Identification of unique reciprocal and non reciprocal cross packaging relationships between HIV-1, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral vector system with highly favourable features for in vivo testing and clinical usage
Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells.
HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors.
This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.
KeywordsViral Vector Gene Transfer Efficiency Mixed Glial Culture Gene Transfer Vector Major Splice Donor
Viral vectors based on primate and non-primate lentiviruses have been shown to be efficient for gene delivery to a variety of cell types both in vitro and in vivo and may offer considerable advantages in gene therapy strategies [1, 2]. Lentiviral vectors can provide stable gene expression following integration into the host chromosome and pseudotyping of these vectors with heterologous envelopes such as the G protein of Vesicular stomatitis virus (VSV) has provided a broad cell tropism . Lentiviral vectors are particularly suited for transduction of non-dividing cells  such as those of the central nervous system  exemplified by successful therapeutic gene transfer to the brain of primates for treatment of experimentally induced Parkinson's disease . Packaging of unspliced vector mRNA in the producer cell line is a key part in process of lentiviral vector production and measures to increase the packaging efficiency and to reduce self packaging of the Gag-Pol or other helper construct have contributed to increased vector titre and biosafety . Lentiviral RNA packaging is achieved by an interaction between an RNA structure known as the packaging signal or psi and the nucleocapsid (NC) domain of the Gag structural polyprotein. This highly specific process results in the selection of unspliced viral mRNA from a high background of cellular mRNA. The packaging signals of several lentiviruses have been mapped by deletion and mutational analysis. For HIV-1, sequences between the major splice donor and the start codon of Gag have been shown to be important for efficient packaging . HIV-1 may be the exception amongst lentiviruses since for HIV-2 and SIV, sequences upstream of the splice donor predominantly contribute to mRNA packaging [9, 10] and in FIV regions in U5 and in the Gag coding sequence appear to be the major signals [11, 12]. RNA packaging in HIV-2 has been shown to involve two novel mechanisms to increase specificity, cotranslational packaging and competition for limiting Gag polyprotein . These differences in the location of the major packaging determinants may contribute to the ability of viral mRNA to be cross packaged by a heterologous Gag protein. The localisation of RNA capture in the cell is unclear although recent evidence suggests that the centrosome may be the primary site  and that the psi signal may act as a subcellular localisatio signal as well as a high affinity binding site for Gag. The resulting RNA-protein complex is then targeted to the plasma membrane where virion budding takes place.
The ability of one lentiviral Gag to cross-package the unspliced mRNA of another lentivirus species has been well demonstrated for HIV-1, which can cross-package HIV-2 , SIV [16, 17] and FIV . Both SIV and FIV Gag-Pol have been shown to cross-package HIV-1 mRNA [16, 18], however HIV-2 Gag-Pol is unable to package HIV-1 mRNA . How closely this reduced efficiency correlates with the effectiveness of gene transfer of cross-packaged vectors has not been assessed, in particular in appropriate primary cells. Cross-packaged lentiviral vectors have been shown to infect predominantly dividing cells in culture but transduction of neurons and CD34+ lymphocytes has only been shown qualitatively . However chimeric vectors based on an SIV genome and an HIV-1 core were unable to transduce dendritic cells and had a reduced ability to transduce primary macrophages .
The production of lentiviral vectors for clinical trials requires that preparations do not contain replication competent lentiviruses (RCL). Development of PCR and sensitive culture based methods for detection of RCLs have confirmed the absence of RCLs in large production lots [20, 21]. Production of RCLs can occur through homologous recombination, thus limiting the sequence similarity between the Gag-Pol construct and gene transfer vector will reduce the possibility of a recombination event. Gag-Pol and gene transfer vectors based on different lentiviruses will significantly reduce the risk of RCL production.
Transduction of the cells of the central nervous system (CNS), both brain and spinal cord, with lentiviral vectors has been well documented and long term therapeutic transgene expression has been reported with only a low level or transient immune/inflammatory response [22, 23]. Furthermore, transduction of neural stem cells with lentiviral and adeno associated viral vectors expressing therapeutic genes that will affect differentiation and serve as markers of cell fate is a promising approach for procuring cells for transplantation into degenerated or damaged areas of the brain. Such cells have the potential to be useful for the treatment of Parkinson's disease, spinal cord injury and other inflammatory or destructive conditions of the CNS[24, 25].
We investigated the cross packaging ability of the Gag-Pol components of HIV-1, HIV-2 and SIV and found a unique non-reciprocal packaging relationship between SIV Gag-pol and vectors based on HIV-2.
In this paper the tropism of these viruses is quantitated by examining the ability of a series of cross-packaged lentiviral vectors based on HIV-1, HIV-2 and SIV to transduce primary mixed glial cells which, are the predominant cell type in the injured brain or spinal cord. Qualitative data is also presented on the transduction of primary neuronal embryonic stem cells with cross-packaged vectors.
Cross-Packaging of lentiviral RNA
Gene transfer efficiency of cross packaged vectors
Transduction of CNS cell types
Both lentiviruses and other retroviruses have shown an ability to cross package other viral genomes with HIV-1 Gag-Pol demonstrating the greatest cross packaging ability. Non-reciprocal packaging relationships such as have been demonstrated in HIV-1 and HIV-2  or spleen necrosis virus and murine leukaemia virus  suggest that individual viruses have different packaging mechanisms relating possibly to the availability of the Gag protein or the position of the RNA packaging signal relative to the major splice donor or other as yet unknown factors. In this study we demonstrate for the first time a non-reciprocal packaging relationship between SIV and HIV-2. Interestingly, the major packaging determinant of both HIV-2 and SIV has been shown to be upstream of the major splice donor [9, 10] and by inference one would expect SIV to demonstrate the same co-translational packaging process as HIV-2 . SIV Gag-Pol has been previously reported to cross package HIV-1 and FIV unspliced vector mRNA [16, 7, 18] however the gene transfer ability of these chimeric vectors has been limited. We could not demonstrate any appreciable gene transfer of an HIV-1 based vector cross-packaged by SIV Gag-Pol, which is in contrast to a previous study , where transduction of both dividing and non-dividing cells was demonstrated. Nor was gene transfer of the HIV-1 GFP seen when packaged by HIV-2 Gag-Pol, in contrast to a previous report .
One advantage of a chimeric lentiviral vector is a reduction in the risk of development of a replication competent retrovirus which may occur through a recombination event due to sequence homology between the Gag-Pol and gene transfer constructs. However it is important to assess the gene transfer capabilities of these chimeric vectors in suitable primary cells. This has been highlighted in a study where a gene transfer vector based on SIV packaged by HIV-1 Gag-Pol showed a reduced transduction efficiency of human dendritic cells associated with a post-entry defect. . A second major advantage of this chimeric system is the ability to deliver a cross-packaged vector to a simian animal model with a vector based on SIV Gag-Pol packaging an HIV-2 genome. The same combination could subsequently be used in humans allowing biosafety and bio-distribution studies to be performed directly without the necessity for surrogate systems. This is not possible with an HIV-1 based system and would give the SIV/HIV-2 system considerable advantages over other primate lentiviral combinations.
Rat astrocytes are the major cell type associated with the glial scar resulting from injury to the CNS  and human fetal embryonic neural stem cells offer the potential for regenerating damaged areas of the CNS . Engraftment of neural stem cells transduced with a lentiviral vector based on HIV-1 has been demonstrated with a high level and duration of transgene expression. Our results demonstrate that both the HIV-1 and HIV-2 homologous GFP lentivectors efficiently transduced rat primary astrocytes. Similar to previous studies on the effect of the cPPT sequence on gene transfer [30, 31] our data shows that the presence of the cPPT sequence in the HIV-1 vector results in a two fold increase in transduction efficiency, similar to the HIV-2 homologous vector system which also contains the HIV-2 cPPT in the pol sequence. The SIV Gag-Pol/ HIV-2 GFP vector also transduced primary astrocytes with efficiency similar to the HIV-1 cPPT homologous vector system, indicating no associated post-entry defects. Efficient transduction of human fetal embryonic neural stem cells was also shown with the cross packaged SIV Gag-Pol/HIV-2 GFP vector highlighting the ability of this vector to transduce human cells.
We have identified a non reciprocal cross packaging relationship between SIV Gag-Pol and a HIV-2 based GFP vector, which demonstrated equivalent transduction efficiencies in 293T cells, rat primary astrocytes and embryonic stem cells as that of homologous HIV-1 and HIV-2 vector systems. The efficiency of the combination correlates with the level of vector RNA packaged indicating that this is a major determinant of vector efficiency. It suggests that there are as yet unidentified differences in the RNA capture mechanisms of HIV-1, HIV-2 and SIV.
The implications for testing of lentiviral vector biosafety are potentially very important. Testing in appropriate animal models is a major concern associated with the use of lentiviral vectors in clinical trials. As HIV-1 only causes AIDS in humans, there is presently no animal model to test the safety of HIV-1 based vectors. However animal models based on Asian macaques and baboons exist for SIV and HIV-2, respectively which may be applicable to testing the biosafety of SIV cross packaged HIV-2 lentiviral vectors.
Construction of minimal HIV-2 based vectors
pSVRΔNBΔH  was digested with BsmBI, and a ClaI linker was inserted into the site. ClaI and EcoRV digestion of this produced two DNA fragments, the smaller of which (nucleotides 1101–6128 encompassing gag and pol sequences) was discarded. The remaining fragment was religated and formed pSVRΔGP. CMVGFP was obtained from SalI digestion of pSVRΔ-CMVGFP and ligated into the SalI linker of dephosphorylated pSVRΔGP to give pSVRΔGP-CMVGFP.
The HIV-2 U3 region contains a TATA box, core enhancer regions, and Sp1, κB and peri-κB binding sites that are responsible for transcription from the 5'LTR. This 141 bp region (nucleotides 9329–9470) was deleted in the 3'LTR to produce a SIN vector as follows. The 3'LTR was removed from pSVRΔGP, by BamHI and XbaI digestion, and subcloned into pBluescript II KS. Site directed mutagenesis introduced BglII restriction sites at the 5' and 3' ends of the 141 bp region that was to be deleted. Mutagenesis was carried out as in two stages using the following primers: stage 1, upstream mutagenesis:- 5'-GGAATACCATTTAGTTAAAGATCTGAACAGCTATACTTGGTCAGGG-3' and :- 5'-CCCTGA CCAAGTATAGCTGTTCAGATCTTTAACTAAATGGTATTCC-3'; for stage 2, downstream mutagenesis, 5'-CGCCCTCATATTCTCTGTATAGATCTACCCGCTAGCTTGCATTG-3' and 5'-CAATGCAAGCTAGCGGGTAGATCTATACAGAGAATATGAGGGCG-3'.
The 141 bp U3 region was removed from the plasmid by BglII digestion and the plasmid religated. BamHI and XbaI digestion of the plasmid and religation of the Δ3'LTR into pSVRΔGP created the pSVRΔSIN vector. CMVGFP was inserted as described to produce the vector pSVRΔSIN-GFP
Lentiviral vector production
Lentiviral vectors were produced by calcium phosphate transfection of 293T cells grown in DMEM media and 10% FCS with 7 μg of the gene transfer vector, 7 μg of the Gag-Pol construct, 3 μg of a Rev expressor and 3 μg of the VSV-G heterologous envelope. For HIV-2 and SIV vector production the Rev expressor was omitted. 24 hours following transfection the media was replaced and supernatant containing recombinant virions was recovered 48 hours post transfection. Virions were concentrated by ultracentrifugation for 2.5 hours at 25,000 RPM in an SW28 Beckmann rotor. The viral pellet was resuspended in 300 μl of tissue culture media, aliquoted and stored at -70°C.
Lentiviral vectors were quantitated using a commercially available RT-assay (Cavid Tech, Uppsala) Vector preparations were measured in duplicate and normalised to a concentration of 8 ng of RT per μl. Although the sensitivity of the assay for different RTs may be slightly different the fact that each Gag-Pol construct is being used to package each vector provides an internal control.
Levels of RNA packaging were assessed by RT-PCR of Virion associated RNA. Virion RNA was extracted using the Qiagen Viramp kit from 10 ng of virus (RT levels). Following extraction the RNA was also treated with RNase Free DNase for 10 mins at 37°C and the DNase was in activated by incubation at 70°C for a further 10 mins. An aliquot of RNA was reverse transcribed to cDNA using the Promega Improm RT system with an antisense GFP primer (AAGTCGTGCTGCTTCATGTG). The cDNA was then serially diluted and amplified using a sense primer (GACGTAAACGGCCACAAGTT) and the antisense primer. Amplified products were resolved by agarose gel electrophoresis and EtBR staining.
The transduction efficiency of cross-packaged vectors was assessed by FACS analysis of GFP positive cells. A range of viral vector concentration from 40 ng to 4 ng was used to transduce 1 × 106 of fibroblast SV2C cells in a six well plate. Viral vector was diluted in DMEM containing 6 μg/ml polybrene and cells were exposed to virus for 5 hours. The media was then replaced and GFP expression was assessed at time periods after 72 hours post transduction.
Glial cell Culture and Stem cell culture
Primary mixed glial cultures were prepared from the brains of newborn rats > 3 days old by dissociation of whole cortex in trypsin, then cultured in poly-D-lysine coated flasks in DMEM/10% FCS. Mixed glial cultures were derived from these cells, once they were confluent, by trypsinisation. The cells were then resuspended in DMEM containing 10% FCS and 1% PSF and centrifuged at 10,000 RPM for 5 minutes. The supernatant was removed and cells were resuspended in DMEM/10% FCS and plated onto Poly-D-Lysine coated coverslips in 24 well plates. Transduction of glial cultures with lentiviral vectors was carried out as described for SV2C cultures. 72 hours post transduction; glial cultures were fixed in 4% paraformaldehyde and stored in PBS at 4°C prior to immunostaining.
Human fetal neuronal stem cell culture was performed as described previously . Transduction of Stem cell cultures of cortical origin was performed with 20 ng of viral vector in DMEM/ HAMS F12 (2:1), 1% N2, EGF (20 ng/ml) FGF-2 (20 ng/ml) and heparin (5 mg/ml) for four hours followed by replacement of the media. Cells were allowed to differentiate on poly-L-lysine/laminin coated coverslips followed by replacement of the media 72 hours post transduction. Cells were fixed in 4% paraformaldehyde after a further 96 hours, followed by immunostaining for GFP, GFAP and β-Tubulin III.
Lentiviral vector transduced mixed glial cultures were first blocked using 3% goat serum in TXTBS (0.2% triton X-100, in Tris Buffered Saline) for one hour. Monoclonal anti GFAP (Sigma, 1:500) and polyclonal goat anti rabbit GFP (Molecular Probes), 1: 1000) were diluted in TXTBS with 1% normal goat serum (NGS) for 2 hours. Cells were then washed in TBS for 3 × 10 minutes. Cells were then incubated with secondary antibodies, goat anti mouse Alexa (Molecular Probes, 1:500) and biotinylated goat anti rabbit (Amersham Biosciences, 1:500) for 90 minutes. Following a second 3 × 10 minute wash in TBS, Streptavidin-FITC (Serotec, 1:100) was added in TBS with 1% NGS and Bis-benzamide (Sigma, 1:5000). Coverslips were then mounted in Fluorosave reagent (Calbiochem). Cell counts of immunostained mixed glial cultures were performed from one edge of the coverslip all the way across to the other, horizontally and vertically. A 0.5 mm2 area was counted every 1.5 mm.
This work was supported by a programme grant from the Medical Research Council (UK)
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