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Gene therapy is one of the strategies that may reduce or reverse progressive neurodegeneration in retinal neurodegenerative diseases. However, efficiently delivering transgenes to retinal ganglion cells (RGCs) remains hard to achieve. In this study, we innovatively investigated transduction efficiency of adeno-associated virus (AAV)-PHP.eB in murine RGCs by retro-orbital venous sinus injection. Five doses of AAV-PHP.eB-EGFP were retro-orbitally injected in venous sinus in adult C57/BL6J mice. Two weeks after administration, RGCs transduction efficiency was quantified by retinal flat-mounts and frozen section co-labeling with RGCs marker Rbpms. In addition, safety of this method was evaluated by RGCs survival rate and retinal morphology. To conform efficacy of this new method, AAV-PHP.eB-CNTF was administrated into mature mice through single retro-orbital venous injection after optic nerve crush injury to evaluate axonal elongation. Results indicated that AAV- PHP.eB readily crossed the blood-retina barrier and was able to transduce more than 90% of RGCs when total dose of virus reached 5 1010 vector genomes (vg). Moreover, this technique did not affect RGCs survival rate and retinal morphology. Furthermore, retro-orbital venous delivery of AAV-PHP.eB-CNTF effectively transduced RGCs, robustly promoted axonal regeneration after optic nerve crush injury. Thus, novel AAV-PHP.eB retro-orbital injection provides a minimally invasive and efficient route for transgene delivery in treatment of retinal neurodegenerative diseases.
Reverse-transcribed gene copies (retrocopies) have emerged as major sources of evolutionary novelty. MicroRNAs (miRNAs) are small and highly conserved RNA molecules that serve as key post-transcriptional regulators of gene expression. The origin and subsequent evolution of miRNAs have been addressed but not fully elucidated.
In this study, we performed a comprehensive investigation of miRNA origination through retroduplicated mRNA sequences (retro-miRs). We identified 17 retro-miRs that emerged from the mRNA retrocopies. Four of these retro-miRs had de novo origins within retrocopied sequences, while 13 retro-miRNAs were located within exon regions and duplicated along with their host mRNAs. We found that retro-miRs were primate-specific, including five retro-miRs conserved among all primates and two human-specific retro-miRs. All retro-miRs were expressed, with predicted and experimentally validated target genes except miR-10527. Notably, the target genes of retro-miRs are involved in key biological processes such as metabolic processes, cell signaling, and regulation of neurotransmitters in the central nervous system. Additionally, we found that these retro-miRs play a potential oncogenic role in cancer by targeting key cancer genes and are overexpressed in several cancer types, including liver hepatocellular carcinoma and stomach adenocarcinoma.
Our findings demonstrated that mRNA retrotransposition is a key mechanism for the generation of novel miRNAs (retro-miRs) in primates. These retro-miRs are expressed, conserved, have target genes with important cellular functions, and play important roles in cancer.
Remarkably, it has been shown that the genome incorporation of novel miRNAs is a pivotal and instrumental step in the evolution of organismal complexity [5]. It is clear that the expression of miRNAs modulates the transcript variability of their target coding genes, fine-tuning the protein molecules produced by the coding gene, and therefore, conferring robustness to cellular pathways and gene networks. Consequently, the robustness of gene expression may have contributed to a decrease in phenotypic variation, maintaining an invariant phenotype despite endogenous and exogenous perturbations. This increased the heritability of species-specif