| dc.description.abstract |
β-hemoglobinopathies, including β-thalassemia and sickle cell disease
(SCD), are autosomal recessive inherited disorders caused by various
mutations in the β-globin gene. The most effective curative therapy involves
allogenic hematopoietic stem cell transplantation (HSCT) from an
immunologically-matched donor. However, this approach presents some
limitations in terms of finding a suitable donor and transplantation related
risks, such as a graft-versus-host disease (GvHD). In this thesis, we strongly
emphasized that autologous HSCT in combination with gene therapy tools will
develop novel treatments for β-hemoglobinopathies.
A promising technique for gene editing has emerged in recent years based
on the use of Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR) -associated RNA-guided endonuclease Cas9 (CRISPR/Cas9)
technology. This revolutionary tool enables to perform gene disruption, gene
correction, and gene addition in a specific locus of interest with high reliability
and efficiency. Through this technology, in our first study, we demonstrated
that targeting KLF1 and BCL11A, which control the expression of γ-globin, as
well as the promoter region of HBG1 and HBG2 (HBG1/2), resulted in upregulation
of γ-globin gene expression and fetal hemoglobin (HbF).
Furthermore, after a deep comparison between the three gene editing
strategies, we confirmed that BCL11A approach, which is currently in clinical
phase, is the safest gene therapy treatment for β-hemoglobinopathies.
Nevertheless, HBG1/2 strategy also holds potential for clinical translation.
Alternatively, since β-globin gene correction is also a feasible gene therapy
approach, in our second study, we attained successfully gene addition in
HSPCs by inserting a NheI-tag at the common aberrant splicing mutation
point, HBB IVS1-110 by means of Cas9 mRNA and ssODN electroporation.
In the last project, we thoroughly compared three lentiviral transgenes
encoding for IGF2BP1, shRNA BCL11A, and ꝩ-globin to reactivate HbF
production in HSPCs. Also, we assessed whether baboon envelope proteins
(BaEV and BaEV-RLess) have a beneficial advantage over the regularly used
vesicular-stomatitis-virus-G envelope protein (VSV-G). Our results showed
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that all treatments using VSV-G envelope proteins resulted in therapeutic
levels of HbF. In addition, baboon envelopes, especially BaEV-RLess,
achieved decent levels of HbF with less viral particles, which might ameliorate
the symptoms of the disease. Finally, even though IGF2BP1 and BCL11A
approaches induced higher HbF levels than ꝩ-globin strategy, their role in
gene regulation might cause undesired iatrogenic effects. Therefore, we
considered ꝩ-globin the best lentiviral gene therapy strategy for the treatment
of β-hemoglobinopathies.
During this thesis we evidenced that both gene therapy tools, lentiviral
gene transfer and genome editing, provide a successful platform for gene
treatment of blood disorders. In addition to the current clinical trial
approaches, we strongly believe that resurgence of HbF is the most straight
forward strategy together with β-globin gene correction. However, due to the
low occurrence of gene correction events in HSPCs, further investigation is
required. |
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