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. 2008 Mar 3:6:9.
doi: 10.1186/1479-0556-6-9.

Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats

Tracey Graham  1 , Jenny McIntosh, Lorraine M Work, Amit Nathwani, Andrew H Baker
Affiliations

Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats

Tracey Graham et al. Genet Vaccines Ther. .

Abstract

Background: Vectors based on adeno-associated virus-8 (AAV8) have shown efficiency and efficacy for liver-directed gene therapy protocols following intravascular injection, particularly in relation to haemophilia gene therapy. AAV8 has also been proposed for gene therapy targeted at skeletal and cardiac muscle, again via intravascular injection. It is important to assess vector targeting at the level of virion accumulation and transgene expression in multiple species to ascertain potential issues relating to species variation in infectivity profiles.

Methods: We used AAV8 vectors expressing human factor IX (FIX) from the liver-specific LP-1 promoter and administered this virus via the intravascular route of injection into 12 week old Wistar Kyoto rats. We assessed FIX levels in serum by ELISA and transgene expression at sacrifice by immunohistochemistry using anti-FIX antibodies. Vector DNA levels in organs we determined by real time PCR.

Results: Administration of 1 x 10(11) or 5 x 10(11) scAAV8-LP1-hFIX vector particles/rat resulted in efficient production of physiological hFIX levels, respectively in blood assessed 4 weeks post-injection. This was maintained for the 4 month duration of the study. At 4 months we observed liver persistence of vector with minimal non-hepatic distribution.

Conclusion: Our results demonstrate that AAV8 is a robust vector for delivering therapeutic genes into rat liver following intravascular injection.

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Figures

Figure 1
Figure 1
Quantitative analysis of vector biodistribution following intravascular administration of AAV8LP-1FIX into rats. DNA was extracted and processed from tissues of rats, which were dissected 4 months after intravenous injections of scAAV8-LP1-hFIX. Purified DNA was subsequently analysed by quantitative real-time PCR using hFIX specific primers designed to hybridise to the human FIX coding region of the AAV8 vector.12. Data are presented as the means ± standard error of the mean. VG = vector genomes.
Figure 2
Figure 2
Determination of circulating human factor IX levels in blood plasma of AAV8hFIX infused rats. Blood plasma samples from scAAV8-LP1-hFIX infused rats at (black sqaure) 1 × 1010vg/rat, (black triangle) 5 × 1010vg/rat, (open triangle) 1 × 1011vg/rat, (open square) 5 × 1011vg/rat or (black circle) saline were assayed by ELISA to measure circulating hFIX activity. All values represent average hFIX levels with standard error of mean. Percentages refer to the percentage of normal circulating FIX levels.
Figure 3
Figure 3
Histological analysis of human FIX expression in livers of AAV8-LP1-hFIX infused rats. Representative liver sections of immunohistochemical staining for hFIX 4 months after gene transfer in rats infused with scAAV8-LP1-hFIX at (A) 5 × 1011vg/rat, (B) 1 × 1011vg/rat, (C) 5 × 1010vg/rat, (D) 1 × 1010vg/rat or (E) saline. Paraffin liver sections were stained for human FIX expression using goat anti-human FIX conjugated to horse-radish peroxidase and goat IgG was used as the negative control (F). Magnification is × 40.
See this image and copyright information in PMC

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