Somatic hypermutation systems

Abstract

The present application relates to somatic hypermutation (SHM) systems and synthetic genes. Synthetic genes can be designed using computer-based approaches to increase or decrease susceptibility of a polynucleotide to somatic hypermutation. Genes of interest are inserted into the vectors and subjected to activation-induced cytidine deaminase to induce somatic hypermutation. Proteins or portions thereof encoded by the modified genes can be introduced into a SHM system for somatic hypermutation and proteins or portions thereof exhibiting a desired phenotype or function can be isolated for in vitro or in vivo diagnostic or therapeutic uses.

1 .- 41 . (canceled) 
     
     
         42 . A method for preparing a gene product having a desired property, which method comprises:
 (a) expressing a first polynucleotide sequence encoding a gene product of interest in a cell that expresses AID activity or can be induced to express AID activity via the addition of an inducing agent, whereupon expression of AID activity in the cell produces one or more mutated first polynucleotide sequences comprising one or more mutations;   (b) sequencing at least one mutated first polynucleotide sequence produced in step (a), and identifying one or more mutations in the one or more mutated first polynucleotide sequences,   (c) preparing one or more second polynucleotide sequences, each of which differs from the first polynucleotide sequence and contains one or more mutations of the one or more mutated first polynucleotide sequences identified in step (b),   (d) expressing the second polynucleotide sequences in a cell,   (e) identifying one or more second polynucleotide sequences that express a gene product having a desired property and differing from the gene product of interest, and   (f) optionally iteratively repeating steps (a)-(e), wherein an identified second polynucleotide sequence in step (e) becomes the first polynucleotide sequence in step (a).   
     
     
         43 . The method of  claim 42 , wherein the second polynucleotide sequence has an altered density of hot spot motifs as compared to the first polynucleotide sequence. 
     
     
         44 . The method of  claim 42 , wherein the second polynucleotide sequence exhibits an altered rate of AID mediated mutagenesis as compared to the first polynucleotide sequence. 
     
     
         45 . The method of  claim 42 , wherein the gene product of interest is an antibody or antigen-binding fragment thereof, a selectable marker, a reporter protein, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a structural protein, a toxin, a co-factor, or a transcription factor. 
     
     
         46 . The method of  claim 42 , wherein the gene product having a desired property and differing from the gene product of interest is an antibody or antigen-binding fragment thereof, a selectable marker, a reporter protein, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a structural protein, a toxin, a co-factor, or a transcription factor. 
     
     
         47 . The method of  claim 46 , wherein the gene product having a desired property and differing from the gene product of interest is an antibody or antigen-binding fragment thereof. 
     
     
         48 . The method of  claim 47 , wherein the desired property is selected from the group consisting of affinity, cross-reactivity, specificity, expression, stability, and solubility. 
     
     
         49 . The method of  claim 42 , wherein step (f) is carried out at least one time. 
     
     
         50 . An isolated gene product having a desired property, which gene product is produced by a method comprising:
 (a) expressing a first polynucleotide sequence encoding a gene product of interest in a cell that expresses AID activity or can be induced to express AID activity via the addition of an inducing agent, whereupon expression of AID activity in the cell produces one or more mutated first polynucleotide sequences comprising one or more mutations;   (b) sequencing at least one mutated first polynucleotide sequence produced in step (a), and identifying one or more mutations in the one or more mutated first polynucleotide sequences,   (c) preparing one or more second polynucleotide sequences, each of which differs from the first polynucleotide sequence and contains one or more mutations of the one or more mutated first polynucleotide sequences identified in step (b),   (d) expressing the second polynucleotide sequences in a cell,   (e) identifying one or more second polynucleotide sequences that express a gene product having a desired property and differing from the gene product of interest, and   (f) optionally iteratively repeating steps (a)-(e), wherein an identified second polynucleotide sequence in step (e) becomes the first polynucleotide sequence in step (a).   
     
     
         51 . The isolated gene product of  claim 50 , wherein the second polynucleotide sequence has an altered density of hot spot motifs as compared to the first polynucleotide sequence. 
     
     
         52 . The isolated gene product of  claim 50 , wherein the second polynucleotide sequence exhibits an altered rate of AID mediated mutagenesis as compared to the first polynucleotide sequence. 
     
     
         53 . The isolated gene product of  claim 50 , wherein the gene product of interest is an antibody or antigen-binding fragment thereof, a selectable marker, a reporter protein, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a structural protein, a toxin, a co-factor, or a transcription factor. 
     
     
         54 . The isolated gene product of  claim 50 , wherein the gene product having a desired property and differing from the gene product of interest is an antibody or antigen-binding fragment thereof, a selectable marker, a reporter protein, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a structural protein, a toxin, a co-factor, or a transcription factor. 
     
     
         55 . The isolated gene product of  claim 54 , wherein the gene product having a desired property and differing from the gene product of interest is an antibody or antigen-binding fragment thereof. 
     
     
         56 . The isolated gene product of  claim 55 , wherein the desired property is selected from the group consisting of affinity, cross-reactivity, specificity, expression, stability, and solubility. 
     
     
         57 . The isolated gene product of  claim 50 , wherein step (f) is carried out at least one time.