10 research outputs found
The Ongoing Challenge of Hematopoietic Stem Cell-Based Gene Therapy for β-Thalassemia
β-thalassemia is characterized by reduced or absence of β-globin production, resulting in anemia. Current therapies include blood transfusion combined with iron chelation. BM transplantation, although curative, is restricted by the matched donor limitation. Gene therapy, on the other hand, is promising, and its success lies primarily on designing efficient globin vectors that can effectively and stably transduce HSCs. The major breakthrough in β-thalassemia gene therapy occurred a decade ago with the development of globin LVs. Since then, researchers focused on designing efficient and safe vectors, which can successfully deliver the therapeutic transgene, demonstrating no insertional mutagenesis. Furthermore, as human HSCs have intrinsic barriers to HIV-1 infection, attention is drawn towards their ex vivo manipulation, aiming to achieve higher yield of genetically modified HSCs. This paper presents the current status of gene therapy for β-thalassemia, its success and limitations, and the novel promising strategies available involving the therapeutic role of HSCs
Repression of hedgehog signal transduction in T-lineage cells increases TCR-induced activation and proliferation
Hedgehog proteins signal for differentiation, survival and proliferation of the earliest thymocyte progenitors, but their functions at later stages of thymocyte development and in peripheral T-cell function are controversial. Here we show that repression of Hedgehog (Hh) pathway activation in T-lineage cells, by expression of a transgenic repressor form of Gli2 (Gli2DeltaC2), increased T-cell differentiation and activationin response to TCR signalling. Expression of the Gli2DeltaC2 transgene increased differentiation from CD4(+)CD8(+) to single positive thymocyte, and increased peripheral T cell populations. Gli2DeltaC2 T-cells were hyper-responsive to activation by ligation of CD3 and CD28: they expressed cell surface activation markers CD69 and CD25 more quickly, and proliferated more than wild-type T-cells. These data show that Hedgehog pathway activation in thymocytes and T-cells negatively regulates TCR-dependent differentiation and proliferation. Thus, as negative regulators of TCR-dependent events, Hh proteins provide an environmental influence on T-cell fate
The Ongoing Challenge of Hematopoietic Stem Cell-Based Gene Therapy for beta-Thalassemia
beta-thalassemia is characterized by reduced or absence of beta-globin
production, resulting in anemia. Current therapies include blood
transfusion combined with iron chelation. BM transplantation, although
curative, is restricted by the matched donor limitation. Gene therapy,
on the other hand, is promising, and its success lies primarily on
designing efficient globin vectors that can effectively and stably
transduce HSCs. The major breakthrough in beta-thalassemia gene therapy
occurred a decade ago with the development of globin LVs. Since then,
researchers focused on designing efficient and safe vectors, which can
successfully deliver the therapeutic transgene, demonstrating no
insertional mutagenesis. Furthermore, as human HSCs have intrinsic
barriers to HIV-1 infection, attention is drawn towards their ex vivo
manipulation, aiming to achieve higher yield of genetically modified
HSCs. This paper presents the current status of gene therapy for
beta-thalassemia, its success and limitations, and the novel promising
strategies available involving the therapeutic role of HSCs
Gene Therapy for Malignant and Benign Gynaecological Disorders: A Systematic Review of an Emerging Success Story
Despite the major advances in screening and therapeutic approaches, gynaecological malignancies still present as a leading cause of death among women of reproductive age. Cervical cancer, although largely preventable through vaccination and regular screening, remains the fourth most common and most lethal cancer type in women, while the available treatment schemes still pose a fertility threat. Ovarian cancer is associated with high morbidity rates, primarily due to lack of symptoms and high relapse rates following treatment, whereas endometrial cancer, although usually curable by surgery, it still represents a therapeutic problem. On the other hand, benign abnormalities, such as fibroids, endometriosis, placental, and embryo implantation disorders, although not life-threatening, significantly affect women’s life and fertility and have high socio-economic impacts. In the last decade, targeted gene therapy approaches toward both malignant and benign gynaecological abnormalities have led to promising results, setting the ground for successful clinical trials. The above therapeutic strategies employ both viral and non-viral systems for mutation compensation, suicide gene therapy, oncolytic virotherapy, antiangiogenesis and immunopotentiation. This review discusses all the major advances in gene therapy of gynaecological disorders and highlights the novel and potentially therapeutic perspectives associated with such an approach
The Optimized γ-Globin Lentiviral Vector GGHI-mB-3D Leads to Nearly Therapeutic HbF Levels In Vitro in CD34+ Cells from Sickle Cell Disease Patients
We have previously demonstrated that both the original γ-globin lentiviral vector (LV) GGHI and the optimized GGHI-mB-3D LV, carrying the novel regulatory elements of the 3D HPFH-1 enhancer and the 3’ β-globin UTR, can significantly increase HbF production in thalassemic CD34+ cells and ameliorate the disease phenotype in vitro. In the present study, we investigated whether the GGHI-mB-3D vector can also exhibit an equally therapeutic effect, following the transduction of sickle cell disease (SCD) CD34+ cells at MOI 100, leading to HbF increase coupled with HbS decrease, and thus, to phenotype improvement in vitro. We show that GGHI-mB-3D LV can lead to high and potentially therapeutic HbF levels, reaching a mean 2-fold increase to a mean value of VCN/cell of 1.0 and a mean transduction efficiency of 55%. Furthermore, this increase was accompanied by a significant 1.6-fold HbS decrease, a beneficial therapeutic feature for SCD. In summary, our data demonstrate the efficacy of the optimized γ-globin lentiviral vector to improve the SCD phenotype in vitro, and highlights its potential use in future clinical SCD trials
Activation of the Hedgehog signaling pathway in T-lineage cells inhibits TCR repertoire selection in the thymus and peripheral T-cell activation
TCR signal strength is involved in many cell fate decisions in the T-cell lineage. Here, we show that transcriptional events induced by Hedgehog (Hh) signaling reduced TCR signal strength in mice. Activation of Hh signaling in thymocytes in vivo by expression of a transgenic transcriptional-activator form of Gli2 (Gli2ΔN2) changed the outcome of TCR ligation at many stages of thymocyte development, allowing self-reactive cells to escape clonal deletion; reducing transgenic TCR-mediated positive selection; reducing the ratio of CD4/CD8 single-positive (SP) cells; and reducing cell surface CD5 expression. In contrast, in the Shh−/− thymus the ratio of CD4/CD8 cells and both positive and negative selection of a transgenic TCR were increased, demonstrating that Shh does indeed influence TCR repertoire selection and the transition from double-positive (DP) to SP cell in a physiological situation. In peripheral T cells, Gli2ΔN2 expression attenuated T-cell activation and proliferation, by a mechanism upstream of ERK phosphorylation
Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation
Gene therapy utilizing lentiviral vectors (LVs) constitutes a real
therapeutic alternative for many inherited monogenic diseases.
Therefore, the generation of functional vectors using fast,
non-laborious and cost-effective strategies is imperative. Among the
available concentration methods for VSV-G pseudo-typed lentiviruses to
achieve high therapeutic titers, ultracentrifugation represents the most
common approach. However, the procedure requires special handling and
access to special instrumentation, it is time-consuming, and most
importantly, it is cost-ineffective due to the high maintenance expenses
and consumables of the ultracentrifuge apparatus.
Here we describe an improved protocol in which vector stocks are
prepared by transient transfection using standard cell culture media and
are then concentrated by ultrafiltration, resulting in functional vector
titers of up to 6 x 10(9) transducing units per millilitre (TU/ml)
without the involvement of any purification step. Although
ultrafiltration per se for concentrating viruses is not a new procedure,
our work displays one major novelty; we characterized the nature and the
constituents of the viral batches produced by ultrafiltration using
peptide mass fingerprint analysis. We also determined the viral
functional titer by employing flow cytometry and evaluated the actual
viral particle size and concentration in real time by using laser-based
nanoparticle tracking analysis based on Brownian motion.
Vectors generated by this production method are contained in intact
virions and when tested to transduce in vitro either murine total bone
marrow or human CD34(+) hematopoietic stem cells, resulted in equal
transduction efficiency and reduced toxicity, compared to lentiviral
vectors produced using standard ultracentrifugation-based methods. The
data from this study can eventually lead to the improvement of protocols
and technical modifications for the clinical trials for gene therapy.
(C) 2013 Elsevier B.V. All rights reserved
Indian hedgehog (Ihh) both promotes and restricts thymocyte differentiation.
We show that Indian Hedgehog (Ihh) regulates T-cell development and homeostasis in both fetal and adult thymus, controlling thymocyte number. Fetal Ihh(-/-) thymi had reduced differentiation to double-positive (DP) cell and reduced cell numbers compared with wild-type littermates. Surprisingly, fetal Ihh(+/-) thymi had increased thymocyte numbers and proportion of DP cells relative to wild type, indicating that Ihh also negatively regulates thymocyte development. In vitro treatment of thymus explants with exogenous recombinant Hedgehog protein promoted thymocyte development in Ihh(-/-) thymi but inhibited thymocyte development in Ihh(+/-), confirming both positive and negative regulatory functions of Ihh. Analysis of Rag(-/-)Ihh(+/-) thymi showed that Ihh promotes T-cell development before pre-T-cell receptor (pre-TCR) signaling, but negatively regulates T-cell development only after pre-TCR signaling has taken place. We show that Ihh is most highly expressed by the DP population and that Ihh produced by DP cells feeds back to negatively regulate the differentiation and proliferation of their double-negative progenitors. Thus, differentiation from double-negative to DP cell, and hence the size of the DP population, is dependent on the concentration of Ihh in the thymus. Analysis of Ihh conditional knockout and heterozygote adult mice showed that Ihh also influences thymocyte number in the adult
A Novel BaEVRless-Pseudotyped gamma-Globin Lentiviral Vector Drives High and Stable Fetal Hemoglobin Expression and Improves Thalassemic Erythropoiesis In Vitro
It has previously been demonstrated that the self-inactivating
gamma-globin lentiviral vector GGHI can significantly increase fetal
hemoglobin (HbF) in erythroid cells from thalassemia patients and thus
improve the disease phenotype in vitro. In the present study, the GGHI
vector was improved further by incorporating novel enhancer elements and
also pseudotyping it with the baboon endogenous virus envelope
glycoprotein BaEVRless, which efficiently and specifically targets human
CD34(+) cells. We evaluated the hypothesis that the newly constructed
vector designated as GGHI-mB-3D would increase hCD34(+) cell tropism and
thus transduction efficiency at low multiplicity of infection, leading
to increased transgene expression. High and stable HbF expression was
demonstrated in thalassemic cells for the resulting GGHI-mB-3D/BaEVRless
vector, exhibiting increased transduction efficiency compared to the
original GGHI-mB-3D/VSVG vector, with a concomitant 91% mean HbF
increase at a mean vector copy number per cell of 0.86 and a mean
transduction efficiency of 56.4%. Transduced populations also exhibited
a trend toward late erythroid, orthochromatic differentiation and
reduced apoptosis, a further indication of successful gene therapy
treatment. Monitoring expression of ATG5, a key link between autophagy
and apoptosis, it was established that this correction correlates with a
reduction of enhanced autophagy activation, a typical feature of
thalassemic polychromatophilic normoblasts. This work provides novel
mechanistic insights into gene therapy-mediated correction of
erythropoiesis and demonstrates the beneficial role of BaEVRless
envelope glycoprotein compared to VSVG pseudotyping and of the novel
GGHI-mB-3D/BaEVRless lentiviral vector for enhanced thalassemia gene
therapy
Non-redundant role for the transcription factor Gli1 at multiple stages of thymocyte development
The Hedgehog (Hh) signaling pathway influences multiple stages of murine T-cell development. Hh signaling mediates transcriptional changes by the activity of the Gli family of transcription factors, Gli1, Gli2 and Gli3. Both Gli2 and Gli3 are essential for mouse development and can be processed to function as transcriptional repressors or transcriptional activators, whereas Gli1, itself a transcriptional target of Hh pathway activation, can only function as a transcriptional activator and is not essential for mouse development. Gli1-deficient mice are healthy and appear normal and non-redundant functions for Gli1 have been difficult to identify. Here we show that Gli1 is non-redundant in the regulation of T-cell development in the thymus, at multiple developmental stages. Analysis of Gli1-deficient embryonic mouse thymus shows a role for Gli1 to promote the differentiation of CD4−CD8− double negative (DN) thymocytes before pre-TCR signal transduction, and a negative regulatory function after pre-TCR signaling. In addition, introduction of a Class I-restricted transgenic TCR into the adult Gli1-deficient and embryonic Gli2-deficient thymus showed that both Gli1 and Gli2 influence its selection to the CD8 lineage