Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

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.

Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin–related liver disease in mice

“…It will be interesting to see whether similar effects can be achieved in patients with PiZZ at acceptable doses. Treatment with patient-derived induced pluripotent stem cells combined with zinc finger-mediated homologous recombination and in vitro differentiation (50), treatment with siRNA or microRNA to reduce Z protein production (34,51), inhibition of Z protein aggregation by targeting β-sheet A (52), and use of chaperones (53) have all been tested in cell-based or animal models. However, the practical utility of these approaches in patients with a chronic disease like AAT-related liver disease needs to be established.…”

Long-Term Rescue of Retinal Structure and Function by Rhodopsin RNA Replacement with a Single Adeno-Associated Viral Vector in P23HRHOTransgenic Mice

“…For therapy, RNA blockade must be combined with replacement genes containing ''silent'' sequence changes making the resultant mRNA resistant to cleavage. This approach has been taken for treatment of liver disease associated with the PiZ allele of a 1 -antitrypsin (Li et al, 2011;Mueller et al, 2012). O'Reilly and colleagues (2007) used a single adeno-associated viral (AAV) vector to deliver a small interfering RNA (siRNA) and a resistant RHO gene to P23H transgenic mice.…”

Neuron-specific Sumo1–3 knockdown in mice impairs episodic and fear memories

“…27 Moreover, Pol II promoter-mediated expression of miRNAs is believed to be better tolerated than Pol III promoter-based shRNAs. 28,29 Another advantage of this system is the ability to express multiple designed miRNAs from a single construct to silence expression of several genes concurrently, or to increase silencing efficacy, as demonstrated in cell culture studies. 27,30,31 In the present experiment, we provided further evidence of the flexibility of this system by advancing from cell cultures to animals and illustrating that expression of multiple genes can be simultaneously silenced in vivo from 1 transgene driven by a Pol II promoter.…”