ALBERT

Description: A significant percentage of clinical grade safety-enhanced gamma-retrovirus and lentivirus vector supernatants are currently produced by transient transfection. These products contain high concentrations of plasmid DNA that pose the risk of transfer into subjects, and preclude accurate estimation of transduction efficiency. Most envelope pseudotyped vectors are too labile for DNAse treatment or other purification steps and adding non-clinical grade reagents into any production step further complicates product qualification. Here we present development of a method to remove residual plasmid as a part of the transduction procedure of human CD34+ cells or mouse bone marrow progenitors, using the DNAse Pulmozyme®. Using a standard clinical transduction protocol, CD34+ were transduced with SRS11.EFS.IL2RG.pre* or SRS11.EFS.EGFP. pre* gamma-retroviral vectors pseudotyped with GALV envelope or a mock control. Retronectin coated flasks preloaded with vector were either treated with Pulmozyme® or no Pulmozyme®. Cells were transduced on two consecutive days and collected 4 –24 hrs after the 2nd transduction (day1). CFU were plated and enumerated and proportion of transduced progenitors quantified by real time PCR (qPCR) using wPRE primers. For the EGFP containing vector, gene transfer was determined by flow cytometry in cultures that were continued for up to 14 days. DNA was extracted and tested by qPCR on days 1, 7, and 14. Residual plasmid DNA was quantified using GALV primers. Transduction was also estimated on the bulk cultures using qPCR. In all qPCR reactions human ApoB was quantified concurrently to asses the cell number. Pulmozyme® treatment reduced GALV copy numbers in cells one day following transduction by up to 20-fold. After 7 days in culture, there was no residual plasmid detectable in all groups. More importantly, Pulmozyme® treatment did not alter the transduction efficiency of the progenitors in any of the experiments, as evaluated by either wPRE qPCR on Day-7 DNA (4.3 vs. 4.2, 6.8 vs. 7.6 and 2.0 vs. 1.9 vector copies per cell for Pulmozyme® treated and non-treated respectively), colony PCR (69.4 vs. 66.7% vector positive CFUs) or flow cytometry (81.8 vs. 85.7% EGFP positive cells). We also analyzed the engraftment potential of cells transduced using Pulmozyme®-treated versus non-treated vector after transplanting cells in NOD/SCID and NOD/SCID-IL2Rg null (NOG) mice. Analysis of mice at 6 weeks post transplantation showed no significant reduction in human cell engraftment in the Pulmozyme® and the average copy number per human cell in NOG bone marrow was 0.65 vs. 0.52 and in NOD/SCID bone marrow 1.03 vs. 0.67 (Pulmozyme® treated versus non-treated, respectively). We also tested Pulmozyme® treatment in mouse experiments using gamma-retroviral vector pseudotyped with ecotropoc envelope with a similar result. We conclude that treatment of gamma-retroviral vector supernatant with Pulmozyme® after vector preload on Retronectin® coated plates reduces vector and packaging plasmids and does not inhibit transduction, clonogenic potential, or engraftment. The removal of excess plasmid from transiently produced virus supernatant without affecting the virus transducibility allows accurate estimation of transduction efficiencies prior to transplant and will conceivably reduce the toxicity to primary cells from excess plasmid. These studies can be directly translated to preclinical animal safety studies and clinical trials.

Description: Introduction: The DxH 520 is a small hematology analyzer capable of performing CBC and 5-part differential in fresh whole blood samples (venous and capillary) collected in K2EDTA and K3EDTA anticoagulants. Medical care of children and adolescents is significantly dependent on reference intervals to properly interpret laboratory test results. A multi-center study was performed to verify or establish pediatric reference intervals for all parameters (White Blood Cells (WBC), Red Blood Cells (RBC), Hemoglobin (Hgb), Hematocrit (Hct), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), RDW-SD, Platelets (PLT), Mean Platelet Volume (MPV), (Lymphocyte) LY%, LY#, Monocyte% (MO%), MO#, Neutrophil % (NE%), NE#, Eosinophil% (EO%), EO#, Basophil % (BA%), and BA#) with combined genders. Methods: Whole blood samples from healthy children were tested within 8 hours of collection on the DxH 520 analyzer. Samples generating review flags or suspect messages were excluded from the analyses. Even gender distribution was targeted. A total of 208 specimens were enrolled that included 20 neonates (0 to 30 days), 27 infants (31 days to 2 years), 94 children (3 to 12 years) and 67 adolescents (13 to 21 years). Results were analyzed according to CLSI EP20-A3c guidelines. Results: Reference intervals for the neonate age group were verified from existing published ranges using the transference method described in the guideline (Table 1). The robust method was used to calculate two sets of reference intervals partitioned by combining data from the infant and children age groups, and from the adolescent age group (Table 2). Conclusion: Reference Intervals for CBC and differential parameters have been established for pediatric age groups (0 to 30 days, 31days to 12 years, 13 to 21 years) on the DxH 520 Hematology analyzer. *CE marked. Pending 510(k) clearance by the United States Food and Drug Administration; not yet available for in vitro diagnostic use in the U.S. Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. Disclosures Smit: Beckman Coulter: Employment. Seidel:Beckman Coulter: Employment. Rohrbach:Beckman Coulter: Employment. Magari:Beckman Coulter: Employment. Lo:Beckman Coulter: Employment. Martin:Beckman Coulter: Employment. Tejidor:Beckman Coulter: Employment.