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Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

Mueller C, Tang Q, Gruntman A, Blomenkamp K, Teckman J, Song L, Zamore PD, Flotte TR - Mol. Ther. (2012)

Bottom Line: In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed.These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs).This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Gene Therapy Center, UMass Medical School, Worcester, Massachusetts 01605, USA. chris.mueller@umassmed.edu

ABSTRACT
α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

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In vivo optimization of anti-AAT miRNA delivery within rAAV9 vectors. (a) Transgenic mice expressing the human PiZ allele were injected with 5 × 1011 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. (b) Quantitative RT-PCR for artificial miRNA was quantified from total RNA obtained from mouse livers. RT-PCR was used to assay for the presence of the three artificial anti-AAT miRNAs from mice receiving rAAV9-miRNA vectors. * ≤0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; miRNA, microRNA; rAAV, recombinant adeno-associated virus; RT-PCR, reverse transcriptase-PCR.
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fig3: In vivo optimization of anti-AAT miRNA delivery within rAAV9 vectors. (a) Transgenic mice expressing the human PiZ allele were injected with 5 × 1011 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. (b) Quantitative RT-PCR for artificial miRNA was quantified from total RNA obtained from mouse livers. RT-PCR was used to assay for the presence of the three artificial anti-AAT miRNAs from mice receiving rAAV9-miRNA vectors. * ≤0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; miRNA, microRNA; rAAV, recombinant adeno-associated virus; RT-PCR, reverse transcriptase-PCR.

Mentions: We next determined whether the location of the miRNAs within the expression cassette had any effect on their efficiency. We investigated whether cloning the three miRNAs between the 3′ end of the GFP gene and the polyA tail changed the kinetics of AAT knockdown and whether expressing the miRNAs from both locations (intron and polyA) which should double the amount of miRNAs being produced would in turn lead to a further enhancement of AAT knockdown. As in the previous experiments, Z-AAT transgenic mice received 5 × 1011 vps of rAAV9 vectors expressing the miRNAs either from the intron (intronCB-3XmiR), polyA region (PolyA-3XmiR) or at both locations at once (Double-6XmiR) (Figure 3a). By 4 weeks the PolyA-3XmiR and Double-6XmiR were more effective than the intronCB-3XmiR vector at decreasing serum Z-AAT levels by 85–70%, or in some cases with the Double-6XmiR vector by up to 95% (Figure 3a). Real-time quantitative reverse transcriptase-PCR (RT-PCR) analysis of liver tissue from these mice was performed to measure each of the three artificial vector derived miRs (910, 914, and 943). Both the PolyA-3XmiR and Double-6XmiR vectors produced about twice as many copies of each of the miRs than the intronCB-3XmiR (Figure 3b).


Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

Mueller C, Tang Q, Gruntman A, Blomenkamp K, Teckman J, Song L, Zamore PD, Flotte TR - Mol. Ther. (2012)

In vivo optimization of anti-AAT miRNA delivery within rAAV9 vectors. (a) Transgenic mice expressing the human PiZ allele were injected with 5 × 1011 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. (b) Quantitative RT-PCR for artificial miRNA was quantified from total RNA obtained from mouse livers. RT-PCR was used to assay for the presence of the three artificial anti-AAT miRNAs from mice receiving rAAV9-miRNA vectors. * ≤0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; miRNA, microRNA; rAAV, recombinant adeno-associated virus; RT-PCR, reverse transcriptase-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293602&req=5

fig3: In vivo optimization of anti-AAT miRNA delivery within rAAV9 vectors. (a) Transgenic mice expressing the human PiZ allele were injected with 5 × 1011 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. (b) Quantitative RT-PCR for artificial miRNA was quantified from total RNA obtained from mouse livers. RT-PCR was used to assay for the presence of the three artificial anti-AAT miRNAs from mice receiving rAAV9-miRNA vectors. * ≤0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; miRNA, microRNA; rAAV, recombinant adeno-associated virus; RT-PCR, reverse transcriptase-PCR.
Mentions: We next determined whether the location of the miRNAs within the expression cassette had any effect on their efficiency. We investigated whether cloning the three miRNAs between the 3′ end of the GFP gene and the polyA tail changed the kinetics of AAT knockdown and whether expressing the miRNAs from both locations (intron and polyA) which should double the amount of miRNAs being produced would in turn lead to a further enhancement of AAT knockdown. As in the previous experiments, Z-AAT transgenic mice received 5 × 1011 vps of rAAV9 vectors expressing the miRNAs either from the intron (intronCB-3XmiR), polyA region (PolyA-3XmiR) or at both locations at once (Double-6XmiR) (Figure 3a). By 4 weeks the PolyA-3XmiR and Double-6XmiR were more effective than the intronCB-3XmiR vector at decreasing serum Z-AAT levels by 85–70%, or in some cases with the Double-6XmiR vector by up to 95% (Figure 3a). Real-time quantitative reverse transcriptase-PCR (RT-PCR) analysis of liver tissue from these mice was performed to measure each of the three artificial vector derived miRs (910, 914, and 943). Both the PolyA-3XmiR and Double-6XmiR vectors produced about twice as many copies of each of the miRs than the intronCB-3XmiR (Figure 3b).

Bottom Line: In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed.These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs).This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Gene Therapy Center, UMass Medical School, Worcester, Massachusetts 01605, USA. chris.mueller@umassmed.edu

ABSTRACT
α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

Show MeSH
Related in: MedlinePlus