Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G

Lentiviral vectors are a common tool used to introduce new and corrected genes into cell therapy products for treatment of human diseases. Although lentiviral vectors are ideal for delivery and stable integration of genes of interest into the host cell genome, they potentially pose risks to human health, such as integration-mediated transformation and generation of a replication competent lentivirus (RCL) capable of infecting non-target cells. In consideration of the latter risk, all cell-based products modified by lentiviral vectors and intended for patient use must be tested for RCL prior to treatment of the patient. Current Food and Drug Administration (FDA) guidelines recommend use of cell-based assays to this end, which can take up to 6 weeks for results. However, qPCR-based assays are a quick alternative for rapid assessment of RCL in products intended for fresh infusion. We describe here the development and qualification of a qPCR assay based on detection of envelope gene sequences (vesicular stomatitis virus G glycoprotein [VSV-G]) for RCL in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Our results demonstrate the sensitivity, linearity, specificity, and reproducibility of detection of VSV-G sequences, with a low false-positive rate. These procedures are currently being used in our phase 1 clinical investigations

Production of therapeutic enzymes by lentivirus transgenesis

Since ERT for several RSDs treatment has emerged at the beginning of the 1980s with Orphan Drug approval, patients´s expectancy and life quality have been improved. Most LSDs treatment are based on the replacement of mutated or deficient protein with the natural or recombinant protein.One of the main ERT drawback is the high drug prices. Therefore, different strategies trying to optimize the global ERT biotherapeutic production have been proposed. LVs, a gene delivery tool, can be proposed as an alternative method to generate stable cell lines in manufacturing of recombinant proteins. Since LVs have been used in human gene therapy, clinical trials, safety testing assays and procedures have been developed. Moreover, one of the main advantages of LVs strategy to obtain manufacturing cell line is the short period required as well as the high protein levels achieved.In this chapter, we will focus on LVs as a recombinant protein production platform and we will present a case study that employs LVs to express in a manufacturing cell line, alpha-Galactosidase A (rhaGAL), whic is used as ERT for Fabry disease treatment.Fil: Rodríguez, María Celeste. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Ceaglio, Natalia Analia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Antuña, Sebastían. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Tardivo, María Belén. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Prieto, Claudio. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Non-viral precision T cell receptor replacement for personalized cell therapy.

The T cell receptor (TCR) provides the fine specificity of T cells to recognize mutations in cancer cells 1-3. We developed a clinical-grade approach based on CRISPR/Cas9 non-viral precision genome editing to simultaneously knock-out the two endogenous TCR genes, TCRα (TRAC) and TCRβ (TRBC), and insert in the TRAC locus the two chains of a neoantigen-specific TCR (neoTCR), isolated from the patient\u27s own circulating T cells using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with refractory solid cancers received up to three distinct neoTCR-transgenic cell products, each expressing a patient-specific neoTCR, in a cell dose-escalation, first-in-human phase 1 clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome, and one grade 3 encephalitis. All had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease, and the other 11 had disease progression as best response on therapy. NeoTCR-transgenic T cells were detected in tumour biopsies post-infusion at frequencies higher than the native TCRs pre-infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs recognizing mutational neoantigens, the simultaneous knock-out of the endogenous TCR and knock-in of the neoTCRs using single-step, non-viral precision genome editing, the manufacturing of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene edited neoTCR T cell products, and the ability of the transgenic T cells to traffic to the patients\u27 tumours