US 20190270992A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2019 /0270992 A1 BRESNICK et al. (43 ) Pub . Date : Sep . 5 , 2019 ( 54 ) METHODS OF CONTROLLING RED BLOOD (52 ) U . S . CI. CELL PRODUCTION CPC . .. .. C12N 15 / 113 ( 2013 .01 ) ; C12N 5 /0641 (2013 .01 ) ; A61K 33 / 30 ( 2013 .01 ) ; A61K 45 / 06 ( 71 ) Applicant: WISCONSIN ALUMNI RESEARCH ( 2013 . 01 ) ; A61K 31 / 7088 ( 2013 .01 ) ; ACIK FOUNDATION , MADISON , WI (US ) 35 / 18 (2013 .01 ) ; C12N 2310 / 14 ( 2013 . 01 ) ; C12N 2506 / 11 (2013 . 01 ) ; C12N 2501 / 999 (72 ) Inventors : EMERY H . BRESNICK , ( 2013 . 01 ) ; C12N 2501 / 90 ( 2013 .01 ) ; C12N MIDDLETON , WI (US ); NOBUYUKI 2310 / 122 ( 2013 .01 ) ; CI2N 2310 / 531 TANIMURA , KITA -KU ( JP ) ( 2013 .01 ) ; C12N 2506 /03 ( 2013 .01 ) (21 ) Appl. No. : 16 /292 , 922 (57 ) ABSTRACT A method of controlling red blood cell production includes ( 22 ) Filed : Mar. 5 , 2019 contacting red blood cell precursors with a composition including zinc , a composition including an inhibitor of a zinc Related U . S . Application Data exporter protein , or a combination thereof; wherein the (60 ) Provisionalapplication No .62 / 638 , 563, filed on Mar . contacting promotes survival of the red blood cell precur 5 , 2018 sors , promotes terminal differentiation of the red blood cell precursors to mature red blood cells , or a combination Publication Classification thereof; or contacting red blood cell precursors with a ( 51 ) Int. Cl. composition including a zinc chelator, a composition includ C12N 15 / 113 ( 2006 .01 ) ing an inhibitor of a zinc importer protein , or a combination C12N 5 /078 ( 2006 . 01 ) thereof ; wherein the contacting inhibits survival of the red A61K 3330 ( 2006 .01 ) blood cell precursors, inhibits terminal differentiation of the A61K 45 /06 ( 2006 . 01 ) red blood cell precursors to mature red blood cells , or a A61K 31 / 7088 ( 2006 .01 ) combination thereof . A61K 35 / 18 ( 2006 .01 ) Specification includes a Sequence Listing . Erythroid Maturation Zing Transporter Switch Here L . .. .. Exporter Slc30a1 Importer Sic39a8 Intracellular Zinc GATA . gove Heme Importer Experter impostos Exporter { Zinc ) [Zinc ] SurvivalDifferentiation Terminal Differentiation Patent Application Publication Sep . 5 , 2019 Sheet 1 of 31 US 2019 / 0270992 A1 WT Double Mutant miminimin tim timinimiminna n GATA - 1 Alas2 Alas2 ALAS - 2 ALAS - 2 Heme + B - estradiol FIG . IA GATA - 1 -regulated proteins Alas2 - enhancer- regulated proteins Para SLC4A ) H88 -81 GATA - 2 HBE -B1 SLC4A1 month Cutoeoop ** * fanyesd)"*601- BACH1 ALAS - 2 ALAS EF2S1m EF3K ER2S F3K @ AU numpaunanny of 1111111111111111 11 Fold change : log , Est + WTV/ EST - WT1 ) Fold change: 0g , (Est Mutt ! Est + WT1) < 0 .05 and Fold change (log , transfornec ): >> 1 > 0 , 05 P < 0 .05 and Fold change (log , transformed ) < 1 FIG . 1B Patent Application Publication Sep . 5 , 2019 Sheet 2 of 31 US 2019 /0270992 A1 GATA - 1 - activated GATA - 1 - repressed RNA - seq (3431 ) RNA - seq ( 3781) Proteomics 472 | 637 3144 FIG . 1C Alas2 - enhancer- activated Alas2 - enhancer- repressed RNA - Seq (738 ) ANA -Seq (602 ) 1608 _ 35 ) 703 230 135 ) 567 FIG . ID Patent Application Publication Sep . 5 , 2019 Sheet 3 of 31 US 2019 /0270992 A1 EF3K U23 SLC4A1 is EF251 FIG.1E Mut1 w B1-HBB teistele WTT BACHT 600 4Q opens * mutang mengenai angan p ) units Relative( ) units Relative( @ intensities Peptide intensities Peptide Patent Application Publication Sep . 5 , 2019 Sheet 4 of 31 US 2019 / 0270992 A1 ANA - Sea 66 GATA Theme activated genes Proteomics 3323 4 not detected others GATA - 1 / Alas2 - enhancer - activated 3 : GATA - 1 - activated & Alas2 -enhancer activated Globin Ubiquitin (0 . 5 < Fold change (log , transformed ): < 1 ) Fbxo30 Srunt 3 : GATA - 1 - activated Ube20 2 Alas2anhancer- activated Hbb -bh1 Tubb2a Slc30a ) . W . ti proteins " activatedWAW proteins LC30A1 HBB -B1 HBB -B1017 HBA - X SLC30A1 HBA- X ?????? , -109(Pvalue) BX030 . HBB -B200 H BB HBB- BH1 SRXN1 3102 SRXN1 TUBB2A UBE20 KIF2A L TUBB2A 0 2 4 6 8 10 - 10 8 6 4 2 0 Fold change log , (Est WT1 / Est- WT1) log , (Est + Mutt/ Est* WT1 ) P < 0 .05 and Fold change ( log , transformed ) > 1 P < 0 .05 and (Fold change (log , transformed ) < 1 M FIG . 1F Patent Application Publication Sep . 5 , 2019 Sheet 5 of 31 US 2019 /0270992 A1 . o Est + WT1 . O Est- Mutt . O Est + Muti . .. 60 . 0 0 . 20. 60. 60 . FIG . 2 Sic30 faniily ( Zinc exporter ) Sic39 family (Zinc importer ) 1 2 3 4 5 6 7 8 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 WT1 - Est Normalized TPM FIG . 3A Zinc exporter Zinc importer Sla30a1 Sic39a8 MRNALevels (PelativeUnits) winning L 0 .0 5 - ALA : - inti/8Muti? int1/8Mut2 WT int1/8Mutiara int1/8Mut24 WT3 int1/8Mut1 int1/6 - estradiot hy B -estradiol - B - estradiol 6 - estradiol FIG . 3B Patent Application Publication Sep . 5 , 2019 Sheet 6 of 31 US 2019 /0270992 A1 SLC30A10 wi SLC39A10 . SLC30A6 FIG.3C SLC30A5 SLC39A7 w w . olen aina my SLC30A1 olen SLC39A6 - namume namwanammed ) units Relative( intensities peptideestradiol D- intensities Peptide Patent Application Publication Sep . 5 , 2019 Sheet 7 of 31 US 2019 / 0270992 A1 R2 18 .2 R3 > -CD71 SA12 . MUS STRA. * ** W . wuuuuuuuuuuuuuuuuuuu %livecells R1 R2 R3 R4 /5 10 % 0 10 % 10 10 % - Ter119 FIG . 4A Slc30a1 (Zinc exporter ) O Slc39a8 (Zinc importer ) Hbb -b1 MRNALevels(Relativeunits) yyyyy R1 R2 R3 R475 R1 R2 R3 R4/ 5 FIG . 4B Patent Application Publication Sep . 5 , 2019 Sheet 8 of 31 US 2019 / 0270992 A1 20 OOOOOOOSSS hereTotal 20 PRIDR2R3R4 16 Hemsfrom OXYHO,6 13 ) pm( call / Herre 20 Day witament Day:9 Succinylacetone(SA) ) % ( Population 82.0 Differentiation3 Day20(SA) CD235a 54.30940 ?????????????????????????????????????????? TY 56789012121314151617181920 Day20 33.30182 Differentiation2 yyyyR31072 FIG.4C Day16 310R2 9.9902000 Differentiation1 Day13 Sorel02.962 lR3104A2 l Julebe Expansion D CD71 Patent Application Publication Sep . 5 , 2019 Sheet 9 of 31 US 2019 / 0270992 A1 Day 3 : Day 2049A • . - - FIG . 4D F13A1 SLCIQAT SLC29A8 ? MANALevels(Helativeunits) nothese ALL ?lay : in 15ATTAS 200 10 15 A 20 0 15 20 0 .0 FIG . 4E HEAT SLC30A1 SLC39A8 ACIB MANALevels {Relativeunits) 3 .LoL ALL . mirror por t finiiiiier rete day 19 20 39 20 19 20 19 20 FIG . 4F Patent Application Publication Sep . 5 , 2019 Sheet 10 of 31 US 2019 / 0270992 A1 . , , , , , , , , , . .. * . D PoE Ortho . , , , , , , , , , . : : : : : : : : : : : : : : : . Slc30 family ( Zinc exporter ) SQUI Sic39 family (Zinc importer) . .. .. Y . S . $ . ment. W . : : : : : : : : : . R2 . X . $ < . Relativeunits 2000 . 2000 . : : . .. ) . , ' , . ' . , , ' , . powiniinimumininiaisumisieireire C . .. : : : : : : : : : : : : 2 . Ni : : : : : : : : S . .. yolongannnnnnnnnnnnnnn asian ? . 1 2 3 4 5 6 7 8 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ' ' . - . IV N A • • • , Aja S E T ' ' ' • • • 2000 SLC30 family (Zinc exporters Duowo SL439WWWVTORY family (Zinc I importer ) UBUNH Relativeunits 30000 10 . who camerieriguarantee . 1 2 3 5 6 7 8 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 FIG . 5 Patent Application Publication Sep . 5 , 2019 Sheet 11 of 31 US 2019 /0270992 A1 . 9 . " . ' Ter119 . ' . 11 . LOR O NARYONO . CD71 . Y: . 3 R . :. · . .. ASSAS24 . t 122 . Yir . 23 . 2 . 7. : . 2 . R17 5. 21 . 73 . 3 . * . .* . , , , , , , , ' , ' , ' , ' ' ' . ' , , ' , i , ' , * * * * * . - . ' . 2 . Y . V . 3 . V21 .OOO . ??????????????????????????????????????????????????????????????????????????????????????????????????????? - Ter119 CD71high CD71 high FSC.