Methods for modifying the growth rate of a cell

Abstract

Genetically modified cells with at least one codon substituted to a synonymous codon, and with modified replicative fitness as compared to the unmodified cell, wherein a slower translating synonymous codon increases replicative fitness and a faster translating codon decreased replicative fitness are provided. Further, vaccine composition comprising those cells as well as methods for modifying replicative fitness of a cell are provided.

1 . A method of modifying replicative fitness in a cell, the method comprising modifying ribosome density upstream of a ribosome backup on at least one translating sequence in said cell, wherein increasing ribosome density upstream of a ribosome backup decreases replicative fitness and decreasing ribosome density upstream of a ribosome backup increased replicative fitness, thereby modifying replicative fitness in a cell. 
     
     
         2 . The method of  claim 1 , wherein said ribosome backup comprises a region upstream of said backup with a ribosome density at least 5% higher than a ribosome density downstream of said backup. 
     
     
         3 . The method of  claim 1 , wherein said ribosome backup comprises a slowly translating codon. 
     
     
         4 . The method of  claim 3 , wherein said slowly translating codon translates at a slower rate than the average translational rate of codons 11 to 50 of said translating sequence. 
     
     
         5 . The method of  claim 3 , wherein said slowly translating codon is downstream of codon 50 from a translational start site of said translating sequence. 
     
     
         6 . The method of  claim 1 , wherein said modifying ribosome density does not substantially decrease the translation efficiency of said translating sequence. 
     
     
         7 . The method of  claim 6 , wherein a substantial decrease is a decrease of at least 5%. 
     
     
         8 . The method of  claim 6 , wherein said modifying ribosome density comprises substituting a codon with a different codon and further comprises determining whether said substituting would reduce translation efficiency below said threshold, and wherein said determining comprises any one of a Forward Gene Minimization (FGM), Backward Gene Minimization (BGM) and Greedy Gene Minimization (GGM) algorithm. 
     
     
         9 . The method of  claim 1 , wherein modifying ribosome density upstream of a ribosome backup is decreasing ribosome density upstream of a ribosome backup and comprises substituting a codon upstream of said ribosome backup with a slower translating codon. 
     
     
         10 . The method of  claim 9 , wherein said slower translating codon is a synonymous codon. 
     
     
         11 . The method of  claim 9 , wherein said substituting a codon comprises substituting a codon that most greatly increases a free ribosome pool in said cell. 
     
     
         12 . The method of  claim 1 , wherein modifying ribosome density upstream of a ribosome backup is increasing ribosome density upstream of a ribosome backup and comprises substituting a codon upstream of said ribosome backup with a faster translating codon. 
     
     
         13 . The method of  claim 12 , wherein said faster translating codon is a synonymous codon. 
     
     
         14 . The method of  claim 12 , wherein said substituting a codon comprises substituting a codon that most greatly decreases a free ribosome pool in said cell. 
     
     
         15 . The method of  claim 1 , wherein said modifying ribosome density comprises modifying a free ribosome pool in said cell. 
     
     
         16 . The method of  claim 15 , wherein increasing said free ribosome pool increases replicative fitness and decreasing said free ribosome pool decreases replicative fitness. 
     
     
         17 . The method of  claim 15 , comprising modifying ribosome density upstream of a ribosome backup on a plurality of translating sequences in said cell wherein said free ribosome pool in said cell is increased or decreased by at least 10%. 
     
     
         18 . The method of  claim 17 , wherein said plurality is at least 10 translating sequences. 
     
     
         19 . The method of  claim 15 , wherein modifying ribosome density upstream of a ribosome backup comprises substituting a plurality of codons upstream of said ribosome backup, wherein substituting faster translating codons increases ribosome density and substituting slower translating codons decreases ribosome density. 
     
     
         20 . The method of  claim 19 , wherein said plurality of codons is at least 5 codons.