In vitro cell culture methods for beta-thalassemia using activin type II receptor ligand traps

1. A method of treating beta-thalassemia in a subject, comprising administering an ActRII signaling inhibitor to the subject, wherein the patient has been selected by using an in vitro cell culture method comprising the steps of:

(a) co-culturing an erythroid progenitor cell (EPC) and a stromal cell in the presence of an activin type II receptor (ActRII) signaling inhibitor for a period of time; and

(b) determining the level of GYPA, GATA1, GATA2, or alpha-globin in the EPC or determining the level of expansion of the EPC, wherein the stromal cell and/or the EPC has been obtained from the subject;

wherein the selected subject has:

(i) an increase in the level of GYPA in the EPC of the in vitro cell culture method as compared to the level of GYPA in a control EPC;

(ii) an increase in the level of GATA1 in the EPC of the in vitro cell culture method as compared to the level of GATA1 in a control EPC;

(iii) a decrease in the level of GATA2 in the EPC of the in vitro cell culture method as compared to the level of GATA2 in a control EPC;

(iv) a decrease in the level of alpha-globin in the EPC of the in vitro cell culture method as compared to the level of alpha-globin in a control EPC; or

(v) an increase in the level of expansion of the EPC in the in vitro cell culture method as compared to the level of expansion in a control EPC;

or an in vitro cell culture method comprising the steps of:

(a) culturing a stromal cell that has been obtained from the subject in the presence of an activin type II receptor (ActRII) signaling inhibitor for a period of time; and

(b) determining the level of ICAM-1, IL-1Ra, survivin, Bcl-2, Bcl-xL, MCP-1, serpinE1, GRO-a, IL-8, IL-10, IL-2, RANTES, IP-10, IL-1a, IL-1b, MIF, G-CSF, GMCSF, C5a, IL-6, HO-2, HIF-1a, TRAIL R1, cleaved caspase-3, p27, p21, Bax, Bad, CIAP1, or PON2 in the supernatant obtained from the culture of step (a);

wherein the selected subject has:

(i) an increase in the level of ICAM-1 in the supernatant of the in vitro cell culture method as compared to the level of ICAM-1 in a control supernatant

(ii) an increase in the level of IL-1Ra in the supernatant of the in vitro cell culture as compared to the level of IL-1Ra in a control supernatant

(iii) an increase in the level of survivin in the supernatant of the in vitro cell culture method as compared to the level of survivin in a control supernatant

(iv) an increase in the level of Bcl-2 in the supernatant of the in vitro cell culture method as compared to the level of Bcl-2 in a control supernatant;

(v) an increase in the level of Bcl-xL in the supernatant of the in vitro cell culture method as compared to the level of Bcl-xL in a control supernatant;

(vi) an increase in the level of MCP-1 in the supernatant of the in vitro cell culture method as compared to the level of MCP-1 in a control supernatant;

(vii) an increase in the level of serpinE1 in the supernatant of the in vitro cell culture method as compared to the level of serpinE1 in a control supernatant;

(viii) an increase in the level of GRO-a in the supernatant of the in vitro cell culture method as compared to the level of GRO-a in a control supernatant;

(ix) an increase in the level of IL-8 in the supernatant of the in vitro cell culture method as compared to the level of IL-8 in a control supernatant;

(x) an increase in the level of IL-10 in the supernatant of the in vitro cell culture method as compared to the level of IL-10 in a control supernatant;

(xi) an increase in the level of IL-2 in the supernatant of the in vitro cell culture method as compared to the level of IL-2 in a control supernatant;

(xii) an increase in the level of CIAP1 in the supernatant of the in vitro cell culture method as compared to the level of CIAP1 in a control supernatant;

(xiii) an increase in the level of PON2 in the supernatant of the in vitro cell culture method as compared to the level of PON2 in a control supernatant;

(xiv) a decrease in the level of RANTES in the supernatant of the in vitro cell culture method as compared to the level of RANTES in a control supernatant;

(xv) a decrease in the level of IP-10 in the supernatant of the in vitro cell culture method as compared to the level of IP-10 in a control supernatant;

(xvi) a decrease in the level of IL-1a in the supernatant of the in vitro cell culture method as compared to the level of IL-1a in a control supernatant;

(xvii) a decrease in the level of IL-1b in the supernatant of the in vitro cell culture method as compared to the level of IL-1b in a control supernatant;

(xviii) a decrease in the level of MIF in the supernatant of the in vitro cell culture method as compared to the level of MIF in a control supernatant;

(xix) a decrease in the level of G-CSF in the supernatant of the in vitro cell culture method as compared to the level of G-CSF in a control supernatant;

(xx) a decrease in the level of GMCSF in the supernatant of the in vitro cell culture method as compared to the level of GMCSF in a control supernatant;

(xxi) a decrease in the level of C5a in the supernatant of the in vitro cell culture method as compared to the level of C5a in a control supernatant;

(xxii) a decrease in the level of IL-6 in the supernatant of the in vitro cell culture method as compared to the level of IL-6 in a control supernatant;

(xxiii) a decrease in the level of HO-2 in the supernatant of the in vitro cell culture method as compared to the level of HO-2 in a control supernatant;

(xxiv) a decrease in the level of HIF-1a in the supernatant of the in vitro cell culture method as compared to the level of HIF-1a in a control supernatant;

(xxv) a decrease in the level of TRAIL R1 in the supernatant of the in vitro cell culture method as compared to the level of TRAIL R1 in a control supernatant;

(xxvi) a decrease in the level of cleaved caspase-3 in the supernatant of the in vitro cell culture method as compared to the level of cleaved caspase-3 in a control supernatant;

(xxvii) a decrease in the level of p27 in the supernatant of the in vitro cell culture method as compared to the level of p27 in a control supernatant;

(xxviii) a decrease in the level of p21 in the supernatant of the in vitro cell culture method as compared to the level of p21 in a control supernatant;

(xxix) a decrease in the level of Bax in the supernatant of the in vitro cell culture method as compared to the level of Bax in a control supernatant; and/or

(xxx) a decrease in the level of Bad in the supernatant of the in vitro cell culture method as compared to the level of bad in a control supernatant.

2. The method of claim 1 , wherein the patient is being monitored using the in vitro cell culture method.

3. The method of claim 1 , wherein the subject is a human.

4. The method of claim 1 , wherein the stromal cell has been obtained from the bone marrow the subject.

5. The method of claim 1 , wherein the erythroid progenitor cell has been obtained from peripheral blood of the subject.

6. The method of claim 1 , wherein the ActRII signaling inhibitor is a polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) 90% identical to SEQ ID NO:2;

(b) 95% identical to SEQ ID NO:2;

(c) 98% identical to SEQ ID NO:2;

(d) SEQ ID NO:2;

(e) 90% identical to SEQ ID NO:3;

(f) 95% identical to SEQ ID NO:3;

(g) 98% identical to SEQ ID NO:3;

(h) SEQ ID NO:3;

(i) 90% identical to SEQ ID NO:6;

(j) 95% identical to SEQ ID NO:6;

(k) 98% identical to SEQ ID NO:6;

(l) SEQ ID NO:6;

(m) 90% identical to SEQ ID NO:7;

(n) 95% identical to SEQ ID NO:7;

(o) 98% identical to SEQ ID NO:7;

(p) SEQ ID NO:7;

(q) 90% identical to SEQ ID NO:12;

(r) 95% identical to SEQ ID NO:12;

(s) 98% identical to SEQ ID NO:12;

(t) SEQ ID NO:12;

(u) 90% identical to SEQ ID NO:17;

(v) 95% identical to SEQ ID NO:17;

(w) 98% identical to SEQ ID NO:17;

(x) SEQ ID NO:17;

(y) 90% identical to SEQ ID NO:20;

(z) 95% identical to SEQ ID NO:20;

(aa) 98% identical to SEQ ID NO:20;

(bb) SEQ ID NO:20;

(cc) 90% identical to SEQ ID NO:21;

(dd) 95% identical to SEQ ID NO:21;

(ee) 98% identical to SEQ ID NO:21;

(ff) SEQ ID NO:21;

(gg) 90% identical to SEQ ID NO:25;

(hh) 95% identical to SEQ ID NO:25;

(ii) 98% identical to SEQ ID NO:25; and

(jj) SEQ ID NO:25.

7. The method of claim 1 , wherein the ActRII signaling inhibitor is an ActRIIA signaling inhibitor.

8. The method of claim 7 , wherein the ActRIIA signaling inhibitor is a polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) 90% identical to SEQ ID NO:2;

(b) 95% identical to SEQ ID NO:2;

(c) 98% identical to SEQ ID NO:2;

(d) SEQ ID NO:2;

(e) 90% identical to SEQ ID NO:3;

(f) 95% identical to SEQ ID NO:3;

(g) 98% identical to SEQ ID NO:3;

(h) SEQ ID NO:3;

(i) 90% identical to SEQ ID NO:6;

(j) 95% identical to SEQ ID NO:6;

(k) 98% identical to SEQ ID NO:6;

(l) SEQ ID NO:6;

(m) 90% identical to SEQ ID NO:7;

(n) 95% identical to SEQ ID NO:7;

(o) 98% identical to SEQ ID NO:7; and

(p) SEQ ID NO:7.

9. The method of claim 1 , wherein the ActRII signaling inhibitor is a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

10. The method of claim 1 , wherein the ActRII signaling inhibitor is a humanized fusion-protein consisting of the extracellular domain of ActRIIA and the human IgG1 Fc domain.

11. The method of claim 1 , wherein the wherein the ActRII signaling inhibitor is a signaling inhibitor of ActRIIB.

12. The method of claim 11 , wherein the ActRIIB signaling inhibitor is a polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) 90% identical to SEQ ID NO:17;

(b) 95% identical to SEQ ID NO:17;

(c) 98% identical to SEQ ID NO:17;

(d) SEQ ID NO:17;

(e) 90% identical to SEQ ID NO:20;

(f) 95% identical to SEQ ID NO:20;

(g) 98% identical to SEQ ID NO:20;

(h) SEQ ID NO:20;

(i) 90% identical to SEQ ID NO:21;

(j) 95% identical to SEQ ID NO:21;

(k) 98% identical to SEQ ID NO:21;

(l) SEQ ID NO:21;

(m) 90% identical to SEQ ID NO:25;

(n) 95% identical to SEQ ID NO:25;

(o) 98% identical to SEQ ID NO:25; and

(p) SEQ ID NO:25.

13. The method of claim 1 , wherein the ActRII signaling inhibitor is a polypeptide comprising the amino acid sequence of SEQ ID NO:25.

14. The method of any of claim 1 , wherein the ActRII signaling inhibitor is a humanized fusion-protein consisting of the extracellular domain of ActRIIB and the human IgG1 Fc domain.