Self-amplifying RNA (saRNA) is an extremely promising platform because it can potentially produce more protein for less RNA. We used a ‘sort and sequence’ approach to identify host cell factors associated with transgene expression from saRNA; the hypothesis was that cells with different expression levels would have different transcriptomes. We tested this in CDK4/hTERT immortalised human muscle cells transfected with VEEV derived saRNA encoding GFP. Cells with the highest expression levels had very high levels of transgene mRNA (5-10% total reads); the cells sorted with low and negative levels of GFP protein also had detectable levels of both VEEV and GFP RNA. To understand host responses, we performed RNASeq. Differentially expressed gene (DEG) patterns varied with GFP expression; GFP high cells, had many more DEG, these were associated with protein synthesis and cell metabolism. Comparing profiles by an unsupervised approach revealed that negative cells expressed higher levels of cell intrinsic immunity genes such as IFIT1, MX1, TLR3 and MyD88. To explore the role of interferon, cells were treated with the Jak inhibitor Ruxolitinib, this reduced DEG number but differences between cells sorted by expression level remained. These studies demonstrate the complex interplay of factors, some immune related, affecting saRNA transgenes.