Blood analysis for red cell heterogeneity (A) and hemoglobin level (B). Blood samples from IVS2–654 mice before (prebleed) and after treatment with 12 i.v. injections of SSO P005 (n = 5) were analyzed in Heska's animal blood counter. Asterisk indicates statistically significant (one-tailed Wilcoxon signed test) difference between prebled and SSO P005-treated mice. (A and B) P = 0.03. (C and D) RDW and hemoglobin levels of WT mice (n = 3) injected with saline (mean ± SD).

Improvement of RBC morphology after SSO P005 treatment. (A) Wright-Giemsa-stained peripheral blood smears of IVS2–654 mouse were prepared before [day (d) 0] and after 12 i.v. injections of SSO P005 (d 19). WT, wild-type mouse. (B) Differential count of abnormal (ARBC), target (TRBC), hypochromic (HRBC), and normal (NRBC) RBCs. Blood smears before and after 12 i.v. injections of SSO P005 from IVS2–654 mice (n = 4) were stained with Wright-Giemsa. Three different field images were taken from each smear and used for the differential RBC count. Data are presented as mean ± SD. The difference between the numbers of NRBCs before and after SSO P005 treatment is 10% but not significant (P = 0.06; one-tailed Wilcoxon signed test).

(A) Splicing of IVS2–654 β-globin pre-mRNA and its repair by SSO. Boxes, exons; solid lines, introns and correct splicing pathway; dotted lines, aberrant splicing pathway. Thick bar, SSO; arrows, primers used in the RT-PCR assay. The length (in nucleotides) of RT-PCR products representing aberrantly (ab) and correctly (c) spliced mRNAs is shown. (B–D) Restoration of correctly spliced human β-globin mRNA in cultured erythroid progenitor cells. Cells were isolated from the bone marrow of IVS2–654 mouse and treated with PNA-4K (B), morpholino (C), and SSO P005 (D) SSOs. Treatment was by free uptake (FU) of SSO from the culture medium. SSO concentrations and the time of cell harvest are indicated. Total cellular RNA was analyzed by RT-PCR. N, RNA from normal human blood.

Toxicity assay of SSO P005. Serum samples from IVS2–654 mice before [day (d) 0] and after treatment with 16 i.v. injections (d 26) of saline (n = 5) or SSO P005 (n = 4) were analyzed for (A) aspartate aminotransferase (AST, U/L), (B) alanine aminotransferase (ALT, U/L), (C) blood urea nitrogen (BUN, mg/dL), and (D) creatinine (CREA, mg/dL). (E) Body weights of the mice were recorded before (d 0) and after every 4 i.v. injections (d 5, 12, 19, and 26) of saline (n = 5) or SSO P005 (n = 4). (F) Quantification of cytokine mRNAs by RT-qPCR. The peripheral blood RNAs from saline mice (n = 3) and mice treated with 4 (n = 5), 8 (n = 6), and 12 (n = 4) injections of SSO P005 were analyzed for IFN-γ and IL-12a mRNAs. Data presented as mean ± SD.

Repair of human β-globin mRNA in vivo in IVS2–654 thalassemic mice by i.v. injections of SSO P005. (A) Conventional RT-PCR of total RNA from SSO P005-treated mice. RNA was isolated from peripheral blood. Lane 1, normal human blood (N); lane 2, prebleed; lanes 3–5, blood collected after 4, 8, and 12 SSO injections, respectively; lanes 6–7, blood collected from IVS2–654 mice treated with negative control P005 scr SSO; lane 8, no reverse transcription (NRT); lane 9, no template control (NTC). Days of bleeding and percentages of correctly (c) spliced mRNA compared with aberrantly (ab) spliced mRNA are indicated. No product was detectable without the RT step or in the absence of mRNA template. (B) Quantification of RNA repair by RT-qPCR. The mice were prebled and intravenously injected with 4 (n = 4), 8 (n = 3), and 12 (n = 3) doses of SSO P005. Total RNA from blood collected before [day (d) 0] and after every 4 SSO injections (d 5, 12, and 19) was analyzed. Data presented as mean ± SD. P values <0.05 were determined by one-way ANOVA. Asterisks denote statistically significant difference. The correctly spliced human β-globin mRNA was not detectable (ND) in prebled samples because the probe was designed to hybridize exclusively to the correct human β-globin mRNA (Materials and Methods). (C) Chimeric mouse-human hemoglobin (mα₂hβ₂) generated by SSO P005 treatment. IVS2–654 mice (n = 9) were dosed intravenously with SSO P005 for 3 weeks on a schedule: 4 once-daily injections and 3 days off. (Top) Hemoglobin levels were assayed by immunoblots with anti-human β-globin antibody of cellulose acetate electrophoregrams. Blood samples were taken before injections (lanes 1–3) and 1 day after the last injection (d 19) (lanes 4–12). (Bottom) Immunoblots were quantified using ImageQuant TL software.