Efficient Hepatic Delivery and Expression from a Recombinant Adeno-associated Virus 8 Pseudotyped α1-Antitrypsin Vector

Abstract

alpha1-Antitrypsin (AAT) deficiency is a single-gene disorder in which a mutation in the AAT (approved symbol SERPINA1) gene (PI*Z) leads to misfolding of the protein, loss of the protective antiprotease effect of AAT for the lungs, and a toxic effect on hepatocytes. Optimal therapy for AAT deficiency will require a high percentage of hepatocyte transduction to be effective for liver and lung disease. Recently, rAAV genomes pseudotyped with capsids from serotypes 7 and 8 showed efficient hepatic transduction. We hypothesized that upon portal vein injection to target hepatocytes, serotype 8 would better transduce target cells and therefore express hAAT in both a greater percentage of cells and greater amounts. AAV2 and pseudotyped vectors for serotypes 1, 5, and 8 carrying the human AAT transgene were injected at 1 x 10(10) particle doses into C57Bl/6 mice. Circulating hAAT from AAV2/8-injected animals showed a 2-log advantage over AAV2 and 3-log increase over AAV2/1 and 5 for the 24-week study. Most significantly, up to 40% of total liver cells stained positive for the transgene in AAV2/8 subjects while remaining primarily episomal. Therefore, pseudotyped AAV8 provides a vehicle to infect a high percentage of hepatocytes stably and thereby express therapeutic molecules to modify AAT PiZ transcripts.

In vivo post-transcriptional gene silencing of α-1 antitrypsin by adeno-associated virus vectors expressing siRNA

“…19,32 Human Cell Lines The HEK-293 cell line (ATCC no. CRL-1573, Manassas, VA, USA) and the HepG2 liver cell line (ATCC no.…”

“…[34][35][36][37] Injection has been described previously. 19 Work is carried out completely under a sterilized laminar flow hood in SPF animal facility. A portable rodent isoflourane machine is used at 3% mixture.…”

“…[16][17][18][19][20] A variety of different vector approaches have been utilized for this purpose, and AAT expression and secretion can often be accomplished from ectopic sites, such as skeletal muscle. The latter approach is currently under examination in phase I clinical trials.…”

“…19,[24][25][26][27][28] In this report, a number of small-interfering RNA (siRNA) constructs directed against AAT were expressed from rAAV vectors in vitro and in vivo and their ability to decrease the levels of intracellular Z-AAT within the liver was assessed. A tri-functional (3X) construct emerged from these studies as a potential candidate for future studies of gene therapy of AAT liver disease.…”

Potential of AAV vectors in the treatment of metabolic disease

“…Studies in adult mice have shown that partial hepatectomy, which is associated with a burst of hepatocellular proliferation, leads to rapid loss of episomal vector genomes. [14][15][16] Essentially the same phenomenon is seen following vector delivery to neonatal mice, where high rates of hepatocellular proliferation result in almost complete clearance of episomal vector genomes within two doublings of liver mass occurring within the first 1-2 weeks of life. 17,18 A stable basal level of transgene expression, probably reflecting vector integration, persists thereafter in up to 8% of hepatocytes depending on the vector dose used.…”