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 10 9 transducing unit (TU)/mL and 5 × 10 4 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.

Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates

“…Similarly to the acute response previously reported following i.v. LV administration in murine models 17 , 23 , pro-inflammatory cytokines, and chemokines, likely released from innate immune cells, significantly increased in the blood-stream of NHP after LV administration. We observed transient elevation of multiple signals for the recruitment and activation of innate immune effectors, such as neutrophils, eosinophils, monocytes, and dendritic cells, and in particular growth-regulated protein beta (GRO-β), interleukin (IL)-5, monocyte chemoattractant protein 1 (MCP1), IFNγ and interferon gamma-induced protein 10 (IP-10) within the first 1–2 days post-LV, as well as interferon–inducible T-cell alpha chemoattractant (I-TAC) and MCP1 chemokines for the recruitment and activation (IL-6) of T and B lymphocytes.…”

Lentiviral Vector Bioprocessing

“…In addition, determining the relevant parameters which impact the performance of process options will be important in the optimal selection of these options and their operation. Screening studies can provide crucial bioprocess information such as the level of transmembrane pressures [ 65 ] or crossflow rate [ 145 ] in TFF operations, column flow rate in AEX chromatography [ 145 ] or the right molecular weight cut-off (MWCO) or membrane material in TFF operation [ 65 , 145 , 243 , 244 ]. It is also important to determine the impact of using frozen-thawed materials in process development of unformulated LV products (e.g., [ 190 , 216 ]), as opposed to using fresh material, as this step may have a huge influence on the process performance rather than as result of the bioprocess operation itself.…”

Gene Therapy Potential for Genetic Disorders of Surfactant Dysfunction

“…Options for promoter choice include cell type specific or constitutive elements. Production of clinical grade vector for in vivo somatic cell targeting represents a challenge, but advances in lentivirus manufacturing (Valkama et al, 2018;Martínez-Molina et al, 2020;Soldi et al, 2020) make this a promising therapeutic for SP-B deficiency.…”