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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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Long-term in vivo silencing of human AAT by rAAV9 expressed miRNAs. Transgenic mice expressing the human PiZ allele were injected with 1 × 1012 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. (a) Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. Hepatocytes PiZ monomer versus polymer densitometry analysis at 90 days post-rAAV9 delivery. (b) Immunoblot for AAT after the monomer and polymer separation protocol from liver lysates of mice. The 52 kDa Z-AAT was from livers was processed and separated in into a monomer and polymer pool. (c) Densitometric analysis for the monomer and (d) polymer pools was performed using Image J software. Statistical significance was considered when *P ≤ 0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; CB-GFP, chicken β-actin–green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; miRNA, microRNA; rAAV, recombinant adeno-associated virus.
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fig4: Long-term in vivo silencing of human AAT by rAAV9 expressed miRNAs. Transgenic mice expressing the human PiZ allele were injected with 1 × 1012 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. (a) Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. Hepatocytes PiZ monomer versus polymer densitometry analysis at 90 days post-rAAV9 delivery. (b) Immunoblot for AAT after the monomer and polymer separation protocol from liver lysates of mice. The 52 kDa Z-AAT was from livers was processed and separated in into a monomer and polymer pool. (c) Densitometric analysis for the monomer and (d) polymer pools was performed using Image J software. Statistical significance was considered when *P ≤ 0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; CB-GFP, chicken β-actin–green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; miRNA, microRNA; rAAV, recombinant adeno-associated virus.

Mentions: Having achieved a short-term physiologically significant knockdown of more than 50% of Z-AAT serum protein levels, we determined whether this knockdown could be sustained for a longer period of time. The three vector constructs were delivered via the tail vein at a slightly higher titer of 1.0 × 1012 vps/mouse and serum Z-AAT was monitored weekly for 3 months. The onset of the effect of the three vector varied, with the Double-6XmiR vector achieving 90% knockdown 2 weeks after delivery, the PolyA-3XmiR reaching 90% by the third week, while the intronCB-3XmiR vector resulted in 50–65% knockdown for the first 7 weeks (Figure 4a, Supplementary Figure S1). In order to monitor if there was any liver toxicity associated with rAAV9 delivery or miRNA expression, we assessed serum alanine and aspartate aminotransferase concentrations 2 weeks after rAAV9 delivery. As observed in the (Supplementary Figure S2) rAAV9 delivery was not associated with increases in either alanine aminotransferase or aspartate aminotransferase levels, in fact the Double-6XmiR group had a significant reduction in alanine aminotransferase and aspartate aminotransferase serum concentrations and trends in the same direction were seen with the two other groups expressing anti-AAT miRs (Supplementary Figure S1). Although Z-AAT serum concentration rose slightly in animals in the Double-6XmiR and PolyA-3XmiR groups between week 7 and 13, all three vector groups showed stabilized values of serum Z-AAT at a sustained knockdown of 75% for the remainder of the study (Figure 4a). Further analysis of liver homogenates to determine whether this reduction was in the monomer or polymer pools of Z-AAT was performed on all groups. This modified western blot separates the monomer and polymer Z-AAT fractions under nondenaturing conditions after which they are denatured and quantitatively assessed by immunoblotting. As shown in Figure 4b the reduction in the monomer pool 3 months after miRNA is evident in all the groups, densitometric analysis of the bands shows highly significant differences in the PolyA-3XmiR and Double-6XmiR as compared to mice treated with a GFP vector control(Figure 4c). Polymer pool analysis revealed that there was no reduction in the Z-AAT polymers fraction even after 90 days (Figure 4d).


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)

Long-term in vivo silencing of human AAT by rAAV9 expressed miRNAs. Transgenic mice expressing the human PiZ allele were injected with 1 × 1012 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. (a) Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. Hepatocytes PiZ monomer versus polymer densitometry analysis at 90 days post-rAAV9 delivery. (b) Immunoblot for AAT after the monomer and polymer separation protocol from liver lysates of mice. The 52 kDa Z-AAT was from livers was processed and separated in into a monomer and polymer pool. (c) Densitometric analysis for the monomer and (d) polymer pools was performed using Image J software. Statistical significance was considered when *P ≤ 0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; CB-GFP, chicken β-actin–green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; miRNA, microRNA; rAAV, recombinant adeno-associated virus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Long-term in vivo silencing of human AAT by rAAV9 expressed miRNAs. Transgenic mice expressing the human PiZ allele were injected with 1 × 1012 virus particles or rAAV9 expressing miRNAs against AAT under the control of the hybrid chicken β-actin promoter via the tail vein. (a) Serums from each cohort were collected on a weekly basis and were used to assess Z-AAT concentration by ELISA. Hepatocytes PiZ monomer versus polymer densitometry analysis at 90 days post-rAAV9 delivery. (b) Immunoblot for AAT after the monomer and polymer separation protocol from liver lysates of mice. The 52 kDa Z-AAT was from livers was processed and separated in into a monomer and polymer pool. (c) Densitometric analysis for the monomer and (d) polymer pools was performed using Image J software. Statistical significance was considered when *P ≤ 0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; CB-GFP, chicken β-actin–green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; miRNA, microRNA; rAAV, recombinant adeno-associated virus.
Mentions: Having achieved a short-term physiologically significant knockdown of more than 50% of Z-AAT serum protein levels, we determined whether this knockdown could be sustained for a longer period of time. The three vector constructs were delivered via the tail vein at a slightly higher titer of 1.0 × 1012 vps/mouse and serum Z-AAT was monitored weekly for 3 months. The onset of the effect of the three vector varied, with the Double-6XmiR vector achieving 90% knockdown 2 weeks after delivery, the PolyA-3XmiR reaching 90% by the third week, while the intronCB-3XmiR vector resulted in 50–65% knockdown for the first 7 weeks (Figure 4a, Supplementary Figure S1). In order to monitor if there was any liver toxicity associated with rAAV9 delivery or miRNA expression, we assessed serum alanine and aspartate aminotransferase concentrations 2 weeks after rAAV9 delivery. As observed in the (Supplementary Figure S2) rAAV9 delivery was not associated with increases in either alanine aminotransferase or aspartate aminotransferase levels, in fact the Double-6XmiR group had a significant reduction in alanine aminotransferase and aspartate aminotransferase serum concentrations and trends in the same direction were seen with the two other groups expressing anti-AAT miRs (Supplementary Figure S1). Although Z-AAT serum concentration rose slightly in animals in the Double-6XmiR and PolyA-3XmiR groups between week 7 and 13, all three vector groups showed stabilized values of serum Z-AAT at a sustained knockdown of 75% for the remainder of the study (Figure 4a). Further analysis of liver homogenates to determine whether this reduction was in the monomer or polymer pools of Z-AAT was performed on all groups. This modified western blot separates the monomer and polymer Z-AAT fractions under nondenaturing conditions after which they are denatured and quantitatively assessed by immunoblotting. As shown in Figure 4b the reduction in the monomer pool 3 months after miRNA is evident in all the groups, densitometric analysis of the bands shows highly significant differences in the PolyA-3XmiR and Double-6XmiR as compared to mice treated with a GFP vector control(Figure 4c). Polymer pool analysis revealed that there was no reduction in the Z-AAT polymers fraction even after 90 days (Figure 4d).

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