scholarly journals Lentiviral-Mediated Gene Therapy for the Treatment of Adenosine Deaminase 2 Deficiency

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2937-2937
Author(s):  
Alessandra Mortellaro ◽  
Matteo Zoccolillo ◽  
Cristina Mesa Nuñez ◽  
Alessia Brix ◽  
Immacolata Brigida ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Systemic Inflammation ◽  
Severe Neutropenia ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
M1 Macrophage ◽  
Adenosine Deaminase 2

Abstract Adenosine deaminase 2 deficiency (DADA2) is a recently defined inborn error of immunity caused by loss-of-function mutations in the ADA2 gene. Patients suffer from severe manifestations, including early-onset lacunar strokes, intracranial hemorrhages, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic abnormalities. The therapeutic benefit of the current treatments is unsatisfactory. Anti-tumor necrosis factor therapy reduces strokes and systemic inflammation but does not correct cytopenia and bone marrow failure. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Therefore, we proposed that autologous HSPC gene therapy may be an alternative curative option for patients who has no compatible donor or cannot receive intense chemotherapy. We performed an in-depth study, using multiparametric flow cytometry, of the bone marrow (BM) cell composition of three adult patients with the hematological phenotype of DADA2. Compared with healthy donors (HDs), patients' BM exhibited a reduced number of mature and immature populations belonging to different hematopoietic lineages. Patients exhibited a substantial reduction in circulating neutrophils and hematopoietic stem cells and progenitor pools in the BM. Severe neutropenia and HSPC defects are direct causes of DADA2. Indeed, ADA2 knock-down in zebrafish - as rodents do not harbor an ADA2 orthologue gene - caused a significant decrease in neutrophil and HSPC numbers, reminiscent of patients' phenotype. Administration of human recombinant ADA2 effectively corrected both neutropenia and defective hematopoiesis in the zebrafish embryo. We used a third-generation LV to restore constitutive ADA2 expression in HSPCs. Transduction of healthy donors' HSPCs allowed efficient delivery of the functional ADA2 enzyme with no toxicity. Supranormal ADA2 expression in healthy donors' and patients' HSPCs was well-tolerated and did not impact HSPC multilineage differentiation potential in vitro and in vivo. We also assessed whether LV-derived ADA2 could correct the hyperinflammatory M1 macrophage phenotype characteristic of DADA2. ADA2 reconstitution in patients' macrophages led to the normalization of IL-6 and TNF release. Similar results were obtained using M1 macrophages differentiated from ADA2-transduced HSPCs. Altogether, our findings indicate that HSPC gene therapy is a promising approach to re-establish stable ADA2 activity and correct the hematological and inflammatory manifestations in patients with DADA2. Disclosures Aiuti: Orchard Therapeutics: Other: PI of clinical trials sponsored by company.

scholarly journals Lentiviral correction of enzymatic activity restrains macrophage inflammation in adenosine deaminase 2 deficiency

2021 ◽  
Vol 5 (16) ◽  
pp. 3174-3187
Author(s):  
Matteo Zoccolillo ◽  
Immacolata Brigida ◽  
Federica Barzaghi ◽  
Serena Scala ◽  
Raisa Jofra Hernández ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Enzymatic Activity ◽  
Adenosine Deaminase ◽  
Systemic Inflammation ◽  
Lentiviral Vector ◽  
Clinical Manifestations ◽  
Hematopoietic Stem ◽  
Functional Correction ◽  
Adenosine Deaminase 2

Abstract Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti–tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients’ HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients’ macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2.

scholarly journals Analysis of Deficiency of Adenosine Deaminase 2 Pathogenesis Based on Single Cell RNA Sequencing of Monocytes

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2317-2317
Author(s):  
Naoki Watanabe ◽  
Shouguo Gao ◽  
Sachiko Kajigaya ◽  
Carrie Diamond ◽  
Lemlem Alemu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Immune Response ◽  
Single Cell ◽  
Adenosine Deaminase ◽  
Rna Sequencing ◽  
Healthy Donors ◽  
Single Cell Rna Sequencing ◽  
Adenosine Deaminase 2 ◽  
Upregulated Genes

Deficiency of adenosine deaminase 2 (DADA2) is a rare autosomal recessive disease caused by loss-of-function mutations in the ADA2 gene. DADA2 typically presents in childhood and is characterized by vasculopathy, stroke, inflammation, and immunodeficiency as well as hematologic manifestations, such as bone marrow failure and lymphoproliferation. The ADA2 protein is predominantly expressed in stimulated monocytes, dendritic cells and macrophages. ADA2 increases in the setting of inflammation and/or infection conditions. ADA2 has been reported to have a critical role in maintaining the balance between M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages. Macrophages of DADA2 patients are polarized towards M1 subset., DADA2 pathogenesis is not well characterized. To elucidate molecular mechanisms in DADA2 deficiency, we analyzed a gene expression profile of CD14+ monocytes derived from peripheral blood using single cell RNA sequencing (scRNA-seq). Blood was collected from DADA2 patients and age- and sex-matched healthy donors; all patients were studied in a registered research protocol (clinicaltrials.gov NCT00071045). Samples were obtained from 14 DADA2 patients and 6 healthy donors; median age of the DADA2 patients was 23 years old (range, 5 - 57 years). Among the 14 patients, 7 had hematological phenotypes: 5 lymphopenia, 3 neutropenia, 3 thrombocytopenia, and 2 with hypocellular bone marrow histology. Low serum immunoglobulins and cutaneous findings were frequent. Nine of the 14 patients had been treated with TNF inhibitors (etanercept and adalimumab). Mutations were distributed throughout the ADA2 gene; although two siblings had the same mutation, even they showed poor genotype-phenotype correlation. Monocytes were isolated by immunomagnetic positive selection with the EasySep™ positive CD14 selection kit Ⅱ, then subjected to scRNA-seq using Single Cell 3' Reagent Kits v2 (10X Genomics). Libraries for scRNA-seq were sequenced on the HiSeq-3000 instrument. Based on scRNA-seq data, we could classify monocytes into three populations by conventional flow cytometric criteria using cell surface protein expression imputed from scRNA-seq: CD14++CD16- classical, CD14++CD16+ intermediate, and CD14+CD16++ nonclassical monocytes (Figure A). CD16 expression was higher in DADA2 patients than in healthy donors (Figure B). A proportion of nonclassical monocytes among total monocytes were significantly higher in DADA2 patients compared to healthy donors (Figure C). On comparison of gene expression of each monocyte subtypes in DADA2 patients with that of healthy donors, there were 215, 237, and 267 differentially expressed upregulated genes in classical, intermediate, and nonclassical monocytes, respectively (at a threshold avg_logFC > 0.2). Approximately 35% of upregulated genes were overlapped among the three monocyte subtypes of DADA2 patients, including immune response genes such as IFITM1, IFITM2, IFITM3, and C3AR1 (Figure D). Common gene pathways were associated with immune function, such as interferon alpha/beta signaling and interferon gamma signaling. Specific genes to classical and intermediate monocytes were less than 10% of all the upregulated genes. Distinctively, the NF-κB pathway was upregulated in nonclassical monocytes, this might contribute to the pathogenesis of DADA2 as inflammatory disease. Overall, each monocyte subtype of DADA2 patients showed upregulation of immune response gene sets compared to controls. DADA2 patients have increased numbers of nonclassical monocytes which may contribute the immune dysregulation and increased inflammation observed in the disease. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Case Report: Deficiency of Adenosine Deaminase 2 Presenting With Overlapping Features of Autoimmune Lymphoproliferative Syndrome and Bone Marrow Failure

2021 ◽  
Vol 12 ◽  
Author(s):  
Gianluca Dell’Orso ◽  
Alice Grossi ◽  
Federica Penco ◽  
Roberta Caorsi ◽  
Elena Palmisani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Bone Marrow Failure ◽  
Genetic Diagnosis ◽  
Functional Study ◽  
Sequencing Analysis ◽  
Autoimmune Lymphoproliferative Syndrome ◽  
Hematopoietic Stem ◽  
Lymphoproliferative Syndrome ◽  
Adenosine Deaminase 2

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disease associated with a highly variable clinical presentation, such as vasculitis, inflammation, and hematologic manifestations. Some associations of clinical features can mimic autoimmune lymphoproliferative syndrome (ALPS). We report a case of a female patient who fulfilled the 2009 National Institute of Health revised criteria for ALPS and received a delayed diagnosis of DADA2. During her childhood, she suffered from autoimmune hemolytic anemia, immune thrombocytopenia, and chronic lymphoproliferation, which partially responded to multiple lines of treatments and were followed, at 25 years of age, by pulmonary embolism, septic shock, and bone marrow failure with myelodysplastic evolution. The patient died from the progression of pulmonary disease and multiorgan failure. Two previously unreported variants of gene ADA2/CECR1 were found through next-generation sequencing analysis, and a pathogenic role was demonstrated through a functional study. A single somatic STAT3 mutation was also found. Clinical phenotypes encompassing immune dysregulation and marrow failure should be evaluated at the early stage of diagnostic work-up with an extended molecular evaluation. A correct genetic diagnosis may lead to a precision medicine approach consisting of the use of targeted treatments or early hematopoietic stem cell transplantation.

scholarly journals Adenosine Deaminase 2 Deficiency As a Cause of Pure Red Cell Aplasia Mimicking Diamond Blackfan Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3615-3615 ◽  
Author(s):  
Ghadir S. Sasa ◽  
M. Tarek Elghetany ◽  
Katie Bergstrom ◽  
Sarah Nicholas ◽  
Ryan Himes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Pure Red Cell Aplasia ◽  
Red Cell ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Red Cell Aplasia ◽  
Ebv Reactivation ◽  
Adenosine Deaminase 2 ◽  
Red Cell Transfusion

Abstract Diamond Blackfan anemia (DBA) is an inherited pure red cell aplasia. Most cases present in the first year of life with elevation in erythrocyte adenosine deaminase (eADA) and frequently with increased mean corpuscular volume (MCV) and hemoglobin F (hgb F). Approximately 70 percent of cases are due to a mutation in one of several ribosomal protein (RP) genes or in GATA1, whereas the remaining cases are genetically uncharacterized. Here we report a child born with severe anemia and diagnosed with DBA at 2 months of age. His bone marrow was normocellular with a paucity of erythroid progenitors and scattered lymphocytes. An eADA level was not obtained prior to the first red cell transfusion. He was red cell transfusion dependent and his anemia did not respond to a steroid trial. His 4 year old sister, who had normal hemoglobin, MCV, hgb F, and eADA measurements, served as his HLA identical donor for hematopoietic stem cell transplantation (HSCT). HSCT resulted in 100% donor chimerism, but red cell engraftment was not achieved. He subsequently underwent a mismatched unrelated HSCT with trilineage engraftment. Ten years later, at the age of 14 years, the sister presented with profound hypoproductive normocytic anemia. The bone marrow showed absence of erythroid precursors and presence of lymphoid aggregates. Findings of immunodeficiency included numerous cutaneous warts, recurrent aphthous ulcers, Epstein Barr virus (EBV) reactivation, low IgM, and low numbers and percentages of CD19+ and CD3-56+16+ lymphocytes. The anemia and reticulocytopenia persisted despite resolution of EBV reactivation. Upon her presentation, levels of iron, ferritin, transferrin saturation, and liver transaminases were elevated. A liver biopsy obtained after transfusion of a total of 60 ml/kg packed red blood cells demonstrated 4.8 mg Fe/g dry liver weight with stage 2 portal fibrosis. Targeted DNA sequencing studies performed on the affected sister were negative for single nucleotide variants in any of 12 RP genes previously implicated in DBA and a genome wide chromosome microarray was normal. Whole exome analysis of her and her parents demonstrated that she carried compound heterozygous variants in CECR1 (cat eye syndrome chromosome region, candidate 1). The variant p.R169Q had been previously reported as pathogenic, while the p.G358R variant was of uncertain significance. These variants are present at frequencies of 4.9X10-4 and 2.6X10-5 in the Exome Aggregation Consortium database, respectively. Analysis of buccal swab DNA of the proband showed the same biallelic variants. An unaffected 16-year-old sibling had a normal genotype. CECR1 encodes adenosine deaminase 2 (ADA2) and ADA2 levels in the plasma of the affected sister were markedly low, consistent with a deficiency state. CECR1 is highly expressed in cells of myeloid origin and ADA2 is a secreted protein implicated in macrophage differentiation and proliferation. Deficiency of ADA2 (DADA2) results in aberrant monocyte differentiation favoring M1 over M2 macrophages, thereby resulting in a proinflammatory state. Recent descriptions of patients with DADA2 due to CECR1 mutations reported a spectrum of phenotypes including intermittent fevers, lacunar stroke in childhood, livedoid rash, polyarteritis nodosa, and immunodeficiency with B lymphopenia and low IgM levels. Our cases are similar to the report of one of two brothers, homozygous for CECR1 p.R169Q, described by van Montfrans, et al,. (NEJM, 2014). The eldest was given a diagnosis of atypical DBA (refractory pure red cell aplasia) in infancy and underwent a HSCT from his asymptomatic, HLA identical brother. This HSCT resulted in non-engraftment, necessitating a subsequent unrelated donor HSCT. The younger sibling donor later developed hepatosplenomegaly, profound lymphopenia, and evidence of an inflammatory state. Together, these three cases support pure red cell aplasia as a presentation of DADA2 and that this may be confused with DBA, particularly when manifest in infancy. We propose DADA2 should be considered in patients with genetically uncharacterized DBA. Differentiating features to suggest DADA2 may include normal eADA, MCV, and hgb F levels and findings of associated immunodeficiency. Additionally, the macrophage activation due to DADA2 may have played a role in the iron overload observed in our second patient prior to any red cell transfusion. Disclosures No relevant conflicts of interest to declare.

Expression vectors for human adenosine deaminase gene therapy

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 832-839 ◽  
Author(s):  
Kateri A. Moore ◽  
Frederick A. Fletcher ◽  
Raye Lynn Alford ◽  
Deborah K. Villalon ◽  
Dianne H. Hawkins ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Adenosine Deaminase ◽  
Retroviral Vectors ◽  
Expression Vectors ◽  
Mouse Bone Marrow ◽  
Human Genetic Disease ◽  
Hematopoietic Stem

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one of these vectors (pΔNN2ADA), human ADA was detected in 60–85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.Key words: gene therapy, adenosine deaminase, retrovirus vectors, immunodeficiency.

scholarly journals Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marie-Theresa Weickert ◽  
Judith S. Hecker ◽  
Michèle C. Buck ◽  
Christina Schreck ◽  
Jennifer Rivière ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Fate ◽  
Stromal Cells ◽  
Adipogenic Differentiation ◽  
Cell Types ◽  
Bone Marrow Niche ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Bm Niche

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

Hematopoietic stem cell gene therapy for adenosine deaminase deficient-SCID

2009 ◽  
Vol 44 (1-3) ◽  
pp. 150-159 ◽  
Author(s):  
Alessandro Aiuti ◽  
Immacolata Brigida ◽  
Francesca Ferrua ◽  
Barbara Cappelli ◽  
Robert Chiesa ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Adenosine Deaminase ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Cell Gene ◽  
Stem Cell Gene ◽  
Stem Cell Gene Therapy

scholarly journals Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1733-1741 ◽  
Author(s):  
M Kaleko ◽  
JV Garcia ◽  
WR Osborne ◽  
AD Miller
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Dna Sequences ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Helper Virus ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Wide Range

Abstract A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h- ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h- ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for “helper virus” infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.

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