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Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.

The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

In certain embodiments, the modified nucleic acids comprise mRNA. In certain embodiments, the mmRNA comprises at least two nucleoside modifications. In certain embodiments, these nucleoside modifications are 5-methylcytosin and pseudouridine. Further, provided herein are formulations comprising the modified nucleic acids described herein.

In certain embodiments, the formulations further comprise a pharmaceutically acceptable carrier. In certain embodiments, the carrier is formulated for systemic or local administration. In certain embodiments, the administration is oral or topical.

In certain embodiments, the compositions comprise naked modified nucleic acids. In other embodiments, the modified nucleic acids are complexed or encapsulated. Provided herein are methods of treating a subject having or being suspected of having a disease, the methods comprising administering to a subject in need of such treatment a formulation described herein in an amount sufficient to treat the disease.

In certain embodiments, the methods of treating a subject having or being suspected of having a disease comprise administering to the subject in need of such treatment a formulation comprising a modified nucleic acid described herein in an amount sufficient to modulate one or more activities associated with MSH, G-CSF or insulin to treat the disease.

Provided herein are pharmaceutical formulations comprising: In certain embodiments, the G-CSF polypeptide comprises: In some embodiments, the pharmaceutical formulation provided herein further comprise a lipid-based transfection reagent. In some embodiments, the synthetic messenger ribonucleic acid mRNA encoding a granulocyte colony-stimulating factor G-CSF polypeptide lacks at least one destabilizing element.

Further provided herein are pharmaceutical formulations consisting essentially of: In certain embodiments, methods are provided wherein the mRNA or a pharmaceutically acceptable salt thereof is administered to the subject in the dosage range of about 1. In some embodiments, methods are provided wherein the subject is human.

In certain embodiments, compositions are administered to the subject by an intravenous route. In some embodiments, the composition is administered at least twice but fewer than ten times prior to administering the AMD In some embodiments, methods are provided, wherein the composition further comprises a synthetic messenger ribonucleic acid mRNA encoding i a macrophage inflammatory protein MIP polypeptide or ii an antibody that prevents EGF binding by EGFR.

Further provided herein are pharmaceutical formulations comprising: In certain embodiments, pharmaceutical formulations comprising synthetic messenger ribonucleic acid mRNA encoding an insulin polypeptide are provided, wherein the insulin polypeptide comprises: In certain embodiments, the pharmaceutical formulations further comprise a lipid-based transfection reagent.

Further provided herein are methods of regulating carbohydrate and lipid metabolism in a mammalian subject in need thereof, comprising the step of: Provided herein are isolated nucleic acids comprising a translatable region and at least two different nucleoside modifications, wherein the nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid.

In certain embodiments the mRNA does not substantially induce an innate immune response of a cell into which the mRNA is introduced. In certain embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridinone ribonucleoside, 5-aza-uridine, 2-thioaza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethylthio-uridine, 1-taurinomethylthio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thiomethyl-pseudouridine, 2-thiomethyl-pseudouridine, 1-methyldeaza-pseudouridine, 2-thiomethyldeaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxythio-uridine, 4-methoxy-pseudouridine, and 4-methoxythio-pseudouridine.

In certain embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thiomethyl-cytidine, 4-thio-pseudoisocytidine, 4-thiomethyl-pseudoisocytidine, 4-thiomethyldeaza-pseudoisocytidine, 1-methyldeaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-azathio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxymethyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxymethyl-pseudoisocytidine.

In other embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deazaaza-adenine, 7-deazaaminopurine, 7-deazaazaaminopurine, 7-deaza-2,6-diaminopurine, 7-deazaaza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- cis-hydroxyisopentenyl adenosine, 2-methylthio-N6- cis-hydroxyisopentenyl adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In yet other embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deazaaza-guanosine, 6-thio-guanosine, 6-thiodeaza-guanosine, 6-thiodeazaaza-guanosine, 7-methyl-guanosine, 6-thiomethyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyloxo-guanosine, 1-methylthio-guanosine, N2-methylthio-guanosine, and N2,N2-dimethylthio-guanosine.

In some embodiments, nucleic acids are provided herein, wherein at least one of the two different nucleoside modifications are present in the translatable region. In some embodiments, nucleic acids are provided herein are capable of binding to at least one polypeptide that prevents or reduces an innate immune response of a cell into which the nucleic acid is introduced.

Further provided herein are isolated polypeptides produced by translation of the mRNAs described herein. Further provided herein are isolated complexes comprising a conjugate of a protein and a nucleic acid, comprising i a mRNA comprising a translatable region and at least two different nucleoside modifications; and ii one or more polypeptides bound to the mRNA in an amount effective to prevent or reduce an innate immune response of a cell into which the complex is introduced.

Further provided herein are isolated mRNAs comprising i a translatable region, ii at least two different nucleoside modifications, and iii a degradation domain.

Further provided herein are isolated mRNAs comprising i a translatable region and ii at least two different nucleoside modifications, wherein the translatable region encodes a polypeptide variant having an altered activity relative to a reference polypeptide. In certain embodiments, isolated mRNAs are provided, wherein the altered activity comprises an increased activity or wherein the altered activity comprises a decreased activity.

Further provided herein are non-enzymatically synthesized mRNAs comprising at least one nucleoside modification, optionally comprising a translatable region. In certain embodiments, the non-enzymatically synthesized mRNAs comprise at least two different nucleoside modifications. In certain embodiments, the non-enzymatically synthesized mRNAs are substantially not translatable. In certain embodiments, the non-enzymatically synthesized mRNAs are provided in an amount effective as a vaccine when administered to a mammalian subject.

Further provided herein are isolated nucleic acids comprising i a translatable region, ii at least one nucleoside modification, and iii at least one intronic nucleotide sequence capable of being excised from the nucleic acid. Further provided herein are libraries comprising a plurality of polynucleotides, wherein the polynucleotides individually comprise: In certain embodiments, libraries are provided, wherein the polypeptide comprises an antibody or functional portion thereof.

In certain embodiments, libraries are provided, wherein the polynucleotides comprise mRNA. Further provided herein are methods for treating or preventing a symptom of cystic fibrosis in a mammalian subject, comprising contacting a cell of the subject with the nucleic acid of claim 1, wherein the translatable region encodes a Cystic Fibrosis Transmembrane Conductance Regulator CFTR polypeptide, under conditions such that an effective amount of the CFTR polypeptide is present in the cell, thereby treating or preventing a symptom of cystic fibrosis in the subject.

In certain embodiments, the cell is an epithelial cell, an endothelial cell, or a mesothelial cell. In certain embodiments, the nucleic acid comprises an RNA molecule formulated for administration by inhalation. Further provided herein are methods for inducing an alteration in cell fate in a mammalian cell, comprising the steps of: Further provided herein are methods for enhancing protein product yield in a cell culture process, comprising the steps of: In certain embodiments, methods are provided, wherein the increased protein production efficiency comprises increased cell transfection.

In certain embodiments, the increased protein production efficiency comprises increased protein translation from the nucleic acid. In certain embodiments, the increased protein production efficiency comprises decreased nucleic acid degradation. In certain embodiments, the increased protein production efficiency comprises reduced innate immune response of the host cell. Each team has a designated leader and its own performance targets.

Team leaders also belong to a store team, and store-team leaders belong to a regional team. To do its job, every team has access to the kind of information—including sales and even salary figures—that most companies reserve for traditional managers. Companies vary widely in choosing which tasks teams are allowed to manage and which ones are best left to upper-level management only.

As you can see in Figure 1. A cross-functional team is designed to take advantage of the special expertise of members drawn from different functional areas of the company. When the Internal Revenue Service, for example, wanted to study the effects on employees of a major change in information systems, it created a cross-functional team composed of people from a wide range of departments.

The final study reflected expertise in such areas as job analysis, training, change management, industrial psychology, and ergonomics. Typically, team members include not only product designers, marketing specialists, and accountants but also sports-research experts, coaches, athletes, and even consumers.

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Committees and task forces, both of which are dedicated to specific issues or tasks, are often cross-functional teams. Problem-solving teams, which are created to study such issues as improving quality or reducing waste, may be either intradepartmental or cross- functional.

Technologies such as videoconferencing allow people to interact simultaneously and in real time, offering a number of advantages in conducting the business of a virtual team. Team size does not seem to be an obstacle when it comes to virtual-team meetings; in building the F Strike Fighter, U. You can view it online here: Not surprisingly, this is a fairly complex issue. Teams are most effective when the following factors are met: Members depend on each other. When team members rely on each other to get the job done, team productivity and efficiency tend to be high.

Members trust one another. Members work better together than individually. When team members perform better as a group than alone, collective performance exceeds individual performance. When each member is encouraged by other team members to do his or her best, collective results improve. Team members enjoy being on the team. Some of these factors may seem intuitive.

Group Cohesiveness The idea of group cohesiveness refers to the attractiveness of a team to its members. If a group is high in cohesiveness, membership is quite satisfying to its members.

US9447164B2 - Engineered nucleic acids and methods of use thereof - Google Patents

The bigger the team, the less satisfied members tend to be. When teams get too large, members find it harder to interact closely with other members; a few members tend to dominate team activities, and conflict becomes more likely. People usually get along better with people like themselves, and teams are generally more cohesive when members perceive fellow members as people who share their own attitudes and experience. When teams are successful, members are satisfied, and other people are more likely to be attracted to their teams.

The harder it is to get into a group, the happier the people who are already in it. Membership is valued more highly when there is motivation to achieve common goals and outperform other teams. Maintaining team focus on broad organizational goals is crucial. Such tendencies may also encourage a phenomenon known as groupthink—the tendency to conform to group pressure in making decisions, while failing to think critically or to consider outside influences.

Groupthink is often cited as a factor in the explosion of the space shuttle Challenger in January As members of workplace teams, they need motivation, and when motivation is low, so are effectiveness and productivity. The difficulty of maintaining a high level of motivation is the chief cause of frustration among members of teams.

What if, for example, half the members of a product-development team want to create a brand-new product and half want to improve an existing product? The entire team may get stuck on this point of contention for weeks or even months. Lack of cooperation between teams can also be problematic to an organization. Lack of managerial support. Failure of managers to delegate authority.