Complete bibliography: https://www.ncbi.nlm.nih.gov/myncbi/14gVia2i54M5O/bibliography/public/
Development of methods to produce cultured red blood cells from hematopoietic stem cells and from pluripotent stem cells.
Progress in cell culture methods has open up the possibility of manufacturing red blood cells (RBCs) for transfusion. We have developed methods to produce large number of red blood cells from hematopoietic stem cells and from pluripotent stem cells. Using detailed analysis of globin chain expression, we were the first to demonstrate that differentiation of hESCs and iPSCs into erythroid cells faithfully recapitulates the embryonic and fetal stages of human erythropoiesis but do not yield red blood cells with an adult phenotype. We also demonstrated that fetal stage erythroid cells derived from pluripotent cells can enucleate in vitro.Since these early studies, observations that cells produced from pluripotent cells are developmentally immature have been made in many other lineages, by others. Finding methods to produce developmentally mature cells from pluripotent stem cells has become a central focus of many labs including my own. We recently obtained NIH funding to apply our advanced culture methods and translate our results into a commercial product by developing a panel of IPSC lines from patients carrying very rare blood groups that can be used as universal donor for transfusion and as reagent RBCs that will be used to type antibodies in allo-immunized patients with sickle cell disease.
1. Olivier EN, Qiu C, Velho M, Hirsch RE, Bouhassira EE. Large-scale production of embryonic red blood cells from human embryonic stem cells. Exp Hematol. 2006 Dec;34(12):1635-42. PMID: 17157159 Cited by 161
2. Qiu C, Olivier EN, Velho M, Bouhassira EE. Globin switches in yolk sac primitive and fetal definitive RBCs produced from human embryonic stem cells. Blood. 2008; 111(4):2400-8. PMID: 18024790 Cited by 154
3. Wang K, Guzman AK, Yan Z, Zhang S, Hu MY, Hamaneh MB, Yu YK, Tolu S, Zhang J, Kanavy HE, Ye K, Bartholdy B, Bouhassira EE. Ultra-High-Frequency Reprogramming of Individual Long-Term Hematopoietic Stem Cells Yields Low Somatic Variant Induced Pluripotent Stem Cells. Cell Rep. 2019 Mar 5;26(10):2580-2592.e7 PMID: 30840883 Cited by 2
4. Olivier EN, Zhang S, Yan Z, Suzuka S, Roberts K, Wang K, Bouhassira EE. RED, an Albumin-Free Robust Erythroid Differentiation Method to Produce Enucleated Red Blood Cells from Human Pluripotent and Adult Stem Cells. Exp Hematol. 2019 Jul;75:31-52.e15 PMID: 31176681
5. Olivier EN, Wang K, Grossman J, Mahmud N, Bouhassira EE. Differentiation of Baboon (Papio anubis) Induced-Pluripotent Stem Cells into Enucleated Red Blood Cells. Cells. 2019 Oct 19;8(10).PMID: 31635069.
Gene therapy for the hemoglobinopathies
Lentiviral based gene therapy for the hemoglobinopathies is coming of age. We have contributed to the field by studying at the basic science level the major causes of transgene silencing in erythroid cells. Using the RMCE method we have demonstrated that in eukaryotic cells, silencing is not primarily caused by integration near heterochromatin but rather by transcriptional interferences between the transgenes and neighboring sequences. By studying cassettes that were either devoid of any CpG dinucleotides, or that were pre-methylated prior to integration, we were able to demonstrate that DNA methylation is not necessary for silencing but that it confers an epigenetic memory.
We also demonstrated that inclusion of insulators in gene therapy cassettes was a double edged sword since these elements can prevent silencing and insertional mutagenesis at some genetic loci, but can also cause silencing and insertional mutagenesis at other locations. Some of these basic science findings were applied to design the gene therapy cassettes that were used, in collaboration with the Leboulch lab, to provide the first proof of principle in mice that gene therapy could be used to cure sickle cell disease. These vectors are currently tested by BlueBird therapeutics in human clinical trials that have had encouraging results.
1. Feng YQ, Warin R, Li T, Olivier E, Besse A, Lobell A, Fu H, Lin CM, Aladjem MI, Bouhassira EE. (2005): The Human b-Globin Locus Control Region Can Silence as Well as Activate Gene Expression. Mol Cell Biol.25: (10):3864-74. PMID: 15870261 Cited by 44
2. Feng YQ, Desprat R, Fu H, Olivier E, Lin CM, Lobell A, Gowda SN, Aladjem MI, Bouhassira EE. (2006). DNA methylation supports intrinsic epigenetic memory in mammalian cells. PLoS Genet. 2006 Epub 2006 Apr 28. PMID: 16683039 Cited by 75
3. Pawliuk R., KA Westerman, ME Fabry, E Payen, R Tighe, EE Bouhassira, SA Acharya, J Ellis, IM London, CJ Eaves, RK Humphries, Y Beuzard, RL Nagel, P Leboulch (2001): Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 294:2368-71. PMID: 11743206 Cited by 574
4. Boulad F, Maggio A, Xiuyan Wang X, Moi P, Acuto S, Kogel F, Takpradit A, Prockop S, Mansilla-Soto J, Cabriolu A, Odak A. Thummar K, Du F, Shen L, Raso s, Barone R, Di Maggio R, Pitrolo L, Giambona A, Mingoia M, Everett JK, Hokama P, Roche A, Cantu A, Adhikari H, Reddy S, Bouhassira EE, Mohandas N, Bushman FD, Rivière I, Sadelain M (2001) Lentiviral globin gene therapy with reduced-intensity conditioning in adults with β-thalassemia: a phase 1 trial. Nat Med. 2022 Jan;28(1):63-70.
Development of Recombinase-Mediated Cassette Exchange and safe harbor concept
Lentiviral transduction, stable transfection and transgenesis results in random integration of transgenes which leads to positive or negative position-effects. Position-effects greatly complicate gene therapy as well as the interpretation of most stable transfection or transduction experiments. To eliminate these problems, we have developed RMCE, a method that allows site-specific integration of cassettes at predetermined chromosomal locations in mammalian cells. We used the method extensively to better understand the molecular basis of position-effects in erythroid cells (see above). Many other labs, all over the world, have adopted RMCE, and adapted it to many cell types and many organisms. Over 500 studies that take advantage of RMCE have been published.
The use of the RMCE led us to develop the concept of safe harbors as an efficient method to perform gene therapy safely and cost efficiently, without having to design and test novel vector or gene editing strategy for every mutation in every defective human gene. We demonstrated the concept by correcting alpha-thalassemia in iPSCs by targeting constructs to the AAVS1.
1. Bouhassira EE., K Westerman, P Leboulch: (1997) Transcriptional Behavior Of LCR Elements Integrated At The Same Chromosomal Locus By RMCE. Blood, 90: 3332-3244. PMID: 9345015 Cited by 177
2. Feng YQ, Seibler J, Alami R, Eisen A, Fiering SN, Bouhassira EE: (1999) Site-Specific Chromosomal Integration In Mammalian cells: Highly Efficient CRE Recombinase-Mediated Cassette Exchange. J. Mol. Biol. 292 (4): 779-785. PMID: 10525404 Cited by 249
3. Chang CJ and Bouhassira EE. Zinc-finger nuclease mediated correction of α-thalassemia in iPS cells. Blood. 2012; Nov 8;120(19):3906-14. PMID: 23002118 Cited by 100
Molecular basis of Thrombotic Thrombocytopenic Purpura
In collaboration with Dr. Han-Mou Tsai we have demonstrated that ADAMTS13 is responsible for congenital TTP, that auto-antibody resistant forms of the protein could be generated, that ADMTS13 is expressed predominantly in stellate cells, and we have explored some of the molecular mechanisms associated with low levels of ADAMTS13 expression in human population.
1. Levy GG, Nichols WC, Lian EC, Foroud T, McClintick JN, McGee BM, Yang AY, Siemieniak DR, Stark KR, Gruppo R, Sarode R, Shurin SB, Chandrasekaran V, Stabler SP, Sabio H, Bouhassira EE, Upshaw JD Jr, Ginsburg D, Tsai HM. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature. 2001; 413(6855):488-94. PMID: 11586351. Cited by 1683
2. Zhou W, Dong L, Ginsburg D, Bouhassira EE, Tsai HM. Enzymatically active ADAMTS13 variants are not inhibited by anti-ADAMTS13 autoantibodies: a novel therapeutic strategy? The Journal of biological chemistry. 2005; 280(48):39934-41. PMID: 16203734 Cited by 59
3. Zhou W, Inada M, Lee TP, Benten D, Lyubsky S, Bouhassira EE, Gupta S, Tsai HM. ADAMTS13 is expressed in hepatic stellate cells. Laboratory investigation; a journal of technical methods and pathology. 2005; 85(6):780-8. PMID: 15806136 Cited by 162
4. Zhou W, Bouhassira EE, Tsai HM. An IAP retrotransposon in the mouse ADAMTS13 gene creates ADAMTS13 variant proteins that are less effective in cleaving von Willebrand factor multimers. Blood. 2007; 110(3):886-93. PMID: 17426255 Cited by 38
Characterization of replication timing and replication origins in human primary erythroid cells
Gene transcription is regulated by transcription factors and by chromatin structure. Using the Recombinase-Mediated Cassette Exchange (RMCE) method and b-globin transgenes as a model, we demonstrated that replication timing plays a critical role in gene expression at some genetic loci in erythroid cells. This prompted us to investigate the mechanisms that regulate replication timing in basophilic erythroblasts. In collaboration with the Schildkraut and Mieg labs, we developed the TimEX and TimEX-seq method to measure replication timing genome-wide using tiling micro-arrays and massively parallel sequencing. We applied these methods to generate genome-wide maps of replication timing in several cell types that demonstrated that replication timing is very tightly regulated in mammalian cells and that the timing of replication and gene expression levels are very closely linked.
In collaboration with the Aladjem lab, we also developed methods to generate allele-specific profiles of replication timing and replication origins. Using these maps and a novel analytic approach, we show that the two chromosome homologs replicate within a few minutes of each other in about 92% of the genome but that about 8% of the genome replicate asynchronously. Asynchrony is associated with imprinting random mono-allelic expression and proximity to large structural variants. We also showed that an asymmetry in nucleotide distribution, which increases the propensity of origins to unwind and adopt non-B DNA structure, rather than the ability to form G4-quadruplexes, is directly associated with origin activity. This work also led to the development of GenPlay, a powerful, JAVA, open-source genome browser and analyzer application that is available online and currently utilized by over 100 labs worldwide.
1. Fu H., Lixin W., Lin CH, Singhania S, Bouhassira EE, Aladjem MI. Human Replicators Can Prevent Gene Silencing. Nature Biotech. 2006, 24(5):572-6. PMID: 16604060 Cited by 37
2. Desprat R, Thierry-Mieg D, Lailler N, Lajugie J, Schildkraut C, Thierry-Mieg J, Bouhassira EE. Predictable dynamic program of timing of DNA replication in human cells. Genome Research. 2009 Dec;19(12):2288-99. PMID: 19767418 Cited by 117
3. Mukhopadhyay R, Lajugie J, Fourel N, Selzer A, Schizas M, Bartholdy B, Mar J, Lin CM, Martin MM , Ryan M, Aladjem MI and Bouhassira EE. Allele-Specific Genome-wide Profiling in Human Primary Erythroblasts Reveal Replication Program Organization. PLoS Genet. 2014 May 1;10(5):e1004319. PMID: 24787348 Cited by 42
4. Bartholdy B, Mukhopadhyay R, Lajugie J, Aladjem MI, Bouhassira EE. Allele-specific analysis of DNA replication origins in mammalian cells. Nat Commun. 2015 May 19;6:7051. PMID: 25987481 Cited by 32