Table 2.

Environmental Impact of Five Nucleotide Variants Identified in the TEG001 Insert

Mutation	Element	Impact
Position 415 C to G	junction of U3 to R region in 5′ LTR	The mutation occurred outside the promoter region and therefore does not affect promoter activity. The mutation does not lead to the introduction of an additional start codon and will not lead to a new open reading frame (ORF). The mutation does not lead to the introduction of an additional stop codon and as such has no influence on protein transcription. It is unlikely that this mutation affects packaging, integration, and transcription, and is therefore considered neutral to the biology of the retroviral vector. In conclusion, there is no altered or additional environmental risk as a result of this mutation.
Position 568 T to C	primer binding site	Because the mutations at positions 568 and 578 are both just after the 5′ LTR but in the primer binding site, their impact is considered together. The primer binding site is essential for replication because it is the site where the tRNA from the host cell binds as primer to initiate the inverse transcription and replication process. In the pMP71 vector, the primer binding site was derived from the murine embryonic stem cell virus-5′ untranslated region47 to provide a non-methylated 5′ UTR to improve stability of transgene expression in murine stem cells compared with the Moloney murine leukemia virus primer binding site.48 The mutations found may abolish this function in murine stem cells. The effect of this specific primer binding site on protein stability in human differentiated T cells is not known. High virus titers measured by transgene expression on indicator cells indicate that not only transcriptional activity was intact but also protein stability and expression were not affected. The mutations do not lead to the introduction of an additional start codon and as such will not lead to a new ORF. The mutations do not lead to the introduction of an additional stop codon and as such have no influence on protein transcription. It is unlikely that this mutation affects packaging, and integration resulted from their position. In conclusion, there is no altered or additional environmental risk as a result of these two mutations.
Position 578 T to C	primer binding site
Position 606 A +1C	after primer binding site and before packaging sequence (Ψ)	The insertion occurred outside the primer binding site and packaging sequence, and therefore it is unlikely that it affects packaging, integration, and transcription. The insertion does not lead to the introduction of an additional start codon and will not lead to a new ORF. The insertion does not lead to the introduction of an additional stop codon and as such has no influence on protein transcription. In conclusion, there is no altered or additional environmental risk as a result of this insertion.
Position 3552 C to G	junction of U3 to R region in 3′ LTR	This mutation is identical to the mutation at position 415; see above. In conclusion, there is no altered or additional environmental risk as a result of this mutation.