Biosafety Studies of a Clinically Applicable Lentiviral Vector for the Gene Therapy of Artemis-SCID

Abstract

Genetic deficiency of the nuclease DCLRE1C/Artemis causes radiosensitive severe combined immunodeficiency (RS-SCID) with lack of peripheral T and B cells and increased sensitivity to ionizing radiations. Gene therapy based on transplanting autologous gene-modified hematopoietic stem cells could significantly improve the health of patients with RS-SCID by correcting their immune system. A lentiviral vector expressing physiological levels of human ARTEMIS mRNA from an EF1a promoter without post-transcriptional regulation was developed as a safe clinically applicable candidate for RS-SCID gene therapy. The vector was purified in GMP-comparable conditions and was not toxic in vitro or in vivo. Long-term engraftment of vector-transduced hematopoietic cells was achieved in irradiated Artemis-deficient mice following primary and secondary transplantation (6 months each). Vector-treated mice displayed T and B lymphopoiesis and polyclonal T cells, had structured lymphoid tissues, and produced immunoglobulins. Benign signs of inflammation were noted following secondary transplants, likely a feature of the model. There was no evidence of transgene toxicity and no induction of hematopoietic malignancy. In vitro, the vector had low genotoxic potential on murine hematopoietic progenitor cells using an immortalization assay. Altogether, these preclinical data show safety and efficacy, and support further development of the vector for the gene therapy of RS-SCID.

Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

“…No free plasmids were detected 4 days post-transduction by this method, indicating that detection of stable integration in the cell can be measured from that day. Across the literature, analysis of VCN is performed at different timepoints post-transduction form 7 days [ 12 , 93 , 94 ], 9 days [ 52 , 54 ], and up to 14 days [ 9 , 95 , 96 ]. It is important to note that; VCN values may differ when analyzed on different days, and while the differences can be subtle, it can hamper the comparison between trials.…”

“…This is of high importance in multi-center studies where there might be a need to agree on a release VCN value across countries and different regulatory agencies. Furthermore, it would be interesting to introduce new techniques for further characterization of the therapeutic gene product such as the abundant heterogeneity regarding stem cell subpopulations and the actual percentage of transduced cells (by flow cytometry or colony-forming assays); these are key features for the success of the gene therapy outcome that is currently being assessed differently in different trials [ 9 , 12 , 37 , 54 , 96 ].…”

“…Successful use of TEs will allow treating more patients with one viral batch, which will help to implement gene therapy as a standard protocol. In the past, to get a sufficient proportion of gene-corrected cells in the therapeutic product, high vector doses (2 transduction hits) and prolonged ex vivo culture (3 days in total) were needed [ 12 , 96 ]. Various TE compounds can be added to the culture media to increase lentiviral transduction efficiency, VCN, and transgene expression; they include Cyclosporin and Rapamycin [ 95 ], Prostaglandin E2 [ 93 ], Staurosporine [ 94 ], or LentiBOOST TM [ 96 , 102 ].…”

“…For example, Schott et al (2019) [ 96 ] showed the combinatorial use of protamine sulfate and LentiBOOST TM that allows to adjust their clinical protocol by reducing the amount of virus needed and shortening the culturing time (from 2 hits strategy to 1 hit), preserving at least similar transduction. Accordingly, gene therapy for Artemis-SCID, for which preclinical studies described a 2 hits approach [ 12 ], may benefit from a similar strategy to enable adaptation to a more efficient protocol. Overall, this strategy makes gene therapy more accessible due to reduced production costs.…”

“…Moreover, over 3000 clinical trials have been reported worldwide [ 1 ], with the majority addressing human cancer (CAR-T cells) and inherited monogenic diseases like primary immunodeficiencies [ 2 ]. Clinical trials with both self-inactivating (SIN) gamma-retroviruses and SIN-lentiviruses (10% of clinical trials) are currently ongoing for various primary immunodeficiencies like ADA Severe Combined Immunodeficiency (SCID) [ 3 , 4 , 5 ], X-linked SCID [ 6 , 7 , 8 , 9 ], Artemis SCID [ 10 , 11 , 12 ], Wiskott–Aldrich Syndrome (WAS) [ 13 , 14 , 15 ] or X-linked chronic granulomatous disease (CGD) [ 16 , 17 ] ( Table 1 ). Severe combined immunodeficiencies (SCIDs) are a group of rare inherited disorders in which both the humoral and cell-mediated immunities fail to function.…”