T cell responses in BALB/c-HA mice.

<p><b>A.</b> 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 <i>in vitro</i> 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 ±<u>SD</u>; “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>B.</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 <u>unchallenged</u> 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 ±<u>SD</u>.</p

Depletion of B cells combined with gene therapy in BL/6-129/sv-HA mice.

<p><b>A.</b> 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 ±<u>SD</u> for n = 5 per experimental group. Two weeks after completion of the FVIII challenge, antibody formation against FVIII was measured: <b>B.</b> Total FVIII-specific IgG1 levels as determined by ELISA. <b>C</b>. Inhibitory antibody titers as measured by Bethesda assay (in BU). Values in panels B and C are shown for individual animals and as averages ±<u>SD</u>. Statistically significant differences between groups are indicated.</p

Codon-optimized hFVIII to induce tolerance and correction.

<p>BALB/c-HA and BL/6-129/sv-HA mice were injected with 10¹¹vg/mouse of an AAV8 vector expressing codon-optimized hFVIII. <b>A.</b> Coagulation times (aPTT in sec) and <b>B.</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 <b>C.</b> as HFVIIII-specific IgG1 titers, and <b>D.</b> 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.</p

Anti-CD20 treatment to prevent antibody formation in hFVIII protein replacement therapy.

<p>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 (<b>A</b>) and anti-hFVIII IgG1 ELISA (<b>B</b>) two weeks after each 4-week challenge. Control mice did not receive αCD20. Gray triangles represent B cell recovery. Data are averages ±<u>SD</u> for n = 3–5/group.</p

Depletion of B cells using anti-CD20.

<p><b>A.</b> 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>B.</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.</p

Immune Responses and Hypercoagulation in ERT for Pompe Disease Are Mutation and rhGAA Dose Dependent

<div><p>Enzyme replacement therapy (ERT) with recombinant human acid-α-glucosidase (rhGAA) is the only FDA approved therapy for Pompe disease. Without ERT, severely affected individuals (early onset) succumb to the disease within 2 years of life. A spectrum of disease severity and progression exists depending upon the type of mutation in the GAA gene (<i>GAA</i>), which in turn determines the amount of defective protein produced and its enzymatic activity. A large percent of the early onset patients are also cross reactive immunological material negative (CRIM-) and develop high titer immune responses to ERT with rhGAA. New insights from our studies in pre-clinical murine models reveal that the type of <i>Gaa</i> mutation has a profound effect on the immune responses mounted against ERT and the associated toxicities, including activation of clotting factors and disseminated intravascular coagulation (DIC). Additionally, the mouse strain affects outcomes, suggesting the influence of additional genetic components or modifiers. High doses of rhGAA (20 mg/kg) are currently required to achieve therapeutic benefit. Our studies indicate that lower enzyme doses reduce the antibody responses to rhGAA, reduce the incidence of immune toxicity and avoid ERT-associated anaphylaxis. Therefore, development of rhGAA with increased efficacy is warranted to limit immunotoxicities.</p></div

Vital signs measured by pulse oxymetry, prior to and 5 minutes after the 8^th rhGAA IV injection in wt BALB/c and wt 129SVJ mice (n = 5).

<p><b>A</b>) Percentage oxygen saturation <b>B</b>) Heart rate <b>C</b>) Breath distention <b>D</b>) Pulse distention, p<0.05 *, ns = not significant, (inj; injection).</p

Antibody responses to varying doses of rhGAA in null mutation (n = 6) or P545L mutant mice (n = 5).

<p><b>A</b>) Anti-rhGAA IgG1 in 1 mg/kg rhGAA injected GAA-/- 129SVE mice tested weekly <b>B</b>) Anti-rhGAA IgG1 in 5 mg/kg rhGAA injected GAA-/- 129SVE mice <b>C</b>) Anti-rhGAA IgG1 in 20 mg/kg rhGAA injected GAA-/- 129SVE mice <b>D</b>) Anti-rhGAA IgG1 response in 20 mg/kg rhGAA injected P545L mice <b>E</b>) Anti-rhGAA IgG2a in 1 mg/kg rhGAA injected GAA-/- 129SVE mice <b>F</b>) Anti-rhGAA IgG2a in 5 mg/kg rhGAA injected GAA-/- 129SVE mice <b>G</b>) Anti-rhGAA IgG2a in 20 mg/kg rhGAA injected GAA-/- 129SVE mice <b>H</b>) Anti-rhGAA IgG2a in 20 mg/kg rhGAA injected P545L mice. Arrows indicate fold decrease over corresponding 20 mg/kg cohort time point. p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p

Immune Responses and Hypercoagulation in ERT for Pompe Disease Are Mutation and rhGAA Dose Dependent - Figure 6

<p><b>A</b>) Reduction of clotting times measured in activated partial thromboplastin time (aPTT) in GAA-/- 129SVE and P545L mice. <b>B</b>) D-Dimer levels in GAA-/-129SVE naïve and rhGAA injected mice. Changes in hemorheologic values from complete blood counts in 129SVE GAA-/- mice and P545L naive and 20 mg/kg rhGAA injected mice (5 min post rhGAA injection). <b>C</b>) Platelet count, <b>D</b>) Mean platelet volume, <b>E</b>) Platelet width distribution, p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p

Comparison of pulse oxymetry measurements of vital signs prior to and post the 4^th rhGAA (1 mg/kg, 5 mg/kg or 20 mg/kg) ERT IV injection in GAA-/- 129SVE (n = 6) and P545L mice (20 mg/kg rhGAA; n = 5).

<p>A) oxygen saturation B) heart rate C) pulse distention D) breath distention, E) Time taken for the formation of a platelet plug post-injury (tail-snip) prior to and post-rhGAA IV administration. Changes in hemorheologic values from complete blood counts in 129SVE GAA-/- mice and P545L naive and 20 mg/kg rhGAA injected mice (5 min post rhGAA injection). F) Hematocrit, G) Hemoglobin, p<0.05 *, p<0.005 **, p<0.0005 ***, ns = not significant.</p