Enhanced Expression Of RNA Vectors

Abstract

Compositions and method for treating a folate-resistant disease in a subject are disclosed. The methods involve administering to the subject an effective amount of a composition containing a formate. For example, the method can be used to reducing the risk of neural tube defects during pregnancy. The method can also be used to treat other conditions normally treatable by folate supplementation.

1 . A synthetic RNA expression vector comprising a sequence encoding an expression product, the nucleic acid comprising at least one region in which the nucleotide composition has been modified such that the frequency of at least one of CpG and UpA dinucleotides is reduced relative to normal frequency. 
     
     
         2 .- 3 . (canceled) 
     
     
         4 . The synthetic RNA expression vector of  claim 1  which is a recombinant RNA viral vector. 
     
     
         5 . The synthetic RNA expression vector of  claim 1  in which the frequency of both CpG and UpA dinucleotides is reduced relative to normal frequency. 
     
     
         6 . The synthetic RNA expression vector of  claim 1  in which the frequency of CpG dinucleotides in the at least one region in which the nucleotide composition has been modified is reduced by at least 50%. 
     
     
         7 . The synthetic RNA expression vector of  claim 1  in which the frequency of UpA dinucleotides in the at least one region in which the nucleotide composition has been modified is reduced by at least 50%. 
     
     
         8 . (canceled) 
     
     
         9 . The synthetic RNA expression vector of  claim 1  in which the frequency of at least one of CpG and UpA dinucleotides in the at least one region in which the nucleotide composition has been modified such that it contains no CpG and/or UpA dinucleotides. 
     
     
         10 . The synthetic RNA expression vector of  claim 1  in which, considering the synthetic RNA expression vector as a whole, the frequency of at least one of CpG and UpA dinucleotides is reduced by at least 20%. 
     
     
         11 . (canceled) 
     
     
         12 . The synthetic RNA expression vector of  claim 1  in which the frequency ratio of the relevant dinucleotide is 0.4 or lower. 
     
     
         13 . The synthetic RNA expression vector of  claim 1  in which the region of nucleic acid with reduced frequency of CpG or UpA dinucleotides is in a sequence which encodes an expression product. 
     
     
         14 . (canceled) 
     
     
         15 . The synthetic RNA expression vector of  claim 1  in which substantially all non-constrained coding sequences of the vector have been modified to reduce the frequency of at least one of CpG and UpA dinucleotides. 
     
     
         16 . The synthetic RNA expression vector of  claim 1  in which substantially all of the coding regions of the synthetic RNA expression vector have been modified to have a reduced at least one of CpG and UpA dinucleotide frequency. 
     
     
         17 . The synthetic RNA expression vector of  claim 1  in which regions totaling at least 50% of the total length of the synthetic RNA expression vector have been modified to reduce the at least one of CpG and UpA dinucleotide frequency. 
     
     
         18 .- 22 . (canceled) 
     
     
         23 . The synthetic RNA expression vector of  claim 1  wherein the synthetic RNA expression vector comprises a recombinant single stranded RNA (ssRNA) virus genome. 
     
     
         24 . (canceled) 
     
     
         25 . The synthetic RNA expression vector of  claim 1  which comprises a RNA virus adapted for expression in a suitable expression system for the production of a virus vaccine. 
     
     
         26 .- 28 . (canceled) 
     
     
         29 . A virion comprising a synthetic RNA expression vector according to  claim 1 . 
     
     
         30 . (canceled) 
     
     
         31 . A host cell comprising a synthetic RNA expression vector according to  claim 1 . 
     
     
         32 .- 34 . (canceled) 
     
     
         35 . An RNA virus vaccine composition, the composition comprising a virion according to  claim 29 , which comprises a sequence encoding one or more antigens. 
     
     
         36 . A method of producing an expression product, the method comprising:
 providing a host cell comprising a synthetic RNA expression vector according to  claim 1 ;   incubating said cell under suitable conditions to induce expression from the vector; and   recovering the expression product.   
     
     
         37 . (canceled) 
     
     
         38 . The method of  claim 36  wherein the expression product is a virion. 
     
     
         39 . The method of  claim 36  wherein the method includes the following steps:
 providing an RNA sequence encoding an expression product; 
 altering the nucleotide composition of said sequence to reduce the frequency of at least one of CpG and UpA dinucleotides; and 
 introducing a synthetic RNA expression vector comprising said nucleic acid into a host cell. 
 
     
     
         40 . (canceled) 
     
     
         41 . The method of  claim 36  which is a method for production of a viral vaccine. 
     
     
         42 .- 44 . (canceled) 
     
     
         45 . A method of producing a synthetic RNA expression vector, the method comprising:
 providing a primary nucleotide sequence of interest;   identifying at least one region of said primary nucleotide sequence susceptible to modification to reduce the frequency of at least one of CpG and UpA dinucleotides;   identifying one or more sequence modifications in said at least one region which will reduce the frequency of at least one of CpG and UpA dinucleotides;   providing a modified nucleotide sequence comprising some or all of said sequence modifications;   producing a synthetic RNA expression vector comprising said modified sequence which has a reduced frequency of at least one of CpG and UpA dinucleotides compared to a corresponding synthetic RNA expression vector which comprises the primary nucleotide sequence.   
     
     
         46 .- 49 . (canceled)