β-Globin Lentiviral Vectors Have Reduced Titers due to Incomplete Vector RNA Genomes and Lowered Virion Production

Abstract

Summary Lentiviral vectors (LVs) commonly used for the treatment of hemoglobinopathies often have low titers and sub-optimal gene transfer efficiency for human hematopoietic stem and progenitor cells (HSPCs), hindering clinical translation and commercialization for ex vivo gene therapy. We observed that a high percentage of β-globin LV viral genomic RNAs were incomplete toward the 3′ end in packaging cells and in released vector particles. The incomplete vector genomes impeded reverse transcription in target cells, limiting stable gene transfer to HSPCs. By combining three modifications to vector design and production (shortening the vector length to 5.3 kb; expressing HIV-1 Tat protein during packaging; and packaging in PKR−/− cells) there was a 30-fold increase in vector titer and a 3-fold increase in vector infectivity in HSPCs. These approaches may improve the manufacturing of β-globin and other complex LVs for enhanced gene delivery and may facilitate clinical applications.

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

“…16,17 Knocking out PKR in HEK293T cells increased titers of LVs, particularly for vectors with internal promoters in the reverse orientation, a common configuration for b-globin expressing LVs. 18,19 Based on these previous studies, it is conceivable that the constitutively expressed antiviral effectors can still restrict LV production in HEK293T cells. Moreover, RFs are not limited only to antiviral effectors but also include genes that regulate the lentiviral life cycle.…”

Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases

“…Interestingly, the combination of minimal HS2, HS3 and HS4 or that of larger versions of HS2 and HS3 seemed to achieve comparable gene expression levels, while the length of the β-globin promoter and the presence of a 3′ enhancer appeared to have only a marginal impact. Due to their size and complex structure, transduction efficiency of LV globin vectors is relatively low [ 78 ], while a high VCN is mandatory to achieve phenotypic correction due to the relatively low mRNA output of a globin minigene compared to its wild-type counterpart. For this reason, correction of the most extreme forms of β-globin deficiency (homozygous β 0 -thalassemias) is only rarely achievable [ 12 , 77 ].…”

Improved SARS-CoV-2 Spike Glycoproteins for Pseudotyping Lentiviral Vectors

“…Spike-pseudotyped lentiviruses were packaged by transient transfection of PKR -/-293T cells with fixed amounts of HIV Gag/Pol, Rev, and Lentiviral envelope (VSV-G or Spike) expression plasmids and equimolar amounts of either MNDU3-eGFP or roUBC-mCitrine transfer plasmid using TransIT-293 (Mirus Bio, Madison, WI) as described in the Supplementary Materials and Cooper et al (32,33). Viral supernatants were then directly used for titer determination or concentrated by tangential flow filtration, as described by Cooper et al, 2011.…”

“…We generated the SARS-CoV-2 pseudotyped virus using a 3rd generation HIV-1 packaging system (Figure 1B). We transfected PKR -/-293T cells with plasmids encoding for HIV-1 proteins, Gag-Pol and Rev; a plasmid encoding for the full-length spike glycoprotein envelope; and a lentiviral transfer plasmid containing an eGFP reporter cassette (33). The S glycoprotein on the produced lentivirus should improve specificity toward ACE2-expressing cells.…”

“…These included the following glycoprotein envelopes: wildtype spike containing the HA or d19 tail (Ou-HA, Ou-d19), the United Kingdom spike variant (Ou-Alpha, Ou-Alpha-HA, Ou-Alpha-d19) or the South African spike variant (Ou-Beta, Ou-Beta-HA, and Ou-Beta-d19). We packaged each S-pseudotyped vector head-to-head with a GFP reporter cassette driven by a reverse oriented ubiquitin C promoter (roUBC-mCitrine) (33). ACE-293T or HEK-293T cells were transduced with the S-pseudotyped virus at equal amounts of p24 particles.…”