Production of Lentiviral Vectors Using Suspension Cells - PowerPoint PPT Presentation

Title: Production of Lentiviral Vectors Using Suspension Cells


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Production of Lent viral Vectors Using Suspension
Cells
HIV-1-derived lentiviral vectors (LVs) are
efficient gene transfer vehicles used in both
basic and clinical research settings. The ability
of LVs to efficiently shuttle DNA into mammalian
cells enables researchers to explore the function
of various genes of interest.1, 2, 3, 4, 5
Clinically, LVs are used ex vivo to deliver
therapeutic genes or expression cassettes to
primary target cells, such as hematopoietic stem
cells (HSCs), to treat genetic disorders or
infectious diseases.6
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Deriving the SJ293TS Cell Line
In order to convert the adherent HEK293T cell
line to grow in suspension, we employed a
commercially available SFM, Freestyle 293
Expression media (FS293). The cells were adapted
by replacing the standard growth media, DMEM plus
10 fetal bovine serum (FBS) (D10), with FS293
supplemented with 10 FBS and seeded onto both
non-tissue culture-treated and tissue
culture-treated plates.
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Optimization of Transient Transfection Method
We assessed vector production using SJ293TS cells
via our previously established transient
transfection method.42,56 Initially, we aimed to
optimize transfection conditions using a simple
GFP-expressing LV (vector CL40-MND-GFP) in 20-mL
cultures in 125-mL shaker flasks for quick and
easy readout.
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Comparison of LV Production by SJ293TS Cells with
Other HEK293-Based Cell Lines
To evaluate LV production in the
suspension-adapted SJ293TS cell line, we compared
1-L productions of SJ293TS cells in 5-L shaker
flasks against the parent HEK293T cells in
10-stack Cell Factory systems. For this
comparison, three different clinically relevant
LVs were produced two containing shRNAmiR
against human BCL11A with fluorophores (vectors
SJL118 and SJL121) and the other with an
expression cassette for a second generation
anti-CD19 chimeric antigen receptor (vector
aCD19-CAR).
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Improvements to SJ293TS LV Production Method
In order to improve the productivity and
suitability of the SJ293TS cell line for cGMP
manufacturing, we made modifications to the LV
production process. Because there is growing
concern using antibiotics for plasmid
manufacturing, we looked for an alternative.
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Downstream Processing of LVs Derived from SJ293TS
Cells
During vector production, Benzonase, an RNA-DNA
endonuclease, was added to producer cells 24 h
post transfection to reduce plasmid and genomic
DNA levels in vector supernatants. Upon harvest,
1-L LV preparations derived from SJ293TS cells
were purified using scale-down cGMP-like
processes.
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Table 2HOS Cell Vector Copy Number at Days 4 and
7 Post transduction
Lentiviral Vector Sample Day 4 VCN Day 7 VCN Prep
1 unconcentrated 2.62 2.32 Prep 1
finala 2.09 1.83 Prep 2 unconcentrated 1.63 1.42 P
rep 2 finala 2.12 1.84 Prep 3 unconcentrated 2.48
2.31 Prep 3 finala 3.81 3.54 Prep 4
unconcentrated 0.259 0.206 Prep 4
finala 0.954 0.809 Prep 5 unconcentrated 1.32 1.30
Prep 5 finalb 1.83 1.73 Prep 6
unconcentrated 1.83 1.55 Prep 6 finalb 10.7 12.5
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Suitability of SJ293TS-Derived LV for Clinical
Gene and Cell Therapy
During our process development, we have produced
clinically relevant LV, and we wanted to test how
these vectors transduced target cell populations,
mainly human T cells or CD34 HSCs. LV produced
from the SJ293TS cell line efficiently transduced
healthy donor-derived human T cells (Figure 5).
Four distinct vector preparations containing two
different second generation anti-CD123-CAR
vectors, pSJL605 and pSJL643, were used to
transduce primary human T cells at a MOI of 25.
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Materials and Methods
Cell Lines Plasmids LV Production HIV-1 p24
ELISA
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