Lentiviral gene transfer into human and murine hematopoietic stem cells: size matters
© The Author(s) 2016
Received: 19 February 2016
Accepted: 3 June 2016
Published: 16 June 2016
Contemporary biomedical research increasingly depends on techniques to induce or to inhibit expression of genes in hematopoietic stem cells (HSCs) or other primary cells to assess their roles on cellular processes including differentiation, apoptosis and migration. Surprisingly little information is available to optimize lentiviral transduction of HSCs. We have therefore carefully optimized transduction of murine and human HSCs by optimizing vector design, serum-free virus production and virus quantitation. We conclude that the viral RNA length, even in relatively small vectors, is an important factor affecting the lentiviral gene transfer on the level of both the virus production and the cellular transduction efficiency. Efficient transfer of large gene sequences into difficult-to-transduce primary cells will benefit from reducing the lentiviral construct size.
Human immunodeficiency virus-based lentiviral gene transfer has been embraced in contemporary laboratory practice as an efficient procedure to shuttle gene-encoding RNA molecules into target cells, where they are reverse-transcribed and integrated into the host genome. Third-generation lentiviral vector systems have proven to be safe methods in gene therapy with very low risks of ongoing integrations in the host genome or generation of replication-competent viral particles [1, 2]. Lentiviruses infect both dividing and non-dividing cells, making them ideally suited to transduce human and murine hematopoietic stem cells (HSCs) [3–6]. Many fields of research often require lentiviral constructs that drive gene expression from a promoter as well as a fluorescent reporter expressed from an internal ribosomal entry site or secondary promoter. The virus production (tested for vectors that encode viral RNA ranging from 4 to 7.5 kb in length)  and efficiency to transduce adherent cell lines seems dependent on the size of the lentiviral vector that encodes for the viral RNA. Vectors with viral RNA ranging from 5 to 9 kb generally tested decent, whereas those ranging from 10 to 18 kb transduced very poorly . However, the nature of the target cell (cell line or primary cells) is crucial and most of the studies published to date have not addressed the transduction efficiency of primary cells. Additionally, it is important to control the production process of lentiviral particles (in HEK293T cells), and the quantitation method to determine the number of competent viral particles produced. Since little information is available regarding these variables and their effects on lentiviral transduction, we carefully optimized lentiviral transfer by optimizing vector design, serum-free virus production and quantitation, as well as by optimizing transduction of murine and human HSCs.
The human T cell leukemia cell line JURKAT (DSMZ, #ACC-282) identity was confirmed by DNA fingerprinting and cells were regularly tested for mycoplasma contamination.
Virus production, concentration and quantification
We optimized HEK293T transfection in DMEM supplemented with 10 % serum using X-tremeGENE HP DNA Transfection Reagent (Roche, #06 366 236 001) to produce vesicular stomatitis virus-G pseudotyped virus particles without the addition of serum (low-serum Opti-MEM I with Glutamax: Life Technologies, #51985-026), in batches of 40 ml (harvest twice, with 24-h intervals, from two confluent 14-cm dishes starting 2 days after transfection, see Additional file 1). Low serum levels minimize the risk of premature differentiation of HSCs that are subjected to lentiviral transduction. Furthermore, fetal bovine serum can influence transduction , and we found that increasing amounts of serum (ranging from 0 to 10 %) negatively affects transduction rates of human HSCs, perhaps due to the aggregation of virus particles in the presence of serum proteins (data not shown). In relation to the quantitation of intact viral particles, the commonly used p24 protein ELISA method overestimates the number of functional viruses by the detection of incomplete, transduction-deficient viral particles as well as soluble p24 protein in the production medium . Quantitative RT-PCR of encapsulated viral RNA particles is a valuable alternative. We therefore calculate the number of viral particles that we produce by quantification of viral RNA copies using RT-qPCR (two RNA copies per viral particle, see Additional file 1). RT-qPCR is performed using primers flanking the cPPT region: 5′-AGGTGGAGAGAGAGACAGAGAC-3′ and 5′-CTCTGCTGTCCCTGTAATAAAC-3′.
Human CD34+ HSC transduction
Human CD34+ HSCs were positively selected from umbilical cordblood (Miltenyi Biotec, #130-100-453) and stimulated for 16-20 h at a concentration of 1x106 cells/ml in X-VIVO 10 (Lonza, #BE04-743Q) supplemented with 50 ng/ml rhSCF (R&D, #255-SC), 20 ng/ml rhTPO (R&D, #288-TP/CF) and 50 ng/ml rhFlt3L (Miltenyi Biotec, #130-093-855). Prior to transduction, add protamine sulfate (Sigma, #P4020-1G) to the cells to a final concentration of 4 µg/ml and pipet the concentrated virus (IVSS VIVASPIN 20 centrifugation concentration columns, Sartorius AG, Sigma-Aldrich, #Z614653-48EA) into a 50 µg/ml retronectin (r-Fibronectin CH-296: TaKaRa, #T100A)-coated 96-well plate (Falcon, #351172). Add HSCs on top of the virus to a final volume of 200 µl/well and mix by gently tapping the plate. Spinoculation: centrifuge the cells in the virus-containing medium at 1800 rpm, 32 °C for 1 h. Incubate the transduced cells at 37 °C, 5 % CO2 for 24 h before further use.
We conclude that while larger viral RNA size negatively affects both virus production and transduction of target cells, other factors can also influence the transduction efficiency (e.g. sequence). This is evident from the observation that the 5750 bp vector (containing the 1535 bp gene) revealed lower transduction efficiency than the slightly larger triple BFP vector (5995 bp). The transduction efficiency of human or mouse stem cells decreases tremendously for viruses with viral RNAs approaching 6 kb or larger. Lentiviral vectors encoding smaller viral RNA sequences perform better and even a reduction of merely 600 bp (5750 versus 5190 bp) already improves transduction efficiency by more than threefold (Fig. 3). To produce more efficient lentiviruses, reducing the viral RNA backbone size by removal of non-essential sequences may be effective. Codon optimization alters the gene sequence without affecting the protein sequence and may also increase transduction efficiency. Additionally, the development of smaller reporter genes or complete removal of the reporter may further enhance the transduction efficiency. In the absence of a fluorescent reporter, integrated lentiviral constructs into the host genome or expression of lentiviral transgene mRNA can be quantified by qPCR [13, 14] or RT-qPCR , respectively. In conclusion, size reduction of lentiviral constructs will facilitate efficient transfer of large gene sequences into difficult-to-transduce primary cells and will be most helpful in many fields of (basic) research.
KCB, RDM, WKS and YMHW performed experiments and/or analyzed data; KCB and JPPM designed study and wrote manuscript; RP and JPPM supervised the study. All authors read and approved the final manuscript.
We thank Karin Pike-Overzet for helpful discussions. This work was supported by the Children Cancer Free Foundation (Stichting Kinderen Kankervrij); Grants KiKa-2008-29 (RDM, WKS and KC-B), KiKa-2013-116 (KC-B) and KiKa-2014-141 (YMH-W).
The authors declare that they have no competing interests.
Ethics approval and consent to participate
The use of human umbilical cord materials from healthy individuals was approved by the Medical Ethical Review Board of the Erasmus MC Rotterdam (MEC-2009-430) and in accordance with the Declaration of Helsinki. C57Bl/6 mice were housed under specific pathogen free conditions at the animal facility of Erasmus MC according to institutional guidelines. The use of murine bone marrow for the experiments has been approved by the Erasmus MC committee for animal welfare (DEC #103-12-02) and is in compliance with Dutch legislation.
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