CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease

HIV-1–derived lentivectors are promising for gene transfer into hematopoietic stem cells but require preclinical in vivo evaluation relevant to specific human diseases. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice accept human hematopoietic stem cell grafts, providing a unique opportunity for in vivo evaluation of therapies targeting human hematopoietic diseases. We demonstrate for the first time that hematopoietic stem cells from patients with X-linked chronic granulomatous disease (X-CGD) give rise to X-CGD–phenotype neutrophils in the NOD/SCID model that can be corrected using VSV-G–pseudotyped, 3rd-generation, self-inactivating (SIN) lentivector encoding gp91phox. We transduced X-CGD patient-mobilized CD34+ peripheral blood stem cells (CD34+PBSCs) with lentivector–gp91phox or amphotropic oncoretrovirus MFGS–gp91phox and evaluated correction ex vivo and in vivo in NOD/SCID mice. Only lentivector transduced CD34+PBSCs under ex vivo conditions nonpermissive for cell division, but both vectors performed best under conditions permissive for proliferation (multiple growth factors). Under the latter conditions, lentivector and MFGS achieved significant ex vivo correction of X-CGD CD34+PBSCs (18% and 54% of cells expressing gp91phox, associated with 53% and 163% of normal superoxide production, respectively). However, lentivector, but not MFGS, achieved significant correction of human X-CGD neutrophils arising in vivo in NOD/SCID mice that underwent transplantation (20% and 2.4%, respectively). Thus, 3rd-generation SIN lentivector–gp91phox performs well as assessed in human X-CGD neutrophils differentiating in vivo, and our studies suggest that the NOD/SCID model is generally applicable for in vivo study of therapies evaluated in human blood cells expressing a specific disease phenotype.

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299. Efficient Functional Oxidase Correction after CRISPR-Oligomer Gene Repair in X-CGD Patient Hematopoietic Stem Cells (HSC) Using Non-Viral, cGMP Compliant, Scalable, Closed System

Molecular Therapy ◽

10.1016/s1525-0016(16)33908-9 ◽

2015 ◽

Vol 23 ◽

pp. S121

Author(s):

Linhong Li ◽

Suk see De Ravin ◽

Cornell Allen ◽

Pachai Natarajan ◽

Harry L. Malech ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Closed System ◽

Gene Repair ◽

Hematopoietic Stem

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

Blood ◽

10.1182/blood.v126.23.2036.2036 ◽

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.

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Results of allogeneic hematopoietic stem cell transplantation in patients with chronic granulomatous disease at the Russian Children’s Clinical Hospital

Russian Journal of Pediatric Hematology and Oncology ◽

10.21682/2311-1267-2020-7-2-23-34 ◽

2020 ◽

Vol 7 (2) ◽

pp. 23-34

Author(s):

E. B. Machneva ◽

E. A. Pristanskova ◽

L. V. Olkhova ◽

A. V. Mezentseva ◽

V. V. Konstantinova ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Chronic Granulomatous Disease ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Conditioning Regimen ◽

Granulomatous Disease ◽

Hematopoietic Stem

Relevance. Chronic granulomatous disease (CGD) belongs to the group of primary immunodeficiencies. Patients with CGD have an impaired quality of life, severe infections and inflammatory organ damage. Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment method for CGD. The authors of the article presented the experience of HSCT in patients with CGD in the Russian Children’s Clinical Hospital.Materials and methods. 20 (19 primary and 1 repeated) HSCT during the period from 2009 to 2020 were performed in nineteen patients with CGD. All patients had a long history of infections, three or more foci of chronic infection, 9 patients had a generalized BCG infection. Bone marrow (ВМ) from a related HLA-identical donor was the source of hematopoietic stem cells (HSC) for 4 (21 %) patients, peripheral blood stem cells (PBSC) for 2 (10.5 %). ВМ from a unrelated fully HLA-identical donor was performed in 9 (47.4 %) patients, PBSC – 2 (10.5 %). ВМ from a unrelated 9/10 HLA-compatible donor was performed in one (5.3 %) patient. In one case (5.3 %) the HSC source became PBSC from a unrelated 9/10 HLA-compatible donor after TcRαβ/CD19+ depletion. In 68.5 % (n = 13) cases the conditioning regimen included threosulfan, fludarabine, melphalan, and antithymocyte globulin. In 2 (10.5 %) patients, melphalan was excluded from the conditioning regimen; in 4 (21 %), it was replaced by thiotepa.Results. The overall survival (OS) was 88.9 ± 10.5 %, the event-free survival (EFS) was 88.1 ± 7.9 %, and there was no transplant mortality. Transplant rejections were observed in two patients who received HSC from a unrelated 9/10 HLA-compatible donor with a previous conditioning regimen that included only one alkylating agent. In 4 patients (21 %) there was a prolonged persistence of mixed chimerism after HSCT without clinical and laboratory signs of CGD. After successful transplantation all patients were cured of the infectious and inflammatory diseases characteristic of CGD.Conclusion. Results of HSCT in patients with CGD can be considered satisfactory, the OS and EFS are high. Failure of HSCT is associated with transplant rejection, which is most likely due to the donor and patient mismatch, as well as the use of conditioning modes with reduced intensity.

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