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 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 FRI0472 DIAGNOSIS AND MANAGEMENT OF ADENOSINE DEAMINASE 2 DEFICIENCY CHILDREN: THE EXPERIENCE FROM CHINA

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 833.1-833
Author(s):  
W. Wang ◽  
T. Zhang ◽  
L. Wang ◽  
H. Song
Keyword(s):  
Enzyme Activity ◽  
Adenosine Deaminase ◽  
Case Reports ◽  
Clinical Manifestations ◽  
Primary Immunodeficiency Disease ◽  
Enzymatic Analysis ◽  
College Hospital ◽  
Hematopoietic Stem ◽  
Medical College ◽  
Adenosine Deaminase 2

Background:Adenosine deaminase 2 deficiency (DADA2) is a rare antoinflammatory disease caused by mutations in ADA2 gene, few Chinese cases have been reported.Objectives:To describe and compare the clinical features, genotypes, and treatments of Chinese DADA2 patients and foreign cases.Methods:Primary immunodeficiency disease Panel or Whole Exome Sequencing was performed to suspected subjects, and assays for adenosine deaminase 2(ADA2) enzyme activity were also carried out to them and their parents. Case reports of Chinese and foreign patients with DADA2 were searched from PubMed and Chinese domestic databases.Results:Seven unrelated DADA2 children from China were included in our study, 5 were identified at Peking union medical college hospital and 2 had been reported previously (1 on PubMed and 1 in Chinese literatures). 14 mutations in ADA2 were identified, and 9 of which have not been found in other countries. Four children receiving enzymatic analysis had lower ADA2 enzyme activity compared to their parents. Phenotypic manifestations included fever, skin symptoms, vasculitis, neurologic involvement, et al. The treatments varying from steroids, immunosuppressants, and tocilizumab, anti-TNF therapy and hematopoietic stem cell transplantation (HSCT) were effective depending on different phenotype and severity.Conclusion:This study includes the biggest number of Chinese DADA2 patients at present. We recommend combination of enzymatic analysis with gene screening to confirm the diagnosis. Genotypes of patients from China were some different, the clinical manifestations were similar. We suggest anti-TNF therapy may not be necessary for mild case and HSCT should be considered even without hematological phenotype.References:[1]Zhou Q, Yang D, Ombrello AK, Zavialov AV, Toro C, Zavialov AV, et al. Early-onset stroke and vasculopathy associated with mutations in ADA2. N Engl J Med. 2014;370:911-920.[2]Meyts I, Aksentijevich I. Deficiency of Adenosine Deaminase 2 (DADA2): Updates on the Phenotype, Genetics, Pathogenesis, and Treatment. J Clin Immunol. 2018;38:569-578.[3]Wang XN, Zhou ZX, Li SN, Lai JM, Su GX, Kang M, et al. A case report of DADA2. Chin J Rheumatol. 2019;23:476-478.[4]Liu L, Wang W, Wang Y, Hou J, Ying W, Hui X, et al. A Chinese DADA2 patient: report of two novel mutations and successful HSCT. Immunogenetics. 2019;71:299-305.Disclosure of Interests:None declared

scholarly journals In Vivo Enrichment of Genetically Manipulated Platelets for Murine Haemophilia B Gene Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3483-3483
Author(s):  
Yingyu Chen ◽  
Jocelyn A. Schroeder ◽  
Jianda Hu ◽  
Qizhen Shi
Keyword(s):  
Gene Therapy ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Hemoglobin Level ◽  
Control Group ◽  
Selective Treatment ◽  
Hematopoietic Stem ◽  
Promising Strategy

Abstract Hemophilia B (HB) is an X chromosome-linked bleeding disorder resulting from a deficiency of factor FIX (FIX). Our previous studies have demonstrated that platelet-targeted FIX gene therapy can provide sustained therapeutic levels of FIX expression in platelets without inhibitory antibody (inhibitor) development, indicating platelet gene therapy could be a promising strategy for the treatment of HB patients. In this study, we aimed to enhance platelet-FIX expression in HB mice with MGMT P140K-mediated in vivo selection of hematopoietic stem cells (HSCs) under a nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT LV), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the drug-resistance MGMT P140K gene. Platelet-FIX expression was introduced in nonmyeloablative preconditioned HB mice after 2bF9/MGMT LV-mediated HSC transduction and transplantation. After 8 weeks of bone marrow reconstitution, recipients were administered with O6-benzylguanine/1, 3-bis-2-chloroethyl-1- nitrosourea (BG/BCNU) once every 4 weeks for a total of 3 treatments. Animals were analyzed starting at 4 weeks after transplantation by PCR, quantitative real-time PCR (qPCR), FIX assays, inhibitor assays, and tail bleeding test. PCR results showed that 2bF9 proviral DNA was detected in all recipients that received 2bF9/MGMT LV-transduced HSCs. Quantitative PCR results confirmed that 2bF9/MGMT LV-transduced cells were effectively enriched after each round of BG/BCNU selective treatment in vivo. The average copy number of the 2bF9 cassette per cell after three BG/BCNU treatments was 0.50 ± 0.04, which was significantly higher than pre-BG/BCNU treatment (0.13 ± 0.01). There was an approximately 2.9-fold higher FIX antigen (FIX:Ag) (4.11 ± 0.76 vs. 1.43 ± 0.06 mU/108 platelets) and a 3.7-fold FIX activity (FIX:C) (2.56 ± 0.50 vs. 0.69 ± 0.04 mU/108 platelets) level, respectively, post-treatment compared to pre-treatment. There was a small amount of FIX:Ag detected in the 2bF9/MGMT LV-transduced recipient plasma. When we normalized FIX:Ag levels to total whole blood FIX content, the results demonstrated that 96.32 ± 0.32% of whole blood FIX in the BG/BCNU treated animals was stored in platelets. To assess whether the bleeding phenotype was rescued in HB mice after receiving 2bF9/MGMT LV-transduced HSCs, we used a 6-hour tail bleeding test. All 2bF9/MGMT LV-transduced recipients stopped bleeding within 6 hours with a clotting time of 2.6 ± 0.5 hours and a remaining hemoglobin level of 65.1 ± 4.9%, which were not significantly different from those of the wild-type control group (1.7 ± 0.9 hours and 70.6 ± 13.9%). In contrast, none of the FIXnull mice stopped bleeding within 6 hours with a remaining hemoglobin level of 38.8 ± 6.7%. Notably, none of the recipients developed anti-FIX inhibitory antibodies as measured by a modified Bethesda assay. To investigate whether immune tolerance was induced in 2bF9/MGMT LV-transduced recipients, all the recipients were challenged with 200U/kg recombinant human FIX (rhFIX) in the presence of Incomplete Freund's adjuvant (IFA) at 9 months after transplantation. Only one out of four recipients developed a low titer of inhibitors (1.3 BU/ml). In contrast, all of the HB controls developed inhibitors ranging from 17-120 BU/ml after the same challenge (n = 4). When we used the ELISA assay for the quantification of total anti-FIX IgG antibodies, low titer antibodies were found in the recipients after they were challenged with rhFIX plus IFA. However, the antibody levels in the HB control mice under the same conditioning were 213-fold higher than those developed in 2bF9 MGMT LV-transduced recipients. In conclusion, our data demonstrated that using the MGMT-mediated drug-selection system in 2bF9 gene therapy can significantly enhance therapeutic platelet-FIX expression, resulting in sustained phenotypic correction and immune suppression in HB mice. Our studies suggest that platelet-targeted FIX gene therapy together with in vivo enrichment of engineered cells may provide a promising strategy for the treatment of HB patients. Disclosures No relevant conflicts of interest to declare.

A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1139-1150 ◽  
Author(s):  
Cecilia Frecha ◽  
Caroline Costa ◽  
Didier Nègre ◽  
Fouzia Amirache ◽  
Didier Trono ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Humanized Mice ◽  
Hematopoietic Stem ◽  
Cd34 Cells

AbstractIn vivo lentiviral vector (LV)–mediated gene delivery would represent a great step forward in the field of gene therapy. Therefore, we have engineered a novel LV displaying SCF and a mutant cat endogenous retroviral glycoprotein, RDTR. These RDTR/SCF-LVs outperformed RDTR-LVs for transduction of human CD34+ cells (hCD34+). For in vivo gene therapy, these novel RDTR/SCF-displaying LVs can distinguish between the target hCD34+ cells of interest and nontarget cells. Indeed, they selectively targeted transduction to 30%-40% of the hCD34+ cells in cord blood mononuclear cells and in the unfractionated BM of healthy and Fanconi anemia donors, resulting in the correction of CD34+ cells in the patients. Moreover, RDTR/SCF-LVs targeted transduction to CD34+ cells with 95-fold selectivity compared with T cells in total cord blood. Remarkably, in vivo injection of the RDTR/SCF-LVs into the BM cavity of humanized mice resulted in the highly selective transduction of candidate hCD34+Lin− HSCs. In conclusion, this new LV will facilitate HSC-based gene therapy by directly targeting these primitive cells in BM aspirates or total cord blood. Most importantly, in the future, RDTR/SCF-LVs might completely obviate ex vivo handling and simplify gene therapy for many hematopoietic defects because of their applicability to direct in vivo inoculation.

scholarly journals Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Pharmaceutics ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 549
Author(s):  
Laura Garcia-Perez ◽  
Anita Ordas ◽  
Kirsten Canté-Barrett ◽  
Pauline Meij ◽  
Karin Pike-Overzet ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Severe Combined Immunodeficiency ◽  
Good Manufacturing Practice ◽  
Proof Of Concept ◽  
Preclinical Development ◽  
Hematopoietic Stem ◽  
Immune Deficiencies ◽  
Suitable Vector

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

scholarly journals Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Tugba Mehmetoglu-Gurbuz ◽  
Rose Yeh ◽  
Himanshu Garg ◽  
Anjali Joshi
Keyword(s):  
Gene Therapy ◽  
Lentiviral Vector ◽  
Suicide Gene ◽  
Vector System ◽  
Tat Protein ◽  
Ccr5 Gene ◽  
Hematopoietic Stem ◽  
Therapy Strategy ◽  
Hiv Tat ◽  
A Cell

Abstract Background Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. Methods A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. Results Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. Conclusions Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

Retrovirus-mediated gene transfer of human adenosine deaminase: expression of functional enzyme in murine hematopoietic stem cells in vivo

1987 ◽  
Vol 7 (10) ◽  
pp. 3459-3465
Author(s):  
B Lim ◽  
D A Williams ◽  
S H Orkin
Keyword(s):  
Adenosine Deaminase ◽  
Leukemia Virus ◽  
Hematopoietic Cells ◽  
Phosphoglycerate Kinase ◽  
Moloney Murine Leukemia Virus ◽  
Systematic Evaluation ◽  
Hematopoietic Stem ◽  
Specific Sequences

Simplified Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed which transduce human adenosine deaminase (ADA) cDNA. ADA transcription is under the control of the constitutive promoter for the human X chromosome phosphoglycerate kinase (pgk) gene. In these simplified vectors, dominant selectable markers are not included and selection is dependent on overproduction of functional ADA enzyme. Primary murine hematopoietic cells were infected with helper-free recombinant ADA virus generated from Psi-2 packaging cells. Protein analysis revealed that human ADA enzyme was expressed in progenitor-derived hematopoietic colonies in vitro and CFU-S-derived spleen colonies in vivo. Enzyme expression was dependent on transcription from the pgk promoter. ADA expression in primary murine hematopoietic cells directed by the internal promoter was not adversely affected by the presence of the Moloney virus long terminal repeat enhancer sequence. Use of these vectors allows systematic evaluation of the effects of specific sequences in recombinant retrovirus vectors on expression in primary murine hematopoietic cells in vivo.

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