Vitro cell culture methods for beta-thalassemia using activin type ii receptor ligand traps

Abstract

Provided herein are methods of treating beta-thalassemia in a subject comprising administering to the subject an activin type II receptor (ActRII) signaling inhibitor (e.g., an activin ligand trap) and utilizing one or more in vitro cell culture methods provided herein in (i) selection of the subject to be treated according to the methods provided herein; and/or (ii) monitoring of the subject being treated according to the methods provided herein.

What is claimed: 
     
         1 . An in vitro cell culture method, comprising
 (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.   
     
     
         2 . An in vitro cell culture method, comprising
 (a) co-culturing an 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 expansion of the EPC.   
     
     
         3 . An in vitro cell culture method, comprising
 (a) culturing 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 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).   
     
     
         4 . An in vitro cell culture method, comprising
 (a) culturing an EPC in conditioned media for a period of time, wherein the conditioned media has been obtained from a stromal cell cultured in the presence of an ActRII signaling inhibitor; and   (b) determining the level of GYPA, GATA1, GATA2, and/or alpha-globin in the EPC and/or 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).   
     
     
         5 . An in vitro cell culture method, comprising
 (a) culturing an EPC in conditioned media for a period of time, wherein the conditioned media has been obtained from a stromal cell cultured in the presence of an ActRII signaling inhibitor; and   (b) determining the level of expansion of the EPC.   
     
     
         6 . An in vitro cell culture method, comprising
 (a) culturing an EPC in the presence of an ActRII signaling inhibitor for a period of time; and   (b) determining the level of GYPA, GATA1, GATA2, or alpha-globin in the EPC and/or 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).   
     
     
         7 . The method of any one of  claims 1 - 5 , wherein the stromal cell has been obtained from bone marrow of a beta-thalassemic subject. 
     
     
         8 . The method of any one of the preceding claims, wherein the erythroid progenitor cell has been obtained from peripheral blood of a beta-thalassemic subject. 
     
     
         9 . The method of any one of the previous claims, 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;   (1) 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.   
     
     
         10 . The method of any of the preceding claims, wherein the ActRII signaling inhibitor is an ActRIIA signaling inhibitor. 
     
     
         11 . The method of  claim 10 , 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;   (1) 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.   
     
     
         12 . The method of claim any one of the previous claims, wherein the ActRII signaling inhibitor is a polypeptide comprising the amino acid sequence of SEQ ID NO:7. 
     
     
         13 . The method of any of  claims 1 - 8 , wherein the ActRII signaling inhibitor is a humanized fusion-protein consisting of the extracellular domain of ActRIIA and the human IgG1 Fc domain. 
     
     
         14 . The method of any of  claims 1 - 9 , wherein the wherein the ActRII signaling inhibitor is a signaling inhibitor of ActRIIB. 
     
     
         15 . The method of  claim 14 , 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;   (1) 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.   
     
     
         16 . The method of  claim 15 , wherein the ActRII signaling inhibitor is a polypeptide comprising the amino acid sequence of SEQ ID NO:25. 
     
     
         17 . The method of any of  claims 1 - 8 , wherein the ActRII signaling inhibitor is a humanized fusion-protein consisting of the extracellular domain of ActRIIB and the human IgG1 Fc domain. 
     
     
         18 . Use of the in vitro cell culture method of any one of the previous claims for predicting responsiveness of a subject to treatment of beta-thalassemia, wherein the subject has been administered an ActRII signaling inhibitor, wherein the EPC has been obtained from the subject. 
     
     
         19 . Use of the in vitro cell culture method of any one of the previous claims for predicting responsiveness of a subject to treatment with an ActRII signaling inhibitor, wherein the stromal cell has been obtained from the subject. 
     
     
         20 . Use of the in vitro cell culture method of any one of the previous claims for monitoring treatment of beta-thalassemia in a subject administered an initial pharmaceutically effective dose of an ActRII signaling inhibitor, wherein the EPC has been obtained from the subject. 
     
     
         21 . Use of the in vitro cell culture method of any one of the previous claims for monitoring treatment of beta-thalassemia in a subject administered an initial pharmaceutically effective dose of an ActRII signaling inhibitor, wherein the stromal cell has been obtained from the subject. 
     
     
         22 . Use of the in vitro cell culture method of any one of the previous claims for selecting a subject to be administered an ActRII signaling inhibitor, wherein the EPC has been obtained from the subject. 
     
     
         23 . Use of the in vitro cell culture method of any one of the previous claims for selecting a subject to be administered an ActRII signaling inhibitor, wherein the stromal cell has been obtained from the subject. 
     
     
         24 . The use of  claim 22  or  23 , wherein the use comprises selecting the subject if use of the in vitro cell culture method results in achieving one or more outcome parameter, wherein the outcome parameter is selected from a group consisting of:
 (a) 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; 
 (b) 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; 
 (c) 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; 
 (d) 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; 
 (e) 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 
 (f) 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 
 (g) 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 
 (h) 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 
 (i) 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; 
 (j) 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; 
 (k) 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; 
 (l) 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; 
 (m) 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; 
 (n) 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; 
 (o) 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; 
 (p) 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; 
 (q) 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; 
 (r) 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; 
 (s) 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; 
 (t) 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; 
 (u) 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; 
 (v) 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; 
 (w) 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; 
 (x) 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; 
 (y) 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; 
 (z) 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; 
 (aa) 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; 
 (bb) 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; 
 (cc) 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; 
 (dd) 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; 
 (ee) 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; 
 (ff) 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; 
 (gg) 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; 
 (hh) 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 
 (ii) 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. 
 
     
     
         25 . The use of any one of  claims 18 - 24 , wherein the subject has beta-thalassemia. 
     
     
         26 . 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 the in vitro cell culture method of any of  claims 1 - 17 . 
     
     
         27 . A method of treating beta-thalassemia in a subject, comprising administering an ActRII signaling inhibitor to the subject, wherein the patient is being monitored by using the in vitro cell culture method of any of  claims 1 - 17 . 
     
     
         28 . The method of  claim 26  or  27 , wherein the subject is a human.