This proposal continues a highly productive program that is committed to developing new therapeutic strategies for thalassemia and sickle cell disease utilizing endogenous or exogenous genes encoding human embryonic globins. Key analyses have established that heterotetrameric hemoglobins incorporating -globin subunits possess physiologically appropriate O2-binding properties and, unexpectedly, also display highly significant antisickling activity. Additional studies indicate the essential role that post-transcriptional mechanisms play in regulating -globin expression, through effects on the functional properties of its encoding mRNA. This critical work is extended in three new Specific Aims that investigate how the native structure of embryonic ?-globin mRNA dictates its stability and translation when expressed in adult-stage, definitive erythroid cells.
The Aims utilize techniques that capitalize on the experience of the applicant laboratory, as well as established methods that have been modified to address the experimental imperatives of this proposal.
Aim I will define specific cis-acting elements that dictate the stability of ?-globin mRNA, through a novel cell- culture based saturation mutagenesis strategy. Sequence substitutions that optimize the function of these elements--producing hyperstable ?-globin mRNAs--will subsequently be identified using an innovative in vivo SELEX approach.
Aim II will identify primary and secondary structural motifs within the -globin 5'UTR and coding region that suppress its translation in definitive erythroid cells by inhibiting 40S ribosomal scanning or 80S ribosomal processivity. These elements will then be altered in a manner that maximizes the efficiency with which ?-globin protein is translated from its cognate mRNA in vivo in intact cells.
Aim III will test two independent methods for augmenting ?-globin expression in primary definitive human erythroid cells, by combining the site-specific function-enhancing sequence substitutions identified in Aims I and II, and by engineering an increase in the cellular levels of tRNAs required for efficient ?-globin translation. The results from all three Aims will (a) detail the critical role that post-transcriptional processes play in regulating the developmental expression of human globins, (b) generate a ?-globin mRNA that is post-transcriptionally optimized to produce a globin protein with demonstrated therapeutic utility and, most importantly, (c) describe a highly innovative approach that can be used to augment the expression of other globin and nonglobin genes. The proposed research comprises a crucial step in an evolving understanding of the role that post- transcriptional processes play in regulating gene expression, and the manner in which they can be manipulated to therapeutic advantage in patients with thalassemia and sickle cell anemia.

Public Health Relevance

Despite recent improvements in clinical care, congenital genetic disorders affecting the normal expression of human globins--including those responsible for sickle cell disease and thalassemia--remain a major cause of morbidity and premature mortality both in the United States and abroad. Future therapeutic advances are likely to require new treatment paradigms based upon innovative molecular strategies. The current proposal describes one new approach, in which processes that regulate the stability and translational efficiency of globin mRNAs are manipulated to therapeutic advantage. This strategy is particularly attractive because it can be applied to augment the effectiveness of existing therapies, including gene-reactivation and gene-replacement approaches.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Qasba, Pankaj
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Pennsylvania
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Safo, Martin K; Ko, Tzu-Ping; Schreiter, Eric R et al. (2015) Structural basis for the antipolymer activity of Hb ?2?(s)2 trapped in a tense conformation. J Mol Struct 1099:99-107
van Zalen, Sebastiaan; Lombardi, Alyssa A; Jeschke, Grace R et al. (2015) AUF-1 and YB-1 independently regulate ?-globin mRNA in developing erythroid cells through interactions with poly(A)-binding protein. Mech Dev 136:40-52
He, Zhenning; Song, Decheng; van Zalen, Sebastiaan et al. (2014) Structural determinants of human ?-globin mRNA stability. J Hematol Oncol 7:35
Safo, Martin K; Ko, Tzu-Ping; Abdulmalik, Osheiza et al. (2013) Structure of fully liganded Hb ?2?2s trapped in a tense conformation. Acta Crystallogr D Biol Crystallogr 69:2061-71
Abdulmalik, Osheiza; Lombardi, Alyssa A; Russell, J Eric (2012) A reverse time-course method for transcriptional chase analyses of mRNA half-lives in cultured cells. PLoS One 7:e40827
van Zalen, Sebastiaan; Jeschke, Grace R; Hexner, Elizabeth O et al. (2012) AUF-1 and YB-1 are critical determinants of ýý-globin mRNA expression in erythroid cells. Blood 119:1045-53
Manning, James M; Popowicz, Anthony M; Padovan, Julio C et al. (2012) Intrinsic regulation of hemoglobin expression by variable subunit interface strengths. FEBS J 279:361-9
Liu, Xingge; Jiang, Yong; Russell, J Eric (2010) A potential regulatory role for mRNA secondary structures within the prothrombin 3'UTR. Thromb Res 126:130-6
Manning, Lois R; Popowicz, Anthony M; Padovan, Julio et al. (2010) Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces. Protein Sci 19:1595-9
Liu, X; Russell, J E (2010) Cytoplasmic stabilities of 3'UTR-polymorphic prothrombin mRNAs. J Thromb Haemost 8:2580-3

Showing the most recent 10 out of 20 publications