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Lentiviral gene therapy for X-linked chronic granulomatous disease

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Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34⁺ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4–1.8 copies per neutrophil) and the persistence of 16–46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.
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Letters
https://doi.org/10.1038/s41591-019-0735-5
1University of California, Los Angeles, CA, USA. 2Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Foundation Trust,
London, UK. 3Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD, USA. 4Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA. 5University of California, San Francisco, CA, USA. 6University
College London Hospitals NHS Foundation Trust, London, UK. 7Eurofins Genomics Sequencing Europe, Konstanz, Germany. 8Leidos Biomedical Research,
Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA. 9University of Pennsylvania, Philadelphia, PA, USA. 10Genethon, Evry, France.
11University of Massachusetts Medical School, Worcester, MA, USA. 12Georg-Speyer Haus, Frankfurt, Germany. 13Orchard Therapeutics, London, UK.
14Inserm, University of Evry, Université Paris Saclay Genethon, Evry, France. 15Present address: City of Hope, Beckman Research Institute, Duarte, CA, USA.
16A list of members and affiliations appears at the end of the paper. *e-mail: dkohn@mednet.ucla.edu; a.thrasher@ucl.ac.uk
Chronic granulomatous disease (CGD) is a rare inherited dis-
order of phagocytic cells1,2. We report the initial results of
nine severely affected X-linked CGD (X-CGD) patients who
received ex vivo autologous CD34+ hematopoietic stem and
progenitor cell-based lentiviral gene therapy following mye-
loablative conditioning in first-in-human studies (trial reg-
istry nos. NCT02234934 and NCT01855685). The primary
objectives were to assess the safety and evaluate the efficacy
and stability of biochemical and functional reconstitution in
the progeny of engrafted cells at 12 months. The secondary
objectives included the evaluation of augmented immunity
against bacterial and fungal infection, as well as assessment
of hematopoietic stem cell transduction and engraftment.
Two enrolled patients died within 3 months of treatment from
pre-existing comorbidities. At 12 months, six of the seven
surviving patients demonstrated stable vector copy numbers
(0.4–1.8 copies per neutrophil) and the persistence of 16–46%
oxidase-positive neutrophils. There was no molecular evi-
dence of either clonal dysregulation or transgene silencing.
Surviving patients have had no new CGD-related infections,
and six have been able to discontinue CGD-related antibiotic
prophylaxis. The primary objective was met in six of the nine
patients at 12 months follow-up, suggesting that autologous
gene therapy is a promising approach for CGD patients.
The cause of X-linked chronic granulomatous disease
(X-CGD) is mutations in CYBB encoding the gp91phox subunit
of the phagocyte nicotinamide adenine dinucleotide phosphate
(NADPH)-oxidase (NOX2). Patients are susceptible to recur-
rent life-threatening infections, impacting their quality of life and
life expectancy. Allogeneic hematopoietic stem cell transplanta-
tion (HSCT) can be curative, and recent advances have improved
the results from this treatment considerably3,4. Clinical trials of
hematopoietic stem and progenitor cell (HSPC) gene therapy were
first initiated with gammaretroviral (γ-RV) vectors5,6. However,
clonal expansion of gene-corrected cells mediated by potent enhancer
elements in the γ-RV long-terminal repeats (LTRs)5,7 eventually led
to leukoproliferative complications. In addition, CpG dinucleotide
promoter methylation led to silencing of transgene expression7. To
retain the efficacy of gene therapy for X-CGD while minimizing
mutagenic risk, a self-inactivating lentiviral vector called G1XCGD
was developed (Fig. 1a; full sequence shown in Extended Data
Fig. 1). To enhance the safety of this vector, a novel chimeric inter-
nal promoter was used to preferentially drive gp91phox expression
at high levels in phagocytes8,9. Complementary clinical studies were
initiated, including a multicenter trial in the United States, a United
Kingdom study and a compassionate-use program with nearly iden-
tical clinical protocols, eligibility criteria, myeloablative condition-
ing, stem cell product manufacturing methods, vector batches and
post-transplant analyses.
Results
Recovery of functional oxidase activity in patients. Ongoing clini-
cal studies of gene therapy with G1XCGD were initiated in both the
United Kingdom (n = 3, plus one compassionate-use patient) and
the United States (n = 5). Dates of therapy are provided in Table 1.
Patients 1, 3, 5 and 9 were treated in the United Kingdom; patients 2,
4, 6, 7 and 8 were treated in the United States. The patients were
all male, with severe deficiency of gp91phox and absent NADPH-
oxidase activity. They ranged in age from 2 to 27 years, and six of
the nine were >18 years of age at entry. All patients had a clinical
history of severe X-CGD-related infections, some active at the time
of treatment, and several had chronic inflammatory complications
(Table 1). Patients were followed up until death or for a minimum
of 12 months, with a maximum follow-up of 36 months. Drug
Lentiviral gene therapy for X-linked chronic
granulomatous disease
Donald B. Kohn  1*, Claire Booth2, Elizabeth M. Kang3, Sung-Yun Pai4, Kit L. Shaw1, Giorgia Santilli2,
Myriam Armant4, Karen F. Buckland  2, Uimook Choi3, Suk See De Ravin3, Morna J. Dorsey5,
Caroline Y. Kuo1, Diego Leon-Rico2, Christine Rivat2, Natalia Izotova2, Kimberly Gilmour2, Katie Snell2,
Jinhua Xu-Bayford Dip2, Jinan Darwish2, Emma C. Morris  6, Dayna Terrazas1, Leo D. Wang4,15,
Christopher A. Bauser  7, Tobias Paprotka7, Douglas B. Kuhns8, John Gregg9, Hayley E. Raymond9,
John K. Everett9, Geraldine Honnet10, Luca Biasco2, Peter E. Newburger11, Frederic D. Bushman  9,
Manuel Grez12, H. Bobby Gaspar2,13, David A. Williams4, Harry L. Malech3, Anne Galy10,14,
Adrian J. Thrasher  2* and the Net4CGD consortium16
NATURE MEDICINE | VOL 26 | FEBRUARY 2020 | 200–206 | www.nature.com/naturemedicine
200
Content courtesy of Springer Nature, terms of use apply. Rights reserved
... Lentivirus-based hematopoietic stem cell gene therapy has been used to treat X-linked adrenoleukodystrophy (X-ALD) [75], the lysosomal storage disease metachromatic leukodystrophy (MLD) [76,77], β-hemoglobinopathies like β-thallassemia [78][79][80] and sickle-cell disease [81], and not least primary immunodeficiencies including ADA-SCID [82], SCID-X1 [83], Wiskott-Aldrich Syndrome (WAS) [84][85][86], and X-linked chronic granulomatous disease (X-CGD) [87]. It is beyond the scope of this review to cover details leading to clinical translation of gene therapies, and the reader is referred to several excellent reviews by authors who are or have been key players in the clinical trials [26,82,[88][89][90][91]. ...
... Based on the experience with this vector, a pCCL-type vector for clinical use for treatment of ADA-SCID has been produced by exchanging CYBB gene encoding gp91 phox , a catalytic subunit of the phagocyte NADPH-oxidase. For treatment of patients, a pCCL-type lentiviral vector containing a codonoptimized CYBB cDNA version was expressed from a chimeric promoter allowing high levels of expression in myeloid cells [87] (Fig. 7E). This promoter, a fusion of 5'-flanking regions of the genes encoding cathepsin G and c-Fes, is highly active in granulocytes and was found in this vector context to effectively restore NAPDH-oxidase activity [100]. ...
... Due to technical improvements of next-generation sequencing methodologies, the number of vector integration sites identified in individual patients has steadily increased from the first clinical trials, and the combined number of mapped vector integration sites (for each study counting only sites that are unique for that particular study) now totals more than 1.5 million integration sites. Most recently, Kohn and co-workers identified 724,685 unique lentiviral integration sites in nine X-CGD patients [87]. Analyses of vector integration site distribution allows monitoring of the extent of polyclonal hematopoiesis, and integration sites can be used to track clonal behavior and potential clonal expansion. ...
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Viruses are naturally endowed with the capacity to transfer genetic material between cells. Following early skepticism, engineered viruses have been used to transfer genetic information into thousands of patients, and genetic therapies are currently attracting large investments. Despite challenges and severe adverse effects along the way, optimized technologies and improved manufacturing processes are driving gene therapy toward clinical translation. Fueled by the outbreak of AIDS in the 1980s and the accompanying focus on human immunodeficiency virus (HIV), lentiviral vectors derived from HIV have grown to become one of the most successful and widely used vector technologies. In 2022, this vector technology has been around for more than 25 years. Here, we celebrate the anniversary by portraying the vector system and its intriguing properties. We dive into the technology itself and recapitulate the use of lentiviral vectors for ex vivo gene transfer to hematopoietic stem cells and for production of CAR T-cells. Furthermore, we describe the adaptation of lentiviral vectors for in vivo gene delivery and cover the important contribution of lentiviral vectors to basic molecular research including their role as carriers of CRISPR genome editing technologies. Last, we dwell on the emerging capacity of lentiviral particles to package and transfer foreign proteins.
... Autologous hematopoietic stem cell (HSC) gene therapy abrogates any risk of alloreactivity due to GvHD making it an extremely attractive potential therapy. For over 20 years, autologous HSC gene therapy has been a promising treatment option for specific immune disorders [11][12][13][14][15][16], and this success continues to promote the development of new gene therapy approaches for other monogenic immune disorders including several forms of familial HLH [17][18][19][20]. ...
... Hematopoietic stem cell gene therapy has been used successfully to treat a number of monogenic immunological, hematological and metabolic diseases [11][12][13][14][15][16][65][66][67], and offers a curative treatment option for patients lacking a suitable donor for HSCT. XIAP deficiency is an inborn error of immunity with a range of severe manifestations, and even when diagnosed early, provision of prophylactic therapies and close monitoring may not prevent fatal complications such as HLH. ...
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... Over the past several decades, marked progresses have been made on the development of ex vivo manipulation of HSCs via viral gene vectors, and the subsequent autologous HSCT in clinical settings (8,9,20). Currently, the use of self-inactivating lentiviral vectors has shown good promise as a safe and effective platform for HSC gene therapy, as demonstrated in clinical trials for X-linked SCID, adenosine deaminase (ADA)-deficient SCID, Fanconi anemia (FA), CGD and WAS (21)(22)(23)(24)(25). Moreover, similar gene therapy approaches for other IEIs including RAG1-deficient SCID and Artemis -deficient SCID have also entered clinical stages (see NCT04797260 and NCT03538899), based on favorable outcomes from preclinical studies (26, 27). ...
... Previously, a lentiviral vector with a myeloid-specific chimeric promoter has been developed for gene rescue in X-CGD (99,100). Recently, a clinical trial on such a X-CGD gene therapy platform has shown promising efficacy in 12 months of follow-up (21). Nevertheless, a gene correction strategy at the endogenous locus would more closely mimic physiological expression of the corrected genes, potentially enhancing the treatments' efficacy/toxicity profiles. ...
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Background Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent β-thalassemia. After previously establishing that lentiviral transfer of a marked β-globin (βA-T87Q) gene could substitute for long-term red-cell transfusions in a patient with β-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent β-thalassemia. Methods In two phase 1–2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent β-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. Results At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non–β0/β0 genotype had stopped receiving red-cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a β0/β0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed. Conclusions Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe β-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526.) FREE QUICK TAKE VIDEO SUMMARY LentiGlobin Gene Therapy for β-Thalassemia 02:03
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Chronic granulomatous disease (CGD) is a debilitating primary immunodeficiency affecting phagocyte function due to the absence of nicotinamide dinucleotide phosphate (NADPH) oxidase activity. The vast majority of CGD patients in the Western world have mutations within the X-linked CYBB gene encoding for gp91phox (NOX2), the redox center of the NADPH oxidase complex (XCGD). Current treatments of XCGD are not entirely satisfactory, and prior attempts at autologous gene therapy using gammaretrovirus vectors did not provide long-term curative effects. A new strategy was developed based on the use of the lentiviral vector G1XCGD expressing high levels of the gp91phox transgene in myeloid cells. As a requisite for a clinical trial approval, standardized non-clinical studies were conducted in vitro and in mice in order to evaluate the pharmacodynamics and biosafety of the vector and the biodistribution of G1XCGD-transduced cells. Transduced CD34+ cells derived from XCGD patients engrafted and differentiated similarly to their non-transduced counterparts in xenograft mouse models and generated therapeutically relevant levels of NADPH activity in myeloid cells expressing gp91phox. Expression of functional gp91phox in hematopoietic cells did not affect their homing properties, which engrafted at high levels in mice. Extensive in vitro and in vivo genotoxicity studies found no evidence for adverse mutagenesis related to vector treatment. These studies paved the way for the approval of clinical trials in Europe and in the United States for the treatment of XCGD patients with G1XCGD gene-modified autologous hematopoietic cells.
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Background: Chronic granulomatous disease (CGD) is characterized by recurrent life-threatening bacterial and fungal infections and aberrant inflammation. Mutations in CYBB cause X-linked CGD and account for 65%-70% of cases in western countries. Objective: To understand the clinical manifestations associated with the X-linked CGD carrier state. Methods: We undertook a comprehensive retrospective study of 162 affected females. We examined dihydrorhodamine oxidation (DHR) data for percent (%) X chromosome inactivation. We correlated lyonization (%DHR+) with clinical features. Where possible, we followed %DHR+ levels over time. Results: Clinical data were available for 93 females: The %DHR+ was 46% (mean) and 47% (median)(SD=24). Using %DHR+ as the criterion for X inactivation, 78% of patients had levels of inactivation 20-80%, suggesting random inactivation that was independent of age. In contrast, carriers with CGD-type infections had median %DHR+ of 8% (n=14, range 0.06-48 %); those with only autoimmune or inflammatory manifestations (AIM) had median %DHR+ of 39% (n=31, range 7.4-74%). Those with both infections and autoimmunity had low %DHR+ (n=6, range=3-14%). A %DHR+ <10 % was strongly associated with infections (OR:99). Strong association persisted when the %DHR+ was <20% (OR=12) Autoimmunity was not associated with %DHR+. In two sets of identical twins the %DHR+ populations tracked closely over time. While the %DHR+ populations were very similar between sisters, those between mothers and daughters were unrelated. Conclusions: A low %DHR+ strongly predicts infection risk in X-linked CGD carriers, while the carrier state itself is associated with autoimmunity.
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