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 .- 31 . (canceled) 
     
     
         32 . A method for preparing a gene product having a desired property, comprising:
 a) expressing in a population of cells at least one somatic hypermutation (SHM) resistant synthetic gene encoding a gene product, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding an unmodified gene product has been replaced by one or more second SHM motifs having a lower probability of SHM, the synthetic gene having a greater density of cold spots and/or a lower density of hot spots than the unmodified polynucleotide sequence, and wherein the population of cells express AID activity, or can be induced to express AID activity via the addition of an inducing agent; and   b) selecting a cell or cells within the population of cells which express a modified gene product having the desired property.   
     
     
         33 . The method of  claim 32 , optionally further comprising activating or inducing the expression of AID activity in the population of cells. 
     
     
         34 . The method of  claim 32 , further comprising establishing one or more clonal populations of cells from the cell or cells identified in (b). 
     
     
         35 . The method of  claim 32 , wherein the at least one SHM resistant synthetic gene encodes an enzyme involved in SHM, a synthetic selectable marker gene, or a synthetic reporter gene. 
     
     
         36 . The method of  claim 32 , wherein the population of cells further comprises a gene product encoded by a SHM susceptible synthetic gene, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding an unmodified gene product has been replaced by one or more second SHM motifs having a higher probability of SHM, the synthetic gene having a greater density of hot spot motifs and/or a lower density of cold spots than the unmodified polynucleotide sequence. 
     
     
         37 . A method for preparing a gene product having a desired property, comprising:
 a) preparing a somatic hypermutation (SHM) susceptible synthetic gene encoding a gene product, wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding an unmodified gene product has been replaced by one or more second SHM motifs having a higher probability of SHM, the synthetic gene having a greater density of hot spot motifs and/or a lower density of cold spots than the unmodified polynucleotide sequence,   b) expressing the SHM susceptible synthetic gene in a population of cells; wherein the population of cells expresses AID activity, or can be induced to express AID activity via the addition of an inducing agent; and   c) selecting a cell or cells within the population of cells which express a modified gene product having the desired property.   
     
     
         38 . The method of  claim 37 , optionally further comprising activating or inducing the expression of AID activity in the population of cells. 
     
     
         39 . The method of  claim 37 , further comprising establishing one or more clonal populations of cells from the cell or cells identified in (c). 
     
     
         40 . The method of  claim 37 , wherein the SHM susceptible synthetic gene encodes an antibody or antigen-binding fragment thereof, a neurotransmitter, a hormone, a cytokine, a chemokine, an enzyme, a receptor, a toxin, a co-factor, or a transcription factor. 
     
     
         41 . The method of  claim 37 , wherein the population of cells further comprises a gene product encoded by a SHM resistant synthetic gene wherein one or more first SHM motifs in an unmodified polynucleotide sequence encoding an unmodified gene product has been replaced by one or more second SHM motifs having a lower probability of SHM, the synthetic gene having a greater density of cold spots and/or a lower density of hot spots than the unmodified polynucleotide sequence.