Judy C. Chang

Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

β-thalassemia, one of the most common genetic diseases worldwide, is caused by mutations in the human hemoglobin beta (HBB) gene. Creation of human induced pluripotent stem cells (iPSCs) from β-thalassemia patients could offer an approach to cure this disease. Correction of the disease-causing mutations in iPSCs could restore normal function and provide a rich source of cells for transplantation. In this study, we used the latest gene-editing tool, CRISPR/Cas9 technology, combined with the piggyBac transposon to efficiently correct the HBB mutations in patient-derived iPSCs without leaving any residual footprint. No off-target effects were detected in the corrected iPSCs, and the cells retain full pluripotency and exhibit normal karyotypes. When differentiated into erythroblasts using a monolayer culture, gene-corrected iPSCs restored expression of HBB compared to the parental iPSCs line. Our study provides an effective approach to correct HBB mutations without leaving any genetic footprint in patient-derived iPSCs, thereby demonstrating a critical step toward the future application of stem cell-based gene therapy to monogenic diseases.

Targeted disruption of the ubiquitous CNC-bZIP transcription factor, Nrf-1, results in anemia and embryonic lethality in mice

The CNC‐basic leucine zipper (CNC‐bZIP) family is a subfamily of bZIP proteins identified from independent searches for factors that bind the AP‐1‐like cis‐elements in the β‐globin locus control region. Three members, p45‐Nf‐e2, Nrf‐1 and Nrf‐2 have been identified in mammals. Expression of p45‐Nf‐e2 is largely restricted to hematopoietic cells while Nrf‐1 and Nrf‐2 are expressed in a wide range of tissues. To determine the function of Nrf‐1, targeted disruption of the Nrf‐1 gene was carried out. Homozygous Nrf‐1 mutant mice are anemic due to a non‐cell autonomous defect in definitive erythropoiesis and die in utero.

A Sensitive New Prenatal Test for Sickle-Cell Anemia

METHODS for prenatal diagnosis of sickle-cell anemia are improving rapidly.1 Initially the diagnostic procedure required fetal blood sampling, but new techniques for restriction-endonuclease analys...

Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection

Significance Patients homozygous for the C-C chemokine receptor type 5 (CCR5) gene with 32-bp deletions (Δ32) are resistant to HIV infection. Using the piggyBac technology plus transcription activator-like effector nucleases or clustered regularly interspaced short palindromic repeats-Cas9, the authors report, to our knowledge, for the first time in induced pluripotent stem cells (iPSCs) the efficient and seamless derivation of a homozygous CCR5Δ32 mutation, exactly mimicking the natural mutation. Monocytes and macrophages differentiated from these mutated iPSCs in vitro are resistant to HIV infection. This approach can be applied in the future toward the functional cure of HIV infection. The findings are also of great interest to researchers in many fields who wish to correct or introduce mutations in specific genes. Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9–mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

Generation of induced pluripotent stem cells using site-specific integration with phage integrase.

To date, a large number of reports have described reprogramming many somatic cell types into induced pluripotent stem (iPS) cells, using different numbers of transcription factors and devising alternate methods of introducing the transcription factor genes or proteins into the somatic cells. Here, we describe a method using bacteriophage ΦC31 integrase to reprogram mouse embryonic fibroblasts and human amniotic fluid cells into iPS cells. These iPS cells showed morphology, surface antigens, gene expression, and epigenetic states similar to ES cells and formed teratomas with three germ layers in nonobese diabetic/severely compromised immunodeficient mice. Importantly, these iPS cells have only a single integration site in each cell line. The locations of integration favor the intergenic regions, and their distances from the adjacent genes extended from several hundred to >1 million bp. The effect of the insertion on the expression of these genes can be studied by RT-PCR. No insertion into microRNA gene loci was detected. Hence, it is possible to select cells in which adjacent gene functions are not affected, or the inserts can be removed if necessary. We conclude that phage integrase-mediated site-specific recombination can produce iPS cells that have undisturbed endogenous gene function and could be safe for future human therapeutic application.

Fetal gene therapy of α-thalassemia in a mouse model

Fetuses with homozygous α-thalassemia usually die at the third trimester of pregnancy or soon after birth. Hence, the disease could potentially be a target for fetal gene therapy. We have previously established a mouse model of α-thalassemia. These mice mimic the human α-thalassemic conditions and can be used as preclinical models for fetal gene therapy. We tested a lentiviral vector containing the HS 2, 3, and 4 of the β-LCR, a central polypurine tract element, and the β-globin gene promoter directing either the EGFP or the human α-globin gene. We showed that the GFP expression was erythroid-specific and detected in BFU-E colonies and the erythroid progenies of CFU-GEMM. For in utero gene delivery, we did yolk sac vessel injection at midgestation of mouse embryos. The recipient mice were analyzed after birth for human α-globin gene expression. In the newborn, human α-globin gene expression was detected in the liver, spleen, and peripheral blood. The human α-globin gene expression was at the peak at 3–4 months, when it reached 20% in some recipients. However, the expression declined at 7 months. Colony-forming assays in these mice showed low abundance of the transduced human α-globin gene in their BFU-E and CFU-GEMM and the lack of its transcript. Thus, lentiviral vectors can be an effective vehicle for delivering the human α-globin gene into erythroid cells in utero, but, in the mouse model, delivery at late midgestation could not transduce hematopoietic stem cells adequately to sustain gene expression.

Assigning the polymorphic human insulin gene to the short arm of chromosome 11 by chromosome sorting

SummaryWe have determined the subchromosomal location of the human insulin gene by analyzing DNA isolated from sorted human metaphase chromosomes. Metaphase chromosome suspensions were sorted into fractions according to relative Hoechst fluorescence intensity by the fluorescence activated chromosome sorter. The chromosomal DNA in each fraction was characterized by restriction endonuclease analysis. Initial sorts indicated that the insulin gene-containing fragment resided in a fraction containing chromosomes 9, 10, 11 and 12. Studies of cell lines that contained chromosome translocations permitted the assignment of the insulin gene to a derivative chromosome that contains portions of the short arm of chromosome 11. Simultaneous sorting of the normal homolog from this small derivative chromosome separated the two different sized insulin gene-containing restriction fragments in this individual. These data indicate that the two restriction fragments represent insulin gene polymorphism and not duplicate gene loci.

Suppression of the nonsense mutation in homozygous beta 0 thalassaemia.

The common form of β thalassaemia associated with elevated haemoglobin A2 levels can be broadly classified as β+ or β0 type according to the presence or absence of β-globin chain synthesis in the homozygous state1–4. The molecular pathology of each type is heterogeneous3–9. Apart from a subgroup of Indo-Pakistani patients10, the β-globin structural gene is intact in the majority of patients with β0 thalassaemia2–4. The amount of β-globin mRNA present in the reticulocytes of these patients varies5–7: in some it is absent or barely detectable; in others, a substantial amount is present, but it is nonfunctional. We recently demonstrated that the molecular lesion in a Chinese patient with nonfunctional β-globin mRNA11,12 was due to the mutation of the normal lysine codon AAG at amino acid 17 to the amber terminator codon UAG, which prematurely terminates the β-globin chain13. In the present study we demonstrate the first example of a nonsense mutation in humans which can be suppressed in vitro by the suppressor tRNA, as has been found in other eukaryotic cells and viruses14,15.

TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

Molecular diagnosis of hemoglobinopathies and thalassemia

The idea of investigating prenatal diagnosis of the hemoglobinopathies and thalassemia began with some key observations on hemoglobin synthesis early in life. First, biosynthetic studies in a sample of cord blood showed that β-globin chain synthesis in the cord blood of a newborn was significantly lower than normal (Kan and Nathan, 1968). Follow-up studies confirmed that the child was heterozygous for β-thalassemia. Subsequently, Hollenberg and coworkers reported that adult hemoglobin synthesis could be detected in fetuses at midtrimester and suggested that prenatal diagnosis of sickle-cell anemia and related hemoglobinopathies might be possible (Hollenberg et al., 1971). This was followed by detection of the βS-globin chain in the blood of a fetus (Kan et al., 1972). Fetal blood sampling by placentocentesis or fetoscopy was then introduced (Hobbins and Mahoney, 1974; Kan et al., 1974), and prenatal diagnosis became a clinical test using fetal blood analysis and practiced in this way for the next 5 years or so.