Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver

Abstract

Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here we investigated if incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3′ untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that 5 copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and β-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23–fold in the liver. However, gene expression in other tissues including the heart was not inhibited. Similarly, we inserted 4 copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3′ UTR of the AAV-luciferase vector. We wished to see if they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly due to promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that live-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in non-hematopoietic tissues resulted from promoter shut-off.

Persistence, Localization, and External Control of Transgene Expression After Single Injection of Adeno-Associated Virus into Injured Joints

“…The transgene expression from the soft tissues was limited for both OCD-and ACLT-injured joints. This can also be due to other reasons, such as increased volume of synovial fluid in injured joints diluting the intraarticular viral concentrations, injury-activated macrophages clearing up the virus prior to effective transduction, or the viral gene promoter shutting off in response to the injury (Qiao et al, 2011).…”

Endothelial transcription factor KLF2 negatively regulates liver regeneration via induction of activin A

“…29. In detail, the spleen focus forming virus (SFFV) promoter of the pSEW (30) transfer vector plasmid was exchanged by the endothelial-cell-specific CD31 promoter and two triple miRNA-detargeting sequences matching miRNA142-3P (31) and miRNA122 target sites (29,32) were inserted 3′ to the expression cassette (Fig. S1).…”

K137R Mutation on Adeno-Associated Viral Capsids Had Minimal Effect on Enhancing Gene DeliveryIn Vivo

“…K-R mutant and corresponding original AAV vectors (2 · 10 11 vg/mouse) were injected into 3-month-old C57BL/6 mice via tail vein injection. This time the less vector particles were utilized to rule out gene expression saturation issue, and the treated mice were euthanized earlier to avoid the concern of CMV promoter shutting off in the liver because of longer gene expression time (Qiao et al, 2011). The treated mice were euthanized 1 week postinjection, and various tissues were cryo-preserved.…”