Dataset Information

Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering.

ABSTRACT: Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 10⁹ transducing unit (TU)/mL and 5 × 10⁴ TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo, as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing.

SUBMITTER: Soldi M

PROVIDER: S-EPMC7683235 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Publications

Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced <i>Ex Vivo</i> and <i>In Vivo</i> Genetic Engineering.

Soldi Monica M Sergi Sergi Lucia L Unali Giulia G Kerzel Thomas T Cuccovillo Ivan I Capasso Paola P Annoni Andrea A Biffi Mauro M Rancoita Paola Maria Vittoria PMV Cantore Alessio A Lombardo Angelo A Naldini Luigi L Squadrito Mario Leonardo ML Kajaste-Rudnitski Anna A

Molecular therapy. Methods & clinical development 20201020

Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental stu ...[more]

PMID: 33294490

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Project description:BackgroundSevere combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency.MethodsWe treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up.ResultsOverall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade.ConclusionsTreatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).

Dataset Information

Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering.

Publications

Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced <i>Ex Vivo</i> and <i>In Vivo</i> Genetic Engineering.

Similar Datasets

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