Methods for assessing the presence or absence of replication competent virus

Abstract

Provided are methods of detecting replication competent retrovirus in a sample containing a cell transduced with a viral vector particle encoding a recombinant and/or heterologous molecule, e.g., heterologous gene product. The methods may include assessing transcription of one or more target genes, such as viral genes, that are expressed in a retrovirus but not expressed in the viral vector particle. Replication competent retrovirus may be determined to be present if the levels of RNA of the one or more target genes is higher than a reference value, which can be measured directly or indirectly, including from a positive control sample containing RNA from the respective target gene at a known level and/or at or above the limit of detection of the assay.

1 . A primer comprising an oligonucleotide comprising a sequence set forth in any of SEQ ID NOs: 1-24 or 35-41. 
     
     
         2 . The primer of  claim 1 , further comprising a fluorescent moiety or label. 
     
     
         3 . A kit comprising one or more primers according to  claim 1 . 
     
     
         4 . The kit of  claim 3 , further comprising one or more of nuclease-free water, a reverse transcriptase, a polymerase, deoxynucleotide triphosphates, a buffer, and a DNase. 
     
     
         5 . A primer set for amplification of a viral gene that is GaLV env, wherein the set comprises a forward and reverse primer, respectively, comprising the sequences set forth in SEQ ID NOs: 4-5. 
     
     
         6 . The primer set of  claim 5 , which further comprises a hydrolysis probe comprising the sequence set forth in SEQ ID NO: 6 
     
     
         7 . A primer set for amplification of a viral gene that is MMLV gag, wherein the set comprises a forward and reverse primer, respectively, comprising the sequences set forth in SEQ ID NOs. 16-17, SEQ ID Nos: 19-20 or SEQ ID Nos: 22-23. 
     
     
         8 . The primer set of  claim 7 , which further comprises a hydrolysis probe comprising the sequence set forth in SEQ ID NO: 18, 21, or 24. 
     
     
         9 . A primer set for amplification of a viral gene that is VSV-G env, wherein the set comprises a forward and reverse primer, respectively, comprising the sequences set forth in SEQ ID NOs: 35-36. 
     
     
         10 . The primer set of  claim 9 , which further comprises a hydrolysis probe comprising the sequence set forth in SEQ ID NO: 37. 
     
     
         11 . A primer set for amplification of a viral gene that is rev, wherein the set comprises a forward and reverse primer, respectively, comprising the sequences set forth in SEQ ID NOs: 38-39. 
     
     
         12 . The primer set of  claim 11 , which further comprises a hydrolysis probe comprising the sequence set forth in SEQ ID NO: 40. 
     
     
         13 . The primer set of  claim 5 , which further comprises one or more oligonucleotide primers for the amplification of β-actin, wherein the primers for the amplification of β-actin individually comprises a forward primer and reverse primer, respectively comprising the sequences set forth in any one of SEQ ID NOs: 1 and 2, 1 and 8, 13 and 14, 10 and 11, or 41 and 2. 
     
     
         14 . The primer set of  claim 7 , which further comprises one or more oligonucleotide primers for the amplification of β-actin, wherein the primers for the amplification of β-actin individually comprises a forward primer and reverse primer, respectively comprising the sequences set forth in any one of SEQ ID NOs: 1 and 2, 1 and 8, 13 and 14, 10 and 11, or 41 and 2. 
     
     
         15 . The primer set of  claim 9 , which further comprises one or more oligonucleotide primers for the amplification of β-actin, wherein the primers for the amplification of β-actin individually comprises a forward primer and reverse primer, respectively comprising the sequences set forth in any one of SEQ ID NOs: 1 and 2, 1 and 8, 13 and 14, 10 and 11, or 41 and 2. 
     
     
         16 . The primer set of  claim 11 , which further comprises one or more oligonucleotide primers for the amplification of β-actin, wherein the primers for the amplification of β-actin individually comprises a forward primer and reverse primer, respectively comprising the sequences set forth in any one of SEQ ID NOs: 1 and 2, 1 and 8, 13 and 14, 10 and 11, or 41 and 2. 
     
     
         17 . The primer set of  claim 13 , which further comprises a hydrolysis probe comprising a sequence set forth in SEQ ID NO: 3, 9, 7, 12, or 15. 
     
     
         18 . The primer set of  claim 14 , which further comprises a hydrolysis probe comprising a sequence set forth in SEQ ID NO: 3, 9, 7, 12, or 15. 
     
     
         19 . The primer set of  claim 15 , which further comprises a hydrolysis probe comprising a sequence set forth in SEQ ID NO: 3, 9, 7, 12, or 15. 
     
     
         20 . The primer set of  claim 16 , which further comprises a hydrolysis probe comprising a sequence set forth in SEQ ID NO: 3, 9, 7, 12, or 15.