Prevention of Transcriptional γ-globin Gene Silencing by Inducing
The Hereditary Persistence of Fetal Hemoglobin Point Mutation
Using Chimeraplast-Mediated Gene Targeting
Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies.
Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy
to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced
Materials and Methods
In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively.
Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation
confirmed erythroid differentiation in HSCs and K562 cells.
Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both
EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit
Beta-thalassemia is one of the most common monogenic diseases, which turns out to be a significant public health concern due to its global burden and several complications associated with its homozygous form. Currently, thalassemia major patients are treated with regular blood transfusion, iron chelation and judicious splenectomy, all of which are temporary strategies accompanied by complications and life threatening side effects (1, 2).
Currently, allograft hematopoietic stem cell
transplantation (HSCT) is thought to be the only definitive
treatment for ß-thalassemia patients. Although, the
success of transplantation, under ideal conditions, is more
than 90%, the major limitation is lack of HLA-matched
donors and therewith an outbreak of acute and chronic
graft-versus-host disease (3). Several gene therapy
techniques have been introduced which insert a normal
copy of the
Chimeraplasty is a non-viral gene therapy technique in which single nucleotide conversion is executed via an artificial chimeric oligonucleotide. This oligonucleotide is synthesized from DNA and RNA fragments and is complementary to a specific sequence except for a single mismatch nucleotide at the substitution position of interest. Accordingly, this approach is exclusive to site- specific gene correction of point mutations (7-9).
Recently, hemoglobin F (HbF) inducers, both
pharmaceutical and genetic agents, have attracted a vast
interest for their potential therapeutic characteristics
in ameliorating the severity of symptoms in Cooley’s
anemia and sickle cell disease. Non-deletional hereditary
persistence of fetal hemoglobin (HPFH) is a genetic
disorder mainly caused by point mutations in the
Taking advantage of the HPFH genetic mechanism,
we tried to induce the HPFH-like point mutation (
Materials and Methods
Chimeric oligonucleotide designing
In this experimental study, a 68-base synthesized
chimeric RNA/DNA oligonucleotide (RDO) (now termed
chimeraplast) was designed comprising a central core of 5
DNA-based nucleotides flanked by 10 2´-O-Methyl RNA
sequences. To obtain stability in the chimeraplast structure,
two nuclease-resistant hairpin caps of 4 T-residues were
also designed followed by 25 complementary nucleotides
to both the central DNA and the surrounding 2´-O-Methyl
RNA sequences at the 5 end. Additionally, short regions
with high melting temperature sequences were inserted
at the 3´ end (9). The chimeraplast sequence was entirely
matched to the corresponding genomic sequence of
Erythroid series were differentiated from HSCs existing in peripheral blood mononuclear cells (PBMNCs) using one-phase liquid medium culture system. Anti- coagulated whole blood samples, collected from normal volunteers with informed consent, were mixed with equal volume of phosphate buffered saline (PBS) and gently layered at a ratio of 3:1 onto the mononuclear separation medium (Lymphodex, Inno-Train, Germany). Following centrifugation, the mononuclear layer at the interface was harvested and cultured in a 6-well culture plate at a density of 6×106 cells per ml of Iscove’s Modified Dulbecco’s Medium (IMDM, Caisson, USA) containing 30% fetal bovine serum (not heat-inactivated, Gibco, USA), 1% bovin serum albumin, ß-mercaptoethanol (10-5 M, Sigma, USA), dexamethasone sodium phosphate (10-6 M, Sigma, USA), human hollo-transferrin (0.3 mg/mL, Sigma, USA) and StemSpan™ Erythroid Expansion supplement which contained recombinant human stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO) (STEMCELL Technologies, Canada).
Cells were then incubated for 28 days at 37°C with 5% CO2 and the differentiation medium was refreshed on day 14. At four time points over a 28-day period (days 7, 14, 21 and 28) cells were harvested from culture media and prepared for morphology assessment (Wright staining). Consequently, the expression of the surface marker, CD235a, was assessed by flow cytometry and the expression of globin gene was quantified using quantitative reverse transcription polymerase chain reaction (RT-qPCR).
K562 cells (ATCC, USA) were also differentiated
with analogous conditions in 7 days and were similarly
assessed for erythroid differentiation as well as
Erythroid differentiation was tracked by evaluating the percentage of CD235a (Glycophorin A) positive cells per well of each individual 6-well culture plate. Cells were harvested, transferred to a 1.5 ml microtube and spun down at 500 xg for 5 minutes. To prevent the interference of pre-existing red blood cells (RBCs), the cell pellet was washed in 1 ml of RBC lysing solution and incubated with 5 µl of monoclonal anti-Glycophorin A-phycoerythrin (PE) (Dako, Denmark). After, the cells were washed in PBS and the single cell suspension, prepared in 500 µl PBS, was subjected to a flow cytometer (BD FACSCalibur, USA) versus PE labeled isotype control. Data were analysed using the FlowJo 7.6 software (Tree Star Inc., USA).
Upon erythroid colony growth on day 16, following culture initiation, once erythroid cells were strikingly increased in number, cells were transfected with RDO using a polycationic vector, polyethyleneimine (PEI, Sigma, USA). PEI (30 µl of 1 mg/ml) and RDO (10 µg) had been previously diluted in 120 µl of serum and antibiotic free Opti-MEM media (Gibco, USA), and incubated at room temperature for 10 minutes to form RDO-PEI complexes. Subsequently, 850 µl of supplemented IMDM was added to the complex and the mixture was then added dropwise to 6-well plates and mixed by gently rocking. The same trend was performed for the K562 cell line with the transfection time being on day 1.
To evaluate transfection efficiency, FAM-RDOtransfected EPCs and K562 cells were detected with flow cytometry and fluorescent microscopy on the second day of transfection. Cells were initially harvested and washed in PBS, and then assessed prior and after labeling with anti CD235a-PE antibody through the FL1 and FL2 channels of a BD FACSCalibur flow cytometer. Additionally, a number of cells were evaluated for nuclear entry of chimeric oligonucleotides by Fluorescence Microscope Axiostar Plus (Gottingen, Germany).
Polymerase chain reaction-restriction-fragment length polymorphism
Presence of the point mutation in the
|Target||Sequence primer (5ˊ-3ˊ)|
|BCL11a-xl cDNA||F: GTCTCGCCGCAAGCAAGG|
|AS-qPCR wild type||F: AAACTGGAATGACTGAATCG|
|AS-qPCR mutant||F: AAACTGGAATGACTGAATCG|
RT-qPCR; Quantitative reverse transcription polymerase chain reaction and AS-qPCR; Allele specific-quantitaive PCR.
Digested fragments were isolated and extracted by GEL DNA recovery kit (Vivantis, Malaysia), and then ligated with T4 DNA ligase (Vivantis, Malaysia) for 4 hours at 16ºC. Eventually, ligated DNA was reamplified and directly sequenced along with undigested wild-type fragment using the same primer pairs used in PCR- restriction-fragment length polymorphism (PCR-RFLP) (Table 1,).
Chimeraplasty efficiency by allele specific-quantitative polymerase chain reaction
Allele-specific quantitative PCR (AS-qPCR) was carried out by using a real-time PCR system (QIAGEN, Germany) to quantify the relative allelic rate of mutant HBG promoter. Wild type and mutant HBG promoters were amplified by a single common forward primer and reverse allele-specific primers (Table 1,). Amplification efficiency of the intended amplicons were determined by the standard curve of each allele through logarithmic dilution of PCR products of each amplicon. Eventually, the ratio of mutant alleles versus wild-type was quantified. Subsequently, amplified products were verified by 2% agarose gel electrophoresis.
Quantitative reverse transcription polymerase chain reaction
Total RNA was purified from erythroid and K562
cells using the TRIzol reagent (Life Technologies, USA)
and quantified using the NanoDrop spectrophotometer.
Subsequently, DNaseI treated RNA was reverse
transcribed to complementary DNA (cDNA) by using the
one-step SYBR PrimeScript RT Reagent Kit (TaKaRa,
Japan). Relative quantification of
Data are presented as mean ± SD based on replicate experiments. Independent t test was used to compare unpaired groups. The results with P<0.05 was considered as statistically significant. All data were statistically analysed and visualized using the GraphPad Prism software (version 6.04, GraphPad Software, CA).
Erythroid differentiation of peripheral blood hematopoietic stem cells and the K562 cell line
HSCs from PBMNCs were successfully differentiated to mature erythroid cells. Flow cytometry assessment revealed an approximately two-fold increase in the percentage of CD235a positive cells (1.4 to 2.38%) after one-week of differentiation. Two weeks after differentiation, CD235a positive cells reached the highest value of 27.6% and remained rather constant (26.8%) within the next 7 day interval even though a slight decrease was observed at the experimental end point (21.9%) (Fig .1A,).
The first colonies of erythroid series were discernible under the inverted microscope 3 days following the culture initiation. Between days 7 and 14, cells displayed early normoblast morphology (pronormoblast and basophilic normoblast) with Wright’s stain. Colonies expanded and gradually spread over a period of 21 days. On day 21, a considerable number of erythroid cells transformed into polychromatophilic normoblasts, which gave rise to orthochromatophilic normoblasts on day 28 (Fig .1B, C,).
Different stages of erythroid maturation in growth factor-stimulated K562 cells were also observed with Romanowsky stain. After 7 days of treatment with erythroid differentiation factors, K562 cells differentiated into orthochromatophilic normoblasts and the percentage of CD235 positive cells changed from 3.26% on day 0 to an average of 42.2% on day 7.
After 24 and 48 hours, the percentage of transfected cells including CD235a-positive cell population was found to be more than 70% (ranging from 70 to 80%) by flow cytometry assessment. However, the green fluorescent signal visualized from FAM-labeled RDOs in nuclei showed that only one third of the cells were successfully nucleofected at the final time point (Fig .2,). Nucleofection was assessed after adequate resting time including 24 and 48 hours following transfection. Nevertheless, it might be assumed that even after 2 days, RDOs could still introduce into the cells, leading to an elevation of nucleofection efficiency.
Polymerase chain reaction- restriction-fragment length polymorphism
A 223 bp DNA fragment spanning the target region
Gel-extracted 151 bp and 72 bp fragments were ligated,
re-amplified and sequenced with the Sanger method,
confirming the PCR-RFLP result by showing the G→A
substitution at position -117 in the
To assess the quantification of gene conversion, AS-qPCRwas used. Amplification efficiency (E) was determined to be
0.98 for the wild-type allele, and 1.0 for the mutant alleleand the housekeeping gene. The mean cycle of threshold(CT) value of mutant (A) allele, non-mutant (G) allele and
Gamma and ß-globin gene transcript levels
Adecreasing trend in the ratio of
Along with cell maturation, relative expression level
Effect of the inducible variant on the expression level
γ and ß-globin
Subsequent to nucleotide substitution in the genomicregion of interest,
expression levels of
Accordingly, the relative expression of γ-globin to the
housekeeping gene (
Although the rate of success of chimeraplasty is still under debate, it has been so far used for inducing or modifying point mutation in various studies. This rate varies substantially in previous studies from 0.05% reported by Igoucheva et al. (12) to 50% reported by Cole-strauss et al. (13). Surveys have shown that numerous factors including RDO structure, quality, concentration and size along with type of cell and delivery system may significantly influence the success or conversion rate of this method (14).
Here, we used chimeraplasty for a G→A nucleotide
substitution at position -117 of the
Although our results showed a more efficient rate of
nucleotide conversion in K562 cells in comparison with
EPCs, in contrast to Addya et al. (15) and Isoda et al. (16),
A number of investigations have reported that the
inability of erythroid growth factors (IL-3, EPO and SCF)
in mediating the
In a recent similar work by Chin et al. (24) triplex-forming
peptide nucleic acids were utilized to mediate targeted
gene conversion of -117 HPFH and hypoxia response
element (HRE) donor DNA in expansion conditions of
CD34+ cells. This resulted in significant
Secondly, if the effect of such variants results in elevated
It is worth noting that in contrast to our study, which directly targeted the cell genome, Li et al. (29) designed a chimeric oligonucleotide to trigger gene conversion in a plasmid, however, it resulted in a lower efficiency associated with plasmid instability in subcloning cells.
In the present study, the
This article was extracted from a Ph.D. thesis and was financially supported by Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. The authors declare no conflict of interest.
R.R., M.N.Z., H.G.; Contributed to conception and design. R.R., M.N.Z.; Contributed to all experimental work, data and statistical analysis, and interpretation of data. S.S., A.A.P.; Were responsible for overall supervision. R.R.; Drafted the manuscript, which was revised by M.N.Z. and A.A.P. All authors read and approved the final manuscript.