Limits...
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.

Show MeSH

Related in: MedlinePlus

In vivo knockdown of Z-AAT with simultaneous augmentation of M-AAT after rAAV9 dual-function vector delivery. Transgenic mice expressing the human PiZ allele were injected with 1 × 1012 virus particles or rAAV9 expressing miRNAs against AAT and a de-targeted cMyc tagged wild-type M-AAT cDNA 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. Serum Z-AAT levels at each timepoint are expressed as a percent knockdown as compared to the rAAV9-GFP cohort by using a Z-specific AAT ELISA and M-AAT levels are calculated by using an ELISA to quantify the cMYC tag on the wild-type protein. Data are expressed as group means ±SEM (n = 6). Statistical significance was set at * ≤0.05 as determined by a two-way ANOVA comparing each treatment group to the control rAAV-GFP group. Blue dashed line in the upper panel indicates therapeutic levels of wild-type PiM AAT as determined by the FDA and therapeutic knockdown of PiZ protein in the lower panel as determined by achieving levels expected in a PiZ heterozygous status. Total RNA from mouse livers was used to assay for the presence of the either (b) Z-AAT mRNA or (c) M-AAT mRNA by qRT-PCR. Data are expressed as group means ±SEM (n = 6). * ≤0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; ANOVA, analysis of variance; cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; CB-GFP, chicken β-actin–green fluorescent protein; FDA, Food and Drug Administration; mRNA, messenger RNA; miRNA, microRNA; ND, not detected; qRT-PCR, quantitative reverse transcriptase-PCR; rAAV, recombinant adeno-associated virus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3293602&req=5

fig5: In vivo knockdown of Z-AAT with simultaneous augmentation of M-AAT after rAAV9 dual-function vector delivery. Transgenic mice expressing the human PiZ allele were injected with 1 × 1012 virus particles or rAAV9 expressing miRNAs against AAT and a de-targeted cMyc tagged wild-type M-AAT cDNA 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. Serum Z-AAT levels at each timepoint are expressed as a percent knockdown as compared to the rAAV9-GFP cohort by using a Z-specific AAT ELISA and M-AAT levels are calculated by using an ELISA to quantify the cMYC tag on the wild-type protein. Data are expressed as group means ±SEM (n = 6). Statistical significance was set at * ≤0.05 as determined by a two-way ANOVA comparing each treatment group to the control rAAV-GFP group. Blue dashed line in the upper panel indicates therapeutic levels of wild-type PiM AAT as determined by the FDA and therapeutic knockdown of PiZ protein in the lower panel as determined by achieving levels expected in a PiZ heterozygous status. Total RNA from mouse livers was used to assay for the presence of the either (b) Z-AAT mRNA or (c) M-AAT mRNA by qRT-PCR. Data are expressed as group means ±SEM (n = 6). * ≤0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; ANOVA, analysis of variance; cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; CB-GFP, chicken β-actin–green fluorescent protein; FDA, Food and Drug Administration; mRNA, messenger RNA; miRNA, microRNA; ND, not detected; qRT-PCR, quantitative reverse transcriptase-PCR; rAAV, recombinant adeno-associated virus.

Mentions: We next used both of these miRNA configurations to test dual-function vectors in vivo. Three cohorts of seven mice each were given 1.0 × 1012 vp with either a GFP control, Double-6XmiR-CB-AAT or a PolyA-3XmiR-CB-AAT rAAV9 vectors. Serum was harvested from the mice on a weekly basis for 13 weeks and was analyzed for Z-AAT serum concentrations with a PiZ specific ELISA and for M-AAT concentrations with an ELISA detecting the cMYC tag on the M-AAT complementary DNA. Changes in serum Z-AAT were comparable to those in previous experiments, with a sustained knockdown around 75–85% for both vectors (Figure 5a, bottom panel). A more rapid knockdown was seen with the Double-6XmiR vector but the PolyA-3XmiR vector achieved similar values by the fourth week. As serum Z-AAT decreased a concomitant rise in circulating M-AAT was observed in mice receiving the dual-function vectors (Figure 5a, upper panel). Although the amount of knockdown for both vectors was similar for 4 weeks after delivery, the production of M-AAT was significantly different. The PolyA-3XmiR-CB-AAT vector produced 8–10 times more M-AAT than did the Double-6XmiR-CB-AAT vector. Liver RNA was extracted from these mice at the end of the study to quantify the mRNA levels of Z-AAT and M-AAT. As expected there was a precipitous decrease in Z-AAT mRNA in both cohorts of mice receiving vectors with miRNAs as compared to mice receiving a rAAV9-CB-GFP control. Quantitative RT-PCR for M-AAT was also performed to verify production of M-AAT at the RNA level and to determine if the difference in M-AAT production between dual-function vectors was related to mRNA transcription. Despite the clear difference in M-AAT serum protein, there was no statistically significant difference in the M-AAT mRNA between the two groups (Figure 5c). This suggests that mRNA translation of M-AAT but not level of transcription may be affected in the Double-6XmiR-CB-AAT group.


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 knockdown of Z-AAT with simultaneous augmentation of M-AAT after rAAV9 dual-function vector delivery. Transgenic mice expressing the human PiZ allele were injected with 1 × 1012 virus particles or rAAV9 expressing miRNAs against AAT and a de-targeted cMyc tagged wild-type M-AAT cDNA 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. Serum Z-AAT levels at each timepoint are expressed as a percent knockdown as compared to the rAAV9-GFP cohort by using a Z-specific AAT ELISA and M-AAT levels are calculated by using an ELISA to quantify the cMYC tag on the wild-type protein. Data are expressed as group means ±SEM (n = 6). Statistical significance was set at * ≤0.05 as determined by a two-way ANOVA comparing each treatment group to the control rAAV-GFP group. Blue dashed line in the upper panel indicates therapeutic levels of wild-type PiM AAT as determined by the FDA and therapeutic knockdown of PiZ protein in the lower panel as determined by achieving levels expected in a PiZ heterozygous status. Total RNA from mouse livers was used to assay for the presence of the either (b) Z-AAT mRNA or (c) M-AAT mRNA by qRT-PCR. Data are expressed as group means ±SEM (n = 6). * ≤0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; ANOVA, analysis of variance; cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; CB-GFP, chicken β-actin–green fluorescent protein; FDA, Food and Drug Administration; mRNA, messenger RNA; miRNA, microRNA; ND, not detected; qRT-PCR, quantitative reverse transcriptase-PCR; rAAV, recombinant adeno-associated virus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: In vivo knockdown of Z-AAT with simultaneous augmentation of M-AAT after rAAV9 dual-function vector delivery. Transgenic mice expressing the human PiZ allele were injected with 1 × 1012 virus particles or rAAV9 expressing miRNAs against AAT and a de-targeted cMyc tagged wild-type M-AAT cDNA 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. Serum Z-AAT levels at each timepoint are expressed as a percent knockdown as compared to the rAAV9-GFP cohort by using a Z-specific AAT ELISA and M-AAT levels are calculated by using an ELISA to quantify the cMYC tag on the wild-type protein. Data are expressed as group means ±SEM (n = 6). Statistical significance was set at * ≤0.05 as determined by a two-way ANOVA comparing each treatment group to the control rAAV-GFP group. Blue dashed line in the upper panel indicates therapeutic levels of wild-type PiM AAT as determined by the FDA and therapeutic knockdown of PiZ protein in the lower panel as determined by achieving levels expected in a PiZ heterozygous status. Total RNA from mouse livers was used to assay for the presence of the either (b) Z-AAT mRNA or (c) M-AAT mRNA by qRT-PCR. Data are expressed as group means ±SEM (n = 6). * ≤0.05 as determined by a two-way unpaired Student's t-test. AAT, α-1 antitrypsin; ANOVA, analysis of variance; cDNA, complementary DNA; ELISA, enzyme-linked immunosorbent assay; CB-GFP, chicken β-actin–green fluorescent protein; FDA, Food and Drug Administration; mRNA, messenger RNA; miRNA, microRNA; ND, not detected; qRT-PCR, quantitative reverse transcriptase-PCR; rAAV, recombinant adeno-associated virus.
Mentions: We next used both of these miRNA configurations to test dual-function vectors in vivo. Three cohorts of seven mice each were given 1.0 × 1012 vp with either a GFP control, Double-6XmiR-CB-AAT or a PolyA-3XmiR-CB-AAT rAAV9 vectors. Serum was harvested from the mice on a weekly basis for 13 weeks and was analyzed for Z-AAT serum concentrations with a PiZ specific ELISA and for M-AAT concentrations with an ELISA detecting the cMYC tag on the M-AAT complementary DNA. Changes in serum Z-AAT were comparable to those in previous experiments, with a sustained knockdown around 75–85% for both vectors (Figure 5a, bottom panel). A more rapid knockdown was seen with the Double-6XmiR vector but the PolyA-3XmiR vector achieved similar values by the fourth week. As serum Z-AAT decreased a concomitant rise in circulating M-AAT was observed in mice receiving the dual-function vectors (Figure 5a, upper panel). Although the amount of knockdown for both vectors was similar for 4 weeks after delivery, the production of M-AAT was significantly different. The PolyA-3XmiR-CB-AAT vector produced 8–10 times more M-AAT than did the Double-6XmiR-CB-AAT vector. Liver RNA was extracted from these mice at the end of the study to quantify the mRNA levels of Z-AAT and M-AAT. As expected there was a precipitous decrease in Z-AAT mRNA in both cohorts of mice receiving vectors with miRNAs as compared to mice receiving a rAAV9-CB-GFP control. Quantitative RT-PCR for M-AAT was also performed to verify production of M-AAT at the RNA level and to determine if the difference in M-AAT production between dual-function vectors was related to mRNA transcription. Despite the clear difference in M-AAT serum protein, there was no statistically significant difference in the M-AAT mRNA between the two groups (Figure 5c). This suggests that mRNA translation of M-AAT but not level of transcription may be affected in the Double-6XmiR-CB-AAT group.

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