Regenerative Research 3(1) 2014 1-7
TRANSGENE EXPRESSION IS TRANSIENT IN NON-INTEGRATING LENTIVIRAL-BASED TRANSDUCTION SYSTEM: AN ALTERNATIVE APPROACH FOR SAFETY GENE
THERAPY APPLICATION
Fazlina Nordin1,2, Noralisa Abdul Karim2, S Fadilah A Wahid2
1Immunology Group, Department of Molecular Medicine, King’s College London, UK, The Rayne Institute, London, UK
2Cell Therapy Centre (CTC), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Kuala Lumpur, Malaysia
1.0 Introduction
The development of vectors that can effectively mediate transfer of nucleic acids into target cells has facilitated both gene and cell-based therapies techniques as a new approach in the treatment of diseases. It is clear that a profound
understanding of the mechanisms employed by the vectors is necessary to achieve efficient gene delivery and expression without causing toxicities [1].
Viruses have evolved to efficiently transfer their genome to the host cell and these viral vectors have provided more Gene therapy
Integrase enzymes Lentiviral vector Transduction Transgene expression Spleen focus-forming virus
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E Y W O R D SPublished online: 5th June, 2014
*Corresponding Author:
Fazlina Nordin Email:
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Lentiviral vectors have been extensively analysed and used for clinical gene therapy application. The transduction efficiency is attributable by integration into the target cell genome which carries the risk of insertional mutagenesis, thus can lead to malignant transformation. Non-integrating lentiviral vector (NILV) is produced through mutation in the integrase enzymes to interrupt viral RNA integration in the host genome. However, the transgene expression remains episomal which minimized the genomic alteration.
The aim of this study is to investigate the efficiency of the NILV in expressing the transgene in transduced cells.
The expression of the transgene was driven by the spleen focus-forming virus (SFFV).
Integrase-proficient SFFV-GFP (wild-type) and NILV-SFFV-GFP were produced and the viral titres were determined by qRT-PCR. U937 cells were transduced with both NILVs and their wild type counterparts at multiplicity of infection (MOI) 5 and 10. GFP expression was determined by fluorescence microscopy and FACS analysis.
Based on qRT-PCR analysis, the NILV-GFP titres obtained were lower than the wild-type virus (62-fold reduction). NILV transduced target cells with at least 50% lower efficiency than wild-type virus. Although GFP expressing cells significantly reduced over time as the NILV-transduced cells proliferated, the expression was sustained albeit at a continuously decreasing level, for about 11 days.
These data confirmed that transgene expression in the NILV-transduced cells is transient in dividing cells, thus can be an alternative transgene expression for safer clinical applications.
promising system of gene delivery with various advantages over physical and chemical methods [2, 3]. Lentiviruses, a genus of retroviruses which include HIV, have been extensively analysed and used for clinical gene therapy applications [4, 5, 6, 7, 8]. Lentiviral vector constructs have proven to be very productive in terms of transduction due to their ability to infect both dividing and non-dividing cells, including stem cells [9].
Furthermore, lentiviral vectors have several advantageous properties over other viral vectors due to the larger packaging capacity [10, 11], the ability to transfect different cell types including quiescent cells, reduced immunogenicity upon in vivo administration [12], stable gene expression and similar transduction efficiency to adeno-associated virus vectors [13].
Despite its many advantageous, the potential clinical application of lentiviral vectors is limited by the integration of viral transgenes into the host genome that can increase risk of tumorigenicity [14]. This integration property carries the risk of provirus-mediated insertional mutagenesis leading to disruption of host gene expression and malignant transformations of cells which was observed in a clinical trial [15].
Therefore, the non-integrating lentiviral vectors (NILVs) have been developed to overcome these problems. NILVs can be produced by interrupting the function of virus encoded enzyme integrase (IN) through point mutation, which normally mediates its integration into host genomes [16]. IN mutations can be divided into two classes. Class I mutations result in the vector being defective specifically for integration whereas Class II mutations result in reverse transcription defects or impair multiple stages of the viral life cycles [17].
Most studies favour Class I over Class II mutations because the pleotropic effects (where a single gene influences multiple phenotype traits) make Class II mutations unsuitable for vector transgene expression. Moreover, the amount of viral DNA produced is not affected in Class I mutation [18]. IN consists of three functional protein domains: (i) N-terminal domain, (ii) the catalytic core domain, and (iii) the C-terminal domain. Introduction of point mutation into IN at position D64 within the catalytic core domain are commonly used to inactivate the IN protein functions [19, 20, 21].
NILVs have shown to be capable of efficient gene expression without viral integration in cell cultures [22, 23]. An efficient gene expression from NILVs has been demonstrated in the spinal cord, whereby, it is also resulted in efficient RNA interference in dorsal root ganglia neuron culture [24].
Another study has shown that the NILVs gene transfer is stable in dividing cells and also prolonged expression in non- dividing cells of myotubes [25]. NILVs still maintain the positive attributes associated with the integrating lentiviral gene transfer except that the non-integrating lentiviral DNA
accumulates in non-dividing cells [22]. Therefore, NILVs is useful in achieving transient transgene expression in dividing cell without stable integration into host genome, thus could be a useful alternative for safer therapeutic applications.
In this study, we investigated the efficiency of the NILV in expressing the transgene in transduced cells. The expression of the transgene was driven by the spleen focus-forming virus (SFFV). Integrase-proficient SFFV-GFP (wild-type) and NILV-SFFV-GFP were produced and the expression of GFP was determined by infecting U937 cells. These studies were undertaken as a prelude to the future potential use of NILV for the expansion of haematopoietic stem cells (HSCs) without their permanent genetic modification.
2.0 Materials and Methods
2.1 Plasmids
GFP cDNA was cloned into Rous Sarcoma lentivirus (RSV) vector backbone which contains two promoter sites; RSV promoter located upstream of the HIV-1 Rev Response Element (RRE) site and Spleen-focus forming virus (SFFV) located downstream of the RRE site. SFFV promoter drove the expression of the transgene, which is located at 5’LTR of the transgene sequences (Figure 1).
A four-plasmid transfection system was used to produce the vectors which include the vector plasmid, MDG, Rev and MDLg/pRRE (wild type) or IN-MDLg/pRRE (NILV). IN- MDLg/pRRE plasmid has a mutation at D64V site (catalytic core domain region), a Class I IN mutation that abolishing the integrase activity, thus was used to produce NILV encoding GFP.
2.2 Cell lines
Human embryo kidney (HEK) 293T cell lines were cultured in DMEM (Dulbecos’sModified Eagle Medium) (Sigma Aldrich) supplemented with 10% (v/v) heat-inactivated FCS (feotal calf serum) (PAA, Laboratories), 100 μg/mL penicillin-streptomycin (Sigma Aldrich). 293T cells were used as producer cells for effective production of lentiviral particles. Cells were grown in 37ºC incubators in a 5% CO2 atmosphere. Human monocyte (U937) cell lines were cultured
Fig.1 Schematic representation of the RLSV/SFFV-GFP lentiviral construct.
Full length GFP were cloned into BamHI and XhoI sites of the RRL expression vector, in-frame between spleen focus forming viral (SFFV) promoter and the WPRE sequence.
in RPMI-1640 (Roswell’s Park Memorial Institute-1640) (Sigma Aldrich) with the same supplements as mentioned for 293T cell lines. U937 cell lines are used for viral transduction to determine the virus titre for subsequent experiments.
2.3 Virus production
All lentiviruses that were used in this study were produced using the 293T cell line. A total of 4 million cells in 80 mL of complete media (DMEM supplemented with 10% heat in activated FBS (v/v) and 100 μg/mL penicillin-streptomycin) were seeded in 75cm2 culture flasks. A standard calcium phosphate (Ca-PO4) co-precipitation transfection protocol was used as describe elsewhere [26]. The transfection was carried out the following day with 1 ml of DNA Ca-PO4 co- precipitation mixture containing 20 μg of plasmid DNA per culture dish, 0.5 M CaCl2 and 2XHEBS (HEPES Buffered Saline) at pH 6.7. The transfection medium was left to stand for 30 minutes at room temperature to form a fine opalescent precipitation. The DNA Ca-PO4 co-precipitation mixture was added dropwise to the surface of the media containing the cells (50-60% confluent by the time of transfection). The culture dish was gently swirled to mix the media evenly and incubated at 37ºC with 5% CO2 for 72 hours.
Fresh complete media (pre-warmed at 37°C) was replaced 24 hours post-transfection. The culture medium containing the virus particles was harvested 48 hours post-transfection by centrifugation at 300 x g for 10 minutes at 4°C followed by filtration through 0.45 μM syringe filter.
2.4 Determination of viral titres
The filtered culture medium containing the lentiviral vectors was further concentrated by centrifugation at 3900 x g overnight at 4°C. The pellet (which contains the lentiviral vectors) was re-dissolved in 1mL of serum-free media (X- Vivo 15), and aliquoted in 0.2 mL tubes. The lentiviral vectors were then kept in -80°C and the titre was determined by qRT-PCR.
The isolation of viral RNA was performed according to manufacturer’s protocols (Macherey-Nagel). The RNA pellet was eluted in RNase-free H2O, whereby the final concentration ranged between 55-90 μg and kept at -80°C.
qRT-PCR was performed according to manufacturer’s protocols (Clontech,US) using Lenti-X™ qRT-PCR Titration Kit. qRT-PCR amplification of lentiviral genomic RNA with SYBR probe was performed in duplicate with 4 different dilutions (1x, 0.1x, 0.01x, and 0.001x) for each vector, including the control template (which served as a standard).
2.5 Evaluation of GFP expressing cells by FACS Analysis
A total of 200,000 U937 cells per mL were infected with different volumes of concentrated virus in 10 μg/mL DAEA- Dextran added prior to infection. The viral titre was determined 3 days post-infection by analysis of cultures with 5-20% GFP expressing cells. This experiment was performed in triplicate for statistical analysis.
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2.6 Transduction of U937 cells
A total of 100,000 U937 cells per 2 mL were seeded in 6-well plate. The cells were incubated overnight and replaced with fresh media the next day. 10 μg/mL of DAEA-Dextran was added in each culture to induce infectivity. Lentivirals encoding GFP gene were added at MOI 1, 5, and 10 and
Fig.2 Determination of viral titre for non-integrating lentivirus (NILV) by qRT-PCR. Data shown are the amplification plots for lentiviral genomic RNA of (A) Lenti-X RNA control template (standard) provided in the kits, (B) SFFV-GFP (Wild-type), and (C) NILV-SFFV-GFP.
incubated for 48 hours. The transduced target cells were then analyzed by FACS.
3.0 Results
3.1 The titre of NILV lentiviral vectors were lower than titres in the wild-type
Based on qRT-PCR analysis, vector concentration was 8.7 x 109 copies/mL, and 1.4 x 108 copies/mL for SFFV-GFP (wild- type), and NILV-SFFV-GFP, respectively (Figure 2). There was a 62-fold reduction in viral titre (copies/mL) in NILV- SFFV-GFP as compared to the wild-type. Statistical analysis showed no significant difference in viral titres between NILV, and wild-type virus (p>0.05).
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3.2 Transduction Efficiency between Wild-type and Non- integrating LV
NILV-SFFV-GFP and SFFV-GFP (wild-type) transduced U937 cells were examined on day 7 post-transduction by fluorescence microscopy for GFP expressing cells (Figure 3).
Less than 5% of NILV-SFFV-GFP transduced U937 cells were GFP positive (Figure 3A). In contrast, the majority of the SFFV-GFP transduced U937 cells were GFP positive (Figure 3B).
3.3 Transgene expression from the NILVs is transient in dividing cells
The percentage of GFP expressing cells in U937 was further investigated at 24 hour intervals by FACS (Figure 4). Two separate experiments were performed at MOI 5, and 10. In experiment 1, GFP expression was higher in the SFFV-GFP transduced U937 cells (~100%) as compared to NILV-SFFV- GFP transduced U937 cells throughout the investigation until day 11 post-transduction when the experiment was terminated (Figure 5). The GFP expression in NILV-SFFV-GFP transduced cells was at highest level (~75%) at day 2 post- transduction, but consistently decreased to less than 5% by day 11 after transduction (Figure 5A). The mean fluorescence intensity (MFI) was also greater in SFFV-GFP transduced cells compared to NILV-SFFV-GFP transduced cells (Figure 5B).
A similar result was obtained in Experiment 2, whereby the MOI was increased to 10. Almost 100% of the SFFV-GFP transduced U937 cells were GFP positive, while the GFP expressing cells consistently decreased from ~70% on day 2 to less than 5% by day 10, when the experiment was terminated (Figure 5C). The MFI value was also greater in the SFFV-GFP transduced cells compared to NILV-SFFV-GFP transduced cells (Figure 5D).
4.0 Discussion
The potential modification of the cellular genome as a result of using integrating viruses during reprogramming remains one of the major obstacles. NILVs which were produced through IN mutation within the catalytic core domain are failed to integrate into host genome due to defective in IN protein functions [21]. It has been reported that IN mutations do result in reduced transduction efficiency in NILV transduced cells but do not affect viral titres [13]. In line with this finding, the NILV titre showed no significant difference to their wild-type counterpart.
The ability of the NILVs to transduce cells was further investigated and we found that NILV transduced target cells with at least 50% lower efficiency than wild-type virus.
Fig.3 GFP expression in NILV-SFFV-GFP and SFFV-GFP transduced U937 cells on day 7 (MOI 5). (A) <5% of NILV-GFP transduced U937 cells showed green fluorescence indicating GFP expression. (B) Virtually all SFFV-GFP transduced U937 cells showed GFP expression
This result contradicts with previous reports, which demonstrated similar levels of positive cells expressing GFP, with similar levels of MFI in 293T cells [23, 27]. The latter study also demonstrated that by reducing the viral concentration the percentage of GFP positive expressing cells were also decreased in NILVs transduced 293T cells in comparison to the integrase-proficient vector [27].
Our data demonstrated that Class I integrase-deficient (IN), and in particular the D64 mutation resulted in low transduction efficiency, and reduced transgene expression throughout continuous culture in haematopoietic cells, compared to its wild-type counter-part. The percentage of GFP positive cells decreased from ~50% to almost 0 in NILVs transduced cells, while it remained consistently at about 100% in wild type transduced cells up to 11 days post- transduction when the experiment was terminated.
Similar results were reported elsewhere, where the GFP expression dropped quickly within the first week after transduction [27, 28]. In addition, a Class I IN mutation (D64V) was reported to result in a small drop of transgene expression when compared to a triple mutation affecting the entire catalytic core domain (D64V, D116A and E152A) [13].
These data confirmed that the transgene expression in the
NILVs transduced cells is transient in dividing cells, thereby implying that the vectors were indeed defective for integration. The progressive loss of the transgenes expression in dividing cells replicates the rapid dilution of the un- integrated provirus DNA. Since NILV vectors lack an origin of replication, the episomal provirus is not amplified during genomic DNA amplification [29]. Therefore, the number of provirus DNA molecules in each cell is reduced by half with every cell division. We also suggest that the reduced transgene expression with NILV-SFFV-GFP was not due to decreased viability of the cells or the reduced cellular uptake of the plasmid. This is because the transduced cells were increased in number as analysed by flow cytometer (data not shown), and the experiment has been repeated 3 times, independently.
Despite having reduced transgene expression, NILVs could be a useful alternative for therapeutic applications. NILVs still preserve some benefits of integrating LVs, but can achieve gene expression without viral integration, thus reducing the potentially detrimental risk of insertional mutagenesis.
However, more optimisations are needed to be performed before this system can be fully applied in gene therapy. For example, identifying which multiple integrase mutation sites will allow higher viral titre and minimise the potential
Fig. 4 FACS analysis of NILV-SFFV-GFP and SFFV-GFP transduced U937 cells on day 7 post-transduction (MOI 5). Percentage and MFI of GFP positive cells were taken from histogram as displayed in row ‘C’. Untransduced U937 cells were used as a negative control. MFI= Mean Fluorescence intensity.
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problem of reversion back to an integrating phenotype.
Moreover, identification of trans- and cis-acting factors for development of lentiviral vectors that are able to increase transduction and transgene expression efficiency in dividing cells would be beneficial for clinical applications. In addition, increased in vivo testing of NILVs should also be considered to assess the differences in physiological changes and the side effects that may occur after NILV administration of a single
vector dose. This is crucial to show the utility of these techniques before they can be applied clinically.
In conclusion, NILVs show promise for achieving transgene expression in target cells and will be a suitable choice for safer clinical applications. NILVs preserve the advantages of the wild type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.
Acknowledgement:
We are grateful to Ministry of Higher Education Malaysia (MOHE) and Universiti Kebangsaan Malaysia (UKM) for supporting this project. The work was carried out at Department of Molecular Medicine, The Rayne Institute, King’s College London, UK, and Cell Therapy Centre (CTC), UKM Medical Centre, Malaysia.
The authors declare no conflict of interest.
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