Orphan designation: Autologous haematopoietic cells genetically modified with a lentiviral vector containing the human gp91(phox) gene, Treatment of X-linked chronic granulomatous disease

2019 ◽  
Author(s):  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Lentiviral Vector ◽  
Genetically Modified ◽  
Granulomatous Disease ◽  
Orphan Designation ◽  
Haematopoietic Cells ◽  
Gene Treatment

scholarly journals High Titer Lentivector from a Stable Lenti-Producer Efficiently Corrects CD34+ Hematopoietic Stem Cells from Patients with p47phox-Deficient Chronic Granulomatous Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2036-2036
Author(s):  
Uimook Choi ◽  
Narda Theobald ◽  
Throm E Robert ◽  
John Gray ◽  
David J. Rawlings ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Chronic Granulomatous Disease ◽  
Lentiviral Vector ◽  
High Titer ◽  
Granulomatous Disease ◽  
Packaging Cell Line ◽  
Hematopoietic Stem ◽  
Nsg Mice

Abstract Chronic granulomatous disease (CGD) is an inherited immune deficiency due to mutations in the genes for the NADPH subunits (the genes for p47phox, p22phox, p67phox, p40phox autosomal chronic granulomatous disease), or gp91phox (X-linked chronic granulomatous disease). This results in a failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs), the major defect in chronic granulomatous disease causing recurrent infections and granulomatous complications. Hematopoietic stem cell transplantation (HSCT) with a suitable donor is potentially curative. However, in the absence of HLA-matched donor, gene therapy using autologous gene-corrected HSC offers potential for significant clinical benefit. To date, despite myeloid conditioning, gene therapy for CGD patients using gamma-retroviral vectors have achieved either minimal long-term gene marking and engraftment, or has been associated with insertional mutagenesis. In contrast, lentivector-mediated gene therapy has successfully treated patients with Wiskott-Aldrich syndrome and Metachromatic Leukodystrophy without any dysregulated clonal expansion. We used a lentivector construct which incorporates an MND internal promoter, a modified self-inactivating MoMuLV LTR U3 region with myeloproliferative sarcoma virus enhancer, and a 650bp single chicken b-globin insulator encoding codon-optimized p47phox gene. Mutations in p47phox accounts for the majority of AR-CGD. The production of large-scale, consistently-high-titer lentivector using a transient 4-plasmid transfection system however, is labor- and cost-prohibitive. To address this, we applied concatemeric array transfection of pCL20cW650 MND-p47-OPT into a stable packaging cell line (GPRTG) for HIV-based lentiviral vectors to create a stable producer of VSV-G pseudotyped pCL20cW650 MND-p47OP. The concatemer array of HIV lentiviral vector construct and bleomycin selectable gene cassette showed 10 copies of lentiviral vector in a stable producer line, capable of producing vector at 10^7 IU/ml. Hematopoietic CD34+ stem cells from p47phox- CGD were transduced with pCL20cW650 MND-p47-OPT vector (MOI 10) with 2 overnight transductions following 24 hours pre-activation with SCF, FLT-3L and TPO (100ng/ml). Following three weeks in vitro culture, non-transduced or transduced p47 CGD HSC versus normal HSC were 0%, 42% and 20% p47phox positive, respectively. To determine functional correction, PMA stimulated oxidant production was measured using the dihydrorhodamine assay, confirmation similar levels of oxidant generation in transduced patient cells compared with normal controls. More than 90% of CFU were vector positive, indicating a high level of gene marking. Transduced and control naïve p47phox-patient CD34+ HSC were transplanted into 20 immunodeficient Nodscid-gc deficient (NSG) mice, and at 13 weeks post-transplant the CD13+ human neutrophils arising in mouse bone marrow were assessed for p47phox expression. Over 40% CD13+ neutrophils expressed p47phox protein from NSG mice transplanted with transduced p47-patient CD34 HSC, compared with 74% or 0% in mice transplanted with normal CD34 or p47 patient naive CD34 cells respectively. Detailed histopathology of each transplanted mice confirmed the absence of vector insertion-related myeloid tumors, and deep sequencing of bone marrow CD45+ human cells from each mouse also demonstrated polyclonal distribution of vector integration sites. In conclusion, we provide preclinical data demonstrating the efficacy and safety of high titer VSVg-pseudotyped lentivector (CL20cW650 MND-p47-OPT) generated by our stable GPTRG p47 lenti-producer for correction of p47phox-deficient human CD34 HSC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic Tumors in a Mouse Model of X-linked Chronic Granulomatous Disease after Lentiviral Vector-Mediated Gene Therapy

2020 ◽  
Author(s):  
Raisa Jofra Hernández ◽  
Andrea Calabria ◽  
Francesca Sanvito ◽  
Fabiola De Mattia ◽  
Giada Farinelli ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Mouse Model ◽  
Chronic Granulomatous Disease ◽  
Lentiviral Vector ◽  
Granulomatous Disease

scholarly journals Development of a pCCLChim Lentiviral Vector for Gene Therapy of Patients with Chronic Granulomatous Disease (CGD) due to p47-phox Deficiency

2017 ◽  
Vol 139 (2) ◽  
pp. AB186
Author(s):  
Walmir Cutrim Aragao-Filho ◽  
Andrea Shejtman ◽  
Marta Zinicola ◽  
Ulrich Siler ◽  
Janine Reichenbach ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Chronic Granulomatous Disease ◽  
Lentiviral Vector ◽  
Granulomatous Disease

scholarly journals Chronic granulomatous disease 2018: advances in pathophysiology and clinical management

2019 ◽  
Vol 6 (1) ◽  
pp. 1-16
Author(s):  
Reinhard A. Seger
Keyword(s):  
Chronic Granulomatous Disease ◽  
Clinical Management ◽  
Lentiviral Vector ◽  
Host Cells ◽  
Type I ◽  
Safe Procedure ◽  
Granulomatous Disease ◽  
Phagocytic Cells ◽  
Hematopoietic Stem ◽  
Anti Inflammatory

Chronic granulomatous disease (CGD) is a rare immunodeficiency disorder of phagocytic cells resulting in failure to kill a characteristic spectrum of bacteria and fungi and to resolve inflammation. The last few years have witnessed major advances in pathogenesis and clinical management of the disease: Better understanding of 3 physiologic anti-inflammatory functions of NADPH oxidase-derived reactive oxygen species: Promotion of the clearance of dying host cells, suppression of inflammasomes, and regulation of type I interferon signalling. This insight is opening new avenues for targeted drug interventions. Advances in reduced intensity conditioning (RIC) for allogeneic hematopoietic stem cell transplantation (HSCT) make it a promising and safe procedure even for fragile patients with ongoing severe infection or hyperinflammation. Encouraging early data of a multicenter trial of gene-replacement therapy using a self-inactivated lentiviral vector. Combining targeted anti-infectious/anti-inflammatory measures and considering extended indications for curative HSCT are key to improving patient outcome further. Gene therapy will likely become a viable option for disease correction, but long-term assessment is not yet possible. Statement of novelty: We discuss important advances in pathogenesis and treatment of CGD that will change our approach to clinical management.

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