Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

Abstract

Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome-to-trans-Golgi-network trafficking. To explore the possibility of Rab9-related gene therapy for neurodegenerative diseases, we packed Lentivirus encoding Rab9. The expressing plasmid pCDH1-MCF1-Rab9-EF1-copGFP was constructed by using molecular biological techniques. The Lentivirus encoding Rab9 cDNA was packed by Lifectamine-2000 mediated co-transfection of the plasmid pPACKH1-GAG, pPACKH1-REV and pVSV-G into 293T cells. DNA sequencing proved the successful construction of pCDH1-MCF1-Rab9-EF1-copGFP. After 72 hours, the expression of GFP could be detected in BV-2 cells. Western blotting revealed that the Rab9 gene expression in BALB/c mice brain was up-regulated significantly 4 weeks after injection with Lentivirus encoding Rab9, which evidenced a satisfactory increasing effect of this virus. Administration of Lenti-Rab9 to postnatal day 3 Niemann-Pick disease type C (NPC) mice reduced motor defects and prevented the weight loss associated with female NPC mice, as well as modulating the death rate of Purkinje neurons. It is concluded that the packaging of Lentivirus encoding Rab9 was successful. Lentivirus encoding Rab9 can increase the expression of Rab9 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

Keywords

Rab9 / lentivirus / gene therapy / gene transfer

Introduction

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder of intracellular cholesterol trafficking with tragic consequences, which is of interest in its own right and is a “juvenile” model for Alzheimer disease [1, 2]. The majority of NPC cases arise from mutations in the NPC1 gene [3]. A characteristic of NPC is the massive accumulation of cholesterol and glycosphingolipids within late endosomes and lysosomes [4-6]. In normal cells, endocytosed low density lipoproteins are delivered to endosomes, where they are hydrolyzed and free cholesterol is released. This cholesterol is transported rapidly out of endosomes to the plasma membrane and endoplasmic reticulum [7, 8].

In NPC cells, the cholesterol does not exit the endocytic pathway and it accumulates within lysosomes [9]. So far, no effective therapy has been available. Studies have demonstrated that NPC1 human skin fibroblasts overexpressing endosomal Rab9 protein shows a correction in the storage disease phenotype [10, 11]. To learn more about the role of Rab9 protein in modulating lipid traffic and accumulation in NPC cells, and to develop the therapeutic potential of this system, an alternative method for conventional DNA transfection is required to introduce the Rab9 gene into living cells. The lentiviral vector mediated transgenic technique that developed rapidly in recent years has the advantages of easy-operation, high-rate integration, no selection in cell cycle of host cells compared with traditional microinjection [12-14]. In this study, lentivirus encoding Rab9 gene was generated and introduced into BALB/c mice brain to evaluate the efficacy of gene transfer and its function.

Materials and methods

Materials

The plasmid p-Rab9-EGFP was purchased from Addgene Inc (USA). The plasmid pCDH1-MCF1-EF1-copGFP and the 293T cell line were provided by KangChen Bio-tech Inc (China). T4 DNA ligase and the restriction enzymes of BamHI and NheI were products of New England Biolab (USA). DNA markers were bought from Beijing Tiangen Biochemical Technology Co. (China). The plasmid extract kit was from Qiagen (Germany). The primers were synthesized by Shanghai Bioengineering Co. (China). Fetal bovine serum (FBS) and high glucose DMEM (dulbecco minimum essential medium, DMEM) powder were procured from Hyclone Co., USA and Gibco Co., USA, respectively. Lifectamine-2000 was the product of Invitrogen Inc (USA). The pPACKH1 Packaging Plasmid Mix consists of an optimized mixture of three plasmids — pPACKH1-GAG, pPACKH1-REV and pVSV-G was from System Biosciences (SBI), USA. BALB/c mouse strain was obtained from the Jackson Laboratory (Bar Harbor, ME). Protease inhibitor cocktail was purchased from Sigma Co, USA. Rabbit-anti-mouse Rab9 and β-actin antibody were ordered from Santa Cruz Biotechnologies, USA, and Cell Signaling Co, USA, respectively.

Methods

Preparation of plasmid and Rab9 gene fragment

The Rab9 cDNA was obtained from plasmid p-Rab9-EGFP by polymerase chain reaction (PCR) amplification. The primers of Rab9 for complete CDS (coding sequence) were: 5'-TAGCTAGCGCCACCATGGCAGGAAAATCATCACTTTTTA-3' (sense) (cleavage site of NheI was added to 5') and 5'-TAGGATCCTCAACAGCAAGATGAGCTAGG-3' (anti-sense)(cleavage site of BamHI was added to 5'). The PCR product (678 bp) was analyzed by electrophoresis on a 1% agarose gel, and then extracted and purified according to the protocols of the gel extracted mini-kit.

Ligation of Rab9 fragment with shuttle vector

The Rab9 fragment was ligated with pMD 19-T vector at the NheI and BamHI site. The product was transformed into the competent cells DH5α. The plasmid prepared from the positive clone was digested by restriction enzymes NheI and BamHI and analyzed by electrophoresis on 1% agarose gel containing ethidium bromide (0.5 mmol/L). The extracted and purified Rab9 fragment and the linearized shuttle vector pCDH1-MCF1-EF1-copGFP (digested with NheI and BamHI) were ligated in a mole ratio of 1.5∶1 by T4 DNA ligase at 16°C for 16 h. The construction plasmid was amplified, analyzed and sequenced.

Generation of lentivirus

The packaging procedure was performed according to SBI’s User Manual. 293T cells were cultured in 10% high glucose Dulbecco’s modified Eagle’s medium (DMEM) at 37°C in an incubator with 5% CO₂. The growth status and morphological characteristics were observed under inverted phase contrast microscope. Four plasmids pCDH1-MCF1-Rab9-EF1-copGFP, pPACKH1-GAG, pPACKH1-REV and pVSV-G were co-transfected into 293T cells with approximately 60% confluence by using the Lipofectamine 2000^TM method. The (green fluorescent protein) GFP expression in the cells was observed under an inverted fluorescent microscope so as to confirm the success of co-transfection. After a 72 hour transfection, the medium was harvested and concentrated by centrifugation at 3000 r/min for 5 min at 15°C, and then filtrated with a 0.45 μm filter membrane. The filtrate was concentrated by centrifugation at 25 000 r/min for 2 hours. The supernatant was discarded. The precipitate was diluted with phosphate balanced solution PBS and stored at -80°C. The Lentivirus was named Lenti-Rab9. The final virus titer was determined by dot blot assay.

Expression of recombinant lentivirus in BV-2 cells

Microglioma cells/BV-2 (BV-2 cells) were cultured in 24-well plastic plates at an initial density of 1.5×10⁵ cells/well. Sixteen hours after plating, the old medium was removed, and the cells were washed with high glucose DMEM twice. The BV-2 cells were transfected by Lentivirus-Rab9 at different multiplicities of infection (MOI) (MOI=1×10², 1×10³, 1×10⁴, 1×10⁵) in a pre-mixed incomplete culture medium of 200 μL per well respectively, and incubated for 2 hours at 37°C. Then the medium was removed and replaced with complete culture medium. In the control group, the Lentivirus-Rab9 was replaced by the same volume of culture medium. The expression of GFP in the two groups was observed under an inverted fluorescent microscope 3 days later and the transfection efficiency was calculated.

Cerebella injection of recombinant lentivirus in BALB/c mice

Twelve 4-week-old BALB/c mice were randomly divided into an experimental (virus) group and a control (saline) group (n=6 per group, male∶female=1∶1). Four microliters (2.0×10⁸ TU/mL) of lentivirus suspended in PBS was injected into the cerebella bilaterally in the experimental group. In the control group, 4 μL saline was administered. Mice were sacrificed and the brains were dissected for biochemical analysis 4 weeks after injection. The GFP expression in the brain was observed under a fluorescent microscope.

Western blot analysis

Western blot was performed as reported previously [15]. Briefly, frozen sagittal cerebella halves were homogenized in lysis buffer. Protein concentration was determined by Bradford assay. Aliquots of total protein (100 μg) were electrophoresed in 15% sodium lauryl sulfate (SDS) gel, transferred to nitrocellulose, and probed with the Rab9 antibody (1∶100) or β-actin antibody (1∶2000), then followed by incubation with horseradish peroxidase-conjugated secondary antibody (1∶1000). The bands were detected with enhanced chemiluminescence (ECL) reagents. ECL films were scanned and the appropriate band densities were measured using (National Institutes of Health) NIH Image software, USA. β-actin served as loading control.

Bilateral cerebroventricular injection of recombinant lentivirus in NPC mice

A four μL solution containing Lenti-Rab9 or control virus was injected into the bilateral cerebroventricles of postnatal day 3 NPC mice (n=6) with a microinjector. From four to eight weeks after treatment, mice were weighed and evaluated for limb motor activity using the coat hanger test once a week [16]. The animals were allowed to grab a metal coat hanger suspended 20 cm above a flat surface, observed for 2 minutes, and the time length that the mouse remained on the hanger was measured (hanging time). The ratio of the mouse hanging time at the eighth week after the end of the treatment and that at the fourth week after the treatment served as a measure of the decline (control) or sustenance (Lenti-Rab9) of motor activity. These analyses were performed in a double-blind manner. Purkinje cells were counted in HE-stained sections from the experimental (virus) group and the control (saline) group. Purkinje cell counting was performed by two separate individuals both blinded to the experimental details. To standardize the counting, the same lobe was selected in each section, and all visible Purkinje neurons were counted in that lobe. The number was averaged, and then statistical analyses were performed using the Student’s t-test. The data were analyzed statistically as described below.

Statistical analysis

Statistical differences were observed in both cases so only the combined results are presented here. Throughout, a P value less than 0.05 was considered to be statistically significant.

Results

Identification of pCDH1-MCF1-Rab9-EF1-copGFP plasmid

The pCDH1-MCF1-Rab9-EF1-copGFP was digested by restriction enzymes BamHI and NheI, and two expected fragments (678 bp and 7.5 kb respectively) were generated (Fig. 1).

Sequencing of pCDH1-MCF1-Rab9-EF1-copGFP

DNA sequencing was executed with the recombinant plasmid pCDH1-MCF1-Rab9-EF1-copGFP (Fig. 2). The detected sequence was compared with Rab9 by using Basic Local Alignment Search Tool (Blast), and their coherence was 100% (GenBank: NM_004251).

Observation of microglia cells after 72-h infection with pCDH1-MCF1-Rab9-EF1-copGFP

Seventy-two hours after the infection, GFP could be found in BV-2 cells under the inverted fluorescent microscope (Fig. 3). The transfection efficiency was approximately 80%.

Expression and functional identification of pCDH1-MCF1-Rab9-EF1-copGFP of Rab9 in BALB/c mice brain

Four weeks after injection, GFP and Rab9 proteins were detected in BALB/c mice brain by fluorescent microscope and western blot analysis respectively. Green fluorescence could be observed in the whole brain of the mice 4 weeks after the lentivirus injection (Fig. 4). Compared to the control group, the expression level of Rab9 gene in cerebellum of the lentivirus treated mice was increased by 36.7% (Fig. 5).

Lenti-Rab9 improved motor activity and inhibited weight loss in female NPC mice, but did not decrease weight loss in male NPC mice

NPC mice characteristically experienced weight loss and decline in motor ability beginning at the age of 6 weeks. To determine whether weight loss or motor defects were alleviated in NPC mice administered Lenti-Rab9, the mice were weighed and their motor ability was examined using the coat hanger test once weekly between the ages of 4 and 8 weeks. NPC mice treated with control virus alone displayed the expected decline in motor activity, but this symptom was improved with Lenti-Rab9 treatment (Fig. 6c). Here, the performance of the mice treated with Lenti-Rab9 was improved by almost 240% compared with that of the control mice (Fig. 6c). With respect to body weight, a significant difference was noted between control and Lenti-Rab9-treated female NPC mice (Fig. 6a), but there was no significant difference among male NPC mice (Fig. 6b). These combined results suggest that Lenti-Rab9 inhibits the progression of motor dysfunction and alleviates weight loss in female NPC mice, but does not prevent the weight loss associated with male NPC mice.

Lenti-Rab9 ameliorated but did not spare Purkinje cell loss in NPC mice

The progressive loss of Purkinje cells in NPC mice started from the 6th week, leading to gradual ataxia and paralysis. The number of Purkinje neurons in a selected lobe of the cerebellum was scored in non-operation, control virus and Lenti-Rab9-treated mice. These cells, stained with HE, were barely detected in control virus-treated 8-week-old NPC mice where empty spaces were apparent in locations where Purkinje cells once existed (Fig. 7a arrows). In contrast, Purkinje neurons were apparent in the 8-week-old mice that received Lenti-Rab9 (Fig. 6b). However, in the mice receiving Lenti-Rab9, there was no sparing of Purkinje neuron loss. These results suggest that introducing Lenti-Rab9 in postnatal day 3 NPC^-/- mice modulates the death rate of Purkinje neurons but does not prevent the death of Purkinje neurons.

Discussion

Rab proteins represent a large family of ras-like GTPases (guanosine triphosphate kinase) that regulates distinct vesicular transport events at the level of membrane targeting and/or fusion. Rab9 is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN) in living cells [17]. The bulk of Rab9 is localized to the surface of late endosomes and a small amount of Rab9 at the TGN. This localization is consistent with the observation that Rab9 can facilitate vesicular transport from late endosomes to the TGN.

NPC cellular pathology is characterized by a defect in intracellular vesicular lipid trafficking associated with accumulation of unesterified free cholesterol, glycosphingolipids, and other lipid species in late endosomes/lysosomes of various tissues. A study has documented the reduction of intracellular free cholesterol in NPC^mut cells with overexpressed Rab9 [18]. However, Endogenous Rab9 levels were elevated by 1.8-fold in NPC^mut cells relative to wild type cells, and its half-life increased by 1.6-fold, suggesting that Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes [9]. We propose that endogenous Rab9 may not properly effect on NPC^mut cells and the release of exogenous active Rab9 may be sufficient to restore critical membrane transport, thus allowing the eventual clearance of stored lipids. Presently, the hypothesis has been partly confirmed in some study in vitro. Some researchers demonstrated that intracellular cholesterol was reduced by VP22-Rab9 transduction in cultured mouse NPC1 neurons [10]. A small increase in Rab9 protein level could yield a large increase in transport.

These observations provide important and novel information about the correction of membrane traffic in NPC^mut cells by Rab9 overexpression and may be a basis for new therapeutic approaches for treating NPC. It would require multiple levels of study to develop a suitable therapeutic approach based on the observation that Rab9 can correct storage defects of cells in culture. The feasibility of Rab9 overexpression as a therapeutic treatment for NPC has yet to be tested in the NPC1 mouse model in vivo. Thus, a powerful vector is needed to facilitate gene therapy for NPC.

Viral recombination technology has become an effective tool for gene therapy [19]. In numerous viral delivery systems, lentiviral vectors are the most effective vehicles for transducing and stably expressing different effector molecules (siRNA, cDNA, DNA fragments, antisense, ribozymes, etc.) or reporter constructs in almost any mammalian cell — including non-dividing cells and whole model organisms. The most popular lentiviral system is HIV based [20, 21]. By packaging the lentiviral expression construct into pseudoviral particles, highly efficient transduction (up to 100%) can be obtained — even with the most difficult transfecting cells, such as primary, stem, and differentiated cells. The expression construct transduced in cells is integrated into genomic DNA and provides stable, long-term expression of a reporter gene. Stably integrated constructs with transcriptional reporters are a novel approach to study transcriptional regulation in the natural chromosomal environments and the monitoring of specific signaling pathways. Moreover, lentiviral delivery does not produce the non-specific cell responses typically associated with chemical transfection or use of an adenoviral delivery system [22]. This system is designed to maximize its biosafety, including: a deletion in the enhancer of the U3 region of 3’LTR to ensure self-inactivation of the lentiviral construct after transduction and integration into genomic DNA of the target cells; the number of HIV-1 viral genes necessary for packaging, replication, and transduction is reduced to three (gag, rev, and pol). These genes are from different plasmids which lack packaging signals and have significant homology to pCDH and VSV-G expression vectors, and can prevent generation of a recombinant replication-competent virus. These features provide the lentiviral expression system a superior biosafety rating among gene delivery and expression vectors of viral origin. For these advantages, lentivirus was selected as the vector for gene therapy in this study. After concentration, the titer of the recombinant virus is 2.0×10⁸ TU/mL. The titer should be sufficient for further in vitro and in vivo experiments.

In the viral package process, lentiviral-293T cells were cultured in 150 cm² tissue culture plates. In this way, the viral titer could be increased effectively. In some companies, 293 cells are cultured in a roller bottle cell culture apparatus in order to increase the quantity of virus. Compared with the industrial virus production, our virus production method is more suitable for utilization in the laboratory.

The infective abilities of the various serotype vectors are different. The most reliable way to normalize vector titers is the dot-blot assay, which allows direct comparison of the potency of the different serotype vectors administered to the same cell type [23]. Therefore, more researchers choose the physical titers to indicate the recombinant viral titer. However, it does not indicate virus infectivity or functionality of the expression cassette. We have not only determined physical titers of recombinant virus by dot-blot assay, but also observed the infectivity of the recombinant virus. Lentiviral vector carrying GFP and Rab9 was transfected into BV-2 cells. The GFP expression was observed under a fluorescent microscope. The transfection efficiency was approximately 80% in 2×10⁸ TU/mL. More importantly, the gene carried in the recombinant lentivirus could be effectively delivered into BALB/c mice brain. Results of western blotting showed that the Rab9 level in cerebella was increased by 36.7% compared with that of the control group. Purkinje neurons, weight loss and a decline in motor ability were important characteristics in NPC mice. In this study, by administration of Lenti-Rab9 to postnatal day 3 NPC mice, the motor defects were reduced, the weight loss associated with female NPC mice was prevented, and the death rate of Purkinje neurons were modulated. Although weight loss of male NPC mice was not affected and the death of Purkinje neurons was not prevented, the data showed that Lenti-rab9 had an effect on NPC mice.

The recombinant lentivirus encoding Rab9 gene has been successfully constructed and the viral titers harvested in our experiment are enough for further researches both in vitro and in vivo.

Our work has laid the foundation for gene therapy of NPC in the future. The lentivirus may be a powerful tool to further investigate the role of Rab9 in NPC. However, it needs to be noted that despite improved biosafety features, third generation HIV(human immunodeficiency virus, HIV) cloning vectors still pose a potential biohazard risk due to the possible recombination with endogenous viral sequences to form a self-replicating HIV virus. So, it is necessary to further evaluate biosafety in research. Additionally, there is still a long way for us to evaluate the efficacy of Rab9 expression in animal models as well as the role of Rab9 in NPC mice.

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