Skip to main content



Reporting period: 2018-06-01 to 2020-05-31

β-thalassemias and sickle cell disease (SCD) are caused by mutations in the β-globin gene (HBB) that result in the defective synthesis or the production of an abnormal variant of hemoglobin. Currently, the only curative option is allogeneic transplantation of hematopoietic stem cells (HSCs). Alternative strategies, based on lentiviral (LV) vector-mediated introduction of a functional copy of the HBB gene followed by autologous bone marrow (BM) transplantation, are currently explored. Despite that clinical data are promising, achieving a broad therapeutic benefit remains a major hurdle because of suboptimal gene correction and limited transplantation fitness of corrected HSCs. This proposal intended to confer an in vivo competitive advantage to transduced HSCs by modulating the expression levels of the chemokine receptor CXCR4 by means of two different approaches. On one hand, as increased CXCR4 levels enhance bone marrow (BM) homing, we aimed to develop an innovative LV-based strategy to transiently deliver CXCR4 into HSCs to improve their migration to the BM. Despite we could create LV particles capable to transiently transfer proteins on HSCs, CXCR4 was not functional under the tested experimental conditions and this prevented to demonstrate the feasibility of the proposed approach in vivo. On the other hand, as CXCR4 haploinsufficiency enhances proliferation of HSCs, we aimed to develop LV particles down-regulating CXCR4 expression to endow corrected HSCs with an increased BM repopulating capacity. We developed an artificial LV cassette capable to reduce CXCR4 expression in cell lines, but it failed to function in HSCs. Importantly, as the project was interrupted by an early grant termination, we could not further optimize the described LV systems.
The aim of the project was to improve transplantation of corrected HSCs by modulating CXCR4 expression. In work package 1, as increased CXCR4 levels have been described to promote BM homing upon transplantation, we proposed to generate a multifunctional LV particle encoding the transgene of interest in its genome and capable to transiently deliver CXCR4 as an envelope associated protein (eCXCR4). First, we screened several methods for LV production using GFP and ΔNGFR as markers for transduction efficiency and membrane delivery, respectively. The best of them consisted of overexpressing ΔNGFR in 293T cells to produce LV.GFP.eΔNGFR by conventional transfection of packaging and transgene plasmids. Next, we characterized ΔNGFR delivery in both cell lines and CD34+ cells. In line with the transient nature of the transfer, we observed a rapid reduction of the transferred eΔNGFR protein within 30 hours from cell infection. In addition, we observed that eΔNGFR and GFP deliveries correlated in cell lines, but not in CD34+ cells. Taking into account this limitation of our system, we moved to assess the behaviour of LV.GFP.eCXCR4 particles. For their production, we moved to a retroviral system to reach high CXCR4 expression on the membrane of 293T cells in order to maximize its loading on the envelope of LV.GFP.eCXCR4. Through FACS-based single cell sorting, we isolated a 293T cell clone, which showed the highest CXCR4 expression and could produce multifunctional LV particles with a comparable efficiency as that of 293T wt cells. Using this clone, we generated LV vectors capable to increase CXCR4 expression in CD34+ cells to a similar level as that obtained by stable LV-mediated overexpression. Next, we evaluated the functionality of the membrane-delivered eCXCR4 few hours after infection by performing an in vitro transmigration assay in response to its natural ligand SDF-1. As a positive control, we used CD34+ cells stably overexpressing CXCR4, which were infected 4 days before to allow LV integration and transgene expression. While stable CXCR4 overexpression increased in vitro migration, eCXCR4 did not have any effect. Additional studies using NB4 cells showed that LV transduction had an immediate dramatic effect on in vitro migration, suggesting that this could over-rule the effect of eCXCR4 and contribute to the observed lack of phenotype. In this respect, we extensively tried to adjust the timing of our experimental protocol to evaluate whether prolonged recovery after transduction could reduce the negative effect of transduction on cell migration. However, we could not determine a sufficiently long recovery time before eCXCR4 expression rapidly decreased over time. To increase the permanence of eCXCR4 on the cell surface of CD34+ cells and therefore extend the duration of post-transduction recovery time, we tested a series of CXCR4 mutants that were previously reported to have an increased stability. To this end, we identified a specific mutant that also showed increased in vitro migration of CD34+ cells upon stable overexpression. Additional experimentation will be required to study whether the selected CXCR4 mutant will be suitable for our transient protein-delivery system. A recent study has shown that murine HSCs hemizygous for CXCR4 engraft better than wild type cells in mouse competitive repopulation assays, without long-term side effects. In work package 2, we proposed to generate a LV particle encoding the transgene of interest as well as a cassette for RNA interference mediated knockdown (KD) of CXCR4 expression to mimic CXCR4 haploinsufficiency and enhance engraftment of modified HSCs. Initially, we created a LV transfer plasmid containing GFP as a transgene to identify transduced cells upon transduction. This vector was further upgraded by coupling GFP gene transcription with that of an artificial miR (amiR). Initial screening was performed in jurkat cells that express CXCR4 at high expression levels. We tested more than fifteen candidates that allowed the identification of 2 short-hairpin sequences resulting in a reduction of CXCR4 expression down to 50%. When tested in CD34+ cells, however, the selected sequences showed a much lower knockdown efficiency. To improve their potency, we tried to optimize design of the employed amiR, but our adjustments failed to reduce CXCR4 expression further, thereby warranting additional future studies. Data from both work packages was not sufficient to generate manuscripts and material to present in national or international conferences.
The premature termination of the project has prevented to formally prove the feasibility of the proposed approaches to ameliorate the efficacy of LV-mediated gene correction for β-thalassemias and SCD. However, the collected data may serve as a starting point for future studies. On one hand, the KD strategy will clarify whether reducing CXCR4 expression can be used as a safe approach to ameliorate the engraftment of gene corrected HSCs and to what extent. On the other hand, the original strategy of exploiting LV-mediated delivery of CXCR4 can potentially provide a proof-of-concept for the transient transfer of molecules capable to control the activity of HSCs or facilitate their handling. In fact, we were able to create a highly versatile platform that can be virtually adapted to deliver many transmembrane proteins other than CXCR4, to transiently endow transduced HSCs with additional functions. Notably, both approaches can potentially benefit many diseases amenable to HSC gene therapy and be possibly implemented and patented in currently used LV design, thereby highly increasing their potential clinical and impact.