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. 2012;7(5):e37671.
doi: 10.1371/journal.pone.0037671. Epub 2012 May 24.

Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy

Brandon K Sack  1 , Sherin Merchant, David M Markusic, Amit C Nathwani, Andrew M Davidoff, Barry J Byrne, Roland W Herzog
Affiliations

Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy

Brandon K Sack et al. PLoS One. 2012.

Abstract

The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors) against factor VIII (FVIII). The current method for eradicating inhibitors, termed immune tolerance induction (ITI), is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8) gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Depletion of B cells using anti-CD20.
A. Treatment schedule. Hemophilia A (HA) mice in “αCD20” groups received a dose of 10 mg/kg IgG2a αCD20 i.v. on day 0 and day 21. One week following the first αCD20 injection, mice in “AAV8” groups received 1011 vg/mouse of AAV8-hFVIII. Blood samples were collected at indicated time points. Mice were challenged with intravenous hFVIII (1 IU/mouse, weekly for 4 weeks) at indicated time points. B. Representative examples of B cell depletion in different lymphoid organs of BALB/c-HA mice 1 day and 7 weeks after the second injection second αCD20 injection. Numbers in each histogram represent percent CD19+ lymphocytes (as shown by forward and side scatter gating in left panel) for both untreated control (in black) and αCD20-treated animals (red). Cells were stained with anti-CD19 antibody conjugated to V450 fluorochrome at 1 day post second injection, or to APC-Cy7 for the 7-weeks post-αCD20 time point.
Figure 2
Figure 2. Depletion of B cells combined with gene therapy in BL/6-129/sv-HA mice.
A. Activated partial thromboplastin time (aPTT) of mice receiving 1011vg/mouse of AAV8-hFVIII either alone (“AAV8-F8”) or in combination with αCD20 therapy (“AAV8-F8+CD20”). “FVIII challenge” indicates a period of weekly HFVIII injections (see Fig. 1A). “AAV8-unchallenged” group is mice that received vector but were not challenged with hFVIII protein. Range of aPTT for untreated mice and coagulation time for HA mouse plasma corrected to 1% HFVIII activity are also shown. Data are average values ±SD for n = 5 per experimental group. Two weeks after completion of the FVIII challenge, antibody formation against FVIII was measured: B. Total FVIII-specific IgG1 levels as determined by ELISA. C. Inhibitory antibody titers as measured by Bethesda assay (in BU). Values in panels B and C are shown for individual animals and as averages ±SD. Statistically significant differences between groups are indicated.
Figure 3
Figure 3. B Cell depletion combined with gene therapy in BALB/c-HA mice.
A. Activated partial thromboplastin time (aPTT) of mice receiving 1011vg/mouse of AAV8-hFVIII either alone (“AAV8-F8”) or in combination with αCD20 therapy (“AAV8-F8+CD20”). “FVIII challenge” with weekly hFVIII protein is indicated (see also Fig. 1A). “AAV8-unchallenged” mice received vector but no hFVIII protein. “αCD20” mice received B cell depletion and hFVIII challenge in the absence of gene transfer. Range of aPTT for untreated mice and coagulation time for HA mouse plasma corrected to 1% HFVIIII activity are also shown. Data are average values ±SD for n = 7–9 mice per experimental group. Two weeks after completion of the FVIII challenge, antibody formation against HFVIIII was measured: B. Total FVIII-specific IgG1 levels. C. Inhibitor titers (in BU). Values in panels B and C are shown for individual animals and as averages ±SD. Statistically significant differences between groups are indicated. D. Starting in week 22, mice were given a second round of hFVIII protein challenge (see Fig. 1A). Five min after the last HFVIIII injection, mice were bled via tail vein, and collected plasma assayed by aPTT (n = 3–4 per experimental group). “Naïve” mice were also treated with hFVIII and bled 5 min later, but had no prior exposure to hFVIII. “FVIII-only” mice were mice that had been challenged previously with hFVIII (without αCD20 treatment or gene therapy) and thus had developed inhibitors. “αCD20+FVIII” mice had initially received αCD20 but no gene therapy and developed inhibitors in subsequent protein therapy (labeled as “αCD20” group in panels A–C). NS: not significant.
Figure 4
Figure 4. T cell responses in BALB/c-HA mice.
A. Following the final tail vein bleed (Fig. 3D), mice were sacrificed and spleens collected. Splenocyte cultures for individual mice (n = 4 per group) were stimulated in vitro with 10 µg/mL of hFVIII for 48 h. Subsequently, CD3+CD4+ T cells were purified by flow cytometry and analyzed by quantitative RT-PCR for expression of several immune-regulatory genes. Shown are data for indicated groups (averages ±SD; “fold increase” is change in RNA transcripts of hFVIIII- vs. mock-stimulated). The dotted horizontal line indicates the minimally required increase of 2.5-fold for a statistically significant difference. B. Evidence for Treg induction in mice that had received gene transfer or a combination of αCD20 and gene transfer. Following one round of hFVIII challenge, CD4+CD25+ splenocytes were purified from each treatment group via magnetic sorting, and 106 cells/mouse were adoptively transferred to naïve BALB/c-HA recipients via tail vein injection. Control cells were from unchallenged naïve mice of the same strain. Twenty-four hours later, all recipient mice (n = 3 per group) were challenged with 1 IU hFVIII in adjuvant. Anti-FVIII IgG titers were measured 1 month later by ELISA. Data are averages ±SD.
Figure 5
Figure 5. Anti-CD20 treatment to prevent antibody formation in hFVIII protein replacement therapy.
BL/6-129/sv-HA and BALB/c-HA mice were treated with αCD20 antibody as outlined in Fig. 1A (indicated with large arrows) followed by 4 weeks of hFVIII challenge (indicated by small arrows) beginning at 4 weeks after the second αCD20 administration. Mice were treated with hFVIII twice more following the same schedule. Antibody formation against HFVIIII was measured by Bethesda assay (A) and anti-hFVIII IgG1 ELISA (B) two weeks after each 4-week challenge. Control mice did not receive αCD20. Gray triangles represent B cell recovery. Data are averages ±SD for n = 3–5/group.
Figure 6
Figure 6. Codon-optimized hFVIII to induce tolerance and correction.
BALB/c-HA and BL/6-129/sv-HA mice were injected with 1011vg/mouse of an AAV8 vector expressing codon-optimized hFVIII. A. Coagulation times (aPTT in sec) and B. HFVIIII activity were measured as a function of time after vector administration. Data are averages ±SD for n = 4/group. Mice were challenged (starting at week 10 after gene transfer) with hFVIII at the same dose and schedule as in previous experiments. Anti-hFVIII formation was measured C. as HFVIIII-specific IgG1 titers, and D. by Bethesda assay. Values in panels C and D are shown for individual animals and as averages ± SD and plotted on the same scale as in Fig. 2 to compare magnitude of responses.
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References

    1. Di Michele DM. Immune tolerance induction in haemophilia: evidence and the way forward. J Thromb Haemost. 2011;9(Suppl 1):216–225. - PubMed
    1. Odeyemi IA, Dano AM. Optimising immune tolerance induction strategies in the management of haemophilia patients with inhibitors: a cost-minimisation analysis. Curr Med Res Opin. 2009;25:239–250. - PubMed
    1. Verweij CL, Vosslamber S. New insight in the mechanism of action of rituximab: the interferon signature towards personalized medicine. Discov Med. 2011;12:229–236. - PubMed
    1. Weiner GJ. Rituximab: mechanism of action. Semin Hematol. 2010;47:115–123. - PMC - PubMed
    1. Aleem A, Saidu A, Abdulkarim H, Al-Diab AR, Al-Sagheer A, et al. Rituximab as a single agent in the management of adult patients with haemophilia A and inhibitors: marked reduction in inhibitor level and clinical improvement in bleeding but failure to eradicate the inhibitor. Haemophilia. 2009;15:210–216. - PubMed

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