Vector genome loss and epigenetic modifications mediate decline in transgene expression of AAV5 vectors produced in mammalian and insect cells.

Recombinant adeno-associated virus (rAAV) vectors are often produced in HEK293 or Spodoptera frugiperda (Sf)-based cell lines. We compared expression profiles of "oversized" (∼5,000 bp) and "standard-sized" (4,600 bp) rAAV5-human α1-antitrypsin (rAAV5-hA1AT) vectors manufactured in HEK293 or Sf cells and investigated molecular mechanisms mediating expression decline. C57BL/6 mice received 6 × 1013 vg/kg of vector, and blood and liver samples were collected through week 57. For all vectors, peak expression (weeks 12-24) declined by 50% to week 57. For Sf- and HEK293-produced oversized vectors, serum hA1AT was initially comparable, but in weeks 12-57, Sf vectors provided significantly higher expression. For HEK293 oversized vectors, liver genomes decreased continuously through week 57 and significantly correlated with A1AT protein. In RNA-sequencing analysis, HEK293 vector-treated mice had significantly higher inflammatory responses in liver at 12 weeks compared with Sf vector- and vehicle-treated mice. Thus, HEK293 vector genome loss led to decreased transgene protein. For Sf-produced vectors, genomes did not decrease from peak expression. Instead, vector genome accessibility significantly decreased from peak to week 57 and correlated with transgene RNA. Vector DNA interactions with active histone marks (H3K27ac/H3K4me3) were significantly reduced from peak to week 57, suggesting that epigenetic regulation impacts transgene expression of Sf-produced vectors.

Prophylactic Prednisolone Promotes AAV5 Hepatocyte Transduction Through the Novel Mechanism of AAV5 Coreceptor Platelet-Derived Growth Factor Receptor Alpha Upregulation and Innate Immune Suppression.

Adeno-associated virus (AAV) vectors are used to deliver therapeutic transgenes, but host immune responses may interfere with transduction and transgene expression. We evaluated prophylactic corticosteroid treatment on AAV5-mediated expression in liver tissue. Wild-type C57BL/6 mice received 6 × 1013 vg/kg AAV5-HLP-hA1AT, an AAV5 vector carrying a human α1-antitrypsin (hA1AT) gene with a hepatocyte-specific promoter. Mice received 4 weeks of daily 2 mg/kg prednisolone or water starting day -1 or 0 before vector dosing. Mice that received prophylactic corticosteroids had significantly higher serum hA1AT protein than mice that did not, starting at 6 weeks and persisting to the study end at 12 weeks, potentially through a decrease in the number of low responders. RNAseq and proteomic analyses investigating mechanisms mediating the improvement of transgene expression found that prophylactic corticosteroid treatment upregulated the AAV5 coreceptor platelet-derived growth factor receptor alpha (PDGFRα) on hepatocytes and downregulated its competitive ligand PDGFα, thus increasing the uptake of AAV5 vectors. Evidently, prophylactic corticosteroid treatment also suppressed acute immune responses to AAV. Together, these mechanisms resulted in increased uptake and preservation of the transgene, allowing more vector genomes to be available to assemble into stable, full-length structures mediating long-term transgene expression. Prophylactic corticosteroids represent a potential actionable strategy to improve AAV5-mediated transgene expression and decrease intersubject variability.

Young mice administered adult doses of AAV5-hFVIII-SQ achieve therapeutic factor VIII expression into adulthood.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5-hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice <1 week old initially exhibited high hFVIII-SQ plasma levels and maintained meaningful levels into adulthood, despite a partial decline potentially due to age-related body mass and blood volume increases. Spatial transduction patterns differed between mice dosed as neonates versus adults. No features of hepatotoxicity or endoplasmic reticulum stress were observed with dosing at any age. These data suggest that young mice require the same total vector genomes as adult mice to sustain hFVIII-SQ plasma levels.