title: Combination therapy utilizing shRNA knockdown and an optimized resistant transgene for rescue of diseases caused by misfolded proteins.
abstract: Molecular knockdown of disease proteins and restoration of wild-type activity represent a promising but challenging strategy for the treatment of diseases that result from the accumulation of misfolded proteins (i.e., Huntington disease, amyotrophic lateral sclerosis, and α-1 antitrypsin deficiency). In this study we used alpha-1 antitrypsin (AAT) deficiency with the piZZ mutant phenotype as a model system to evaluate the efficiency of gene-delivery approaches that both silence the piZZ transcript (e.g., shRNA) and restore circulating wild-type AAT expression from resistant codon-optimized AAT (AAT-opt) transgene cassette using adeno-associated virus (AAV) vector delivery. After systemic injection of a self-complimentary AAV serotype 8 (scAAV8) vector encoding shRNA in piZZ transgenic mice, both mutant AAT mRNA in the liver and defected serum protein level were inhibited by 95%, whereas liver pathology, as monitored by dPAS and fibrosis staining, reversed. To restore blood AAT levels in AAV8/shRNA-treated mice, several strategies to restore functional AAT levels were tested, including using AAV AAT-opt transgene cassettes targeted to muscle and liver, or combination vectors carrying piZZ shRNA and AAT-opt transgenes separately, or a single bicistronic AAV vector. With these molecular approaches, we observed over 90% knockdown of mutant AAT with a 13- to 30-fold increase of circulating wild-type AAT protein from the shRNA-resistant AAT-opt cassette. The molecular approaches applied in this study can simultaneously prevent liver pathology and restore blood AAT concentration in AAT deficiencies. Based on these observations, similar gene-therapy strategies could be considered for any diseases caused by accumulation of misfolded proteins.
authors.affiliation: Gene Therapy Center, Department of Pediatrics, University of North Carolina, Chapel Hill, NC 27599, USA. chengwen@med.unc.edu
authors.forename: Chengwen
authors.initials: C
authors.lastname: Li
authors.forename: Pingjie
authors.initials: P
authors.lastname: Xiao
authors.forename: Steven James
authors.initials: SJ
authors.lastname: Gray
authors.forename: Marc Scott
authors.initials: MS
authors.lastname: Weinberg
authors.forename: R Jude
authors.initials: RJ
authors.lastname: Samulski
mesh_terms: D000818:Animals; D003131:Combined Modality Therapy; D000229:Dependovirus / Q000235:genetics; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D007273:Injections, Intramuscular; D008099:Liver / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D057165:Proteostasis Deficiencies / Q000097:blood / Q000628:therapy*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000378:metabolism*; D012703:Serotyping; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics*; D000515:alpha 1-Antitrypsin / Q000097:blood / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000515:alpha 1-Antitrypsin