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Katherine High

Publications and source records attributed to Katherine High.

6 recordsLinked to original sources
PubMed2004

Intracellular viral processing, not single-stranded DNA accumulation, is crucial for recombinant adeno-associated virus transduction.

Adeno-associated virus (AAV) is a unique gene transfer vector which takes approximately 4 to 6 weeks to reach its expression plateau. The mechanism for this slow-rise expression profile was proposed to be inefficient second-strand DNA synthesis from the input single-stranded (ss) DNA viral genome. In order to clarify the status of ss AAV genomes, we generated AAV vectors labeled with bromodeoxyuridine (BrdU), a nucleotide analog that can be incorporated into the AAV genome and packaged into infectious virions. Since BrdU-DNA can be detected only by an anti-BrdU antibody when DNA is in an ss form, not in a double-stranded (ds) form, ss AAV genomes with BrdU can be readily tracked in situ. Although ss AAV DNA was abundant by Southern blot analysis, free ss AAV genomes were not detectable after AAV transduction by this new detection method. Further Southern blot analysis of viral DNA and virions revealed that ss AAV DNA was protected within virions. Extracted cellular fractions demonstrated that viral particles in host cells remained infectious. In addition, a significant amount of AAV genomes was degraded after AAV transduction. Therefore, we conclude that the amount of free ss DNA is not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV ss DNA release (i.e., uncoating) before second-strand DNA synthesis can occur. AAV ss DNA released from viral uncoating is either converted into ds DNA efficiently or degraded by cellular DNA repair mechanisms as damaged DNA. This study elucidates a mechanism that can be exploited to develop new strategies to improve AAV vector transduction efficiency.

Bromodeoxyuridine↗
PubMedDate not supplied

AAV-mediated gene transfer for hemophilia.

The goal of our work has been to establish an experimental basis for gene transfer as a method of treating hemophilia, an inherited bleeding disorder that results from the absence of functional factor VIII or factor IX. Using an adeno-associated viral vector derived from AAV serotype 2, we have shown in mice and in hemophilic dogs that we can achieve long-term expression (>3 years) of clotting factor at levels that would result in an improvement of clinical symptoms of the disease. A phase I trial of intramuscular injection of AAV-F.IX showed no evidence of local or systemic toxicity in any of the subjects. Muscle biopsies showed evidence for gene transfer and expression by polymerase chain reaction, Southern blot, and immunohistochemistry. We have also shown that AAV-F.IX can be delivered into the portal veins of hemophilic dogs and that this results in high circulating levels of factor IX, on the order of 5% to 14%, whereas delivery of similar doses to skeletal muscle results in factor levels of only 1% to 2%. Based on these results, a trial of AAV-mediated liver-directed gene transfer for hemophilia B has been proposed and is reviewed here.

Animals↗