Browsing by Author "Firrman, Jenni A."

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    Effects of Thermally Induced Configuration Changes on rAAV Genome’s Enzymatic Accessibility
    (Elsevier, 2020-09-11) Xu, Yinxia; Guo, Ping; Zhang, Junping; Chrzanowski, Matthew; Chew, Helen; Firrman, Jenni A.; Sang, Nianli; Diao, Yong; Xiao, Weidong; Pediatrics, School of Medicine
    Physical titers for recombinant adeno-associated viral (rAAV) vectors are measured by quantifying viral genomes. It is generally perceived that AAV virions disassemble and release DNA upon thermal treatment. Here, we present data on enzymatic accessibility of rAAV genomes when AAV virions were subjected to thermal treatment. For rAAV vectors with a normal genome size (≤4.7 kb), thermal treatment at 75°C–99°C allowed only ∼10% of genomes to be detectable by quantitative real-time PCR. In contrast, greater than 70% of AAV genomes can be detected under similar conditions for AAV vectors with an oversized genome (≥5.0 kb). The permeability of virions, as measured by ethidium bromide (EB) staining, was enhanced by thermal stimulation. These results suggest that in rAAV virions with standard-sized genomes, the capsid and DNA are close enough in proximity for heat-induced “crosslinking,” which results in inaccessibility of vector DNA to enzymatic reactions. In contrast, rAAV vectors with oversized genomes release their DNA readily upon thermal treatment. These findings suggested that the spatial arrangement of capsid protein and DNA in AAV virions is genome-size dependent. These results provide a foundation for future improvement of vector assays, design, and applications.
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    Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency
    (Elsevier, 2020-10-22) Cao, Wenjing; Dong, Biao; Horling, Franziska; Firrman, Jenni A.; Lengler, Johannes; Klugmann, Matthias; de la Rosa, Maurus; Wu, Wenman; Wang, Qizhao; Wei, Hongying; Moore, Andrea R.; Roberts, Sean A.; Booth, Carmen J.; Hoellriegl, Werner; Li, Dong; Konkle, Barbara; Miao, Carol; Reipert, Birgit M.; Scheiflinger, Friedrich; Rottensteiner, Hanspeter; Xiao, Weidong; Pediatrics, School of Medicine
    One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.