Improved lentiviral vector titers from a multi-gene knockout packaging line

Abstract

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2 0 -5 0 -oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to $2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded $7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by $2fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to $11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.

Production of rAAV by plasmid transfection induces antiviral and inflammatory responses in suspension HEK293 cells

“…A recent study improved the production of lentiviral vector in HEK293 cells by 6.7-fold after knocking out constitutively expressed antiviral effectors including PKR and 2′-5′-oligoadenylate synthetase 1 (OAS1), suggesting that host pathways were capable of inhibiting vector production. 54 This is consistent with our finding on the induction of an ntiviral response during rAAV production. Together, the time course transcriptomic analysis revealed the cellular response and potential pathways that the host cells use to recognize exogenous stimuli throughout the process of rAAV vector production in suspension HEK293 cells at manufacturing scale for the first time.…”

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

“… 28 This producer line is designed to yield higher titers from the vectors with reverse orientation expression cassettes and has been shown to increase titers of β AS3 -globin vectors by 2- to 5-fold. 28 , 29 We saw a minimal decrease in the unconcentrated viral titers with any of the five UV1-shmiR vectors in comparison to the UV1 control ( Figure 1 B). In particular, the shmiR in the end IVS2 position retained a comparable titer to the parental UV1 vector.…”

Coengineering specificity, safety, and function into T cells for cancer immunotherapy

“…We have tested our dual inverted lentiviral vector in the context of both CARs and TCRs and various gene‐cargo, both in vitro and in vivo 35 . We next plan to test our dual inverted lentiviral vector in the context of modified HEK293T packaging cells developed by Han et al coined CHEDAR cells (CRISPRed HEK293T to Disrupt Antiviral Response) in which the genes OAS1 , LDLR , and PKR are knocked out (factors that impede lentiviral titers) and transcription elongation factors, SPT4 and SPT5 are overexpressed, all‐together leading to 11‐fold increases in lentiviral titers 87 …”

“…Right: solutions to overcome low viral titer are to coexpress an RNA interference suppressor protein (NovB2), and to favor ssRNA transcription by replacing the RSV-based truncated 5′LTR with the complete CMV promoter which harbors four 4 NF-κB binding motifs and including TNFα in the culture supernatant. developed by Han et al coined CHEDAR cells (CRISPRed HEK293T to Disrupt Antiviral Response) in which the genes OAS1, LDLR, and PKR are knocked out (factors that impede lentiviral titers) and transcription elongation factors, SPT4 and SPT5 are overexpressed, alltogether leading to 11-fold increases in lentiviral titers 87.…”