In the spleen, transgene expression (green) is excluded from hematopoietic cells, indicated by CD45 immunoreactivity (red).
Gene therapy has promised much, but delivered little. A major obstacle to long-term gene transfer to treat genetic disorders is the immune response to foreign transgenes. Brown et al. eliminate transgene expression in antigen-presenting cells using endogenous microRNA (miRNA) to produce prolonged gene transfer, as reported in a recent article in Nature Medicine.
miRNA is transcribed from DNA and is digested into single strands of 21–25 nucleotides by endogenous RNases. Mature miRNA binds antisense target sequences in mRNA, commonly located in 3' untranslated regions. Once bound, the miRNA recruits an RNA-induced silencing complex that prevents translation and may also induce degradation of the mRNA.
Antigen-presenting cells, like most immune cells, are derived from hematopoietic stem cells. The authors inserted tandem repeats of sequences complementary to mir-142, an miRNA only expressed in hematopoietic cells, into the 3' untranslated region of lentiviral transgenes encoding green fluorescent protein (GFP). Hematopoietic cell lines, including U937 monocytes and human dendritic cells, containing the transgene showed reduced GFP expression relative to those containing transgenes lacking miRNA recognition sites or transgenes with recognition sites for other miRNAs. In contrast, a nonhematopoietic cell line showed similar GFP expression regardless of the GFP transgene used.
To investigate the mechanism of miRNA-induced reduction of transgene expression, the authors constructed a lentiviral transgene with a bidirectional promoter that drove expression of a reporter gene as well as the GFP transgene containing mir-142 recognition sites. U937 cells expressed the reporter gene, but did not express GFP, suggesting that mir-142 reduced transgene expression post-transcriptionally. The authors also found reduced expression of transgene transcripts that contained mir-142 recognition sites relative to those that did not using quantitative RT-PCR and northern blots. These data suggest that mir-142 degraded the mRNA of complementary transgenes.
The authors administered transgenes to adult immunocompetent mice by tail-vein injection. Mice administered transgenes lacking mir-142 recognition sequences expressed GFP in liver hepatocytes and in some hematopoietic cells of the spleen 5 days after transgene delivery. GFP expression was no longer detectable on day 14, suggesting that the immune system cleared transgene expression. In contrast, mice administered the GFP transgenes containing mir-142 recognition sequences showed GFP expression in the liver for at least 120 days. In these mice, GFP expression in the liver did not overlap with the hematopoietic markers CD8 and F4/80, which are expressed in T-cells and monocytic Kupffer cells, respectively. The authors observed GFP expression in fibroblasts, but not hematopoietic cells, in the spleen.
Therefore, the use of miRNA to limit immune response to transgenes shows great potential to improve gene therapy. This is surprising considering that the endogenous targets for many miRNAs, including mir-142, are unknown.