Genetically engineered enucleated erythroid cells comprising a phenylalanine ammonia lyase receiver polypeptide

Abstract

A genetically engineered enucleated erythroid cell comprises a phenylalanine ammonia lyase receiver polypeptide that is not associated with the membrane of the erythroid cell, produced by genetically engineering the cell by viral transduction, RNA transfection, or RNA electroporation.

What is claimed is: 
     
         1 . A genetically engineered enucleated erythroid cell comprising a phenylalanine ammonia lyase (PAL) receiver polypeptide that is not associated with the membrane of the erythroid cell, produced by a process comprising:
 introducing, by viral transduction, RNA transfection, or RNA electroporation, a transgenic nucleic acid encoding the PAL receiver polypeptide into a nucleated erythroid cell; and   culturing the nucleated erythroid cell under conditions suitable for enucleation of the nucleated erythroid cell and for production of the PAL receiver polypeptide.   
     
     
         2 . The genetically engineered enucleated erythroid cell of  claim 1 , wherein the introducing step comprises viral transduction. 
     
     
         3 . The genetically engineered enucleated erythroid cell of  claim 1 , wherein the introducing step comprises RNA electroporation. 
     
     
         4 . The genetically engineered enucleated erythroid cell of  claim 1 , wherein the introducing step comprises utilizing one or more of: liposome mediated transfer, adenovirus, adenoassociated virus, herpes virus, a retroviral based vector, lipofection, and a lentiviral vector. 
     
     
         5 . The genetically engineered enucleated erythroid cell of  claim 4 , wherein the process comprises producing the lentivirus vector in a 293T cell line. 
     
     
         6 . The genetically engineered enucleated erythroid cell of  claim 1 , wherein the exogenous nucleic acid comprises DNA or RNA. 
     
     
         7 . The genetically engineered enucleated erythroid cell of  claim 6 , wherein the exogenous nucleic acid comprises RNA and the process further comprises producing the exogenous nucleic acid by in vitro transcription with T3 or T7 RNA polymerase. 
     
     
         8 . The genetically engineered enucleated erythroid cell of  claim 6 , wherein the exogenous nucleic acid comprises RNA and the process further comprises performing lipofection. 
     
     
         9 . The genetically engineered enucleated erythroid cell of  claim 1 , wherein the process further comprises electroporating the erythroid cell. 
     
     
         10 . The genetically engineered enucleated erythroid cell of  claim 1 , wherein the process comprises expanding the erythroid cells by at least 20,000-fold in culture. 
     
     
         11 . The genetically engineered enucleated erythroid cell of  claim 1 , which is in a population of enucleated erythroid cells wherein at least 20% of the cells comprise a PAL receiver polypeptide. 
     
     
         12 . The genetically engineered enucleated erythroid cell of  claim 1 , wherein the PAL receiver polypeptide is glycosylated. 
     
     
         13 . The genetically engineered enucleated erythroid cell of  claim 1 , which exhibits substantially the same osmotic membrane fragility as an isolated, unmodified, cultured or uncultured erythroid cell. 
     
     
         14 . The genetically engineered enucleated erythroid cell of  claim 1 , which comprises at least 50,000 copies of the PAL receiver polypeptide. 
     
     
         15 . The genetically engineered enucleated erythroid cell of  claim 1 , which comprises fetal hemoglobin.