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

Qiao et al. (2010). Gene Ther DOI: 10.1038/gt.2010.157

Leaky liver expression from AAV vectors remained a problem even with tissue-specific promoters; this paper added miR-122 target sequences to the 3′ UTR of AAV cassettes. Five copies cut liver luciferase about 50-fold and LacZ about 70-fold in AAV9-treated mice without lowering skeletal muscle expression.

Parts used

miR-122ts miR-122 AAV9 Serotype bGH PolyA Signal

Key findings

In hydrodynamically injected plasmid, 3 copies of miR-122T in the 3′ UTR of firefly luciferase in the AAV-CB-Luc cassette reduce mouse liver luciferase 10- to 50-fold and 5 copies reduce it 20- to 100-fold, relative to the cassette without miR-122T. The same plasmids give no difference in luciferase in 293 cells, which do not express miR-122.
Five copies of miR-122T (5xmiR-122T) in the 3′ UTR of AAV9-CB-Luc reduce liver luciferase about 50-fold three weeks after intravenous injection of 1×10^12 vg into 6-week-old ICR mice (p < 0.01, n = 5), with no difference in tibialis anterior muscle.
The en-MHC-TNT promoter (mouse α-myosin heavy chain enhancer plus the troponin T promoter) is strong in heart but leaky in liver, where the parental TNT promoter is not. Five copies of miR-122T in the 3′ UTR of AAV9-en-MHC-TNT-LacZ reduce liver LacZ about 70-fold three weeks after 3×10^12 vg intravenous injection in ICR mice, leaving only a few LacZ-positive liver cells, and no LacZ is detected in skeletal muscle with either vector.
Five copies of miR-122T in AAV9 vectors raise cardiac muscle expression: luciferase is significantly higher in AAV9-CB-Luc-5xmiR-122T hearts (p < 0.05, n = 5) and LacZ is higher in AAV9-en-MHC-TNT-LacZ-5xmiR-122T hearts (p = 0.06, n = 4), while skeletal muscle is unchanged. The mechanism is not established; the authors suggest increased mRNA stability or an enhancer-like effect of the target copies in cardiomyocytes.
A perfectly complementary miR-122T reduces expression by mRNA degradation: 5xmiR-122T in the 3′ UTR of the pAAV-CAG-dog-MPRO-Ig plasmid lowers transgene mRNA 96% in mouse liver 24 hours after hydrodynamic injection, measured by RT-PCR across a 900-bp intron. The same 5 copies reduce mRNA of a secretable myostatin propeptide from an AAV plasmid 23-fold in liver.
Each miR-122T copy is a sequence fully complementary to mature miR-122. Copies are separated by short linkers (TCAC and GATATC) in a synthesized block and cloned into NheI and SacI sites in the 3′ UTR of the AAV expression cassette, upstream of the BGH polyA signal.
Four copies of miR-142-3pT or miR-142-5pT in the 3′ UTR of AAV9-CB-Luc (2×10^12 vg per mouse, intravenous) do not prevent the decline of luciferase expression in liver and muscle at three months, and AAV9 vectors with or without miR-142T give only background luciferase in circulating mononuclear cells at 36 and 96 hours. Hematopoietic miR-142 target sequences therefore do not prolong AAV9 transgene expression in non-hematopoietic tissues in mice.
Luciferase expression from AAV9-CB-Luc falls 10- to 100-fold between two weeks and three months after 2×10^12 vg intravenous injection in ICR mice while vector copy number stays constant (10–80 copies per liver cell, 3–11 per muscle nucleus), with no CD4+ T-cell infiltration or abnormal histology. The loss therefore reflects promoter shut-off rather than immune clearance of transduced cells.

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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 liver-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.