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Article

An Integrated Gene Therapy Strategy for γ-Globin Addition and HbF Reactivation in β-Thalassemia

by
Kiriaki Paschoudi
1,2,†,
Ninos Ioannis Vasiloudis
1,2,†,
Fotios Papadopoulos
1,2,
Xenia Nikolaou
1,2,
Anastasia Papadopoulou
1,2,
Pavel Sova
3,
Evangelia Yannaki
2,4 and
Nikoletta Psatha
1,*
1
Department of Genetics, Development & Molecular Biology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
2
Gene and Cell Therapy Center and Hematology Clinic/Bone Marrow Transplant Unit, “George Papanikolaou” Hospital, 57010 Thessaloniki, Greece
3
Altius Institute for Biomedical Sciences, Seattle, WA 98121, USA
4
Division of Hematology, Department of Medicine, University of Washington, Seattle, WA 98195, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Thalass. Rep. 2026, 16(3), 19; https://doi.org/10.3390/thalassrep16030019
Submission received: 15 July 2026 / Revised: 19 August 2026 / Accepted: 21 August 2026 / Published: 1 September 2026
(This article belongs to the Section Innovative Treatment of Thalassemia)
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Abstract

Background: β-Thalassemia and other β-hemoglobinopathies arise from defective β-globin production, and reactivating fetal hemoglobin (HbF) is a well-established strategy to ameliorate disease severity. Two principal gene-therapy approaches—erythroid-specific γ-globin gene addition and shRNA/shmiRNA-mediated knockdown of the γ-globin repressor BCL11A—have each shown clinical promise, but their combination within a single vector remains largely unexplored. Methods: Here we developed and compared three compact, C1-insulated lentiviral vectors driven by the micro-locus control region (μLCR): a BCL11A-targeting shmiRNA vector, a γ-globin cDNA vector, and a dual construct combining both elements. Results: All vectors were produced at high titers, and incorporating both cassettes into a single construct did not compromise vector production or cell viability. In CD34+ hematopoietic stem and progenitor cells from healthy donors and β-thalassemia patients, transduction did not impair proliferation, erythroid differentiation, or progenitor colony formation. All three vectors significantly increased the proportion of HbF-expressing cells, with the combined shmiRNA + γ-globin cDNA vector achieving the highest induction, reaching approximately 25% HbF-positive cells in the enucleated erythroid population of thalassemic cells versus approximately 10% in untransduced controls. Notably, all constructs significantly reduced reactive oxygen species levels, indicating alleviation of oxidative stress. Conclusions: Together, these findings support the feasibility and therapeutic potential of combining complementary HbF-inducing mechanisms within a single lentiviral vector for β-thalassemia.

1. Introduction

Beta-hemoglobinopathies are the most common inherited recessive disorders caused by mutations in the β-globin gene, with about 400,000 affected births each year. More than 300 mutations have been identified, leading either to reduced or absent β-globin production (β+ or β0 thalassemia) or to abnormal β-globin variants such as sickle cell disease (SCD) [1,2].
Disease severity is strongly influenced by genetic modifiers, particularly hereditary persistence of fetal hemoglobin (HPFH), which sustains γ-globin expression and increase fetal hemoglobin (HbF), ameliorating the clinical phenotype [3,4,5]. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was until recently the only established curative treatment for β-thalassemia; however, its broader application is limited by donor availability, and transplant-related complications [6].
Over the last three decades, gene therapy has emerged as a promising curative alternative for patients with β-hemoglobinopathies. These approaches are based on the autologous transplantation of genetically modified hematopoietic stem and progenitor cells (HSPCs). The procedure involves mobilizing CD34+ HSPCs from the bone marrow into the bloodstream using agents such as G-CSF combined with Plerixafor in β-thalassemia patients [7,8,9,10,11,12]. After leukapheresis, the collected HSPCs are genetically corrected ex vivo, by either gene addition or genome editing, and reinfused into the patient following conditioning [13,14].
Several lentiviral vectors have been developed and clinically evaluated for β-thalassemia [15]. Among these, LentiGlobin BB305 [16] is the most clinically advanced gene addition platform, demonstrating durable transgene expression and sustained transfusion independence in the majority of patients [17,18,19,20,21]. The vector carries a modified β-globin gene (βA-T87Q), including coding and non-coding sequences, which produces a distinguishable and fully functional b-globin variant. Expression of the transgene is driven by the β-globin promoter and the core HS2, HS3, and HS4 elements of the β-globin locus control region (named μLCR), enabling high-level, erythroid-specific expression. This platform led to the development of betibeglogene autotemcel, marketed as Zynteglo, an autologous CD34+ cell-based gene therapy approved for transfusion-dependent β-thalassemia.
Despite its clinical success, the broader implementation of this approach has been limited by high cost and reimbursement challenges, which in turn contributed to the withdrawal of Zynteglo from the European market [22]. In parallel, the large LCR-based regulatory architecture, along with the large globin expression cassettes, has been shown to reduce viral titers, increase manufacturing demands, and require larger quantities of vectors for efficient HSPC transduction [23,24,25]. Furthermore, integration-related safety concerns also remain, particularly in vectors lacking additional regulatory elements, such as chromatin insulators.
In addition to β-globin gene addition, γ-globin gene addition was among the earliest gene-therapy strategies investigated for β-thalassemia. Initial studies demonstrated that erythroid-specific γ-globin expression could improve globin-chain balance and correct disease-related phenotypes in murine β-thalassemia models, although efficacy was influenced by vector copy number, integration-site position effects, and the composition of the regulatory elements included in the vector [26,27]. Subsequent studies showed that γ-globin gene transfer could generate therapeutically relevant HbF levels in erythroid progeny derived from β-thalassemia patient CD34+ cells and improve erythroid maturation and survival [28,29,30,31,32].
The emergence of approaches that reactivate the endogenous HBG genes has nevertheless reduced the translational momentum of γ-globin gene addition. In particular, genome editing of the erythroid-specific BCL11A (a key γ-globin repressor) enhancer [33,34,35,36,37,38,39] and erythroid-restricted BCL11A knockdown using lentiviral shRNA or shmiR constructs [40,41,42,43] can induce HbF from the endogenous β-globin locus without requiring the introduction of an additional globin transgene. The latter offers an additional advantage of a highly compact therapeutic sequence, substantially smaller than a genomic β-globin or γ-globin expression cassette thereby facilitating vector production or the incorporation of additional functional elements. The strong preclinical and clinical performance of BCL11A-targeting approaches has increasingly directed the field towards endogenous HbF reactivation. However, their efficacy and safety depend on the magnitude and lineage specificity of BCL11A suppression [37,38,44,45,46,47,48,49]. Ubiquitous or extensive depletion can adversely affect hematopoietic stem and progenitor cell function, while complete or profound loss in erythroid cells has been associated with impaired precursor expansion, increased apoptosis, and defective terminal maturation or enucleation [48,50]. These observations highlight the need for erythroid-restricted and carefully controlled BCL11A knockdown and provide a rationale for directly comparing this approach with γ-globin addition, both individually and in combination.
In this study, we performed a direct side-by-side comparison of lentiviral strategies based on γ-globin addition, BCL11A-targeting shmiRNA, or their combination within a single vector under the control of the μLCR. To limit vector size and facilitate incorporation of both therapeutic components, γ-globin was delivered as a compact cDNA rather than as the entire genomic sequence. Across healthy donor- and β-thalassemia patient-derived erythroid cultures, all three vectors increased the proportion of HbF-expressing cells without adversely affecting progenitor capacity or erythroid differentiation. The bifunctional vector produced the highest mean proportion of HbF-positive cells and γ-globin chain production in patient derived erythroid cells, whereas γ-globin cDNA addition alone achieved a comparable response and was associated with robust erythroid expansion. These findings support the feasibility of integrating direct γ-globin provision and endogenous HbF reactivation within a single compact lentiviral platform for β-thalassemia.

2. Materials and Methods

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Research Ethics and Deontology Committee of the Aristotle University of Thessaloniki (OGETHERA: 326425/2023) on 13 December 2023.

2.1. Viral Vectors

To systematically evaluate the therapeutic efficacy of HbF reactivation, we engineered a panel of three compact, self-inactivating lentiviral vectors (Figure 1A). The constructs were designed to test three distinct genetic modalities: (1) indirect HbF induction via post-transcriptional silencing of the BCL11A repressor using a specific shmiRNA (BCL11A shmiRNA vector) [43]; (2) direct γ-globin gene addition via a cDNA expression cassette (γ-globin cDNA vector) [51]; and (3) a dual-functional, combinatorial approach integrating both the BCL11A-targeting shmiRNA and the γ-globin cDNA into a single vector architecture (BCL11A shmiRNA + γ-globin cDNA vector). All transgene cassettes were cloned within a pRRLSIN.cPPT.PGK-GFP.WPRE plasmid (a gift from Didier Trono; Addgene plasmid # 12252), modified to incorporate the C1 dual-function chromatin insulator [52], a beta globin promoter and the HS2,HS3, HS4 μLCR as previously described [53].

2.2. Lentiviral Production and Titration

VSV.G-pseudotyped lentiviral vectors (LVs) were produced by transient co-transfection of 293FT cells using the calcium phosphate precipitation method. For lentiviral production we used a plasmid mixture consisting of the packaging plasmid psPAX2, the envelope plasmid pVSV-G, and the transfer vector carrying the corresponding transgene (BCL11A shmiRNA, γ-globin cDNA, BCL11A shmiRNA + γ-globin cDNA).
After viral production the viral titers were quantified by quantitative real-time PCR (qPCR) using an ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The following primers and probes were used for viral genome quantification:
  • Gag Forward: 5′-GGAGCTAGAACGATTCGCAGTTA-3′
  • Gag Reverse: 5′-GGTTGTAGCTGTCCCAGTATTTGTC-3′
  • Gag Probe: 5′-FAM-ACAGCCTTCTGATGTTTCTAACAGGCCAGG TAMRA-3′
  • hAlb Forward: 5′-TGAAACATACGTTCCCAAAGAGTTT-3′
  • hAlb Reverse: 5′-CTCTCCTTCTCAGAAAGTGTGCATAT-3′
  • hAlb Probe: 5′-VIC-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3′
PCR reactions were conducted in a total volume of 25 μL using TaqMan Universal PCR Master Mix (Applied Biosystems, Waltham, MA, USA). Amplification was carried out with an initial incubation at 50 °C for 2 min, followed by 95 °C for 10 min, and 40 amplification cycles consisting of 95 °C for 15 s and 60 °C for 1 min. Lentiviral vector copy number (VCN) per cell was determined by normalization to the endogenous albumin (ALB) gene.

2.3. Cell Culture of Primary CD34+ Culture

Human CD34+ hematopoietic stem and progenitor cells a HSPCs were obtained from normal donors (n = 3) and patients with intermedia β-thalassemia (n = 2) β+/β+ and severe β-thalassemia (n = 1) β0/β0 (Table 1).
Patient-derived cells were harvested during prior mobilization clinical trials at George Papanikolaou Hospital in Thessaloniki, Greece [9,10]. Cells were cultured at a concentration of (3–5) × 105 cells/mL in (IMDM + Glutamax, GibcoTM, Grand Island, NY, USA) supplemented with 1% Penicillin/Streptomycin (pen/strep, GibcoTM, Grand Island, NY, USA), 1% Glutamine (L-glutamine, GibcoTM, Grand Island, NY, USA), 5% AB plasma (obtained from “G. Papanikolaou” Hospital, Thessaloniki, Greece), 10 ug/mL Insulin (Sigma Aldrich, St. Louis, MO, USA), 2 IU/mL Heparin (Sigma Aldrich, St. Louis, MO, USA), 330 ug/mL Holo-Transferrin (Sigma-Aldrich, St. Louis, MO, USA) This formulation is hereafter referred to as the basal medium. The basal medium was supplemented with 100 ng/mL h-SCF (R&D Systems, Minneapolis, MN, USA), 100 ng/mL h-TPO (Peprotech, Cranbury, NJ, USA), and 100 ng/mL h-Flt3 (Peprotech, Cranbury, NJ, USA), together with the small molecules 35 nM UM171 (Stem Cell Technologies, Vancouver, BC, Canada), 100 nM LY2228820 (Stem Cell Technologies, Vancouver, BC, Canada), and 1 μM SR1 (Stem Cell Technologies, Vancouver, BC, Canada) [54]. Lentiviral transduction was performed at an MOI of 30 on Retronectin-coated plates (Takara Bio, Kusatsu, Shiga, Japan). The transduction medium was supplemented with 8   μ g / m L protamine sulfate (Sigma-Aldrich, St. Louis, MO, USA) and 0.5   m g / m L Synperonic (Sigma-Aldrich, St. Louis, MO, USA). Each donor-derived sample was processed and transduced independently, using separately produced batches of the corresponding lentiviral vectors. Repeated cultures or technical measurements from the same donor were not considered independent biological replicates.

2.4. In Vitro Erythroid Differentiation (ED) of CD34+ Cells

In vitro erythroid differentiation of CD34+ cells was carried out in three sequential stages as previously described [55,56]. The basal medium was supplemented with hydrocortisone, IL-3 (Peprotech, Cranbury, NJ, USA), hSCF (R&D Systems, Minneapolis, MN, USA), and EPO (R&D Systems, Minneapolis, MN, USA) during Stage I. In Stage II, only hSCF and EPO were added, while Stage III utilized EPO as the sole supplement.

2.5. Colony Forming Unit (CFU) Assay

The colony-forming potential of C D 34 + cells was evaluated using a methylcellulose assay. In brief, 2500 cells in 300   μ L of AMEM (Corning, Corning, NY, USA) were mixed with 2   m L of MethoCult H4434 (Stemcell Technologies, Vancouver, BC, Canada). After 14 days of incubation, CFU-GM and BFU-E colonies were classified and enumerated.

2.6. Flow Cytometry

To assess erythroid differentiation during ex vivo culture, 50.000 cells were stained with anti-human CD235a-PE (ExBio, Vestec, Czech Republic) and anti-human CD36-APC (ExBio, Vestec, Czech Republic) antibodies based on manufacturer instruction and analyzed by flow cytometry. To detect intracellular human γ -globin, cells were fixed in 4% paraformaldehyde and permeabilized by sequential washes of 1:1 acetone/water, 100% acetone, and 1:1 acetone/water. Cells were then labeled with an anti-human γ -globin antibody (clone 51.7, Santa Cruz Biotechnology, Dallas, TX, USA). Enucleated erythroid cells were identified by the absence of staining using Nuclear Red (Thermo Fisher Scientific, Waltham, MA, USA).

2.7. Analysis of Oxidative Stress Species (ROS)

Intracellular ROS production was determined using the CellROX Deep Red Flow Cytometry Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, at the final stage of ex vivo differentiation, 1 × 10 6 thalassemic cells were stained with CellROX Deep Red reagent at a final concentration of 500   n M and incubated for 45 min at 37 °C. Following probe oxidation by intracellular ROS, fluorescence intensity was quantified via flow cytometry.

2.8. VCN

Vector copy number (VCN) was quantified by real-time quantitative PCR (qPCR) using an ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Genomic DNA was analyzed using TaqMan assays targeting the vector gag sequence, with the endogenous human albumin (hALB) gene serving as the reference for normalization. The following primers and probes were used:
  • Gag F: 5′-GGAGCTAGAACGATTCGCAGTTA-3′
  • Gag R: 5′-GGTTGTAGCTGTCCCAGTATTTGTC-3′
  • Gag probe: 5′-FAM-ACAGCCTTCTGATGTTTCTAACAGGCCAGG-TAMRA-3′
  • hALB F: 5′-TGAAACATACGTTCCCAAAGAGTTT-3′
  • hALB R: 5′-CTCTCCTTCTCAGAAAGTGTGCATAT-3′
  • hALB probe: 5′-VIC-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3′
Each DNA sample was analyzed in a 25 μL reaction containing TaqMan Universal PCR Master Mix (Applied Biosystems). The thermal cycling conditions consisted of an initial incubation at 50 °C for 2 min, followed by denaturation at 95 °C for 10 min and 40 amplification cycles of 95 °C for 15 s and 60 °C for 1 min. VCN was calculated based on the ratio of vector gag copies to the endogenous hALB gene, allowing estimation of the number of vector copies per cell.

2.9. HPLC

Globin chain expression in ex vivo–differentiated thalassemic cells (day 18) was analyzed by liquid chromatography using a Shimadzu LC-2060C 3D system (Shimadzu Corporation, Kyoto, Japan). Fitted with a GmbH MultiHigh Bio 300 column (250 mm × 3 mm). Separation was performed using a 38–60% gradient of 0.1% trifluoroacetic acid in water/acetonitrile at a flow rate of 1 mL/min.

2.10. Statistical Analysis

All quantitative data are presented as mean ± standard error of the mean (SEM) of at least three independent replicates. Statistical analyses were performed using R (version 4.4.3). To compare the efficacy of the gene therapy strategies, we employed one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons among treatment groups, as well as two-tailed Student’s t-tests where appropriate (such as comparing individual treatment groups directly against the UNTD control). A p-value of less than 0.05 was considered statistically significant. All data visualization and statistical evaluations were conducted within the RStudio environment.

3. Results

3.1. Development of Erythroid Specific Lentiviral Vectors for HbF Reactivation

We developed three compact lentiviral vectors designed to reactivate fetal hemoglobin (HbF) expression through distinct gene therapy strategies (Figure 1A). The first vector (BCL11A shmiRNA) encodes a BCL11A-targeting shmiRNA to suppress the γ-globin repressor BCL11A [43]. The second vector (γ-globin cDNA) contains the γ-globin cDNA, enabling direct gene addition [51]. The third vector combines both the BCL11A-targeting shmiRNA and the γ-globin cDNA within a single construct (BCL11A shmiRNA + γ-globin cDNA).
In all vectors, transgene expression is driven by the μLCR, comprising the HS2, HS3, and HS4 regulatory elements, in combination with a minimal β-globin promoter. This design ensures robust erythroid-specific expression. The dual-function chromatin insulator C1 [52] was incorporated in all vectors, to provide enhancer-blocking and barrier activity and to reduce integration-site-dependent effects on transgene expression and potentially mitigate enhancer-mediated insertional genotoxicity.
Lentiviral vector production was efficient, yielding high titers across all constructs (BCL11A shmiRNA: 1.72 ± 0.14 × 108 TU/mL; γ-globin cDNA: 1.30 ± 0.21 × 108 TU/mL; combined BCL11A shmiRNA + γ-globin cDNA: 1.30 ± 0.28 × 108 TU/mL) (Figure 1B). Notably, no significant differences in viral titers were observed among the three vectors, indicating that the incorporation of both the BCL11A-targeting shmiRNA and γ-globin cDNA within a single construct did not adversely affect vector production. One-way ANOVA showed no significant difference in titer among the three conditions (p = 0.86), and Tukey’s HSD post hoc test found no significant pairwise differences (all adjusted p > 0.05), indicating that construct design (shRNA alone, γ-cDNA alone, or the combined cassette) did not measurably affect viral packaging or production efficiency.

3.2. Evaluation of Gamma Globin Induction in Healthy Donor Derived CD34+ Primary Cells

To evaluate the performance of the three lentiviral vectors in primary cells, mobilized CD34+ hematopoietic stem and progenitor cells from healthy donors were transduced at a multiplicity of infection (MOI) of 30, with untransduced cells serving as controls. Forty-eight hours post-transduction, cells were cultured under erythroid differentiation conditions (Figure 2A). Transduction with any of the three lentiviral vectors did not affect cell expansion compared with untransduced controls throughout the culture period (Figure 2B). Erythroid differentiation and maturation were assessed by flow cytometry through the analysis of the erythroid markers CD36 and CD235a on days 7 and 14 of differentiation. No significant differences were observed between transduced and untransduced cells at either time point, indicating that the overall erythroid differentiation profile was preserved following transduction (Figure 2C,D).
The therapeutic impact of the lentiviral vectors was assessed by quantifying the frequency of HbF-expressing cells within the NucRed- and NucRed+ populations. Across both populations, all three vectors (BCL11A shmiRNA, γ-globin cDNA, and the combined BCL11A shmiRNA + γ-globin cDNA) demonstrated a consistent increase in HbF+ cells compared to the untransduced (UNTD) control (Figure 2E). Within the NucRed- population, all three vectors showed a statistically significant increase in HbF+ cells relative to the UNTD control (NucRed- HbF+ cells: UNTD 18.63 ± 1.60%; BCL11A shmiRNA 30.28 ± 1.34%, p = 0.005; γ-globin cDNA 33.30 ± 4.51%, p = 0.037; BCL11A shmiRNA + γ-globin cDNA 38.93 ± 7.05%, p = 0.048). Furthermore, within the NucRed+ population, the BCL11A shmiRNA vector also exhibited a statistically significant increase in the percentage of HbF+ cells compared to the control, whereas the increases observed with the other two vectors did not reach statistical significance (NucRed + HbF+ cells: UNTD 23.57 ± 1.86%; BCL11A shmiRNA 31.09 ± 0.70%, p = 0.019; γ-globin cDNA 25.77 ± 2.46%; BCL11A shmiRNA + γ-globin cDNA 29.59 ± 4.60%) (Figure 2E). To assess whether BCL11A knockdown and/or γ-globin cDNA expression affected terminal erythroid maturation, we quantified the proportion of enucleated cells (CD235a+/NucRed−) by flow cytometry in healthy donor-derived erythroid cultures. Untransduced (UNTD) cells enucleated at a mean rate of 32.0% ± 0.7% (n = 3). Enucleation was significantly reduced in cells transduced with the BCL11A-targeting shRNA vector alone (BCL11AsmiR: 20.7% ± 1.2%, n = 3; p = 0.0014, unpaired two-tailed t-test) and in cells receiving the combined BCL11AsmiR + γ-cDNA vector (21.8% ± 2.5%, n = 3; p = 0.012) relative to UNTD controls. In contrast, cells transduced with the γ-cDNA vector alone did not differ significantly from UNTD (28.9% ± 2.6%, n = 3; p = 0.21). Together, these data suggest that BCL11A knockdown, whether alone or combined with γ-globin cDNA overexpression, is associated with a modest reduction in enucleation efficiency, whereas γ-globin cDNA expression alone does not appreciably impair terminal maturation in this system.

3.3. Assessment of Vector Efficacy in Thalassemia Patient-Derived CD34+ Hematopoietic Stem and Progenitor Cells

To evaluate the performance of the three lentiviral vectors in a disease model, mobilized CD34+ hematopoietic stem and progenitor cells from patients with β-thalassemia were transduced at a multiplicity of infection (MOI) of 30, with untransduced cells serving as controls. Forty-eight hours post-transduction, cells were cultured under erythroid differentiation conditions to assess maturation and phenotypic expression. Concurrently, a portion of the transduced cells was subjected to colony-forming unit (CFU) assays to evaluate myeloid and erythroid progenitor function, specifically quantifying burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM) colonies (Figure 3A). Absolute numbers of BFU-E and CFU-GM colonies per 2500 seeded cells were quantified for each experimental condition. Colony formation was maintained following transduction with all three vectors, with no reduction in either erythroid or myeloid colony output compared with the UNTD control, indicating preservation of progenitor capacity (Figure 3B).
The proliferative capacity of the cells was monitored over an 18-day culture period across all experimental conditions, including the untransduced (UNTD) control, BCL11A shmiRNA, γ-globin cDNA, and the combined BCL11A shmiRNA + γ-globin cDNA (Figure 3C). The expansion of the cells was similar across all conditions (Day 18 cell counts: UNTD 117.5 ± 14.1 × 106; BCL11A shmiRNA 121.4 ± 17.0 × 106; γ-globin cDNA 175.6 ± 3.6 × 106; combined 119.9 ± 7.8 × 106 cells), demonstrating that transduction with the three vectors did not exert a negative effect on cell growth. Notably, the γ-globin cDNA vector resulted in the highest cell yields at Day 18, reaching approximately 1.5-fold that of the untransduced control (175.6 ± 3.6 × 106 vs. 117.5 ± 14.1 × 106 cells; p = 0.007) with markedly low inter-experiment variability. This increased erythroid output was specific to the γ-globin cDNA condition, whereas the BCL11A shmiRNA and combined-vector groups remained comparable to UNTD cell.
To determine whether differences in vector performance could be attributed to differences in gene transfer efficacy, vector copy number (vcn) was quantified at different time points during erythroid differentiation. VCN was comparable across all three conditions at both timepoints (BCL11AsmiR: 0.82 ± 0.30 at day 7, 0.27 ± 0.09 at day 11; γ-cDNA: 0.59 ± 0.14 at day 7, 0.43 ± 0.09 at day 11; BCL11AsmiR + γ-cDNA: 0.51 ± 0.13 at day 7, 0.45 ± 0.15 at day 11). One-way ANOVA showed no significant difference in VCN among conditions at either day 7 (p = 0.15) or day 11 (p = 0.10), and Tukey’s HSD post hoc test found no significant pairwise differences at either timepoint (all adjusted p > 0.05), indicating that transduction efficiency was similar across the three vector conditions (Figure 3D).
Flow cytometric analysis of CD235a and CD36 expression was utilized to assess the impact of vector transduction on the erythroid differentiation and maturation of thalassemic CD34+ cells (Figure 3E). The data indicates that lentiviral transduction with either vector did not fundamentally alter the lineage commitment of these cells, as the distribution of cell populations remained relatively consistent across experimental conditions. Furthermore, the differentiation trajectory remained normal across all groups, mirroring the observations in healthy primary cells, with no notable differences in the maturation profile compared to the untransduced (UNTD) control (Day 14 CD235a+/CD36- cells: UNTD 13.2 ± 1.9%; BCL11A shmiRNA 19.0 ± 5.0%; γ-globin cDNA 16.2 ± 3.0%; combined 11.7 ± 2.5%). These findings suggest that the genetic modifications do not negatively impact the erythroid differentiation or maturation potential of the patient-derived cells.

3.4. Fetal Hemoglobin Induction and Oxidative Stress in Transduced Patient-Derived Cells

The therapeutic efficacy of the BCL11A shmiRNA and γ-globin cDNA vectors, as well as their combined effect, was evaluated by quantifying the percentage of HbF-expressing cells within the NucRed− and NucRed + populations following erythroid differentiation (Figure 4A). In the NucRed − population, all experimental conditions demonstrated a statistically significant increase in HbF expression compared to the untransduced (UNTD) control, which exhibited a baseline of 9.92 ± 0.63%. Specifically, the BCL11A shmiRNA, γ-globin cDNA, and combined BCL11A shmiRNA + γ-globin cDNA conditions yielded mean HbF levels of 19.74 ± 3.36% (p = 0.038), 22.77 ± 1.78% (p < 0.001), and 24.85 ± 1.65% (p < 0.001), respectively. Within the NucRed + population, the UNTD control baseline was 29.79 ± 5.69%, while the BCL11A shmiRNA, γ-globin cDNA, and combined vector conditions reached mean levels of 43.78 ± 8.11%, 45.72 ± 3.52%, and 53.55 ± 1.45%, respectively. While these differences did not reach conventional statistical significance, the γ-globin cDNA and combined vector conditions approached significance (p = 0.055 and p = 0.050, respectively). Thus, the combined construct produced the highest mean percentage of HbF+ cells, although its effect was not statistically superior to that of individual vectors.
To directly evaluate globin-chain composition, HPLC analysis was performed in terminally differentiated β-thalassemia patient derived-erythroid cells. HPLC analysis showed increased γ-globin/α-globin ratios following vector treatment (Figure 4C). The BCL11A shmiRNA vector alone and the γ-globin cDNA alone did not produce a statistically significant increase (p = 0.14 and p = 0.07, respectively), whereas the combined BCL11A shmiRNA + γ-globin cDNA vector reached statistical significance compared to UNTD cells (0.51 ± 0.11 vs. 0.72 ± 0.08, p = 0.053, p = 0.05). Analysis of the total β-like-globin/α-globin ratio did not reveal statistically significant differences among conditions; nevertheless, all three vector-treated groups showed mean ratios closer to 1:1 than UNTD cells, consistent with a shift toward a more balanced non-α/α-globin chain balance.
Terminal erythroid maturation was further assessed by quantifying the proportion of enucleated (CD235a+/NucRed−) cells at the end of the differentiation culture (Figure 4C). All three lentiviral vectors supported enucleation at levels comparable to the untransduced control, with no statistically significant differences between conditions (CD235a+/NucRed− cells: UNTD 15.90 ± 2.23%; BCL11A shmiRNA 18.24 ± 1.87%, p = 0.444; γ-globin cDNA 20.06 ± 2.40%, p = 0.251; BCL11A shmiRNA + γ-globin cDNA 16.21 ± 1.29%, p = 0.931).
To assess the impact of vector transduction on the redox status of thalassemic erythroid cells, Reactive Oxygen Species (ROS) levels were quantified across all groups (Figure 4D). The assessment of oxidative stress revealed that the untransduced (UNTD) control cells exhibited the highest mean ROS MFI levels at 26,376.8 ± 712.54. In contrast, all transduced conditions demonstrated a statistically significant reduction in mean ROS levels compared to the UNTD control. Specifically, the BCL11A shmiRNA, γ-globin cDNA, and combined dual-function-vector conditions resulted in mean ROS levels of 17,020.5 ± 3686.44 (p = 0.047), 18,107.0 ± 2276.63 (p = 0.011), and 21,176.0 ± 1778.02 (p = 0.027), respectively. Thus, increased γ-globin expression was associated with reduced intracellular ROS across all three vector-treated groups. However, the magnitude of HbF induction did not directly correlate with the extent of ROS reduction, as the combined vector produced the highest mean HbF-positive fraction but not the greatest reduction in ROS.

4. Discussion

Gene therapy has now entered clinical practice for β-hemoglobinopathies, establishing autologous HSPC modification as a viable therapeutic approach. In β-thalassemia, lentiviral β-globin gene addition [16,57] has demonstrated durable clinical benefit and led to the approval of betibeglogene autotemcel [58,59]. Erythroid-restricted BCL11A silencing using a lentiviral shmiRNA vector [16,42,43,49] has also progressed to clinical evaluation, providing proof of principle for therapeutic HbF reactivation. γ-Globin gene addition has likewise been investigated for several decades and has shown efficacy in preclinical models and patient-derived cells [16,30,60,61,62]; however, its recent translational development has focused predominantly on sickle cell disease rather than β-thalassemia [63]. Against this background, a direct comparison of these HbF-inducing strategies in a β-thalassemia context, both individually and in combination, remains particularly relevant. In the present proof-of-concept study, we directly compared three lentiviral strategies designed to increase γ-globin expression in β-thalassemia: γ-globin cDNA addition, BCL11A-targeting shmiRNA-mediated endogenous HbF reactivation, and the combination of both mechanisms within a single vector.
A central concern in the development of multifunctional lentiviral vectors is that increasing cassette size and regulatory complexity may impair vector production and transduction efficiency. This limitation is particularly relevant to globin gene therapy, where the inclusion of large LCR-derived regulatory regions and genomic globin sequences has historically contributed to reduced viral titers and increased manufacturing demands. Accordingly, several efforts have focused on reducing vector size while preserving high-level erythroid-specific expression. These have included systematic minimization of the β-globin LCR, as exemplified by the development of novel compact HS2-, HS3-, and HS4-derived regulatory elements by Kohn and colleagues, which improved vector titer and gene-transfer efficiency compared with conventional β-globin vectors [25]. Other approaches have sought to replace the canonical LCR altogether with alternative erythroid enhancers. In our recent study, high-throughput functional screening identified compact erythroid regulatory elements capable of replacing the μLCR in a therapeutic β-globin vector, increasing viral titers and HSPC transducibility while maintaining correction of the β-thalassemic phenotype [53]. A complementary strategy is to reduce the size of the therapeutic cassette itself. In the present study, this was achieved by using either a compact BCL11A-targeting shmiRNA, a γ-globin cDNA rather than the full genomic γ-globin gene, or both elements within a single construct. Importantly, incorporation of both elements did not significantly reduce viral titer, and vcn analysis in β-thalassemia patient-derived cells showed comparable gene-transfer levels among the three vectors at Days 7 and 11. Thus, the higher mean HbF induction observed with the combined construct cannot be attributed simply to increased vector integration or transduction efficiency. The compact cassette design also enabled incorporation of the C1 chromatin insulator, which provides enhancer-blocking and barrier activities and may help limit integration-site-dependent effects on transgene expression and enhancer-mediated effects on neighboring genes [52].
Multifunctional lentiviral vectors have previously been explored as a means of combining complementary therapeutic mechanisms within a single construct. For example, ATM1.1 couples βA-T87Q gene addition with an intron-embedded miR-E targeting BCL11A, thereby increasing total functional hemoglobin while simultaneously reactivating endogenous HbF [64]. Other strategies have combined two HbF-inducing RNA-interference mechanisms, such as the simultaneous targeting of BCL11A and ZNF410 [65], or paired globin gene addition with suppression of a disease-causing globin transcript [66,67]. These studies support the feasibility of incorporating multiple therapeutic activities into a single lentiviral platform. In the present study, the combined construct showed the highest mean HbF induction in patient-derived erythroid cells, although its advantage over the individual strategies was modest and did not establish statistical superiority. Interestingly, HPLC analysis was consistent with a modest additional effect of the combined strategy on γ-globin production, with the combined vector showing the strongest increase in the γ-globin/α-globin ratio. The performance of the individual constructs is also informative. Importantly, γ-globin cDNA addition alone performed comparably to BCL11A silencing, supporting direct γ-globin provision as a relevant therapeutic strategy in β-thalassemia. Although differences in the total β-like-globin/α-globin ratio were not statistically significant, all vector-treated conditions shifted toward a more balanced 1:1 ratio. Together, these findings suggest that combining direct γ-globin addition with endogenous HbF reactivation may provide a modest benefit in globin-chain output, while the present data do not support a claim of clear superiority over either individual approach. Beyond HbF induction, vector treatment was associated with changes in globin-chain composition and reduced intracellular ROS in β-thalassemia patient-derived cells. Although these findings are consistent with an improvement in the underlying erythroid phenotype, the relationship between γ-globin induction, globin-chain balance, and oxidative stress cannot be established from the present data.
Overall, our findings demonstrate that γ-globin cDNA addition, erythroid-restricted BCL11A silencing, and their combination are all capable of increasing HbF without adversely affecting HSPC expansion, progenitor capacity, or erythroid differentiation. The combined vector retained high viral titers and comparable vcn, and produced the highest mean HbF induction, supporting the concept that simultaneous direct γ-globin expression and endogenous HBG reactivation can be integrated into a single compact lentiviral platform. However, the advantage over the single-function vectors was modest and does not establish superiority across all biological readouts. Given the proof-of-concept nature and limited patient cohort of the present study, further evaluation in larger and genetically diverse cohorts, together with assessment of integration-site distribution, long-term HSC function and engraftment, and in vivo efficacy, will be required to determine the therapeutic relevance of these findings. Nevertheless, this work provides a foundation for multifunctional HbF-inducing vectors and supports their further development as a potential strategy for β-thalassemia.

Author Contributions

K.P. and N.I.V.: Investigation; methodology; formal analysis; visualization; writing—original draft. F.P. and X.N.: Investigation. A.P.: validation; supervision. P.S.: Methodology; Investigation. E.Y.: Resources; validation; supervision; writing—review and editing. N.P.: Conceptualization; methodology; project administration; supervision; funding acquisition; writing—original draft. All authors have read and agreed to the published version of the manuscript.

Funding

This project was carried out within the framework of the National Recovery and Resilience Plan Greece 2.0, funded by the European Union—NextGenerationEU (Implementation body: HFRI, Grant no: 15358 to NP).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Research Ethics and Deontology Committee of the Aristotle University of Thessaloniki (OGETHERA: 326425/2023) on 13 December 2023.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

All data generated or analyzed during this study are included in this published article. Additional information is available upon request.

Conflicts of Interest

The authors have no conflicts of interest to declare.

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Figure 1. Design and production of BCL11A shmiRNA and γ-globin cDNA lentiviral vectors. (A) Schematic of the lentiviral vector constructs. Each contains 5′- and 3′-LTRs flanking a micro-locus control region (µLCR; HS2, HS3, HS4) driving expression from the β-globin promoter (Beta-p), followed by a WPRE element and chromatin insulator (C1). Three constructs were generated: a BCL11A shmiRNA vector (BCL11AsmiR), a γ-globin cDNA vector (γ-cDNA), and a combined dual-expression vector (BCL11AsmiR + γ-cDNA) co-expressing both elements from a single cassette. (B) Viral titers (TU/mL) obtained for each construct following vector production. Box plots show median and interquartile range with whiskers to min/max; dots represent individual production runs. Titers were comparable across all three constructs. Statistical comparisons across the three conditions were performed using a one-way ANOVA, followed by Tukey’s HSD test for all pairwise post hoc comparisons.
Figure 1. Design and production of BCL11A shmiRNA and γ-globin cDNA lentiviral vectors. (A) Schematic of the lentiviral vector constructs. Each contains 5′- and 3′-LTRs flanking a micro-locus control region (µLCR; HS2, HS3, HS4) driving expression from the β-globin promoter (Beta-p), followed by a WPRE element and chromatin insulator (C1). Three constructs were generated: a BCL11A shmiRNA vector (BCL11AsmiR), a γ-globin cDNA vector (γ-cDNA), and a combined dual-expression vector (BCL11AsmiR + γ-cDNA) co-expressing both elements from a single cassette. (B) Viral titers (TU/mL) obtained for each construct following vector production. Box plots show median and interquartile range with whiskers to min/max; dots represent individual production runs. Titers were comparable across all three constructs. Statistical comparisons across the three conditions were performed using a one-way ANOVA, followed by Tukey’s HSD test for all pairwise post hoc comparisons.
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Figure 2. Evaluation of safety and efficacy of lenti-viral vectors in healthy donor HSPCs. Erythroid expansion, fetal hemoglobin induction, and differentiation profile in normal donor-derived cells. (A) Experimental workflow for lentiviral transduction and erythroid differentiation. Normal donor CD34+ cells were transduced with lenti vectors at an MOI of 30 and subjected to an 18-day erythroid differentiation protocol. Samples were collected across multiple time points (Days 0, 4, 7, 11, 14, and 18) to assess cell expansion, immunophenotype, and HbF expression. (B) Cell expansion rate over an 18-day culture period. Normal donor cells were cultured and evaluated at indicated timepoints following treatment with indicated vectors (UNTD, BCL11A shmiRNA vector (BCL11AsmiR), γ-globin cDNA vector (γ-cDNA), BCL11A shmiRNA + γ-globin cDNA vector (BCL11AsmiR + γ-cDNA)). Data points represent the mean ± standard error (SE). (C,D) Flow cytometric analysis of erythroid differentiation at Day 7 (C) and Day 14 (D). Stacked bar plots depict the relative percentages of cell populations defined by CD235a and CD36 surface marker expression: double negative (CD235a-/CD36-, grey), early erythroid (CD235a-/CD36+, light blue), mid-erythroid (CD235a+/CD36+, Fmedium blue), and late erythroid (CD235a+/CD36-, dark navy blue). Bars represent the mean percentage of replicates per condition. (E) Percentage of fetal hemoglobin (HbF)-positive cells evaluated in nucleated erythroid populations (NucRec- and NucRed+). (F) Percentage of enucleated cells referred to as the %CD235A+/NucRed- cells. Statistical significance was determined compared to the UNTD control (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 2. Evaluation of safety and efficacy of lenti-viral vectors in healthy donor HSPCs. Erythroid expansion, fetal hemoglobin induction, and differentiation profile in normal donor-derived cells. (A) Experimental workflow for lentiviral transduction and erythroid differentiation. Normal donor CD34+ cells were transduced with lenti vectors at an MOI of 30 and subjected to an 18-day erythroid differentiation protocol. Samples were collected across multiple time points (Days 0, 4, 7, 11, 14, and 18) to assess cell expansion, immunophenotype, and HbF expression. (B) Cell expansion rate over an 18-day culture period. Normal donor cells were cultured and evaluated at indicated timepoints following treatment with indicated vectors (UNTD, BCL11A shmiRNA vector (BCL11AsmiR), γ-globin cDNA vector (γ-cDNA), BCL11A shmiRNA + γ-globin cDNA vector (BCL11AsmiR + γ-cDNA)). Data points represent the mean ± standard error (SE). (C,D) Flow cytometric analysis of erythroid differentiation at Day 7 (C) and Day 14 (D). Stacked bar plots depict the relative percentages of cell populations defined by CD235a and CD36 surface marker expression: double negative (CD235a-/CD36-, grey), early erythroid (CD235a-/CD36+, light blue), mid-erythroid (CD235a+/CD36+, Fmedium blue), and late erythroid (CD235a+/CD36-, dark navy blue). Bars represent the mean percentage of replicates per condition. (E) Percentage of fetal hemoglobin (HbF)-positive cells evaluated in nucleated erythroid populations (NucRec- and NucRed+). (F) Percentage of enucleated cells referred to as the %CD235A+/NucRed- cells. Statistical significance was determined compared to the UNTD control (* p < 0.05, ** p < 0.01, *** p < 0.001).
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Figure 3. Therapeutic evaluation of vector-treated Thalassemia patients-derived erythroid cells. (A) Beta-thalassemia patients derived CD34+ cells were transduced with the lenti viral vectors (MOI 30) and subsequently seeded in erythroid differentiation medium and in semi-solid methylcellulose culture (B) Clonogenic capacity of treated cells evaluated by CFU-GM and BFU-E colony-forming assays. (C) Erythroid expansion kinetics over an 18-day culture period. Thalassemia cells treated with indicated vectors (BCL11A shmiRNA vector (BCL11AsmiR), γ-globin cDNA vector (γ-cDNA) and BCL11A shmiRNA + γ-globin cDNA vector (BCL11AsmiR + γ-cDNA)) were compared to untransduced (UNTD) controls. Data represents the mean ± standard error (SE). (D) Vector copy number (VCN) in transduced cells at day 7 and day 11 post-transduction. Vector copy number was determined by quantitative PCR at day 7 and day 11 post-transduction in cells receiving the BCL11A-targeting micro-RNA-embedded shRNA vector alone (BCL11AsmiR), the γ-globin cDNA vector alone (γ-cDNA), or the combined vector (BCL11AsmiR + γ-cDNA); untransduced (UNTD) cells were excluded as a reference given the absence of vector integration. Bars represent the mean VCN for each condition, error bars denote the standard deviation, and individual points represent independent biological replicates (n = 4 per condition at each timepoint). Statistical comparisons across the three conditions were performed using a one-way ANOVA at each timepoint, followed by Tukey’s HSD test for all pairwise post hoc comparisons. VCN did not differ significantly among conditions at either day 7 (one-way ANOVA, p = 0.26) or day 11 (p = 0.10), and no pairwise comparison reached significance by Tukey’s HSD post hoc test (all adjusted p > 0.05). (E) Flow cytometric analysis of erythroid differentiation progression at Day 7, Day 11, and Day 14. FACS analysis denotes the relative frequencies of distinct differentiation stages based on CD235a and CD36 expression: double-negative (CD235a-/CD36-), early erythroid (CD235a-/CD36+), mid-erythroid (CD235a+/CD36+), and late erythroid (CD235a+/CD36-). For all bar graphs, bars represent the mean ± SE. Statistical significance was determined (** p < 0.01).
Figure 3. Therapeutic evaluation of vector-treated Thalassemia patients-derived erythroid cells. (A) Beta-thalassemia patients derived CD34+ cells were transduced with the lenti viral vectors (MOI 30) and subsequently seeded in erythroid differentiation medium and in semi-solid methylcellulose culture (B) Clonogenic capacity of treated cells evaluated by CFU-GM and BFU-E colony-forming assays. (C) Erythroid expansion kinetics over an 18-day culture period. Thalassemia cells treated with indicated vectors (BCL11A shmiRNA vector (BCL11AsmiR), γ-globin cDNA vector (γ-cDNA) and BCL11A shmiRNA + γ-globin cDNA vector (BCL11AsmiR + γ-cDNA)) were compared to untransduced (UNTD) controls. Data represents the mean ± standard error (SE). (D) Vector copy number (VCN) in transduced cells at day 7 and day 11 post-transduction. Vector copy number was determined by quantitative PCR at day 7 and day 11 post-transduction in cells receiving the BCL11A-targeting micro-RNA-embedded shRNA vector alone (BCL11AsmiR), the γ-globin cDNA vector alone (γ-cDNA), or the combined vector (BCL11AsmiR + γ-cDNA); untransduced (UNTD) cells were excluded as a reference given the absence of vector integration. Bars represent the mean VCN for each condition, error bars denote the standard deviation, and individual points represent independent biological replicates (n = 4 per condition at each timepoint). Statistical comparisons across the three conditions were performed using a one-way ANOVA at each timepoint, followed by Tukey’s HSD test for all pairwise post hoc comparisons. VCN did not differ significantly among conditions at either day 7 (one-way ANOVA, p = 0.26) or day 11 (p = 0.10), and no pairwise comparison reached significance by Tukey’s HSD post hoc test (all adjusted p > 0.05). (E) Flow cytometric analysis of erythroid differentiation progression at Day 7, Day 11, and Day 14. FACS analysis denotes the relative frequencies of distinct differentiation stages based on CD235a and CD36 expression: double-negative (CD235a-/CD36-), early erythroid (CD235a-/CD36+), mid-erythroid (CD235a+/CD36+), and late erythroid (CD235a+/CD36-). For all bar graphs, bars represent the mean ± SE. Statistical significance was determined (** p < 0.01).
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Figure 4. Efficient HbF reactivation in beta-thalassemia patient-derived HSPCs. (A) Percentage of fetal hemoglobin (HbF)-expressing cells within the nucleated erythroid population at Day 14. (B) γ-globin and β-like-globin relative to α-globin, determined by reverse-phase HPLC. Globin chain composition was quantified by reverse-phase high-performance liquid chromatography (HPLC) in untransduced (UNTD) cells and cells transduced with the BCL11AsmiR vector alone or the γ-cDNA vector alone. Two ratios are shown for each condition: total γ-globin (the sum of the Gγ and Aγ isoforms) relative to α-globin, and total β-like-globin relative to α-globin. Bars represent the mean ratio per condition, error bars denote the standard deviation, and individual points represent independent biological replicates (n = 4 per condition). Statistical significance was assessed by two-tailed unpaired Student’s t-test against the UNTD reference group, performed separately within each ratio type; asterisks denote statistical significance and non-significant comparisons are not annotated. (C) Erythroid differentiation of normal human CD34+ cells. Flow cytometric analysis of CD235a expression versus nuclear condensation (NucRed) following transduction with the indicated vectors, demonstrating the maturation kinetics of normal donor-derived cells. (D) Intracellular accumulation of reactive oxygen species (ROS) measured by mean fluorescence intensity (MFI) at Day 18, assessing the mitigation of oxidative stress. For all bar graphs, bars represent the mean ± SE. Statistical significance was determined * p < 0.05, *** p < 0.001.
Figure 4. Efficient HbF reactivation in beta-thalassemia patient-derived HSPCs. (A) Percentage of fetal hemoglobin (HbF)-expressing cells within the nucleated erythroid population at Day 14. (B) γ-globin and β-like-globin relative to α-globin, determined by reverse-phase HPLC. Globin chain composition was quantified by reverse-phase high-performance liquid chromatography (HPLC) in untransduced (UNTD) cells and cells transduced with the BCL11AsmiR vector alone or the γ-cDNA vector alone. Two ratios are shown for each condition: total γ-globin (the sum of the Gγ and Aγ isoforms) relative to α-globin, and total β-like-globin relative to α-globin. Bars represent the mean ratio per condition, error bars denote the standard deviation, and individual points represent independent biological replicates (n = 4 per condition). Statistical significance was assessed by two-tailed unpaired Student’s t-test against the UNTD reference group, performed separately within each ratio type; asterisks denote statistical significance and non-significant comparisons are not annotated. (C) Erythroid differentiation of normal human CD34+ cells. Flow cytometric analysis of CD235a expression versus nuclear condensation (NucRed) following transduction with the indicated vectors, demonstrating the maturation kinetics of normal donor-derived cells. (D) Intracellular accumulation of reactive oxygen species (ROS) measured by mean fluorescence intensity (MFI) at Day 18, assessing the mitigation of oxidative stress. For all bar graphs, bars represent the mean ± SE. Statistical significance was determined * p < 0.05, *** p < 0.001.
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Table 1. Clinical characteristics of the β-thalassemia donors included in the study. Genotype and mobilization regimen are shown for each independent patient-derived CD34+ HSPC sample.
Table 1. Clinical characteristics of the β-thalassemia donors included in the study. Genotype and mobilization regimen are shown for each independent patient-derived CD34+ HSPC sample.
DonorsGenotypeMobilization Scheme
b-thal 1B0/B0G-CSF + Plerixafor
b-thal 2IVSI-110/IVSI-110G-CSF + Plerixafor
b-thal 3IVSI-110/IVSI-110G-CSF + Plerixafor
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Paschoudi, K.; Vasiloudis, N.I.; Papadopoulos, F.; Nikolaou, X.; Papadopoulou, A.; Sova, P.; Yannaki, E.; Psatha, N. An Integrated Gene Therapy Strategy for γ-Globin Addition and HbF Reactivation in β-Thalassemia. Thalass. Rep. 2026, 16, 19. https://doi.org/10.3390/thalassrep16030019

AMA Style

Paschoudi K, Vasiloudis NI, Papadopoulos F, Nikolaou X, Papadopoulou A, Sova P, Yannaki E, Psatha N. An Integrated Gene Therapy Strategy for γ-Globin Addition and HbF Reactivation in β-Thalassemia. Thalassemia Reports. 2026; 16(3):19. https://doi.org/10.3390/thalassrep16030019

Chicago/Turabian Style

Paschoudi, Kiriaki, Ninos Ioannis Vasiloudis, Fotios Papadopoulos, Xenia Nikolaou, Anastasia Papadopoulou, Pavel Sova, Evangelia Yannaki, and Nikoletta Psatha. 2026. "An Integrated Gene Therapy Strategy for γ-Globin Addition and HbF Reactivation in β-Thalassemia" Thalassemia Reports 16, no. 3: 19. https://doi.org/10.3390/thalassrep16030019

APA Style

Paschoudi, K., Vasiloudis, N. I., Papadopoulos, F., Nikolaou, X., Papadopoulou, A., Sova, P., Yannaki, E., & Psatha, N. (2026). An Integrated Gene Therapy Strategy for γ-Globin Addition and HbF Reactivation in β-Thalassemia. Thalassemia Reports, 16(3), 19. https://doi.org/10.3390/thalassrep16030019

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