the disease.

70. Unfolded protein response

The unfolded protein response (UPR) is a cellular stress response related to the endoplasmic reticulum. It is conserved between all mammalian species, as well as yeast and worm organisms.

The UPR is typically activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum. In this scenario, the UPR has two primary aims: initially to restore normal function of the cell by halting protein translation and activate the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these objectives are not achieved within a certain time lapse or the disruption is prolonged, the UPR aims to apoptosis.

71. Ubiquitin related

Ubiquitin is a small regulatory protein that has been found in almost all tissues

(ubiquitously) of eukaryotic organisms. Attachment to proteins directs protein recycling, protein destruction, or movement to other areas in the cell. Among other functions, it directs protein recycling.

72. Vaccine

A vaccine or like terms is any preparation that when administered increases immunity to a pathogen. A vaccine typically contains an antigen, such as a virus which has been attenuated or killed or mutated, or a protein or peptide of a pathogen.

73. Vector, recombinant vector

The term "vector" or "plasmid" "recombinant vector" or like terms refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term "expression vector" includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). "Plasmid" and "vector" are used interchangeably, as a plasmid is a commonly used form of vector. Moreover, disclosed are other vectors which serve equivalent functions. 74. Virus

There are many types of viruses including RNA viruses (including negative stranded RNA viruses, positive stranded RNA viruses, double stranded RNA viruses and retroviruses), and DNA viruses. All strains, types, and subtypes of RNA viruses and DNA viruses are contemplated herein.

Examples of RNA viruses include, but are not limited to picornaviruses, which include aphthoviruses (for example, foot and mouth disease virus O, A, C, Asia 1, SAT1, SAT2 and SAT3), cardioviruses (for example, encephalomycarditis virus and Theiller's murine encephalomyelitis virus), enteroviruses (for example polioviruses 1, 2 and 3, human

enteroviruses A-D, bovine enteroviruses 1 and 2, human coxsackieviruses A1-A22 and A24, human coxsackieviruses B 1 -B5, human echoviruses 1-7, 9, 11-12, 24, 27, 29-33, human enteroviruses 68-71 , porcine enteroviruses 8-10 and simian enteroviruses 1 -18), erboviruses (for example, equine rhinitis virus), hepatovirus (for example human hepatitis A virus and simian hepatitis A virus), kobuviruses (for example, bovine kobuvirus and Aichi virus), parechoviruses (for example, human parechovirus I and human parechovirus 2), rhinovirus (for example, rhinovirus A, rhinovirus B, rhinovirus C, HRVi _6i HRV, ₆ (VR-1 1757), HRV _H (VR-284), or HRV[ _A (VR-1559), human rhinovirus 1-100 and bovine rhinoviruses 1-3) and teschoviruses (for example, porcine tescho virus).

Additional examples of RNA viruses include caliciviruses, which include noroviruses (for example, Norwalk virus), sapoviruses (for example, Sapporo virus), lagoviruses (for example, rabbit hemorrhagic disease virus and European brown hare syndrome) and vesiviruses (for example vesicular exanthema of swine virus and feline calicivirus).

Other RNA viruses include astroviruses, which include mamastorviruses and avastroviruses. Togaviruses are also RNA viruses. Togaviruses include alphaviruses (for example, Chikungunya virus, Sindbis virus, Semliki Forest virus, Western equine encephalitis, Getah virus, Everglades virus, Venezuelan equine encephalitis virus and Aura virus) and rubella viruses. Additional examples of RNA viruses include the the flaviviruses (for example, tick- borne encephalitis virus, Tyuleniy virus, Aroa virus, Dengue virus (types 1 to 4), Kedougou virus, Japanese encephalitis virus (JEV), West Nile virus (WNV), Kokobera virus, Ntaya virus, Spondweni virus, Yellow fever virus, Entebbe bat virus, Modoc virus, Rio Bravo virus, Cell fusing agent virus, pestivirus, GB virus A, GBV-A like viruses, GB virus C, Hepatitis G virus, hepacivirus (hepatitis C virus (HCV)) all six genotypes), bovine viral diarrhea virus (BVDV) types 1 and 2, and GB virus B). Other examples of RNA viruses are the coronaviruses, which include, human respiratory coronaviruses such as SARS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. Coronaviruses also include bat SARS-like CoV, turkey coronavirus, chicken coronavirus, feline coronavirus and canine coronavirus. Additional RNA viruses include arteriviruses (for example, equine arteri virus, porcine reproductive and respiratory syndrome virus, lactate dehyrogenase elevating virus of mice and simian hemorraghic fever virus). Other RNA viruses include the

rhabdoviruses, which include lyssaviruses (for example, rabies, Lagos bat virus, Mokola virus, Duvenhage virus and European bat lyssavirus), vesiculoviruses (for example, VSV-Indiana, VSV-New Jersey, VSV-Alagoas, Piry virus, Cocal virus, Maraba virus, Isfahan virus and Chandipura virus), and ephemeroviruses (for example, bovine ephemeral fever virus, Adelaide River virus and Berrimah virus). Additional examples of RNA viruses include the filoviruses. These include the Marburg and Ebola viruses (for example, EBOV-Z, EBOV-S, EBOV-IC and EBOV-R.

The paramyxoviruses are also RNA viruses. Examples of these viruses are the rubulaviruses (for example, mumps, parainfluenza virus 5, human parainfluenza virus type 2, Mapuera virus and porcine rubulavirus), avulaviruses (for example, Newcastle disease virus), respoviruses (for example, Sendai virus, human parainfluenza virus type 1 and type 3, bovine parainfluenza virus type 3), henipaviruses (for example, Hendra virus and Nipah virus), morbilloviruses (for example, measles, Cetacean morvilliirus, Canine distemper virus, Peste- des-petits-ruminants virus, Phocine distemper virus and Rinderpest virus), pneumo viruses (for example, human respiratory syncytial virus A2, B 1 and S2, bovine respiratory syncytial virus and pneumonia virus of mice), metapneumoviruses (for example, human metapneumo virus and avian metapneumovirus). Additional paramyxoviruses include Fer-de-Lance virus, Tupaia paramyxovirus, Menangle virus, Tioman virus, Beilong virus, J virus, Mossman virus, Salem virus and Nariva virus.

Additional RNA viruses include the orthomyxoviruses. These viruses include influenza viruses and strains {e.g., influenza A, influenza A strain A/Victoria/3/75, influenza A strain

A/Puerto Rico/8/34, influenza A HlNl (including but not limited to A/WS/33, A/NWS/33 and

A California/04/2009 strains), influenza B, influenza B strain Lee, and influenza C viruses)

H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3 and H10N7), as well as avian influenza (for example, strains H5N1, H5N1 Duck MN/ 1525/81, H5N2, H7N1, H7N7 and

H9N2) thogotoviruses and isaviruses. Orthobunyaviruses (for example, Akabane virus,

California encephalitis, Cache Valley virus, Snowshoe hare virus,) nairoviruses (for example,

Nairobi sheep virus, Crimean-Congo hemorrhagic fever virus Group and Hughes virus), phleboviruses (for example, Candiru, Punta Toro, Rift Valley Fever, Sandfly Fever, Naples, Toscana, Sicilian and Chagres), and hantaviruses (for example, Hantaan, Dobrava, Seoul, Puumala, Sin Nombre, Bayou, Black Creek Canal, Andes and Thottapalayam) are also RNA viruses. Arenaviruses such as lymphocytic choriomeningitis virus, Lujo virus, Lassa fever virus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, SABV and WWAV are also RNA viruses. Borna disease virus is also an RNA virus. Hepatitis D (Delta) virus and hepatitis E are also RNA viruses.

Additional RNA viruses include reoviruses, rotaviruses, birnaviruses, chrysoviruses, cystoviruses, hypoviruses partitiviruses and totoviruses. Orbiviruses such as African horse sickness virus, Blue tongue virus, Changuinola virus, Chenuda virus, Chobar Gorge Corriparta virus, epizootic hemorraghic disease virus, equine encephalosis virus, Eubenangee virus, Ieri virus, Great Island virus, Lebombo virus, Orungo virus, Palyam virus, Peruvian Horse Sickness virus, St. Croix River virus, Umatilla virus, Wad Medani virus, Wallal virus, Warrego virus and Wongorr virus are also RNA viruses.

Retroviruses include alpharetroviruses (for example, Rous sarcoma virus and avian leukemia virus), betaretroviruses (for example, mouse mammary tumor virus, Mason-Pfizer monkey virus and Jaagsiekte sheep retrovirus), gammaretroviruses (for example, murine leukemia virus and feline leukemia virus, deltraretroviruses (for example, human T cell leukemia viruses (HTLV-1, HTLV-2), bovine leukemia virus, STLV-1 and STLV-2), epsilonretriviruses (for example, Walleye dermal sarcoma virus and Walleye epidermal hyperplasia virus 1), reticuloendotheliosis virus (for example, chicken syncytial virus, lentiviruses (for example, human immunodeficiency virus (HIV) type 1, human

immunodeficiency virus (HIV) type 2, human immunodeficiency virus (HIV) type 3, simian immunodeficiency virus, equine infectious anemia virus, feline immunodeficiency virus, caprine arthritis encephalitis virus and Visna maedi virus) and spumaviruses (for example, human foamy virus and feline syncytia-forming virus).

Examples of DNA viruses include polyomaviruses (for example, simian virus 40, simian agent 12, BK virus, JC virus, Merkel Cell polyoma virus, bovine polyoma virus and

lymphotrophic papovavirus), papillomaviruses (for example, human papillomavirus, bovine papillomavirus, adenoviruses (for example, adenoviruses A-F, canine adenovirus type I, canined adeovirus type 2), circoviruses (for example, porcine circovirus and beak and feather disease virus (BFDV)), parvoviruses (for example, canine parvovirus), erythro viruses (for example, adeno-associated virus types 1-8), betaparvoviruses, amdoviruses, densoviruses, iteraviruses, brevidensoviruses, pefudenso viruses, herpes viruses 1,2, 3, 4, 5, 6, 7 and 8 (for example, herpes simplex virus 1 , herpes simplex virus 2, varicella-zoster virus, Epstein-Barr virus,

cytomegalovirus, Kaposi's sarcoma associated herpes virus, human herpes virus-6 variant A, human herpes virus-6 variant B and cercophithecine herpes virus 1 (B virus)), poxviruses (for example, smallpox (variola), cowpox, monkeypox, vaccinia, Uasin Gishu, camelpox, psuedocowpox, pigeonpox, horsepox, fowlpox, turkeypox and swinepox), and hepadnaviruses (for example, hepatitis B and hepatitis B-like viruses). Chimeric viruses comprising portions of more than one viral genome are also contemplated herein.

Respiratory viruses include, but are not limited to, picornaviruses, orthomyxoviruses, paramyxoviruses, coronaviruses and adenoviruses. More specifically, and not to be limiting, the respiratory virus can be an influenza virus, a parainfluenza virus, an adenovirus, a rhmovirus or a respiratory syncytial virus (RSV).

Gastrointestinal viruses include, but are not limited to, picornaviruses, filoviruses, flaviviruses, calciviruses and reoviruses. More specifically, and not to be limiting, the gastrointestinal virus can be a reovirus, a Norwalk virus, an Ebola virus, a Marburg virus, a rotavirus, an enterovirus, a Dengue fever virus, a yellow fever virus, or a West Nile virus.

Also disclosed are viruses considered a pox virus, BVDV, a herpes virus, HIV, an RSV virus, an influenza virus, a hepatitis C virus, a hepatitis B virus, Epstein Barr Virus, Human Papilloma Virus, CMV, West Nile virus, a rhinovirus, an adenovirus, measles virus, Marburg virus, Ebola virus, Rift Valley Fever Virus, LCM, Junin virus, Machupo virus, Guanarito virus, Lassa Fever virus, Hantavirus, SARS virus, Nipah virus, Caliciviruses, Hepatitis A, LaCrosse, California encephalitis, VEE, EEE,WEE, Japanese Encephalitis Virus, Kyasanur Forest Virus, Yellow Fever, Rabies, Chikungunya virus or a Dengue fever virus.

For animals, in addition to the animal viruses listed above, viruses include, but are not limited to, the animal counterpart to any above listed human virus. The provided compounds can also decrease infection by newly discovered or emerging viruses. Such viruses are continuously updated on http://en.wikipedia.org/wiki/Virus and www.virology.net.

75. Weight %

References in the specification and concluding claims to parts by weight, of a particular element or component in a composition or article, denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

76. Zinc finger nuclease

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms, in for example, making a knockout of a gene.

B. Technical basis

Disclosed are cells comprising at least two modulated gene products, wherein the gene products are DCP1A, LPP, PHF15, RPL41, EIF1, SFRS3, NUMA1, PKM2, CLDND1, TES, ZNF131, CEP170, PPPIRIO, ENOl, ZBTB37, MRPS18B, HNRNPC, ΑΖΓΝ1, ANXA1, RPS10, GAS5, MALAT1, MIR21, THRAP3, TAF1D, ANXA2, RCC1,PDCD4, NFkB, IL10, DDIT4 or ATF4. in a specific example the cells modulate the mir21 gene product. The cell utilized in this invention can be any cell or cell line suitable for vaccine and/or protein production, examples of cells include MDCK, Vero, C3A, PerC.6, EBx, CAP, EB66, Hep3G, Hela, or CHO cell. The cells can exist in compositions, for example, comprising viruses, gene constructs for expression of a polypeptide and/or an exogenous polypeptides.

The cells can also comprise a third, forth or more modulated gene products. For example, the third modulated gene product is of the gene mir21. In a specific example the modulated gene products are of the genes of mir21, Gas5, and RPL41. In another example, the modulated gene products are of the genes IFITM3, RPL41, and Gas5. The effect of modulating certain of the listed genes was tested using small interfering RNAs (siRNA) combined with an miRNA mimic for mir21. The results are given in Example 1.

The cells can, for example, comprise the gene products from RPL41, GAS5, ENOl, PKM2, PPPIRIO, where the gene products are decreased. The cells can also comprises the gene products from HNRNPC, SFRS3, MIR21, MALAT1, IL10, ATF4, ANXA1, ANXA2, AZIN1, EIF1 where the gene products are increased. The cells can more generally comprise at least one gene product increased and at least one gene product decreased. These cells can comprise for example, mixtures of increased and decreased gene products noted above and herein.

The cells disclosed herein can for example include modulation comprising providing a functional nucleic acid targeting the gene or gene product. For example, the functional nucleic acid can comprise a siRNA, antisense, or ribozyme. In another example, the modulation can comprise knocking out the gene.

The cells can comprise an exogenous gene construct expressing a gene construct expression product. In one example, the gene construct expression product decreases the gene products. The gene construct expression product can encode an exogenous protein.

Also disclosed are compositions comprising the disclosed modulated cells. The compositions can further comprise, for example, viruses, vectors, and/or recombinant polypeptides.

A method of producing a virus is disclosed comprising culturing a cell comprising at least one modulated gene product, wherein the gene product is DCP1A, LPP, PHF15, RPL41, EIFl, SFRS3, NUMAl, PKM2, CLDNDl, TES, ZNF131, CEP170, PPP1R10, ENOl, ZBTB37, MRPS18B, HNRNPC, AZIN1, ANXA1, RPS10, GAS 5, MALAT1, MIR21, THRAP3, TAF1D, ANXA2, RCC1, PDCD4, NFkB, IL10, DDIT4 or ATF4, and isolating the virus from the culture. Also provided is a method of making a vaccine comprising formulating the virus produced by the disclosed cells with an excipient for administration to a subject.

Also provided is a method of producing proteins comprising culturing a cell comprising at least one modulated gene product, wherein the gene product is DCP1A, LPP, PHF15, RPL41, EIFl, SFRS3, NUMAl, P M2, CLDNDl, TES, ZNF131, CEP170, PPP1R10, ENOl, ZBTB37, MRPS18B, HNRNPC, AZIN1, ANXA1, RPS10, GAS5, MALAT1, MIR21, THRAP3, TAF1D, ANXA2, RCC1, PDCD4, NFkB, IL10, DDIT4 or ATF4, and isolating the polypeptide from the culture. The cells can also comprise a second, third, forth, or more, modulated gene products.

Disclosed is a method of producing cells provided herein comprising obtaining a cell and transforming the cell with a vector comprising a functional nucleic acid targeting one of

DCP1A, LPP, PHF15, RPL41, EIFl, SFRS3, NUMAl, PKM2, CLDNDl, TES, ZNF13I,

CEP170, PPP1R10, ENOl, ZBTB37, MRPS18B, HNRNPC, AZIN1, ANXA1, RPS10, GAS5,

MALAT1, MIR21, THRAP3, TAF1D, ANXA2, RCC1,PDCD4, NFkB, IL10, DDIT4 or ATF4.

In one example, the cell is produced comprising obtaining a cell and transforming the cell with a vector comprising a gene construct that will knockout the gene of the gene product, and isolating or culturing a cell having one allele of the gene knocked out. In one example, the vector can comprise a sequence for homologous recombination with the gene. In another example the vector can comprise a sequence encoding a zinc finger nuclease. In another example, the gene knock out can be accomplished using a sequence specific transcription activator like nuclease (TALEN). The TALEN creates a double stranded break in the gene which is then repaired but usually adds additional nucleotides, disrupting the reading frame (Hockemeyer, et al. Nat. Biotechnoi. 29:731-734 (201 1), Mussolino, et al. Nucleic Acids Res. 39:9283-9293 (201 1)).

Also disclose are cells in a composition, wherein the cells comprise at least one modulated gene product, wherein the gene product is DCP1A, LPP, PHF15, RPL41, EIFI , SFRS3, NUMA1 , PKM2, CLDND1 , TES, ZNF131, CEP170, PPP1R10, ENOl, ZBTB37, MRPS 18B, HNRNPC, AZINl , ANXAl, RPS10, GAS5, MALATI , MIR21, THRAP3, TAFID, ANXA2, RCC1, PDCD4, NFkB, IL10, DD1T4 or ATF4. The composition can comprise a microcarrier, a suspension media, and/or a fluidized bed bioreactor. Also disclosed is a method of culturing a cell comprising growing the cells on a microcarrier, in suspension, with a fluidized bed reactor, and/or at a variable temperature. In one embodiment, the culture temperature is 31 °C or 37°C. The compositions can comprise an additive, wherein the additive is concentrated amino acids, glucose, sparging agents, lipid supplements. In another embodiment, the composition volume can be at least 2, 5, 10, 50, 100, 500, 1000, 2000, 5000, or 10,000 liters. In another embodiment, the compositions comprise serum free medium. In another embodiment, the composition is free or substantially free of non-human components. In another embodiment, the compositions comprise a synthetic based media. The compositions also can comprise cell culture beads, microcarrier cultures, microbeads. The compositions can further comprise a fermenter. In a further embodiment, the cells are in suspension culture. Also disclosed are systems comprising the cells disclosed herein and a fermenter of at least 2 liters.

Further disclosed are compositions comprising a cell and a modulator of DCP1A, LPP, PHF15, RPL41, EIFI, SFRS3, NUMA1 , P M2, CLDND1, TES, ZNF131, CEP170, PPP1RI0, ENOl, ZBTB37, MRPS18B, HNRNPC, AZINl , ANXAl , RPS 10, GAS5, MALATI, MIR21, THRAP3, TAFID, ANXA2, RCC 1, PDCD4, NFkB, IL10, DDIT4 or ATF4. There are a variety of numbers, values, and ranges disclosed, and it is understood that about each of these or similar is also disclosed.

There are variety of compositions disclosed, herein, and it is understood that the activity, of each is also disclosed, such as if p53, ATF4, mTOR, NF-kB, is disclosed, then p53, ATF4, mTOR, NF-kB activity is also disclosed. Also disclosed for each is the basal activity.

The various compositions, molecules, and materials, and substances can be administered.

Disclosed are variety of conditions, such as apoptosis, poly ADP ribosylation, RNA metabolism, RNA splicing, ubiquitin related pathways, RNAi, unfolded proteins response, interferon response, trafficking pathways, signaling pathways, as well as others, and these are disclosed for all cells, as disclosed herein.

There are a variety of cellular targets, modulators, modulated genes, modulated gene products, knockdowns and knockouts disclosed herein.

It is understood that any composition or component as disclosed herein can also be inhibited, reduced, prevented, decreased, as well as increased or activated.

The various cells can be cultured using any means, and controls can be identified for any assay or activity disclosed herein.

It is understood that excipients can be added to any composition or component disclosed herein.

For each of the cells as well as nucleic acids and polypeptide, both endogenous and exogenous, as well as homologous and heterologous versions are disclosed herein.

The various nucleic acids can be identified using hybridization or selective hybridization.

Any of the compositions or components disclosed herein can be isolated.

Disclosed are mammalian cell based systems capable of dramatically more virus or protein production than current systems by altering the expression of stress response genes. Disclosed are sets of genes that can be modified in any cell line to increase virus and protein production. These genes are disclosed with any cell, but a human liver cell line is used as an exemplary system, and is related to influenza production as well as blood product production, such as Factor VIII and Factor IX, as well as BChE.

Mammalian cell based systems are often the only method to produce the quantities of virus and recombinant proteins required for vaccines and therapeutics but these systems suffer from several limitations including long lead time, low yield, lack of generality and expense (1). Current technology utilizing empirical selection of the production cell lines contributes to this problem by necessitating new selections for each product without a firm basis for understanding why the selected line was effective. Through insertional mutagenesis followed by lytic virus challenge, we have discovered a series of genes that control multiple aspects of the stress responses of the cell. By manipulating a selection of these genes through knockdown or overexpression as indicated, we can create cell lines that are better suited to the environment of large scale cell culture and which can synthesize substantially more virus or protein per cell. This will have the effect of lowering the volumes of cell culture necessary for production, thereby lowering the cost and increasing the yield, as well as decreasing the time to

identification of production lines in response to a bioterror or pandemic threat.

One of the disclosed cell lines utilizing this information comes from the C3A cell line disclosed in United States Patent No. 5,290,684. This line was chosen because of its innate ability to synthesize large quantities of protein due to its liver derivation. This C3A cell line is easily infectable with influenza virus. This allows for production of a cell that can both be a protein production cell (both endogenous as well as exogenous, or recombinantly produced) or a virus production cell or both. C3A cells produce a mammalian glycoslation that is unique. Thus, this invention provides proteins and polypeptides having the glycosylation pattern of C3A cells.

Disclosed are cell lines having had stable or transient modification of one or more of the disclosed modification genes. Using standard methods and protocols the production of protein or virus can be monitored and assayed, as well as standard characteristics of the cells, such as growth or metabolism. The methods can also be used to produce systems from any cell, such as MDCK, Vero, C3A, and CHO. The disclosed cells can have one or more of the modification genes modified or effected, in any combination.

The genes were selected across viruses, and therefore, it some subset of the genes defined here can be suitable to most viruses since they were originally selected across multiple viruses and cell lines, as well as to many cells. However, given the information herein, specific gene selection can be tailored to the virus of interest.

1. Pathways central to production

Mir21, RPL41, and Gas5 represent genes, which have been shown to control the ability of a cell to either make virus or to make protein. These genes central in certain signal pathways were identified multiple times in selections designed to identify genes which when mutated allowed ceils to live, and additionally allowed them to live in the presence of a viral onslaught. Mir21 was identified 30 times, RPL41 was identified 10 times, and Gas5 was identified 24 times. Genes that were identified multiple times are associated with certain signal pathways associated with cell health, allowing them to live even under viral onslaught. Certain signal pathways, including apoptosis (Gas5 for example), the unfolded protein response, and the interferon response pathway (mir21, as well as IFITM3 in a different selection) are implicated.

2. Modification genes

A large insertional mutagenesis experiment and bioinformatics analysis was performed to define host genes that are required for the completion of the viral life cycle. In this experiment, libraries of cells were created that had random mutations created throughout their genome by insertion of a gene trap retrovirus. These libraries were then challenged with a lytic virus to kill the cells. Clones that survived had, by definition, inactivated a gene necessary for the virus to kill the cell. Equally importantly, the cell was able to survive with the gene inactivation (2,3). In this way, genes were selected that were essential for virus but

nonessential for the cell. After clone selection, the inactivated gene was identified by rescue of the inserted retrovirus and some of its surrounding DNA. The rescued plasmid was then sequenced and compared to the human genome to identify the insertion point.

This complete insertional mutagenesis project was carried out with multiple cell lines and 14 different viruses, including influenza, Ebola, Marburg, and Dengue fever virus. Over 3000 individual insertion events were analyzed and over 1000 different genes were identified. The bulk of these genes were hit (trapped) once with a single virus, as would be expected by the idiosyncratic nature of viral infection. Some genes, however, were trapped multiple times by multiple viruses. The genes given in Table 1 were trapped over ten times, each by multiple viruses. These multi-hit genes indicate a generality to their mechanism of action.

The disclosed cells are cells that have been modified so that they produce more polypeptides or virus in culture. The cells are modified so that particular genes, identified as genes involved in parts of the cell health allowing increased polypeptide and viral production. In general the cells have had the gene product of a target gene, such as Gas5, decreased, or such as mir21, increased.

Decreasing the gene product of a gene can occur through any means, such as functional nucleic acids targeting either the transcription gene product or the translation gene product, or small molecules or even other peptides targeting either the transcription gene product or the translation gene product. Alternatively, the gene within the chromosome, for example, can be interfered with, through, for example, knockout technology, such as homologous recombination, Talens, or zinc finger nucleases. Certain genes, such as mir21, when their gene product is increased will increase the polypeptide or viral production. The increase can occur by for example, direct administration of either the transcription gene product or the translation gene product, or through using recombinant biotechnology and exogenous gene constructs which will produce either or both the transcription gene product and/or translation gene product.

A variety of cell manipulations involving exogenous gene constructs are disclosed. These constructs can be either stably or transiently introduced to the cell, using any known means or vectors.

The cells disclosed herein can be used to produce virus for vaccine production as well as to produce polypeptides, for therapeutic or industrial purposes.

The viruses and polypeptides produced using the disclosed cells are also disclosed, as well as the various systems, such as the cells and bioreactor, or cells and microbeads, for producing the virus or polypeptide.

Methods for making the cells as well as the viruses and polypeptides produced by the cells are also disclosed.

C. Compositions

1. Delivery of the compositions to cells

There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, piasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A., et ah, Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991)Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modifed to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier. a) Nucleic acid based delivery systems

Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as a nucleic acid encoding a functional nucleic acid into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Viral vectors are , for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promoter cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans. (1) Retroviral Vectors

A retrovirus is an anima! virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer. In Microbiology-1985, American Society for Microbiology, pp. 229-232, Washington, (1985), which is incorporated by reference herein. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868, 1 16 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. The removal of the gag, pol, and env genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

(2) Adenoviral Vectors

The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61 :1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj- Ahmad et al„ J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest.

92:1580-1586 (1993); irshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessier, J. Clin. Invest. 92:1085-1092 (1993); oullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman,

Circulation Research 73: 1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650- 655 (1984); Seth, et a!., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061- 6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.

(3) Adeno-associated viral vectors

Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B 19 parvovirus.

Typically the AAV and B 19 coding regions have been deleted, resulting in a safe, non- cytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United States Patent No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.

The disclosed vectors thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.

The inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

(4) Large payload viral vectors

Molecular genetic experiments with large human herpes viruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpes viruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter and Robertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable The maintenance of these episomes requires a specific EBV nuclear protein, EBNAl, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpes virus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.

b) Non-nucleic acid based systems

The disclosed compositions can be delivered to the target cells in a variety of ways. For example, the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosed vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1 :95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as

macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECT AMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc.

Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

The materials may be in solution, suspension (for example, incorporated into

microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem.. 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281 , (1989); Bagshawe, et al., Br. J. Cancer. 58:700-703, (1988); Senter, et al., Bioconjugate Chem.. 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.

Reviews. 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol. 42:2062-2065, (1991)). These techniques can be used for a variety of other speciifc cell types. Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang. Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, and type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of

receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral integration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can become integrated into the host genome.

Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art.

c) In vivo/ex vivo

As described above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject's cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

2. Expression systems

The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

a) Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment (Greenway, P.J. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein. Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et ai., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3: 1 108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

The promoter and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

In certain embodiments the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTF.

It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as

polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.

b) Markers

The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. Coli lacZ gene, which encodes β-galactosidase, and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P.. J. Molec. Appl. Genet. 1 : 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden. B. et al„ Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

3. Pharmaceutical carriers Delivery of pharamceutical products

As described above, the compositions produced from the cells described herein, can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermal ly, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.

Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, .D., Br. J. Cancer. 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.. 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother.. 35:421-425, (1992); Pietersz and McKenzie. Immunolog.

Reviews, 129:57-80, (1992); and Roffier, et a!., Biochem. Pharmacol. 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1 104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, and type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

The compositions, including antibodies, produced from the cells of disclosed herein, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.

Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oieate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycotic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

Effective dosages and schedules for administering the compositions produced by the cells disclosed herein, may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder is affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

Guidance can be found in the literature for appropriate dosages for given classes of

pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303- 357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 μg kg to up to 100 mg kg of body weight or more per day, depending on the factors mentioned above.

4. Kits

Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include primers to perform the amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended.

5. Compositions with similar functions

It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures which can perform the same function which are related to the disclosed structures, and that these structures will ultimately achieve the same result, for example modulation of a gene product.

D. Methods of making the compositions

The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.

1. Methods of modulating a gene

The disclosed compositions and methods can be used for targeted gene disruption and modification in any animal that can undergo these events. Gene modification and gene disruption refer to the methods, techniques, and compositions that surround the selective removal or alteration of a gene or stretch of chromosome in an animal, such as a mammal, in a way that propagates the modification through the germ line of the mammal, if for example, an animal was to be a producer using the cells disclosed herein. In general, a cell is transformed with a vector which is designed to homologously recombine with a region of a particular chromosome contained within the cell, as for example, described herein. This homologous recombination event can produce a chromosome which has exogenous DNA introduced, for example in frame, with the surrounding DNA. This type of protocol allows for very specific mutations, such as point mutations, to be introduced into the genome contained within the cell. Methods for performing this type of homologous recombination are disclosed herein.

One of the preferred characteristics of performing homologous recombination in mammalian cells is that the cells should be able to be cultured, because the desired

recombination event occurs at a low frequency. TALENS can be used in combination with a targeting vector to insert sequences in a desired genomic location. When a targeting plasmid containing sequences homologous to the desired insertion point and also containing the desired insertion itself are cotransfected with plasmids encoding a sequence specific TALEN, desired sequences can be inserted in a sequence specific manner. In such a way, genes can be modified by insertion or deletion.

Once the cell is produced through the methods described herein, an animal can be produced from this cell through either stem cell technology or cloning technology. For example, if the cell into which the nucleic acid was transfected was a stem cell for the organism, then this cell, after transfection and culturing, can be used to produce an organism which will contain the gene modification or disruption in germ line cells, which can then in turn be used to produce another animal that possesses the gene modification or disruption in all of its cells. In other methods for production of an animal containing the gene modification or disruption in all of its cells, cloning technologies can be used. These technologies generally take the nucleus of the transfected cell and either through fusion or replacement fuses the transfected nucleus with an oocyte which can then be manipulated to produce an animal. The advantage of procedures that use cloning instead of ES technology is that cells other than ES cells can be transfected. For example, a fibroblast cell, which is very easy to culture can be used as the cell which is transfected and has a gene modification or disruption event take place, and then cells derived from this cell can be used to clone a whole animal.

2. Nucleic acid synthesis

For example, the nucleic acids, such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Ikuta et ah, Ann. Rev. Biochem. 53:323-356 (1984), (phosphotnester and phosphite-triester methods), and Narang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et ah, Bioconjug. Chem. 5:3-7 (1994). 3. Peptide synthesis

One method of producing the disclosed proteins, such as SEQ ID NO:23, is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenyImethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY (which is herein incorporated by reference at least for material related to peptide synthesis). Alternatively, the peptide or polypeptide is independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides may be linked to form a peptide or fragment thereof via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science,

266:776-779 (1994)). The first step is the chemo selective reaction of an unprotected synthetic peptide—thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J.Biol.Chem., 269: 16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)). Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).

E. Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1 Multi-hit genes are stress response genes

The genes in Fig. 1 fall into a variety of categories: translation (e.g. SFRS3), splicing (e.g. HNRNPC), and metabolism (e.g. PKM2), for example. Closer analysis reveals that each of the genes is linked to one of the various stress response pathways of the cell: the interferon response (mir21), apoptosis (GAS5) and the unfolded protein response (RPL41) as examples.

Interferon response: mir-21 - MicroRNA 21 has been widely investigated for its role in cancer. More recently, it has been understood to play a central role in shutting down the cell's interferon response (4). PDCD4 is a major target for mir-21. PDCD4 activates NFkB and suppresses IL-10. Loss of mir-21 in the gene trap experiment raises PDCD4, activating NFkB and suppressing IL-10, potentiating the interferon response. In effect, knocking out mir-21 primes the interferon response, suppressing the ability of the virus to replicate. Overexpression of mir-21 can hinder the ability of the cell to fight off the virus, thereby increasing the rapidity of infection.

Apotosis: GAS5 - Growth arrest specific 5 has been shown to mediate the arrest of ceil division by rapamycin inhibition of mTOR (5). GAS5 is a noncoding RNA gene that is also a small nucleolar RNA host gene (SNHG) (6). Interestingly, RCCl , our most highly trapped gene is also an SNHG. Knockout of GAS5 disables the ability of mTOR to arrest T cell division. In the trapping experiment, GAS5 knockout does not prevent virus infection. In fact, subsequent experiments showed that GAS5 knockout cells are verv heavilv infected with virus. The GAS5 knockout came through the original selection because it suppresses the ability of the virus to kill the cell.

Another mechanism to knock down expression of inhibitors of mTOR via DDIT4.

Knockdown of DDIT4 via siRNA increases albumin synthesis in C3A several fold. TALENS can be used to specifically knock out DDIT4 gene expression, resulting in a permanently elevated level of synthesis.

Unfolded protein response: RPL41 - Ribosomal protein L41 has recently been demonstrated to control the degradation of ATF4 (7). ATF4, along with ATF6, is the central activator of the unfolded protein response (UPR). Inactivation of RPL41 in the trapping experiment elevated ATF4, increasing the UPR, allowing the cell to better survive viral infection, similar to GAS5.

Interferon response: IFITM3 - Interferon inducible transmembrane 3 was identified in a parallel siRNA screen looking for host genes involved in viral replication (8). This experiment also identified several genes which, when knocked down, increased virus production, and West Nile virus and Dengue fever virus are increased when IFITM3 is knocked down.

These genes, and the categories they represent, can be mixed and matched, and represent the methods for the disclosed cells and systems. Overexpression or increase, of MIR21 combined with knockdown or decrease of IFITM3, RPL41 and GAS5 can create a cell that is highly susceptible to infection (the interferon response), that synthesizes virus effectively (UPR) and that is resistant to death (apoptosis). Similarly, RPL41 and GAS5 knockout can have positive effects on protein synthesis in the setting of large scale cell culture (9).

2. Cells for producing blood products

a) C3A Human liver cells

C3A is a human liver cell line originally developed for use in a liver assist device to support patients waiting for transplantation (10, 11). It was specifically selected for adult liver specific function including a high albumin to alphafetoprotein ratio and growth in glucose free medium (12). It synthesizes all of the liver specific serum proteins (Table 3), expresses the P450 drug metabolizing enzymes, and has been cultured to very high densities in hollow fiber bioreactors. Importantly for this project, it has been used to treat patients in FDA sanctioned clinical trials and so has undergone all of the appropriate testing (Table 4). The cells are stored in a Qualified Master Cell Bank, are cultured under GLP conditions and can be grown in serum free medium. The human liver cell line C3A was previously used in FDA regulated clinical trials of a liver assist device directed by the principal investigator of this application both in the US and UK (18, 19).

1. Table 3. Plasma proteins synthesized by C3A and verified by immunoblot

Table 4. Selected testing of the C3A Master Cell Bank

Albumin was produced by C3A cells in a hollow fiber bioreactor cultured over 50 days. During peak production, the device produced approximately l g of albumin per day, which is about 40% of the total protein synthesized. This calculates to about 50 pg/cell/day total protein synthesis. This compares quite favorably with the highest rates obtained in CHO cell culture after 20 years of engineering (13). C3A synthesizes and secretes a wide variety of proteins (Table 3), many of which are glycosylated and which carry secondary modifications, indicating that the secondary processing machinery is matched to the synthetic capability. This suggests C3A as a superior production line for modified proteins such as BChE. Modified with suppression or activation as appropriate, or knockout, of the genes described herein can produce even better protein and virus production.

C3A has several additional features that make it appealing for large scale cell culture. It is highly aerobic so lactate does not build up in the culture; neither does ammonia since it synthesizes both glutamine and urea. As shown in Table 3, it synthesizes many of the proteins that are often added to serum free medium, such as albumin and transferring making these additions unnecessary. C3A expresses ICAM-1, a known receptor for the influenza virus.

b) Experimental Approach

Standard molecular biology and siRNA methods can be used to evaluate the genes and carry out the cell line modifications. Constructs to evaluate the genes, both for knockdown and overexpression (decrease or increase), can be obtained from commercial suppliers. The effect of each modification can be evaluated by examining total protein production, albumin production, BChE production and influenza virus infection. Total protein can be measured using standard assays, albumin via ELISA and influenza via QPCR. BChE can be assayed by standard fluorescent enzyme assay methods. Modified C3A clones can be selected in appropriate antibiotic containing medium. More elaborate, multigene constructs can use piggyBAC cloning and insertion (14).

The collection of 127 multihit candidate genes described herein (defined as >3 hits, >2 viruses) can be evaluated by both siRNA knockdown and transient overexpression for their effect on total protein synthesis, albumin synthesis, BChE synthesis and influenza virus production. All assays can be carried out in biological triplicates and triplicate assay determinations in 96 well plates. From the results of these studies, a series of genes to evaluate in stable transformants can be selected. Stable knockdown and/or overexpression clones can be selected and examined for the same set of characteristics as described above. Clones can also be evaluated for their general cell culture behavior relative to the parent C3A in terms of growth rate, density at confluence and contact inhibition. One can identify a subset of the larger list that can be selected for application in specific instances and a set that are useful in the particular applications described here. Finally, using the results from the earlier experiments, a series of genes can be selected, ideally from different pathways, to be tested in combination with one another for the target effects in stable transformants. These constructs can be made in piggyBAC vectors and inserted into C3A and MDCK.

2. Example 2 - Construction of the BChE overproducing line. BChE is a natural product of the liver and hence the C3A cells. A BChE overproducing cell line can be generated via at least two methods. One is the standard insertion of the cloned gene in combination with an amplifiable marker, such as dihydrofolate reductase followed by selection in methotrexate (15). Another is to use BChE itself as the selection using malathion. Malathion is an organophosphorus insecticide that is metabolized in the liver to malaoxon, which is hepatotoxic (16). Selection of C3A in increasing concentrations of malathion should select for BChE gene amplification. An amplified cell can be selected via one of these two mechanisms. The genes disclosed herein can be modified to further increase production of a BChE overproducer. The utility of the line can be demonstrated in pilot bioreactor studies. A BChE overproducer can be produced by simply amplifying the C3A cells ability to produce protein, as described herein, or additionally, exogenous BChE producing constructs can be added to further amplify.

3. Example 3 - Construction of both an MDCK/influenza vaccine line and a C3A/influenza vaccine line.

The set of genes disclosed herein can be modified in Madin Darby Canine Kidney (MDCK) cells, the standard line for production of influenza (17) and in C3A cells.

Measurement of influenza production can be carried out in comparison to the parental cell lines. The utility of the MDCK and C3A lines can be demonstrated in pilot bioreactor studies.

4. Example 4 - siRNA data.

The effect of modulating certain of the listed genes was tested using small interfering RNAs (siRNA) combined with an miRNA mimic for microRNA 21 (mir21). RNAs were introduced into C3A cells using either lipofection or nucleoporation. Lipofectamine (Life Technologies) was used according to the manufacturer's instruction at an siRNA and/or mimic concentration of 50nM. Nucleoporation was carried out using the Amaxa Nucleofector II using program H-22 and the Amaxa Nucleofector Kit V. Cells were harvested after 48 hrs and assayed for knockdown of gene expression using quantitative RT-PCR. Probes were obtained from Life Technologies. Using either lipofection or nucleoporation, the efficiency of transfection approached 100% as monitored by a fluorescently labelled small RNA probe (Blocklt, life Technologies) or by green fluorescent protein expression (pMAX-GFP, Lonza). Efficient knockdown of IFITM3 or DDIT4 was achieved by lipofection. The negative control was Negative 1 siRNA obtained from Life Technologies. RT-QPCR efficiency was normalized to GAPDH. Similar efficiencies were obtained using nucloporation. To assess the effect of knockdown of DD1T4 alone or in combination with overexpression of mir21 _} C3A cells were infected with influenza virus H1N1 16 hrs after addition of the siRNA or mimic. Plaque size was assessed 24 hrs later using immunofluorescence. Plaque size was significantly increase by DDIT4 knockdown and further enhanced by the combination of DDIT4 and mir-21.

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