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Next generation advanced therapies for fight ß-hemoglobinopathies via rational intervention in ?-globin regulatory network

Applicant Benenson Yaakov
Number 182969
Funding scheme Bilateral programmes
Research institution Computational Systems Biology Department of Biosystems, D-BSSE ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Biochemistry
Start/End 01.08.2019 - 31.07.2023
Approved amount 349'999.00
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All Disciplines (2)


Keywords (4)

Hemoglobinopathies; Synthetic transcription factors; Cell therapy; Gene circuits

Lay Summary (German)

Neue therapeutische Ansätze für Hämoglobinopathien
Lay summary

Hämoglobinopathien wie Thalassämie, Sichelzellenerkrankungen und andere sind monogene Erkrankungen mit hoher Prävalenz in der indischen Bevölkerung. Sie zeichnen sich durch einen niedrigen Hämoglobinspiegel bei Erwachsenen im Blut des Patienten aus, was zu einer Vielzahl von klinischen Symptomen und einer schlechten Lebensqualität für den Patienten führt. Fötales Hämoglobin ist ein Protein, das während der Entwicklung des Fötus aktiv ist und in den ersten Lebensmonaten stillgelegt wird. Die Aktivierung des fetalen Hämoglobins beim Erwachsenen ist ein praktikabler Weg, um die klinischen Symptome der Krankheit zu lösen.

Unser Vorschlag enthält einen Fahrplan für die Reaktivierung des fetalen Hämoglobins durch Eingriffe in andere Gene und Proteine, die es normalerweise abschalten. Die Ausschaltkomponenten von fötalem Hämoglobin sind inzwischen bekannt. Unser Ziel ist es, dieses Wissen zu nutzen, um rationale Eingriffe in eine oder mehrere dieser Komponenten vorzunehmen. Wir verwenden eine Reihe von komplementären Ansätzen, um dieses Ziel zu erreichen. Ein von den Partnern in Indien entwickelter Ansatz verwendet Peptidmoleküle mit genregulatorischen Aktivitäten. Der zweite von den Partnern in der Schweiz entwickelte Ansatz zielt darauf ab, mit gentechnisch veränderten Instrumenten in das Netzwerk einzugreifen. Zusammen werden diese Ansätze neue Wege zur Behandlung von Hämoglobinopathien eröffnen

Direct link to Lay Summary Last update: 15.07.2019

Responsible applicant and co-applicants

Gesuchsteller/innen Ausland



Group / person Country
Types of collaboration
Bose Institute India (Asia)
- in-depth/constructive exchanges on approaches, methods or results
JNCASR India (Asia)
- in-depth/constructive exchanges on approaches, methods or results

Associated projects

Number Title Start Funding scheme
175760 Multi-input synthetic gene circuits: reduction to practice 01.08.2018 Project funding (Div. I-III)


Beta-hemoglobinopathies such as beta-thalassemia, sickle cell disease, and others are monogenic disorders with high prevalence in the Indian population. Their clinical management is difficult, expensive and at best partially effective. Fetal hemoglobin (HbF) is a tetramer of two alpha-globin and two beta-globin subunits (the latter being the protein product of HBG1 and HBG2 genes) that is naturally expressed during fetal development but is replaced with adult hemoglobin (HbA, a tetramer of two alpha-globin and two beta-globin subunits) in the first months of life. It was shown that increase of HbF levels via upregulation of gamma-globin expression ameliorates many of the acute symptoms of these pathologies. However, except hydroxyurea against sickle cell anemia, no molecule that acts by up-regulation of fetal hemoglobin has reached the clinic. Allogeneic stem-cell transplantation is so far the only viable curative option, although fraught with enormous post-treatment complications.The proposed research will create new therapeutic options by exploring a number of cutting-edge avenues to implement targeted interference with the gene-regulatory network of gamma-globin genes, with the end goal to upregulate their expression. The first class of agents include synthetic transcription factors (peptide-based molecules suitable for systemic delivery) targeted against a number of regulatory domains controlling the gamma-globin gene so as to lift the repression of the normally silenced gamma-globin gene in the adult. The second class of agents is genetically encoded regulators that are suitable for lentiviral transduction into patient-derived autologous hematopoietic stem cells followed by re-transplantation. These multi-pronged regulators will likewise be designed to relieve the repression of gamma-globin. Further, their expression will be controlled in cell-lineage specific fashion so as not to disrupt the other functions of the regulatory network in non-erythropoietic lineages. Therapeutic candidates will be evaluated in progressively elaborate in vitro assays that mimic the pathophysiology of ß-hemoglobinopathies. We focus on four main regulators of gamma-globin gene expression that comprise the main nodes of the regulatory network, including Klf1, LRF/ZBTB7A, BCL11A, and BP1/DLX-4. ZBTB7A and BCl11A repress gamma-globin via a number of pathways. Notably, naturally occurring genetic mutation in the BCL11A binding domain in gamma-globin promoter creates a phenotype with high constitutive HbF expression that is resistant to beta-hemoglobinopathies. BP1 is also hypothesized to repress gamma-globin, in addition to its well-known role of beta-globin repression. Thus, interference with the DNA binding activity of these four factors, combined with active downregulation of these factors’ expression, has the potential to reactivate gamma-globin expression in adult erythrocytesDirect regulation of gene expression by modulating transcription factor-DNA target site binding is a challenging problem that so far has not yielded a clinically useful molecule. However, recent work has opened up new avenues using synthetic transcription factors (STF). STF are generally understood as small or medium-size synthetic molecules that can bind to specific DNA sequences and regulate targeted genes when applied to live cells. Two major classes of molecules can acts as STFs. Polyamides, developed by Dervan and coworkers, have been the mainstay for decades. Recent work proposed conformationally-constrained synthetic peptides as an alternative with certain advantages. Our team members have extensive experience engineering STF based on conformation-constrained peptides. In recent work, we already developed a highly specific peptide-based synthetic transcription factor targeted against BP1/DLX-4, a homeodomain-containing transcriptional repressor of globin genes. Although BP1 is primarily known as a beta-globin repressor, sequence analysis suggests that it may also act as a repressor of the gamma-globin gene, HBG1. Prof. Roy and coworkers synthesized a peptide-based conformationally constrained synthetic transcription factor targeted against BP1. It was shown to up-regulate the gamma-globin gene in CD34+ hematopoietic stem cells induced with erythropoietin (preliminary data). This expertise will be applied in the current proposal to address additional transcriptional nodes as described above; in addition, the candidate STF against BP-1 will be further explored and improved. This and other STFs developed in this project will be improved via conjugation to a specific minor-groove binding molecule developed by Prof. Govindaraju with the help of suitable computational techniques. The efficiency of the STF will be further enhanced by conjugation of a Histone Acetyltransferase (HAT) activator developed by Prof. Kundu. We have already designed and synthesized a variant of this molecule that has the appropriate functional group for attachment to peptides. Lastly, the delivery to live cells will be improved used cell penetrating and nuclear localization signals for nuclear delivery. Pharmacokinetics and pharmacodynamics properties will be modulated using biodegradable glucose-based nanoparticles developed by Prof. Kundu. Genetically-encoded regulators will include engineered transcriptional regulators to interfere with the TF-promoter binding of the natural gamma-globin network, and artificial microRNAs to downregulate key repressors of gamma-globin expression. One the partners, Prof. Benenson, has extensive experience in both areas with ample track record of engineering novel transcriptional and miRNA regulators. Because the intervention targets have other roles in non-erythropoietic lineages, it is also crucial to only unleash these modifiers in the right cells at the right time. Here, we will utilize Benenson group expertise in designing specific cell targeting tools (so-called “cell classifiers”) and engineer a sensor network that identifies the cell lineage where ?-globin upregulation is required, namely, erythroblasts. For this purpose, available profiling data on hematopoietic system and erythropoietic lineages, supplemented with our own software pipeline, will generate a number of selective signatures that will be employed to restrict gene expression. In particular, because the genetic payload is embedded in CD34+ HSC, care must be taken not to induce HBG expression in all hematopoietic lineages but the erythroid precursors. Building on our expertise with cell classifiers we propose a path toward targeted gene therapy where the expression of gene inducers modeled on the STFs from the first arm, and gene inhibitors such as synthetic miRNAs, and possibly chromatin modifiers developed together with the chromatin expert from our team, will be controlled in a cell-lineage specific fashion. Thus, the expression of the activators, inhibitors, and modifiers will only take place at the right stage of erythropoiesis. Both the peptide-based and genetic-encoded modulators will be tested individually and in combinations in a variety of cell-based assays for their effect on gamma-globin expression as well as possible non-specific effects on other genes, the latter profiled using gene expression and chip-seq analysis. Importantly, the effect of lineage control in cell classifier expression systems will be assessed to validate the improved specificity. The in vitro systems will include erythropoietin induced CD34+ hematopoietic stem cells derived from human cord blood, cell lines such as K562, erythroid precursors isolated from e.g., umbilical cord blood, and recently-developed immortalized erythropoietic cell lines. Promising candidates will be further tested in patient-derived erythroid precursor cells or genome-edited (beta-globin mutated or truncated) hematopoietic stem cells, and in the case of positive results, lead to an in vivo proof-of-concept study in the next phase of the project.