ALBERT

Beschreibung: β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α2β2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34+ cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-thalassemic CD34+ cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency.

Beschreibung: Lentiviral vectors for γ-globin genes are being developed as an efficient tool for the gene therapy of β-chain hemoglobinopathies. The γ-globin gene has been chosen as a therapeutic gene based on the potent anti-sickling properties of γ-globins and on their ability to bind free α-chains. However, their development has been hampered by low titers, variable expression and gene silencing. To address these problems, we have initiated a strategy to exploit novel regulatory elements of the β-cluster conferring high level and sustained globin gene expression. To this end, we have successfully used the HPFH-2 enhancer combined with a 210 bp Aγ-globin gene promoter harboring the Greek HPFH -117 mutation and the HS-40 enhancer from the α-globin locus, in a series of oncoretrovirus vectors (Fragkos et al. Gene Ther12:1591–1600, 2005). Based on the high level of expression of the Aγ-gene (248 ± 99 % per copy of mouse α-globin) and the absence of vector silencing of these vectors and to further exploit the superior transducing efficiency of hematopoietic stem cells by lentiviral vectors, in the present study we have generated two novel self-inactivating lentiviral vectors containing the above regulatory elements. Specifically, vector GGHI contains an expression cassette for Aγ-globin gene linked to the 210 bp Aγ-gene promoter with the Greek HPFH -117 point mutation, the HS-40 enhancer at its 5′ end and the HPFH-2 enhancer at its 3′ end, as well as the cHS4 insulator in the 3′ LTR. The second vector, designated GGHI/PM is essentially similar to GGHI but carries also the MGMT-140K cDNA selectable marker under the control of PGK promoter, to enrich for genetically modified cells. Both vectors exhibited high titers of 108 TU/ml, for GGHI and 107 TU/ml, for GGH/PM. Their efficiency was tested in MEL-585 cells transduced at an MOI of 1–100 and a series of independent clones were generated. The clones were further induced to differentiate using hemin and HMBA and the level of expression of the Aγ-globin transgene was determined by Real Time PCR and by flow cytometry. Vector GGHI was expressed at 237 ± 369 % per copy of mouse α-globin with a mean copy number of 19.3 in 8 individual clones, while GGHI/PM was expressed only at 10 ± 16 % per copy of mouse α-globin, with a mean copy number of 60 in 10 individual clones of unselected cells. FACS analysis using an anti-γ-globin antibody, revealed a pancellular expression of γ-globin (mean MFI 69.7 for GGHI and mean MFI 40.15 for GGHI/PM), while there was no expression of the transgene in undifferentiated MEL-585 cells, suggesting that both vectors are erythroid-specific. Moreover, there was no sign of transgene silencing in any of the above clones. The results for the novel GGHI vector, are consistent with our previous studies and reflect a) the robust synergistic capacity of the HS-40 and HPFH-2 elements to enhance transcription, b) the ability of HPFH-2 to reduce the rate of gene silencing and c) the ability of the -117 point mutation to support the Aγ-globin gene expression in the adult erythroid environment, for the first time, in the context of lentiviral vectors. This extremely high level of expression if achieved in vivo, would clearly exceed the proposed therapeutic threshold for the β-chain hemoglobinopathies. Current studies combine their assessment on CD34+ cells from patients with β-thalassemia as well as their evaluation in vivo using the Hbthal3+/− thalassemic mouse model.

Beschreibung: The recent successes in gene therapy for the treatment of rare blood disorders such as primary immunodeficiencies and hemoglobinopathies have highlighted the need to generate robust and scalable manufacturing processes. Here we compare large scale lentiviral transductions of hemopoietic stem cells performed on the CliniMACS Prodigy® to manual processes in terms of efficacy and viability of the end product. In our first series of experiments, we used a low, non-saturating MOI of 30 and the results showed that transduction efficiency on the CliniMACS Prodigy® was significantly increased from an average of 23.3% (manual) to 50.5% (CliniMACS Prodigy®). These results were further confirmed when we employed an MOI of 100 and showed that the average transduction efficiency was 74% from the CliniMACS Prodigy® compared to 54.1% from the manual steps. Moreover, the total viability of the cells cultured on the CliniMACS Prodigy® remained unaffected after two days of cultivation with an average recovery of 105% but with significantly less variability (SD: 15.4) compared to the manual steps (SD: 35.7). Finally there was no difference to the average VCN which was 2.2 from the manual steps and 2.4 from the CliniMACS Prodigy®. Overall, our results indicate that the CliniMACS Prodigy® generates higher transduction rates, combined with high viability compared to the manual process, but most importantly, with significantly lower variability, suggesting that it represents a closed system able to automatically perform complex processes as successfully as when manual handling steps are performed, but with higher predictability, efficiency and with minimal user interaction. Disclosures Papanikolaou: Miltenyi Biotec: Employment. Bissels:Miltenyi Biotec: Employment. Johnston:Miltenyi Biotec: Employment. Reinartz:Miltenyi Biotec: Employment. Brams:Miltenyi Biotec: Employment. Aivazidou:Miltenyi Biotec: Employment. Krenz:Miltenyi Biotec: Employment. Bomhard:Miltenyi Biotec: Employment. Knöbel:Miltenyi Biotec: Employment. Bosio:Miltenyi Biotec: Employment.

Beschreibung: It has been over 30 years since visionary scientists came up with the term “Gene Therapy,” suggesting that for certain indications, mostly monogenic diseases, substitution of the missing or mutated gene with the normal allele via gene addition could provide long-lasting therapeutic effect to the affected patients and consequently improve their quality of life. This notion has recently become a reality for certain diseases such as hemoglobinopathies and immunodeficiencies and other monogenic diseases. However, the therapeutic wave of gene therapies was not only applied in this context but was more broadly employed to treat cancer with the advent of CAR-T cell therapies. This review will summarize the gradual advent of gene therapies from bench to bedside with a main focus on hemopoietic stem cell gene therapy and genome editing and will provide some useful insights into the future of genetic therapies and their gradual integration in the everyday clinical practice.