1. Factor IX variants improve gene therapy efficacy for hemophilia B
by Joerg Schuettrumpf, Roland W. Herzog, Alexander Schlachterman, Antje Kaufhold, Darrel W. Stafford, and Valder R. Arruda
Blood
Volume 105(6):
March 15, 2005
©2005 by American Society of Hematology

2. Vector design.
Vector design. (A) Skeletal muscle-directed gene transfer: The expression cassette contains the CMV enhancer/promoter, the simian virus 40 (SV40) polyadenylation signal, and is flanked by the AAV-2 inverted terminal repeats (ITRs). The human transgene contains cDNA of exon 1 from the human F.IX gene as WT or variants (K5A or K5A/V10K) interrupted by a 1.4-kb fragment of F.IX intron I, and exons 2 to 8 of the F.IX-WT or variant (R338A). The substitutions introduced in the mature F.IX sequence are indicated. (B) Liver-directed gene transfer: The expression cassette contains the human α1-antitrypsin promoter coupled to the human apolipoprotein E enhancer, exon 1 from the human F.IX gene, a truncated human F.IX intron 1, exons 2 to 8 of the F.IX gene WT or variant (R338A), and the bovine growth hormone polyadenylation signal sequence. The expression cassette is flanked by ITRs derived from AAV type 2.
Joerg Schuettrumpf et al. Blood 2005;105:
©2005 by American Society of Hematology

3. Time course of human F.IX expression in C57Bl/6 Rag-1 knock-out mice.
Time course of human F.IX expression in C57Bl/6 Rag-1 knock-out mice. Mice were injected into skeletal muscle with AAV-1 vectors encoding F.IX-WT (), the F.IX variants K5A (), K5A/V10K (▴), or R338A (▪). Vector was injected at doses of 2 × 1011 vg/kg (A) or 1.2 × 1012 vg/kg (B) at 6 intramuscular sites in the hind limbs. Each line represents average values for the cohort (n = 4 mice). Markers represent mean values (± SD). *P ≤ .002 and **P ≤ .05 of comparison between F.IX-WT and F.IX-K5A/V10K groups.
Joerg Schuettrumpf et al. Blood 2005;105:
©2005 by American Society of Hematology

4. Vector gene copy number and histochemical staining of skeletal muscle of C57Bl/6 Rag-1-deficient mice 14 weeks following injection of AAV vectors encoding F.IX-WT or variant K5A/V10K.
Vector gene copy number and histochemical staining of skeletal muscle of C57Bl/6 Rag-1-deficient mice 14 weeks following injection of AAV vectors encoding F.IX-WT or variant K5A/V10K. (A) Southern blot analysis of genomic DNA extracted from murine skeletal muscle injected with AAV-F.IX-WT or F.IX-K5A/V10K at the doses indicated. Copy number standards were prepared by adding 3.5, 35, and 350 copies of plasmid per murine diploid genomes. (B) Immunofluorescent staining for human factor IX in tibialis anterior muscle. Animals were injected in the hind limbs with 1.2 × 1012 vg/kg AAV-F.IX-WT or K5A/V10K divided equally among 6 injection sites. Representative sections from the injected muscle are shown. Excitation of fluorescence tags revealed both intracellular and extracellular signals in muscle injected with F.IX-WT, but markedly reduced extracellular signals in muscle injected with F.IX-K5A/V10K, while intracellular signals are clearly detectable. Original magnifications, × 200.
Joerg Schuettrumpf et al. Blood 2005;105:
©2005 by American Society of Hematology

5. Specific activity of F. IX-WT, F. IX-K5A/V10K, and F
Specific activity of F.IX-WT, F.IX-K5A/V10K, and F.IX-R338A following intramuscular injection of AAV vectors to C57Bl/6 hemophilia B/CD4 knock-out mice.
Specific activity of F.IX-WT, F.IX-K5A/V10K, and F.IX-R338A following intramuscular injection of AAV vectors to C57Bl/6 hemophilia B/CD4 knock-out mice. (A) F.IX activity is determined in one-stage activated partial thromboplastin assay in mice plasma, and F.IX antigen levels are determined by ELISA. Animals were injected at 6 intramuscular sites in the hind limbs with AAV-F.IX-WT (▦;n = 15) or -R338A (; n = 8) at doses of 4 × 1012 vg/kg or 8 × 1012 vg/kg. AAV-K5A/V10K (▵) was injected at doses of 2 × 1011 vg/kg (n = 4), 1.2 × 1012 vg/kg (n = 4), or 4 × 1012 vg/kg (n = 3). Plasma samples were collected at several time points after vector administration, and results from individual animals are indicated. (B) Factor IX expression in murine tissues harvested at week 18 following injection of an AAV vector under the control of the CMV enhancer/promoter. Shown are RT-PCR products for human factor IX sequences and for the murine hypoxanthine phosphoribosyltransferase (HPRT) as housekeeping gene. Gastrointestinal (GI) tract includes tissues from esophagus, stomach, and intestines. Densitometric analysis of RT-PCR shows that the majority of F.IX expression is derived from skeletal muscle and only a small fraction from the liver.
Joerg Schuettrumpf et al. Blood 2005;105:
©2005 by American Society of Hematology

6. Parameters of hemostasis in vivo following tail-clipping assay of C57Bl/6 hemophilia B/CD4 knock-out mice 8 weeks following intramuscular injection of AAV vectors encoding F.IX-WT or variants.
Parameters of hemostasis in vivo following tail-clipping assay of C57Bl/6 hemophilia B/CD4 knock-out mice 8 weeks following intramuscular injection of AAV vectors encoding F.IX-WT or variants. (A) Tail-bleeding times were determined by visual inspection of blood flow into a saline solution, and time to cessation was recorded. At the time of assay, mean circulating F.IX levels for F.IX-WT- and K5A/V10K-treated mice were 439 ng/mL and 487 ng/mL, respectively. Whereas for R338A, F.IX levels were 248 ng/mL. Control groups consisted of hemostatically normal C57Bl/6 or untreated hemophilia B mice matched by age and sex. The experiment in untreated hemophilia B mice was terminated at 10 minutes. (B) Blood loss (mean ± SD) was determined by measuring the absorbance at A575 of hemoglobin content in the saline solution in which the tail was placed. The number of animals for each group is indicated at the top of each column. *ANOVA was used for statistical analysis with Tukey-Kramer comparison test for all groups; the results are indicated. **WT represents hemophilia B mice injected with AAV-F.IX-WT. NS indicates P value not significant.
Joerg Schuettrumpf et al. Blood 2005;105:
©2005 by American Society of Hematology

7. Human F. IX expression as a function of time and anti-F
Human F.IX expression as a function of time and anti-F.IX antibody measurements.
Human F.IX expression as a function of time and anti-F.IX antibody measurements. (A) Transgenic mice tolerant to human wild-type F.IX received AAV vectors encoding F.IX-WT (▴) or variants (K5A [], K5A/V10K [▪], R338A [×]) at a dose of 1 × 1012 vg/kg by intramuscular injections. Skeletal muscle-derived F.IX levels determined by ELISA are detectable in plasma samples collected 2 weeks after injection and throughout the duration of the experiment. Immunodeficient mice (*; CD4 knock-out mice) as a control group presented similar increase in circulating F.IX after intramuscular injection of AAV-F.IX-WT. When the same vector was administered to normal C57BL/6 (nontolerant [•]) mice, no F.IX was detected after 4 weeks of injection. (B) Detection of specific IgG1 antibodies to F.IX-WT (▪) or variants K5A (▦) or R338A (□) at 4 weeks after vector injection were restricted to nontolerant C57Bl6 mice. The numbers of animals tested are indicated. Data shown are mean values ± SD.
Joerg Schuettrumpf et al. Blood 2005;105:
©2005 by American Society of Hematology

8. F.IX clotting activity and antigen determination in C57Bl/6 hemophilia B mice following liver delivery by tail-vein injection of AAV-F.IX or R338A vectors.
F.IX clotting activity and antigen determination in C57Bl/6 hemophilia B mice following liver delivery by tail-vein injection of AAV-F.IX or R338A vectors. (A) F.IX-specific activity was calculated by dividing the clotting activity by the antigen levels and expressed as unit per milligram. Average of specific activity of liver-synthesized F.IX at expression levels ranging from 100 to 4000 ng/mL following injection of AAV-F.IX-R338A (▪; n = 9) compared with F.IX-WT (□; n = 13). Bars represent mean values ± SD. (B) Graphic data plot showing individual test samples for F.IX-specific activity determination in animals receiving F.IX-WT (□) or F.IX-R338A (). P value was calculated by Student t test. Trendlines were derived from linear regression analysis.
Joerg Schuettrumpf et al. Blood 2005;105:
©2005 by American Society of Hematology