Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation

The therapeutic potential of genome editing for β-thalassemia.

The rapid advances in the field of genome editing using targeted endonucleases have called considerable attention to the potential of this technology for human gene therapy. Targeted correction of disease-causing mutations could ensure lifelong, tissue-specific expression of the relevant gene, thereby alleviating or resolving a specific disease phenotype. In this review, we aim to explore the potential of this technology for the therapy of β-thalassemia. This blood disorder is caused by mutations in the gene encoding the β-globin chain of hemoglobin, leading to severe anemia in affected patients. Curative allogeneic bone marrow transplantation is available only to a small subset of patients, leaving the majority of patients dependent on regular blood transfusions and iron chelation therapy. The transfer of gene-corrected autologous hematopoietic stem cells could provide a therapeutic alternative, as recent results from gene therapy trials using a lentiviral gene addition approach have demonstrated. Genome editing has the potential to further advance this approach as it eliminates the need for semi-randomly integrating viral vectors and their associated risk of insertional mutagenesis. In the following pages we will highlight the advantages and risks of genome editing compared to standard therapy for β-thalassemia and elaborate on lessons learned from recent gene therapy trials

Animal models of beta-hemoglobinopathies: utility and limitations

The structural and functional conservation of hemoglobin throughout mammals has made the laboratory mouse an exceptionally useful organism in which to study both the protein and the individual globin genes. Early researchers looked to the globin genes as an excellent model in which to examine gene regulation - bountifully expressed and displaying a remarkably consistent pattern of developmental activation and silencing. In parallel with the growth of research into expression of the globin genes, mutations within the β-globin gene were identified as the cause of the β-hemoglobinopathies such as sickle cell disease and β-thalassemia. These lines of enquiry stimulated the development of transgenic mouse models, first carrying individual human globin genes and then substantial human genomic fragments incorporating the multigenic human β-globin locus and regulatory elements. Finally, mice were devised carrying mutant human β-globin loci on genetic backgrounds deficient in the native mouse globins, resulting in phenotypes of sickle cell disease or β-thalassemia. These years of work have generated a group of model animals that display many features of the β-hemoglobinopathies and provided enormous insight into the mechanisms of gene regulation. Substantive differences in the expression of human and mouse globins during development have also come to light, revealing the limitations of the mouse model, but also providing opportunities to further explore the mechanisms of globin gene regulation. In addition, animal models of β-hemoglobinopathies have demonstrated the feasibility of gene therapy for these conditions, now showing success in human clinical trials. Such models remain in use to dissect the molecular events of globin gene regulation and to identify novel treatments based upon the reactivation of developmentally silenced γ-globin. Here, we describe the development of animal models to investigate globin switching and the β-hemoglobinopathies, a field that has paralleled the emergence of modern molecular biology and clinical genetics

Interplay between α‐thalassemia and β‐hemoglobinopathies: Translating genotype–phenotype relationships into therapies

Abstract α‐Thalassemia represents one of the most important genetic modulators of β‐hemoglobinopathies. During this last decade, the ongoing interest in characterizing genotype–phenotype relationships has yielded incredible insights into α‐globin gene regulation and its impact on β‐hemoglobinopathies. In this review, we provide a holistic update on α‐globin gene expression stemming from DNA to RNA to protein, as well as epigenetic mechanisms that can impact gene expression and potentially influence phenotypic outcomes. Here, we highlight defined α‐globin targeted strategies and rationalize the use of distinct molecular targets based on the restoration of balanced α/β‐like globin chain synthesis. Considering the therapies that either increase β‐globin synthesis or reactivate γ‐globin gene expression, the modulation of α‐globin chains as a disease modifier for β‐hemoglobinopathies still remains largely uncharted in clinical studies

A reduced curcuminoid analog as a novel inducer of fetal hemoglobin

Thalassemia is an inherited disorder of hemoglobin molecules that is characterized by an imbalance of α- and β-globin chain synthesis. Accumulation of unbound α-globin chains in erythroid cells is the major cause of pathology in β-thalassemia. Stimulation of γ-globin production can ameliorate disease severity as it combines with the α-globin to form fetal hemoglobin. We examined γ-globin-inducing effect of curcuminoids extracted from Curcuma longa L. and their metabolite reduced forms in erythroid leukemia K562 and human primary erythroid precursor cells. The results showed that curcuminoid compounds, especially bisdemethoxycurcumin are potential γ-globin enhancers. We also demonstrated that its reduced analog, hexahydrobisdemethoxycurcumin (HHBDMC), is most effective and leads to induction of γ-globin mRNA and HbF in primary erythroid precursor cells for 3.6 ± 0.4- and 2.0 ± 0.4-folds, respectively. This suggested that HHBDMC is the potential agent to be developed as a new therapeutic drug for β-thalassemia and related β-hemoglobinopathies

Phenotypic comparison of four thalassemia model mice reconstructed from cryo-banked embryos

A major clinical feature of patients with thalassemia is growth retardation due to anemia, therefore, the hematological parameters, weanling weight and post-weanling weight of pups obtained from vitrifiedwarmed embryo transfers were studied for the first time in this report. Two-cell embryos of four transgenic (TG) thalassemic mouse lines (BKO, 654, E2, and E4) were produced by breeding four lines of TG thalassemic males to wild-type (WT) females (C57BL/6J) and were cryopreserved by vitrification in straws using 35% ethylene glycol. After transfer of vitrified-warmed embryos, hematological parameters, spleen index, weanling and post-weanling weight were determined in TG and WT viable pups. In the BKO and 654 mice significantly abnormal hematological parameters and spleen index were observed compared to WT, E2 and E4 mice. The weanling and post-weanling weights of BKO and 654 pups were significantly less than that of the age-matched WT pups. However, no significant differences in weanling and post-weanling weight were found between WT and E2-TG or E4-TG pups. In conclusion, the four transgenic mice lines produced from cryopreserved embryo transfer retain the phenotype of the natural breeding mice indicating that these banked embryos are appropriate for thalassemia model productions