An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus
- Ranjita Sengupta†1,
- Chandreyee Mukherjee†1,
- Nandita Sarkar2,
- Zhihong Sun1,
- Jacob Lesnik1,
- Joseph Huang1 and
- Biao Lu3Email authorView ORCID ID profile
© The Author(s). 2016
Received: 4 February 2016
Accepted: 15 June 2016
Published: 11 July 2016
A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done.
Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed.
An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells.
Lentiviral vectors provide one of the most effective gene delivery systems that have a broad range of applicationsfrombasic research to gene therapy [1–3]. For instance, HIV-basedlentivectorshave been extensively used for stably expressing different effector molecules, including cDNA, siRNA, long non-coding RNA, DNA fragment, antisense, ribozyme, and transcriptional reporter [4–6]. Recently, lentiviral vectors have been used clinically to express chimeric antigen receptor (CAR) in T lymph cells, enabling CAR-T cells in curing end-stage B cell leukemia [7, 8]. By packaging the lentiviral construct into pseudoviral particles, an efficient transduction can be achieved even with the most difficult cell types, such as primary cells, stem cells and hematopoietic cells [1, 2]. Lentivectorsalsohave a larger gene-cargocapacity (~7–8 kb) with low immunogenicity compared to other delivery vehicles. The major disadvantage of lentivectorsisits ability to integrate into the genome, which can lead to insertion mutations and other side effects [1, 9].
To circumvent this, one can generate non-integrative lentivirusesthat remain in the cell epichromosomally. This episomal vector can retain all the advantages of lentivectorsand express transgene of interest without causinginsertional mutations. There are several methods to generate non-integrative lentivirus and one such approach is to mutate the integrase gene. Although studies have identified several critical amino-acids for generating so called integrase defective lentivirus (IDLV) [10–12], a comparative study to systemically evaluate their relative performance on gene expressionislacking.
To make lentiviral particles, lentivectors which carry the transgene of interest (in this case GFP under a constitutive promoter EF1α, CD511B-1) is mixed with a plasmid packaging mix which includes the integrase, envelope gene and other genes necessary to generate pseudoviralparticles. This plasmid mix is then co-transfected into a 293 T producer cell line. Viral harvest is done 48 h and 72 h post transfection. In this study, we designed and generated a cohort of integrase mutants by site directed mutagenesis. We then evaluated the packaging efficiency of each of the mutants when combined with rest of the lentivirus packaging plasmids and identified one with the highest packaging efficiency. We further evaluated and characterized this mutant for its ability totransduceboth dividing and non-dividing cells. Since the mutation occurs in one of the packaging plasmids, this system is likely compatible with most existing lentivectors, hence providing an efficient and robust system for IDLV production.
Results and Discussion
Lentiviral Packaging System and IDLV Production
We initially generated 5 different mutants in the integrase gene (Fig. 1a) and selected the best performer with respect to gene expression. To achieve this, we used a previously established green fluorescent reporter expressed under EF1α promoter CD511B-1 as a read-out (Fig. 1b). We usedWT Integrase and candidate Integrase mutants along with Gag and VSVG packaging plasmid mix to package CD511 under identical experimental conditions. As shown in Fig. 1c, all mutant candidates express GFP at varying levels, albeit much lower than the GFP expressionfromthe WT control, whichissimilar to published reports [10–12]. Among them, mutant D116Ademonstrated the highest levels of reporter expression (Fig. 1c). Since we used the same amount of infectious viral particles to perform the transduction, we selected mutant D116A for further characterization.
D116A Mutant Produces IDLV with Non-Integration Nature
IDLV Tranducesboth Dividing and Non-Dividing Cells
Lentivirus has been used transducing some primary and hoard-to-transfect cells. Although we did not provide direct evidence in this study, we expect IDLV will follow the tropism of wild-type counterpart, because this change occurs within the integrase domain of POL gene, which will not directly affect the tropism of lentivirus.
IDLV Performance in Relationship with Its Cargo Size
We have identified and validated an integrase mutant that can be used to produce non-integrative lentiviral particles. This IDLV system would allow researchers to use their existing lentivectors to generate non-integrative lentiviral particles with increased safety profile and decreased side effect such as insertional mutation and insertion-caused genome instability respectively.
Integrase Mutagenesis and Reporter Construction
The pPACKH1-GAG plasmid (SBI, Mountain View, CA) contains the structural (gag), replication (pol) and envelop (env) gene. The pol contains a functional domain of integrase, which is responsible for provirus integration. As shown in Fig. 1a, we designed and synthesized primer pairs to carry out site-directed mutagenesis using high fidelity DNA polymerase followed by DpnI (NEB, Ipswich, MA) treatment and transformation. The primers used for D64A (forward 5′- aggaatatggcaactagcttgtacacatttagaag-3′; reverse 5′-ctaaatgtgtacaagctagttgccatattcctggac), D116A (forward 5′- gtaaaaacaatacatacagccaatggcagcaatttcacc-3′; reverse 5′- gaaattgctgccattggctgtatgtattgtttttactggc-3′), D152A (forward 5′-caaagtcaaggagtagtagcatctatgaataaagaattaaagaaaatt; reverse 5′- ctttaattctttattcatagatgctactactccttgactttgggg-3′), and triplet mutation in basic region (forward5′-gtgacataaaagtagtgccagcagcacatgcaaagatcattagggat-3′; reverse primer 5′-atccctaatgatctttgcatgtgctgctggcactacttttatgtcac- 3′). Using different combinations of primer pairs, we have generated five integrase mutants as confirmed by double stranded DNA sequencing (SeqeneTech, Mountain View, California).
Cell Culture and Transduction
Human embryonic kidney HEK293-TN (LV900A-1, SBI, Mountain View, CA) and fibrosarcoma HT1080 (ATCC, Manassas, VA) cells were maintained in high glucose Dulbecco’s Minimal Essential Medium (DMEM) supplemented with 10 % FBS, 2 mMGlutaMax (Life Technologies), 100 U/ml penicillin and 100 U/ml streptomycin. Transduction reagent TransDux (LV850A-1, SBI, Mountain View, CA) was used to infect cells with virus to enhance the transducing efficiency. CD511B-1 (GFP reporter) and LV605A-1 (DualGFP-RFP reporter) from SBI were the constructs used to make WT and integrase defective lentiviral particles for most of the studies. These constructs were assembled by a combination of PCR and seamless fusion method as reported before [15, 16].
STO Culture and Actinomycin Treatment
STO cells were cultured in DMEM high-glucose (Life Technologies), supplemented with 2 mMGlutaMax (Life Technologies), and 1 % nonessential amino acid (Life Technologies). Cells were treated with 10 μg/ml actinomycin C (Sigma) for 3 h. The actinomycin C-treated STO non-dividing cells were extensively washed in PBS and plated at 35, 000 cells/cm2 in gelatin-coated tissue culture dish.
Microscopy and Reporter Gene Assay
All microscopy was performed on live cells with a LEICA DMI3000B microscope following 48 ~ 72 h transduction. Data collection and processing were performed with LAS 3.8 software.
Pseudoviral titer was performed using a real time PCR based kit (LV961A-1, SBI, Mountain View, CA). This kit measures the copy numbers of integrated gene after transduction. Briefly, HT1080 cells were transduced in the presence of 1 × TransDux reagent (LV850A-1, SBI, Mountain View, CA) for 72 h. Subsequently cells were lysed and DNAs were isolated according to manufacturer’s instructions. PCR reactions were performed with primers for amplifying a conserved genomic DNA sequences or WPRE in the transgeneusing Applied Biosystems 7300 Real time PCR System. PCR condition: 50 °C, 2 min; 95 °C, 10 min; followed by 40 cycles of 95 °C, 15 s and 60 °C for 1 min, followed by a dissociation step.
Standard Operating Procedure for Lentiviral Packaging
The expression constructs CD511B-1 and LV605A-1 (SBI, Mountain View, CA) are packaged into VSVG pseudotypedviral particles with proteins produced by the lentiviral packaging plasmid mix (LV500A-1, SBI, Mountain view, CA). The lentiviralpackaging system consists of an optimized mixture of three plasmids, including pPACKH1-GAG, pPACKH1-REV and pVSVG. To produce integrative competent lentiviruse, the pPACKH1-GAG has a wild-type pol gene. To produce non-integrative lentiviruses, the pPACKH1-GAG with a mutated integrase (an integrase domain in pol gene) was used instead of the wild-type. Other reagents include virus producer cell lineHEK293TN (LV900A-1, SBI, Mountain View), transfection reagent PureFection (LV750A-1, SBI, Mountain View), and virus precipitation solution (LV810A-1, SBI, Mountain View).
Additional reagents include DMEM high glucose with sodium pyruvate and l-glutamate (Invitrogen, Cat. #111995073), Fetal bovine serum (Invitrogen, Cat. #16000036), Penicillin/Streptomycin (Invitrogen, Cat. # 15070063), Trypsin-EDTA (Sigma, Cat. #T3924), hematopoietic plate cell counter.
Step-by-step Protocol (bench time 110 min, total time 6 days)
Seed 293 TN producer cells (30 min)
18 ~ 24 h prior to transfection, wash cells with pre-warmed DPBS once and add 2 mlTrypsin-EDTA to the culture flask. Incubate for 5 min or until cells become detached from surface and add 10 ml of complete medium to neutralize the digestion enzyme. Precipitate cells by centrifugation at 300 rpm for 5 min at room temperature. Re-suspend cell pellet in 5 ml complete medium and count cells. Seed 293TN cells at 7 ~ 8 × 106/15 cm2 in 20 ml complete culture medium with antibiotics, until the cell density reaches 70 ~ 80 % confluence for transfection.
Prepare lentivector DNA and packaging plasmid mix
Add 1 ~ 1.6 ml plain DMEM (serum and antibiotic free) to an Eppendorf tube; add 4.5 μg lentivector and 45 μl packaging plasmid DNAinto the DMEM. Mix by pipetting for 10 times.
Prepare transfection mix
Add 55 μl PureFection into the same tube. Vertex for 10 s and incubate the DMEM-Plasmid-PureFectionmixture at room temperature for 15 min.
Apply transfection mix to producer cells
Add DMEM-Plasmid-PureFectionmixture drop-wise to the dish, and swirl to disperse evenly; return the dish to the cell culture incubator at 37 °C with 5 % CO2; change the medium 12 ~ 24 h after transfection (optional).
Day 4 and Day 5
Harvest packaged viruses (bench time 30 min; total 2 h)
48 h after transfection, collect the culture medium containing pseudoviral particles into a 50-ml sterile, capped conical centrifuge tube and store at 4 °C until next harvest. Re-fill producer cells with 10 ml fresh complete medium slowly, and return dishes to the cell culture incubator.
72 h after transfection, collect the culture medium and combined it with the collection at 48 h. Centrifuge at 3000 × g for 15 min at room temperature to pellet cell debris. Transfer the viral supernatant into a new tube and add 1 volume of cold PEG-it Virus Precipitation Solution (4 °C) to every 4 volumes of Lentivector-containing supernatant. Larger volume precipitation of lentiviral particles can be achieved by using the Corning 250 ml polypropylene centrifuge tube (Cat. #430776), following manufacturer’s instructions: refrigerate overnight or for at least 12 h. Lentivirus-containing supernatants mixed with PEG-it Virus Precipitate Solution (LV810A-1, SBI, Mountain View, CA) are stable for up to 4 ~ 5 days at 4 °C.
Collect and concentrate psuedotyped viral particles (bench time 20 min; total 1.2 h)
Centrifuge supernatant/PEG-it mixture at 3000 × g for 30 min at 4°C, and transfer supernatant to a fresh tub. After centrifugation, the lentivector particles will appear as a beige or white pellet at the bottom of the vessel. Spin down residual PEG-it solution by centrifugation at 3000 × g for 5 min; remove all traces of fluid by aspiration, and avoid disturbing the precipitated lentiviral particles in pellet. Re-suspend lentiviral pellets from 1/10 to 1/100 of original volume using cold, sterile PBS or DMEM containing 25 mM HEPES buffer at 4 °C. Aliquot into cryogenic vials and store at −70 °C.
Low viral titer (<105 ifu/mL) can be caused by a number of reasons. The most common ones include:(1) poor transfection efficiency due to too high or too low density of producer cells. (2) Poor quality of lentivector or packaging plasmid DNA. (3) Inadequate ratios of plasmid mix to the transfection reagent. To avoid these, plate cells to achieve 70 ~ 80 % confluency at transfection stage and use endotoxin-free plasmid purification kit to prepare lentivector expressing the transgene and packaging plasmids.
If problem remains, other factors to be consideredare: (1) producer HEK293TN cells are of poor quality due to over confluence; (2) cells may be contaminated by mycoplasma; (3) too many passages (>20) of cells. In those cases, one should revive a new batch of producer cells and maintain them in the logarithmic growth phase.
Other factors which could affect virus quality are: The packaging limit for the lentiviral system, which is ~8.5 kb from 5′ → 3′ LTR. The efficiency of packaging drops significantly when cDNA size increases. For a 3 kb insert, the titers and expression level can be 10-fold less compared to that ofa 1 kb insert. Repeated sequences can also cause problems in transgene expression as recently reported .
Safety Guide to Packaging and Transduction of Target Cells
The use of HIV-based pseudotypedlentiviruses falls within NIH Biosafety Level 2 criteria due to the potential biohazard risk of possible recombination with endogenous viral sequences to form self-replicating virus, or the possibility of insertional mutagenesis. For a description of laboratory biosafety level criteria, consult the Centers for Disease Control Office of Health and Safety Web site. It is also important to check with the health and safety guideline at your institution regarding the use of lentiviruses and always follow standard microbiological practices.
CMV, cytomegalovirus promoter; EF1-α, elongation factor 1- alpha promoter; GFP, green fluorescent protein; IDLV, integrase defective lentivirus; MSCV, murine stem cell virus promoter; NILVP, non-integrative lentiviral packaging; RFP, red fluorescent protein; VSVG, vesicular stomatitis virus envelope glycoprotein
We thank all members of SBI for their helpful discussion and technical helps.
This study is supported in part by internal funding of SBI.
Availability of Data and Materials
All data and materials described in this study can be requested from SBI.
RS, NS, and BL conceived the study design and collaborated on the project development. RS, NS, CM, ZS and BL performed the experiments, data analysis and interpretation. RS, CM and BL drafted the manuscript. All authors contributed the revision of the manuscript; have read and approved the final version of this paper.
NS and BL were former employees at SBI (System Biosciences).
Ethics Approval and Consent to Participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Matrai J, Chuah MK, et al. Recent advances in lentiviral vector development and applications. MolTher. 2010;18(3):477–90.Google Scholar
- Collins M, Thrasher A. Gene therapy: progress and predictions. Proc Biol Sci. 2015;282:1821.View ArticleGoogle Scholar
- Sarkis C, Philippe S, et al. Non-integrating lentiviral vectors. Curr Gene Ther. 2008;8(6):430–7.View ArticlePubMedGoogle Scholar
- Yang X, Boehm JS, et al. A public genome-scale lentiviral expression library of human ORFs. Nat Methods. 2011;8(8):659–61.View ArticlePubMedPubMed CentralGoogle Scholar
- Stegmeier F, Hu G, et al. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl AcadSci U S. 2005;A102(37):13212–7.View ArticleGoogle Scholar
- Scherr M, Venturini L, et al. Lentivirus-mediated antagomir expression for specific inhibition of miRNA function. Nucleic Acids Res. 2007;35(22):e149.View ArticlePubMedPubMed CentralGoogle Scholar
- Maus MV, Grupp SA, et al. Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood. 2014;123(17):2625–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Lipowska-Bhalla G, Gilham DE, et al. Targeted immunotherapy of cancer with CAR T cells: achievements and challenges. Cancer ImmunolImmunother. 2012;61(7):953–62.View ArticleGoogle Scholar
- Montini E, Cesana D, et al. Nat Biotechnol. 2006;24(6):687–96.View ArticlePubMedGoogle Scholar
- Kulkosky J, Jones KS, et al. Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol. 1992;12(5):2331–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Saenz DT, Loewen N, et al. Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants. J Virol. 2004;78(6):2906–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Philippe S, Sarkis C, et al. Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo. Proc Natl AcadSci U S. 2006;A103(47):17684–9.View ArticleGoogle Scholar
- Mendenhall A, Lesnik J, Mukherjee C, Antes T, Sengupta R. Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles. J Vis Exp. 2012;62:e3171.PubMedGoogle Scholar
- Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.View ArticlePubMedGoogle Scholar
- Uhde-Stone C, Huang J, et al. A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors. Biol Proced Online. 2012;14(1):8.View ArticlePubMedPubMed CentralGoogle Scholar
- Uhde-Stone C, Sarkar N, et al. A TALEN-based strategy for efficient bi-allelic miRNA ablation in human cells. RNA. 2014;20(6):948–55.View ArticlePubMedPubMed CentralGoogle Scholar
- Holkers M, Maggio I, et al. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res. 2013;41(5):e63.View ArticlePubMedGoogle Scholar