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Minihepcidins to treat ß-thalassemia major mice
term survival in the absence of transfusion. Using these mice, we demonstrate the potential beneficial effect of minihepcidins in mice affected by TDT which were or were not given transfusions.
Methods
Animal models
Hbbth1/th1 mice (B6.D2-Hbbd3th/BrkJ stock n. 000996) were crossed with Hbbth2/+ animals (B6.129P2-Hbbtm1Unc/J stock n. 002204).34-36 All recipient mice were 8- to 12-week old females transgenic for either green fluorescent protein (C57BL/6- Tg(UBC-GFP)30Scha/J)40 or B6.SJL-Ptprca Pepcb/BoyJ (known as Pep Boy). The Pep Boy mice allow us to discriminate between endogenous cells [which carry the differential Ptprca pan-leuko- cyte marker (commonly known as CD45.1 or Ly5.1)] from the donor fetal liver cells (which carry the CD45.2 or Ly5. variant); similarly, GFP+ donors can be distinguished from GFP- recipient source RBC. Blood samples were analyzed as previously described.3,41
Hematopoietic chimeras and genotyping
Donor fetal liver cells were harvested from embryos (E13.5- 15.5 days) obtained by intercrossing Hbbth1/th1, Hbbth2/+, or WT mice. Embryonic genotypes were screened by DNA extraction (KAPA Biosystems, Kapa Mouse Genotyping Kit hotstart, KK7352) and polymerase chain reaction analysis (see Online Supplementary Tables S1 and S2). Fetal liver cells were kept on ice and resuspended in sterile phosphate-buffered saline (ThermoFisher PBS, Ph 7.4, CAT 10010023). To establish bone marrow chimeras, 2.0-5.0x106 cells were injected retro-orbitally into each of the irradiated female recipients. Recipient mice were irradiated with 10 Gy (split dose of 2 × 5 Gy) on the day of transplantation (ISOVOLT Titan E Series X-Ray Generators).
Blood transfusion
Transfusion was performed as previously described.16 Starting 2 months after transplantation, mice were transfused weekly via retro-orbital venous plexus with 300 μL freshly harvested blood from normal healthy C57BL/6 mice or GFP. The first transfusion was delivered at the same time as the first minihepcidin admin- istration. The last transfusion was delivered 1 week before the last minihepcidin injection.
Mouse serum erythroferrone measurement
The immunoaffinity liquid chromatography-tandem mass spectrometry assay to quantify total erythroferrone protein lev- els in mouse serum was developed in-house using surrogate peptide analysis. Briefly, total erythroferrone from 25 μL serum was enriched using a biotinylated mouse anti-erythroferrone antibody (Drakesmith Lab) by diluting serum into 75 μL of phosphate-buffered saline-Tween and incubating with antibody at 30°C for 4 h with interval mixing at 600 rpm. Magnetic strep- tavidin beads were added and incubated for an additional 30 min with interval mixing at 1200 rpm. The bound erythrofer- rone protein was then eluted from the beads using hydrochloric acid and processed for digestion using Promega trypsin-LysC enzyme at 37°C overnight. The liquid chromatography-tandem mass spectrometry quantification was carried out by monitoring two unique erythroferrone-specific surrogate peptides (EFQLL- LK and SGSHFSAILLGL) using a standard curve generated with a recombinant mouse erythroferrone-Fc protein construct. Levels were measured with a lower limit of quantification (LLOQ) of 0.25 ng/mL.42-44
Statistics
Bars represent standard deviation (SD). When multiple com- parisons were needed, statistical analysis was performed using ordinary one-way or two-way analysis of variance (ANOVA) with the Tukey or Sidak adjustment for multiple comparisons. An unpaired two-tailed Student t-test was used for comparisons between two groups. P values <0.05 are considered statistically significant. All data were analyzed using GraphPad Prism ver- sion 7 (Microsoft GraphPad Software, La Jolla, CA, USA). Data for WT fetal liver cells are presented as a reference.
Animal study approval
All animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committee of The Children’s Hospital of Philadelphia.
Results
Generation of a new mouse model of β-thalassemia major or transfusion-dependent thalassemia
We hypothesized that intercrossing Hbbth1/th1 and Hbbth2/+ mice (Figure 1A, B) could generate animals that are able to produce RBC, but with insufficient levels of adult hemo- globin for long-term survival (Figure 1D). At birth Hbbth1/th2 pups were extremely pale but alive (for up to 8 h) and died despite transfusion (Online Supplementary Figure S1A) likely due to irreversible damage associated with the severe hypoxia in late gestation. We then focused on gen- erating mice through transplantation of Hbbth1/th2 fetal liver cells into recipient transgenic animals expressing GFP or Pep Boy mice [Hbbth1/th2 bone marrow chimeras (Hbbth1/th2BMC)] (Online Supplementary Figures S1B and S2A, B; Online Supplementary Table S1 and S2). The GFP+ and the Pep Boy (CD45.1) mice were utilized to monitor the chimerism of circulating RBC over time (GFP- vs. GFP+ RBC) or bone marrow leukocytes (CD45.2 vs. CD45.1) and assess engraftment of donor cells. The resulting mod- els demonstrate the desired phenotype 2 months after transplantation, including production of GFP- RBC or CD45.2 bone marrow leukocytes and anemia (Online Supplementary Figure S3A, B, Figure 2).
Hbbth1/th2BMC animals showed features
of β-thalassemia major, requiring transfusion for long-term survival
Two months after transplantation, analysis of the hematologic parameters indicated that Hbbth1/th2BMC mice produce few RBC, low hemoglobin levels, but high retic- ulocyte counts (Figure 2A-C). Hbbth1/th2BMC mice showed the largest increase in spleen weight (Figure 2D). Peripheral blood smears confirmed more severe anisocy- tosis, poikilocytosis and hypochromasia (Figure 2E) than in models of NTDT. Because Hbbth1/th2BMC mice do not require transfusion for survival for up to 4 months after transplantation, we analyzed the effect of minihepcidins in the absence of transfusion. After this period, Hbbth1/th2BMC mice showed exacerbation of their anemia, incompatible with survival.
Administration of minihepcidins ameliorated red blood cell lifespan, ineffective erythropoiesis, anemia and splenomegaly in untransfused Hbbth1/th2BMC mice
Hbbth1/th2BMC were treated with two doses of minihep- cidins, 2.625 mg/kg [(low dose (MH_L)] or 5.25 mg/kg
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