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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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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:;
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.
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 |
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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Background X-linked inhibitor of apoptosis protein (XIAP) deficiency is a severe immunodeficiency with clinical features including hemophagocytic lymphohistiocytosis (HLH) and inflammatory bowel disease (IBD) due to defective NOD2 responses. Management includes immunomodulatory therapies and hematopoietic stem cell transplant (HSCT). However, this cohort is particularly susceptible to the chemotherapeutic regimens and acutely affected by graft-vs-host disease (GvHD), driving poor long-term survival in transplanted patients. Autologous HSC gene therapy could offer an alternative treatment option and would abrogate the risks of alloreactivity. Methods Hematopoietic progenitor (Lin −ve ) cells from XIAP y/− mice were transduced with a lentiviral vector encoding human XIAP cDNA before transplantation into irradiated XIAP y/− recipients. After 12 weeks animals were challenged with the dectin-1 ligand curdlan and recovery of innate immune function was evaluated though analysis of inflammatory cytokines, body weight, and splenomegaly. XIAP patient-derived CD14 ⁺ monocytes were transduced with the same vector and functional recovery was demonstrated using in vitro L18-MDP/NOD2 assays. Results In treated XIAP y/− mice, ~40% engraftment of gene-corrected Lin −ve cells led to significant recovery of weight loss, splenomegaly, and inflammatory cytokine responses to curdlan, comparable to wild-type mice. Serum IL-6, IL-10, MCP-1, and TNF were significantly reduced 2-h post-curdlan administration in non-corrected XIAP y/− mice compared to wild-type and gene-corrected animals. Appropriate reduction of inflammatory responses was observed in gene-corrected mice, whereas non-corrected mice developed an inflammatory profile 9 days post-curdlan challenge. In gene-corrected patient CD14 ⁺ monocytes, TNF responses were restored following NOD2 activation with L18-MDP. Conclusion Gene correction of HSCs recovers XIAP-dependent immune defects and could offer a treatment option for patients with XIAP deficiency.
... 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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Rapid advances in high throughput sequencing have substantially expedited the identification and diagnosis of inborn errors of immunity (IEI). Correction of faulty genes in the hematopoietic stem cells can potentially provide cures for the majority of these monogenic immune disorders. Given the clinical efficacies of vector-based gene therapies already established for certain groups of IEI, the recently emerged genome editing technologies promise to bring safer and more versatile treatment options. Here, we review the latest development in genome editing technologies, focusing on the state-of-the-art tools with improved precision and safety profiles. We subsequently summarize the recent preclinical applications of genome editing tools in IEI models, and discuss the major challenges and future perspectives of such treatment modalities. Continued explorations of precise genome editing for IEI treatment shall move us closer toward curing these unfortunate rare diseases.
... Gene therapy is a precision approach to treating IEIs. In a subset of IEI disorders including adenosine deaminase deficiency severe combined immune deficiency (ADA-SCID), X-linked SCID (X-SCID), CGD, and Wiskott-Aldrich syndrome, autologous hematopoietic stem cell transplantation (HSCT) in combination with gene therapy has been evaluated in clinical trials and found to be an effective therapeutic strategy (112)(113)(114)(115)(116)(117)(118)(119) Historically, gene therapy involved the introduction of a corrected gene into affected cells, but integration happened randomly and not necessarily at its native site. However, gene editing tools that allow for site-specific editing have recently and rapidly emerged and been developed to overcome the limitations and risks of conventional gene therapy, such as integration at random sites in the genome, which could result in malignancy (120,121). ...
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Inborn errors of immunity (IEIs) are a group of more than 450 monogenic disorders that impair immune development and function. A subset of IEIs blend increased susceptibility to infection, autoimmunity, and malignancy and are known collectively as primary immune regulatory disorders (PIRDs). While many aspects of immune function are altered in PIRDs, one key impact is on T-cell function. By their nature, PIRDs provide unique insights into human T-cell signaling; alterations in individual signaling molecules tune downstream signaling pathways and effector function. Quantifying T-cell dysfunction in PIRDs and the underlying causative mechanisms is critical to identifying existing therapies and potential novel therapeutic targets to treat our rare patients and gain deeper insight into the basic mechanisms of T-cell function. Though there are many types of T-cell dysfunction, here we will focus on T-cell exhaustion, a key pathophysiological state. Exhaustion has been described in both human and mouse models of disease, where the chronic presence of antigen and inflammation (e.g., chronic infection or malignancy) induces a state of altered immune profile, transcriptional and epigenetic states, as well as impaired T-cell function. Since a subset of PIRDs amplify T-cell receptor (TCR) signaling and/or inflammatory cytokine signaling cascades, it is possible that they could induce T-cell exhaustion by genetically mimicking chronic infection. Here, we review the fundamentals of T-cell exhaustion and its possible role in IEIs in which genetic mutations mimic prolonged or amplified T-cell receptor and/or cytokine signaling. Given the potential insight from the many forms of PIRDs in understanding T-cell function and the challenges in obtaining primary cells from these rare disorders, we also discuss advances in CRISPR-Cas9 genome-editing technologies and potential applications to edit healthy donor T cells that could facilitate further study of mechanisms of immune dysfunctions in PIRDs. Editing T cells to match PIRD patient genetic variants will allow investigations into the mechanisms underpinning states of dysregulated T-cell function, including T-cell exhaustion.
Inborn errors of immunity (IEI) are diseases caused by genetic mutations that affect the immune system’s ability to fight pathogens, cope with the microbiota or regulate autoimmunity and inflammation. More than 500 IEI have been described and many are life-threatening and require curative therapy. Allogeneic haematopoietic stem cell transplantation is an increasingly effective curative strategy, and autologous transplantation of gene-modified haematopoietic stem and progenitor cells is also a treatment option. Gene therapy was first successfully used to restore T cell development in patients with severe combined immunodeficiency, with ex vivo engineered gammaretroviral vectors enabling the sustained correction of T cell immunodeficiency more than 20 years later. The generation of safer and more potent vectors has increased the efficacy and application of this therapy to other IEI, such as Wiskott–Aldrich syndrome and chronic granulomatous disease. Nevertheless, gene therapy based on gene addition has some limitations, the greatest of which is the lack of a physiological gene expression control. This Perspective summarizes the journey of the past 25 years that has led to the successful use of gene therapy for IEI and discusses the next steps for the field. In this Perspective, Alain Fischer reflects on the development of gene therapy for patients with inborn errors of immunity. He discusses the challenges the field has faced as well as the progress seen in the past 25 years.
Heterozygous mutations in CTLA-4 result in an inborn error of immunity with an autoimmune and frequently severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic hematopoietic stem cell transplantation. Here, we designed a homology-directed repair (HDR) gene editing strategy that inserts the CTLA-4 cDNA into the first intron of the CTLA-4 genomic locus in primary human T cells. This resulted in regulated expression of CTLA-4 in CD4 ⁺ T cells, and functional studies demonstrated CD80 and CD86 transendocytosis. Gene editing of T cells isolated from three patients with CTLA-4 insufficiency also restored CTLA-4 protein expression and rescued transendocytosis of CD80 and CD86 in vitro. Last, gene-corrected T cells from CTLA-4 −/− mice engrafted and prevented lymphoproliferation in an in vivo murine model of CTLA-4 insufficiency. These results demonstrate the feasibility of a therapeutic approach using T cell gene therapy for CTLA-4 insufficiency.
Chronic granulomatous disease (CGD) is a rare congenital immunodeficiency characterized by a defect in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase required for phagocytosis. Hematopoietic stem cell (HSC) transplantation is currently the only curative treatment but it is ladened with morbidities and mortality. Gene therapy is a promising treatment for CGD. However, if not properly designed, the gene therapy approach may not be successful. We engineered lentiviral vectors (LVs) carrying a universal promoter (EF1a) and two myeloid-specific promoters (miR223 and CD68) to drive the expression of green fluorescent protein (GFP) or CYBB, one of the key defective genes causing CGD. Tissue-specific LV expression was investigated in vitro and in a CGD mouse model. We compared GFP expression in both myeloid differentiated and undifferentiated HSCs. The CGD mice were transplanted with LV-modified mouse HSCs to investigate expression of CYBB and restoration of reactive oxygen species (ROS). The LV promoters were further compared under low and high transgenic conditions to assess safety and therapeutic efficacy. A pneumonia disease model based on pathogenic Staphylococcus aureus (S. aureus) challenge was established to assess the survival rate and body weight change. All three promoters demonstrated ectopic CYBB expression in vitro and in vivo. The EF1a promoter showed the highest expression of GFP or CYBB in transduced cells including HSCs without cytotoxicity, whereas the LV-miR223 showed the highest transgene delivery efficiency with high myeloid-specificity. Importantly, under low transgenic condition, only the LV-EF1a-CYBB showed high anti-bacterial activity in vivo.
Hematopoietic stem-cell gene therapy has proven to be an effective treatment for several primary immunodeficiencies, and yet companies in this space are withdrawing from the EU market. Technological and regulatory innovations and a change to cost–benefit models are needed so that rare disease patients can receive these life-saving medicines.
Paper 2 of the paediatric regenerative medicine Series focuses on recent advances in postnatal approaches. New gene, cell, and niche-based technologies and their combinations allow structural and functional reconstitution and simulation of complex postnatal cell, tissue, and organ hierarchies. Organoid and tissue engineering advances provide human disease models and novel treatments for both rare paediatric diseases and common diseases affecting all ages, such as COVID-19. Preclinical studies for gastrointestinal disorders are directed towards oesophageal replacement, short bowel syndrome, enteric neuropathy, biliary atresia, and chronic end-stage liver failure. For respiratory diseases, beside the first human tracheal replacement, more complex tissue engineering represents a promising solution to generate transplantable lungs. Genitourinary tissue replacement and expansion usually involve application of biocompatible scaffolds seeded with patient-derived cells. Gene and cell therapy approaches seem appropriate for rare paediatric diseases of the musculoskeletal system such as spinal muscular dystrophy, whereas congenital diseases of complex organs, such as the heart, continue to challenge new frontiers of regenerative medicine.
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Hematopoietic stem and progenitor cells (HSPC) are endowed with the role of generating and maintaining lifelong the extremely diverse pool of blood cells1. Clinically, transplantation of human HSPC from an allogeneic healthy donor or infusion of autologous gene-corrected HSPC can effectively replenish defective blood cell production caused by congenital or acquired disorders2-9. However, due to methodological and ethical constraints that have limited the study of human HSPC primarily to in vitro assays10 or xenotransplantation models11,12, the in vivo activity of HSPC has to date remained relatively unexplored in humans13-16. Here we report a comprehensive study of the frequencies, dynamics and output of seven HSPC subtypes in humans that was performed by tracking 148,093 individual clones in six patients treated with lentiviral gene therapy using autologous HSPC transplantation and followed for up to 5 years. We discovered that primitive multipotent progenitor and hematopoietic stem cell (HSC) populations have distinct roles during the initial reconstitution after transplant, compared with subsequent steady-state phases. Furthermore, we showed that a fraction of in vitro-activated HSC are resilient and undergo a defined delayed activation period upon transplant. Finally, our data support the concept that early lymphoid-biased progenitors might be capable of long-term survival, such that they can be maintained independently of their continuous production from HSC. Overall, this study provides comprehensive data on HSPC dynamics after autologous transplantation and gene therapy in humans.
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Hematopoietic cell transplantation (HCT) is widely performed for neoplastic and non-neoplastic diseases. HCT involves intravenous infusion of hematopoietic progenitor cells from human leukocyte antigen (HLA)-matched donor (allogeneic) or from the patient (autologous). Before HCT, the patient is prepared with high dose chemotherapy and/or radiotherapy to destroy residual malignant cells and to reduce immunologic resistance. After HCT, chemotherapy is used to prevent graft rejection and graft versus host disease (GvHD). Neurological complications are related to the type of HCT, underlying disease, toxicity of the conditioning regimens, immunosuppression caused by conditioning regimens, vascular complications generated by thrombocytopenia and/or coagulopathy, GvHD and inappropriate immune response. In this review, neurological complications are presented according to time of onset after HCT: (1) early complications (in the first month) - related to harvesting of stem cells, during conditioning (drug toxicity, posterior reversible encephalopathy syndrome), related to pancytopenia, (2) intermediate phase complications (second to sixth month) - central nervous system infections caused by prolonged neutropenia and progressive multifocal leukoencephalopathy due to JC virus, (3) late phase complications (after sixth month) - neurological complications of GvHD, second neoplasms and relapses of the original disease.
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Recently, an immunodeficiency syndrome caused by guanine-adenine-thymine-adenine 2 (GATA2) deficiency has been described. The syndrome is characterized by (i) typical onset in early adulthood, (ii) profound peripheral blood cytopenias of monocytes, B lymphocytes, and NK cells, (iii) distinct susceptibility to disseminated non-tuberculous mycobacterial (NTM) and other opportunistic infections (particularly human papillomavirus), and (iv) a high risk of developing hematologic malignancies (myelodysplastic syndromes (MDS); acute myeloid leukemias (AML)). Considerable clinical heterogeneity exists among patients with GATA2 deficiency, but once infectious symptoms occur or MDS/AML arises, survival declines significantly. Allogeneic hematopoietic cell transplantation (HCT) currently provides the only curative treatment option for both MDS/AML and dysfunctional immunity with life-threatening opportunistic infections. Strategies regarding timing of allogeneic HCT, antimicrobial prophylaxis and treatment, intensity of the preparative regimen, and optimal donor and graft source have not been clearly defined due to the rarity of the disease. Here, we provide a comprehensive analysis of the available literature and published case reports on the use of allogeneic HCT in patients with GATA2 deficiency. In addition, a case of a young woman with GATA2 deficiency, who developed an immune reconstitution inflammatory syndrome in her mycobacterial skin lesions post allogeneic HCT is presented and illustrates distinct problems encountered in this disease context.
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PurposeThe purpose of this study was to evaluate engraftment and adverse events with a conditioning and prophylactic regimen intended to achieve high rates of engraftment with minimal graft-versus-host disease (GVHD) in allogeneic transplantation for chronic granulomatous disease in a single center. Methods Forty patients, 37 male, with chronic granulomatous disease were transplanted. Transplant products were matched sibling peripheral blood stem cells (PBSCs) in four and matched unrelated donor (MUD) bone marrow in three, and one patient received mismatched unrelated PBSCs. Thirty-two patients received MUD PBSCs. All patients received a conditioning regimen of busulfan/alemtuzumab (with low-dose total body irradiation for MUD recipients) with sirolimus graft-versus-host disease prophylaxis. ResultsEngraftment occured in 38/40 recipients (95%). Acute or chronic GVHD occurred in 18 (45%) and 5 (12.5%), respectively, with 6 episodes of grades III–IV and/or steroid refractory GVHD. Overall survival was 33/40 (82.5%) and event-free survival was 30/40 (80%). Successful engraftment was associated with myeloid and NK cell, but not CD3+ chimerism. Myeloid engraftment was greater than 70% in 30/32 recipients at mean follow-up of 3.4 years. Evidence of persistent immunodeficiency was not seen in successful transplants. Attempts to rescue failed or poorly functioning grafts were associated with unacceptable morbidity and mortality. ConclusionsA reduced-intensity allogeneic transplant protocol based on alemtuzumab and busulfan with sirolimus GVHD prophylaxis produced high rates of successful engraftment and minimal regimen-related toxicity. Prolonged clinical follow-up has confirmed its efficacy in ameliorating CGD-related disease. Outcomes were not acceptable with donor cell infusion rescue of cause with poor graft function.
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Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in?silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at
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Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an?integration site near the cancer-associated gene CCND2. Software is available at
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 numbers, NCT01745120 and NCT02151526.) FREE QUICK TAKE VIDEO SUMMARY LentiGlobin Gene Therapy for β-Thalassemia 02:03
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.
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.
Sickle cell disease results from a homozygous missense mutation in the beta-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling beta-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling beta-globin remained high (approximately 50% of beta-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease.