Low Bioavailability and High Immunogenicity of a New Brand of E. colil-Asparaginase with Active Host Contaminating Proteins.

The drug l-asparaginase is a cornerstone in the treatment of acute lymphoblastic leukemia (ALL). The native E. colil-asparaginase used in Brazil until recently has been manufactured by Medac/Kyowa. Then a decision was taken by the Ministry of Health in 2017 to supply the National Health System with a cheaper alternative l-asparaginase manufactured by Beijing SL Pharmaceutical, called Leuginase®. As opposed to Medac, the asparaginase that has been in use in Brazil under the trade name of Aginasa®, it was not possible to find a single entry with the terms Leuginase in the Pubmed repository. The apparent lack of clinical studies and the scarcity of safety information provided to the hospitals by the drug distributor created a debate among Brazilian pediatric oncologists about issues of safety and efficacy that culminated eventually in a court decision to halt the distribution of the new drug all over the country. Boldrini Children's Center, a non-profit pediatric oncohematology hospital, has conducted its own evaluation of Leuginase®. Mass spectrometry analyses found at least 12 different contaminating host-cell proteins (HCP) in Leuginase®. The presence of two HCP (beta-lactamase and malate dehydrogenase) was confirmed by orthogonal methodologies. The relative number of HCP peptides ranged from 19 to 37% of the total peptides identified by mass spectrometry. In vivo studies in mice injected with Leuginase® revealed a 3 times lower plasma bioavailability and the development of higher antibody titres against l-asparaginase in comparison to Aginasa®-injected animals. The decision to buy a new drug based on its price alone is not safe. Developing countries are especially vulnerable to cheaper alternatives that lack solid quality assurance.

Gemcitabine intercellular diffusion mediated by gap junctions: new implications for cancer therapy.

BACKGROUND: Solid tumors are often poorly vascularized, with cells that can be 100 microm away from blood vessels. These distant cells get less oxygen and nutrients and are exposed to lower doses of chemotherapeutic agents. As gap junctions allow the passage of small molecules between cells, we tested the possibility that the chemotherapeutic agent gemcitabine can diffuse through gap junctions in solid tumors. RESULTS: We first showed with a dye transfer assay that the glioblastoma and the osteosarcoma cells used in this study have functional gap junctions. These cells were genetically engineered to express the herpes simplex virus thymidine kinase (TK), and induced a "bystander effect" as demonstrated by the killing of TK-negative cells in presence of the nucleoside analogue ganciclovir (GCV). The ability of gemcitabine to induce a similar bystander effect was then tested by mixing cells treated with 3 microM gemcitabine for 24 hours with untreated cells at different ratios. In all cell lines tested, bystander cells were killed with ratios containing as low as 5% treated cells, and this toxic effect was reduced in presence of alpha-glycyrrhetinic acid (AGA), a specific gap junction inhibitor. We also showed that a 2- or a 24-hour gemcitabine treatment was more efficient to inhibit the growth of spheroids with functional gap junctions as compared to the same treatment made in presence of AGA. Finally, after a 24-hour gemcitabine treatment, the cell viability in spheroids was reduced by 92% as opposed to 51% in presence of AGA. CONCLUSION: These results indicate that gemcitabine-mediated toxicity can diffuse through gap junctions, and they suggest that gemcitabine treatment could be more efficient for treating solid tumors that display gap junctions. The presence of these cellular channels could be used to predict the responsiveness to this nucleoside analogue therapy.

Characterization of an alternative packaging system derived from the cat RD114 retrovirus for gene delivery.

BACKGROUND: Retroviral vectors derived from the Moloney murine leukemia virus (MLV) are widely used in gene therapy. Pseudotyping of these vectors with the cat RD114 retrovirus envelope increases their potential for delivering genes into human hematopoietic cells. In the present study, we have further investigated the potential of the RD114 retrovirus in gene therapy. We describe and characterize an alternative retroviral packaging system derived from the RD114 retrovirus. METHODS: RD114-derived recombinant retroviruses were produced transiently by transfection of 293T cells, and viral titers were assessed on TE671 cells by measuring the percentage of infected green fluorescent protein (GFP) positive cells by fluorescence-activated cell sorter (FACS) analysis. Purified human hematopoietic cells (lymphocytes and CD34(+) cells) were activated and transduced on retronectin-coated plates. Two days later, the percentage of GFP positive cells was evaluated by FACS analysis. RESULTS: We demonstrate that RD114 viral particles could package MLV transfer vectors, and that, in addition to its natural envelope, RD114 cores could be efficiently pseudotyped by the Gibbon ape leukemia, the MLV-amphotropic and the vesicular stomatitis virus G protein envelopes. Furthermore, we found that RD114 viral particles were highly efficient to transduce human lymphocytes and CD34(+) cells. CONCLUSIONS: This is the first demonstration that replication-defective RD114 viral particles can be generated and used for efficient gene delivery into human hematopoietic cells. We conclude that RD114-derived vectors could be useful in the field of gene therapy.

Efficient human hematopoietic cell transduction using RD114- and GALV-pseudotyped retroviral vectors produced in suspension and serum-free media.

Retroviral vectors derived from the Moloney murine leukemia virus have been used in successful and promising gene therapy clinical trials. However, platforms for their large-scale production must be further developed. As a proof of principle, we reported the generation of a packaging cell line that produces amphotropic retroviral vectors in suspension and serum-free medium (SFM). In the present study, we have constructed and characterized two retroviral packaging cell lines designed for gene transfer in hematopoietic cells. These cell lines grow in suspension and SFM, and produce high-titer RD114- and gibbon ape leukemia virus (GALV)-pseudotyped vectors for a 3-month culture period. Viral particles released are as robust during repeated freeze-thaw cycles and on thermal inactivation at 37 degrees C as their counterparts produced in cells cultured adherently with serum. We also show that RD114- and GALV-pseudotyped vectors produced in suspension and SFM efficiently transduce human lymphocytes and hematopoietic stem cells. As these retroviral packaging cell lines distinctively maintain high vector titers while growing in suspension and SFM, we conclude that these cell lines are uniquely suitable for large-scale clinical-grade vector production for late-phase clinical trials involving gene transfer into hematopoietic cells.