Kooperativer Bibliotheksverbund

In: Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 2143-2143

Abstract: Canine leukocyte adhesion deficiency (CLAD) represents the canine counterpart of the human disease leukocyte adhesion deficiency (LAD). Children with LAD and puppies with CLAD suffer life-threatening bacterial infections as a result of the failure of their leukocytes to adhere to the endothelial surface and migrate to the site of infection. Molecular defects in the leukocyte integrin CD18 molecule are responsible for both LAD and CLAD. Although myeloablative hematopoietic stem cell transplantation can correct the disease phenotype in LAD, this therapy is accompanied by considerable toxicity. Moreover, it is not clear that full donor chimerism is required for reversal of the disease phenotype. To assess the role of mixed chimerism in reversing the disease phenotype in CLAD, we used a non-myeloablative conditioning regimen consisting of 200 cGy total body irradiation preceding matched littermate allogeneic transplant, and followed by a brief post-transplant regimen consisting of cyclosporine and mycophenolic acid. Six dogs received bone marrow cells, three dogs received CD34+ bone marrow stem cells, and four dogs received mobilized peripheral blood stem cells. Eleven of 13 transplanted CLAD dogs achieved mixed donor-host chimerism resulting in complete reversal of the disease phenotype. Donor-derived CD18+ cells measured by flow cytometric analysis in the peripheral blood of the transplanted CLAD dogs correlated closely with donor chimerism measured by DNA analysis of microsatellite repeats in the peripheral blood leukocytes. The 11 dogs with reversal of the CLAD phenotype have been followed for over one year from the time of transplant and displayed levels of donor leukocyte chimerism ranging from 4 to 95%. Since engraftment, all eleven dogs have been free from infection and live in runs with other dogs. Three dogs with very low levels of donor leukocyte chimerism post-transplant displayed evidence of selective egress of CD18+ donor leukocytes into extravascular sites, indicating that the level of CD18+ donor cells measured in the periperal blood may underestimate the total number of CD18+ donor leukocytes. In the two dogs who did not have complete reversal of the CLAD phenotype post-transplant, one dog died at 3 weeks following transplant from a subcapsular hemorrhage of the liver secondary to thrombocytopenia, and one dog had donor microchimerism following transplant with partial reversal of the phenotype. Three dogs who did not have a matched littermate donor, and did not receive a transplant, died of infection at 2, 4, and 6 months of age, respectively. The fact that correction of the CLAD phenotype was achieved in 11 of 13 dogs with mixed donor-host chimerism and the absence of graft-versus-host disease has implications for allotransplant in LAD when a matched sibling donor exists. The observation that very low levels of donor CD18+ leukocytes reversed the disease phenotype supports the use of the CLAD model for testing the ability of autologous, CD18 gene-corrected hematopoietic stem cells to reverse the CLAD phenotype, since low levels of gene correction are anticipated with gene therapy.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V104.11.2143.2143

Language: English

Publisher: American Society of Hematology

Publication Date: 2004

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Experimental Hematology, Elsevier BV, Vol. 33, No. 6 ( 2005-06), p. 706-712

Type of Medium: Online Resource

ISSN: 0301-472X

URL: Article

DOI: 10.1016/j.exphem.2005.03.010

Language: English

Publisher: Elsevier BV

Publication Date: 2005

detail.hit.zdb_id: 2005403-8

In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 1321-1321

Abstract: Abstract 1321 Poster Board I-343 Wiskott-Aldrich syndrome is characterized by immune deficiency and thrombocytopenia. There is also a qualitative platelet defect in Wiskott-Aldrich syndrome, typically presenting on aggregometry as a failure of the second wave of aggregation. The pathophysiology of this defect has not been studied in detail. To investigate this phenomenon, we enumerated dense bodies and measured serotonin content in the platelets of 7 patients with Wiskott-Aldrich syndrome. Patients ranged in age from 14 to 45 years old. Five of 7 patients had had splenectomy. In these patients, the platelet count ranged from 120,000/mcL to 521,000/mcL. Platelet counts were 20,000/mcL and 46,000/mcL in the 2 patients who had not had splenectomy. The latter 2 patients had occasional petechiae, as did 2 of the splenectomized patients. Three patients had no clinical evidence of platelet dysfunction. One of these 3 had tolerated abdominal surgery without untoward bleeding. Blood specimens were taken in the course of routine follow-up, when patients were otherwise well. Platelets were prepaed for electron microscopy by the whole mount technique. For serotonin content, platelets were counted and lysed in distilled water. Serotonin in the lysate was chemically acetylated and then measured by ELISA. Serotonin levels were normalized to ng of serotonin per 10 9 platelets. The average ± standard deviation dense body count in our patient group was 1.1 ± 0.4 dense bodies per platelet (normal count 6-8 dense bodies/platelet). Serotonin content was measured in three patients, who showed 273 ± 103 ng serotonin per 10 9 platelets (normal value 621-1064 ng/10 9 platelets). Our results are the first quantitative analysis of platelet dense body counts and serotonin level in patients with Wiskott-Aldrich syndrome. In addition to further elucidating the pathophysiology of this disease, these data have implications for understanding the role of the Wiskott-Aldrich syndrome protein in the genesis of dense bodies. Disclosures No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V114.22.1321.1321

Language: English

Publisher: American Society of Hematology

Publication Date: 2009

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 1338-1338

Abstract: Wiskott-Aldrich Syndrome (WAS) is a primary immunodeficiency that is associated with microthrombocytopenia. Autoimmunity is one of the defining characteristics of severe WAS, and can contribute to the decision to proceed to hematopoietic cell transplantation. Autoimmune Hemolytic Anemia (AIHA) is one of the most common autoimmune syndromes found in WAS. Even in the absence of AIHA, some patients with WAS have anemia, and this is often of unclear etiology. We studied 29 patients with WAS, 6 of whom had had splenectomy, on 46 occasions. All patients were found to have anisopoikilocytosis. Common red blood cell (RBC) variants included stomatocytes, xerocytes, eccentrocytes, dacryocytes, spherocytes and target cells. Two WAS knockout mice whose blood smears were studied also had poikilocytosis, with dacryocytes predominating. The average hemoglobin in humans with WAS was 13.3 g/dL. In 19/46 measurements, hemoglobin was below the lower limit of normal for age. LDH was elevated in 1/43 measurements, and bilirubin was elevated in 7 of 46 measurements, 4 measurements being from a patient with molecularly diagnosed Gilbert’s syndrome. Average MCHC was 34.7 g/dL, with MCHC being elevated in 3/43 measurements. Despite these unremarkable screening assays, haptoglobin was decreased in 10/45 measurements and undetectable in 6 of these 10. The average plasma free hemoglobin and plasma free oxyhemoglobin were elevated to 21.4 and 14.7 mg/dL respectively. Analysis of osmotic fragility revealed average numbers near the lower end of the normal range, with fragility in 0.6 g/dL NaCl being abnormally low in 29/35 specimens and fragility in 0.65 g/dL NaCl being abnormally low in 9/35 specimens. A standard Coomb’s test was positive in 6/39 specimens. 4 such assays were positive for IgG, 1 for C3 and 1 for neither molecule despite a positive screening assay. Using enhanced methodology, 1/5 specimens was positive only with polyspecific serum. Overall, immune mediated hemolysis was detected by either method in 6/39 specimens. These data were interpreted to be consistent with a subclinical non-immune hemolysis in 33/41 specimens from 28/29 patients. In order to evaluate these findings further, ektacytometry was performed in 5 specimens from 5 patients and intracellular cations were measured in 4 of these specimens. Ektacytometry revealed Omin of 135.8±5.1 mOsm/kg in patients with WAS and 156.4±5.3 mOsm/kg in control specimens (P=0.0002). Sodium and potassium concentrations in patients were 53.3±12.7 mmol/kg hemoglobin and 282.3±13.1 mmol/kg hemoglobin respectively. In control specimens, the concentrations were 4.4±10.5 mmol/kg hemoglobin and 297.5±14.4 mmol/kg hemoglobin (P=0.3196 and 0.1684). These data are consistent with non-immune hemolysis in most patients with Wiskott-Aldrich syndrome. This hemolysis is associated with a xerocytic ekatacytometry curve, although cation concentrations in the small number of specimens tested only trended to statistical significance. This is the first intrinsic red cell defect described in patients with WAS, emphasizing that this disease truly affects all hematopoietic lineages and not only cells of the immune system. Whether the roles of the WAS protein in actin polymerization or signal transduction are involved in these abnormal functions remains unclear and requires additional research. These findings also have implications for clinical management of WAS as they indicate that hemolysis is commonly present without necessarily defining the autoimmune, severe clinical phenotype in these patients. As autoimmunity is often an important consideration in the decision to proceed to hematopoietic cell transplantation, these data should inform the decision-making process toward this and other therapeutic modalities for WAS. Disclosures No relevant conflicts of interest to declare.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V124.21.1338.1338

Language: English

Publisher: American Society of Hematology

Publication Date: 2014

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 3169-3169

Abstract: Background: Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific, large-animal model for the human disease leukocyte adhesion deficiency (LAD). Puppies with CLAD, like children with LAD, experience recurrent life-threatening bacterial infections due to the inability of their leukocytes to adhere and migrate to sites of infection. Mutations in the gene encoding the leukocyte integrin CD18 are responsible for both CLAD and LAD. Allogeneic bone marrow or hematopoietic stem cell transplantation is currently the only curative therapy for LAD. We recently reported the results of non-myeloablative allogeneic transplants in CLAD dogs and showed that very low levels of CD18+ donor-derived neutrophils (less than 300/microliter) were sufficient to reverse the CLAD disease phenotype. These results indicated that CLAD dogs may be amenable to treatment using gene therapy, where there are frequently low numbers of transduced cells. We report the results of retroviral- mediated transduction in autologous hematopoietic stem cells with the canine CD18 gene. Method: Bone marrow was harvested and CD34+ selected from four dogs with CLAD at approximately 3–4 months of age. The purified CD34+ cells were either used immediately or were frozen and subsequently thawed. Cells were pre-stimulated with cSCF, hFlt3-L, hTPO and cIL-6 for approximately 24 hours, then exposed to two rounds of supernatant from the retroviral vector PG13/MSCV-cCD18 for 24 hours each on recombinant fibronectin. At the end of the transduction, the cells were infused back into the animal that had been conditioned with 200 cGy total body irradiation. Post-transplant immunosuppression consisted of cyclosporine given at a dose of 30 mg/kg from day -1 to day 35, then 15 mg/kg from day 36 to day 60, and mycophenolate mofetil at a dose of 20 mg/kg from day 0 to day 28. Peripheral blood samples, as well as pus samples from one animal, were analyzed by flow cytometry at designated time points post-transplant. Results: The four dogs who received autologous, gene-corrected cells have been followed for 7–12 weeks post-infusion. The number of CD18+ CD34+ cells infused per dog ranged from 0.2 to 0.55 x 106 cells/kg. The post-infusion percentage of CD18+ neutrophils in each dog was 0.09%, 0.13%, 0.62% and 0.02% at 12, 10, 8 and 6 weeks respectively. Clinically all four treated CLAD dogs are alive with marked improvement of their CLAD disease. These dogs are now 6–7 months of age. These results contrast with those seen in untreated CLAD dogs who uniformly die or are euthanized within the first few months of life. The reversal of the severe CLAD phenotype despite the very low levels of CD18+ neutrophils in the peripheral blood is likely due to the selective egress of CD18+ neutrophils into the tissue since one treated CLAD dog who had less than 1% CD18+ neutrophils in the blood had nearly 10% CD18+ neutrophils in pus collected from an inflammatory dental lesion. Conclusion: These data suggest that a non-myeloablative conditioning regimen coupled with a minimal immunosuppressive regimen may enable sufficient CD18+ autologous gene-corrected cells to engraft and result in reversal of the severe CLAD phenotype.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V104.11.3169.3169

Language: English

Publisher: American Society of Hematology

Publication Date: 2004

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Platelets, Informa UK Limited, Vol. 24, No. 4 ( 2013-06), p. 288-296

Type of Medium: Online Resource

ISSN: 0953-7104 , 1369-1635

URL: Article

DOI: 10.3109/09537104.2012.693991

Language: English

Publisher: Informa UK Limited

Publication Date: 2013

detail.hit.zdb_id: 2008783-4

In: Experimental and Molecular Pathology, Elsevier BV, Vol. 81, No. 1 ( 2006-8), p. 1-7

Type of Medium: Online Resource

ISSN: 0014-4800

URL: Article

DOI: 10.1016/j.yexmp.2006.03.004

Language: English

Publisher: Elsevier BV

Publication Date: 2006

detail.hit.zdb_id: 1466769-1

In: Blood, American Society of Hematology, Vol. 108, No. 10 ( 2006-11-15), p. 3313-3320

Abstract: Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent, life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens—200 cGy total body irradiation (TBI) or 10 mg/kg busulfan—with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs, therapeutic levels of CD18+ leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment, and immunosuppression was not required for efficacy. The percentage of CD18+ leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification–mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific, large animal model using 2 clinically applicable conditioning regimens, and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2006-03-006908

Language: English

Publisher: American Society of Hematology

Publication Date: 2006

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 3345-3345

Abstract: Background: Severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a rare disorder caused by ADA gene mutations, leading to lymphotoxic build-up of purine metabolites and profound immunodeficiency. Historically, enzyme replacement therapy (ERT) has been used as a bridge therapy until patients can receive an allogeneic hematopoietic stem cell transplantation (HSCT), ideally from a matched related donor (MRD) or, if none is identified, a non-matched and/or unrelated donor. We developed a self-inactivating lentiviral vector (LV), denoted EFS-ADA LV, encoding the human ADA cDNA sequence under the control of a shortened human elongation factor 1α gene promoter. A fresh or cryopreserved formulation of a drug product (OTL-101), composed of autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with EFS-ADA LV, was evaluated in 2 prospective, non-randomized Phase I/II clinical trials at 2 USA centers. We report on safety and efficacy of OTL-101 in 30 ADA-SCID pediatric gene therapy (GT) subjects treated from 2013-2017 with a median follow up (FU) of 24 months (mo; range 12-26 mo), compared to a historical cohort of 26 ADA-SCID patients treated with HSCT. Methods: UCLA Fresh Study (NCT01852071): Autologous CD34+ HSPCs were isolated from bone marrow and pre-stimulated with cytokines before transduction with EFS-ADA LV to yield OTL-101, which was infused as a fresh formulation in 20 subjects (9 male, 11 female; aged 4 mo-4.3 yrs). Single dose busulfan (4 mg/kg) was administered prior to infusion of OTL-101. Subjects were followed for 24 mo. UCLA Cryo Study (NCT02999984): 10 subjects (4 male, 6 female; aged 5-15 mo) received a cryopreserved formulation of OTL-101, which allowed for an extended shelf-life and full quality control prior to infusion. Busulfan was administered in 2 doses, the first at 3 mg/kg and the second adjusted to target a total area under the curve of 4,900 µM*min (20 ng/mL*hr). At the time of analysis, all subjects reached 12 mo FU (except 1 subject who was withdrawn from the study due to lack of engraftment); 7 subjects reached 18 mo of FU. Historical Control Group: 26 patients (aged 0.2 mo-9.8 yrs) were treated with allogeneic HSCT (MRDs n=12, non-MRDs n=14) at Great Ormond Street Hospital, UK (n=16) or Duke University Children's Hospital, USA (n=10) from 2000-2016. Results: Sustained engraftment of genetically modified HSPCs was observed in 29/30 GT subjects by 6-8 mo and persisted through FU in both studies, based on vector gene marking in granulocytes and CD3+ T cell reconstitution (Figure). Subjects who engrafted maintained long-term metabolic detoxification from deoxyadenosine nucleotides after stopping ERT approximately 1 mo post-GT. At last FU (median 24 mo; range 12-24 mo) in the GT group, overall survival (OS) was 30/30 (100%) and event-free survival (survival in the absence of ERT reinstitution or rescue allogeneic HSCT; EvFS) was 29/30 (97%). OS and EvFS were higher in the GT group at last FU compared with HSCT controls (with or without an MRD) at 2 years (Table). One of 30 OTL-101 subjects (3%) did not engraft and was restarted on ERT; the subject was withdrawn from the study at 5.9 mo and subsequently received a rescue HSCT, whereas 42% of HSCT patients required rescue HSCT, PEG-ADA ERT or died. Among the 20 OTL-101 subjects in the UCLA Fresh Study who reached 2 years FU, 18 (90%) stopped immunoglobin replacement therapy (IgRT), compared to 52% of HSCT patients. Preliminary results were observed in 5/7 (71%) OTL-101 subjects in the UCLA Cryo Study with more limited (18 mo) FU. Twelve OTL-101 subjects experienced one or more serious adverse events, most frequently infections and gastrointestinal events; only 1 of which was considered treatment-related (bacteremia due to product contamination). In the GT group, there were no events of autoimmunity with ≤24 mo FU. Due to the autologous nature of OTL-101, there was no incidence of graft vs host disease (GvHD); in contrast, 8 HSCT patients experienced GvHD events (5 acute, 3 chronic events), 1 of which resulted in death. Conclusions: Based on sustained gene correction and restoration of immune function in all subjects who engrafted, treatment of ADA-SCID with OTL-101 has a favorable benefit-risk profile. Key correlates of engraftment were consistent across the expanded cohort. Importantly, higher rates of OS and EvFS compared with HSCT (with or without an MRD) were observed. Disclosures Kohn: Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor on IP licensed from UC Regents to Orchard Therapeutics. Future royalties may occur., Research Funding; NIH: Research Funding. Shaw:Orchard Therapeutics: Consultancy, Other: Personal fees and non-financial support; NIH: Research Funding. Carbonaro-Sarracino:NIH: Other: Salary while working on project at UCLA 2013-2016, Research Funding; Orchard Therapeutics: Consultancy, Employment. De Oliveira:National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding. Terrazas:California Institute for Regenerative Medicine: Research Funding; Gene Therapy Resource Program, NHLBI/NIH: Research Funding. Hollis:Curative Therapeutics: Consultancy, Other: Personal fees. Trevisan:Orchard Therapeutics: Research Funding. Arduini:Orchard Therapeutics: Employment, Equity Ownership. Lynn:Orchard Therapeutics: Employment, Equity Ownership. Kudari:Orchard Therapeutics: Employment, Equity Ownership. Spezzi:Orchard Therapeutics: Employment, Equity Ownership. Buckley:Duke University: Research Funding. Booth:SOBI: Consultancy; GSK: Honoraria; NovImmune: Consultancy. Thrasher:Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Gaspar:Orchard Therapeutics: Employment, Equity Ownership, Patents & Royalties: Lentiviral vector for gene therapy of ADA-SCID.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2019-123432

Language: English

Publisher: American Society of Hematology

Publication Date: 2019

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Pediatric Research, Springer Science and Business Media LLC, Vol. 55, No. 3 ( 2004-3), p. 363-367

Type of Medium: Online Resource

ISSN: 0031-3998 , 1530-0447

URL: Article

DOI: 10.1203/01.PDR.0000111287.74989.1B

Language: Unknown

Publisher: Springer Science and Business Media LLC

Publication Date: 2004

detail.hit.zdb_id: 2031217-9

SSG: 12