Compositions and methods related to therapeutic cell systems expressing exogenous rna

A method of manufacturing a population of reticulocytes that express an exogenous protein, comprising (a) providing a population of erythroid precursor cells, (b) culturing the population of erythroid precursor cells under differentiating conditions to provide a population of differentiating erythroid cells, (c) contacting the differentiating erythroid cells with an mRNA encoding the exogenous protein, wherein the improvement comprises:

• the contacting is performed when the population of differentiating erythroid cells is between 0.1 and 25% enucleated (e.g., between 0.1 and 20% enucleated, between 0.1 and 15% enucleated, between 0.1 and 12% enucleated, or between 0.1 and 10% enucleated).

An erythroid cell, e.g., an enucleated erythroid cell, comprising:

• an exogenous mRNA comprising a coding region operatively linked to a heterologous untranslated region (UTR), wherein the heterologous UTR comprises a regulatory element.

An erythroid cell, e.g., an enucleated erythroid cell, comprising an exogenous mRNA that comprises one or more chemically modified nucleotides of Table 1, one or more chemical backbone modifications of Table 2, one or more chemically modified caps of Table 3, or any combination thereof.

A method of producing an erythroid cell, e.g., enucleated erythroid cell, comprising:

• a) contacting an erythroid cell, e.g., a nucleated erythroid cell, with an exogenous mRNA comprising a coding region operatively linked to a heterologous UTR comprising a regulatory element, (e.g., isolated RNA or in vitro transcribed RNA), and
• b) maintaining the contacted erythroid cell under conditions suitable for uptake of the exogenous mRNA,
• thereby producing the erythroid cell, e.g., an enucleated erythroid cell.

A method of producing an exogenous protein in an enucleated erythroid cell:

• a) providing an erythroid cell, e.g., a nucleated erythroid cell, comprising an exogenous mRNA comprising a coding region operatively linked to a heterologous UTR comprising a regulatory element, (e.g., isolated RNA or in vitro transcribed RNA), and
• b) culturing the erythroid cell under conditions suitable for production of the exogenous protein,
• thereby producing the exogenous protein.

A method of producing an exogenous protein in an enucleated erythroid cell:

• a) providing an erythroid cell, e.g., a nucleated erythroid cell, comprising one or more chemically modified nucleotides of Table 1, one or more chemical backbone modifications of Table 2, one or more chemically modified caps of Table 3, or any combination thereof, and
• b) culturing the erythroid cell under conditions suitable for production of the exogenous protein,
• thereby producing the exogenous protein.

A method of providing a subject with an exogenous protein, providing a subject with an enucleated erythroid cell which can produce an exogenous protein, or treating a subject, comprising administering to the subject:

• an erythroid cell, e.g., a nucleated erythroid cell, comprising an exogenous mRNA comprising a coding region operatively linked to a heterologous UTR comprising a regulatory element, (e.g., isolated RNA or in vitro transcribed RNA),
• thereby providing a subject with an exogenous protein, providing a subject with an enucleated erythroid cell which can produce an exogenous protein, or treating a subject.

A method of providing a subject with an exogenous protein, providing a subject with an enucleated erythroid cell which can produce an exogenous protein, or treating a subject, comprising administering to the subject:

• an erythroid cell, e.g., a nucleated erythroid cell, comprising an exogenous mRNA comprising one or more chemically modified nucleotides of Table 1, one or more chemical backbone modifications of Table 2, one or more chemically modified caps of Table 3, or any combination thereof,
• thereby providing a subject with an exogenous protein, providing a subject with an enucleated erythroid cell which can produce an exogenous protein, or treating a subject.

A method of evaluating an erythroid cell, e.g., enucleated erythroid cell (or a batch of such cells) comprising:

• a) providing an erythroid cell, e.g., a nucleated erythroid cell, comprising an exogenous mRNA comprising a coding region operatively linked to a heterologous UTR comprising a regulatory element (or a batch of such cells), and
• b) evaluating the erythroid cell, e.g., the nucleated erythroid cell (or batch of such cells) for a preselected parameter,
• thereby evaluating the erythroid cell, e.g., enucleated erythroid cell (or a batch of such cells).

A method of evaluating an erythroid cell, e.g., enucleated erythroid cell (or a batch of such cells) comprising:

• a) providing an erythroid cell, e.g., a nucleated erythroid cell, comprising one or more chemically modified nucleotides of Table 1, one or more chemical backbone modifications of Table 2, one or more chemically modified caps of Table 3, or any combination thereof, and
• b) evaluating the erythroid cell, e.g., the nucleated erythroid cell (or batch of such cells) for a preselected parameter,
• thereby evaluating the erythroid cell, e.g., enucleated erythroid cell (or a batch of such cells).

A method of making an erythroid cell comprising an mRNA that encodes an exogenous protein, comprising:

• providing a reaction mixture comprising an erythroid cell and an mRNA encoding the exogenous protein, under conditions which inhibit degradation of mRNA, e.g., by inclusion in the reaction mixture a ribonuclease inhibitor, and
• maintaining the reaction mixture under conditions that allow uptake of the mRNA by the erythroid cell,
• thereby making an erythroid cell comprising an mRNA encoding an exogenous protein.

A reaction mixture comprising:

• i) an erythroid cell, ii) an mRNA comprising an exogenous protein and iii) a ribonuclease inhibitor.

A method of assaying a reaction mixture comprising enucleated erythroid cells that comprise an exogenous protein for a ribonuclease inhibitor, comprising:

• providing a reaction mixture comprising enucleated erythroid cells that comprise an exogenous protein,
• assaying for the presence or level of a ribonuclease inhibitor, e.g., by ELISA, Western blot, or mass spectrometry, e.g., in an aliquot of the reaction mixture.

A method of making an erythroid cell comprising an mRNA that encodes an exogenous protein, comprising:

• providing a reaction mixture comprising an erythroid cell and an mRNA encoding the exogenous protein, under conditions which inhibit protein degradation, e.g., by inclusion in the reaction mixture a proteasome inhibitor, and
• maintaining the reaction mixture under conditions that allow uptake of the mRNA by the erythroid cell,
• thereby making an erythroid cell comprising an mRNA encoding an exogenous protein.

A reaction mixture comprising:

• i) an erythroid cell, ii) an mRNA comprising an exogenous protein and iii) a proteasome inhibitor.

A method of assaying a reaction mixture comprising enucleated erythroid cells that comprise an exogenous protein for a proteasome inhibitor, comprising:

• providing a reaction mixture comprising enucleated erythroid cells that comprise an exogenous protein,
• assaying for the presence or level of a proteasome inhibitor, e.g., by ELISA, Western blot, or mass spectrometry, e.g., in an aliquot of the reaction mixture.

A method of making an erythroid cell comprising an mRNA encoding a first exogenous protein and a second exogenous protein, comprising:

• a) providing an erythroid cell, e.g., in maturation phase, and
• b) contacting the erythroid cell with an mRNA encoding the first exogenous protein and a second mRNA encoding the second exogenous protein, under conditions that allow uptake of the first mRNA and second mRNA by the erythroid cell,
• thereby making an erythroid cell comprising the first mRNA and the second mRNA.

A population of erythroid cells wherein at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cells in the population express a first exogenous protein and a second exogenous protein, wherein the population was not made by contacting the cells with DNA encoding the first or second exogenous protein.

A method of producing a plurality of erythroid cells comprising a predetermined number of copies of an exogenous protein per cell, comprising contacting the population with a predetermined amount of mRNA encoding the exogenous protein, thereby making the erythroid cell comprising the predetermined amount of the exogenous protein.

A method of evaluating the amount of an exogenous protein in a sample of erythroid cells, e.g., enucleated erythroid cells comprising:

• providing a plurality of erythroid cells comprising a predetermined number of copies of an exogenous protein per cell, which was made by contacting the population with a predetermined amount of mRNA encoding the exogenous protein, and
• determining the amount of the exogenous protein in the plurality of erythroid cells.