A novel gene therapy for neurodegenerative Lafora disease via EPM2A-loaded DLinDMA lipoplexes

Plasmids & cloning

Plasmids pEGFP-N3, pcDNA3.1 N-terminal FLAG tagged (pCNF) laforin and catalytically inactive laforin-encoding pCNF laforin C266S were generous gifts from Dr. Matthew Gentry (University of Kentucky). Plasmids were amplified in Escherichia coli (DH5α strain) and purified using PureLink HiPure Plasmid Maxiprep kit (Invitrogen), according to the manufacturer’s protocol. The concentration and purity of DNA were determined using NanoDrop One^c (Thermo Fisher Scientific).

Cell culture

Human embryonic kidney HEK293 cells were grown in DMEM with high glucose 4.5 gm/l (Corning Cellgro), 5% v/v fetal bovine serum (FBS; Atlanta Biologicals) and 100 μg/ml penicillin/streptomycin/amphotericin B (MP Biomedicals). Human neuroblastoma SK-N-SH cells were cultured in DMEM containing glucose 1 gm/l (Corning Cellgro) and supplemented with 10% v/v FBS and 100 μg/ml penicillin/streptomycin/amphotericin B. All transfection experiments with liposomes were carried out 16 h after subculturing cells to about 70% confluency.

Preparation of DLinDMA liposomes

Cationic liposomes containing DLinDMA (MedChem Express) or DOTAP (Avanti Polar Lipids) at 3 mg/ml were prepared by the ethanol injection method followed by ultrasonication to produce small unilamellar vesicles. As mentioned in Table 1, the required amounts of neutral lipids (DOPE and DOPC), cholesterol and cationic lipid (DLinDMA or DOTAP) were dissolved in ethanol. The resulting organic phase was injected into the aqueous phase (Milli-Q water) to spontaneously form the liposomes. Liposomal suspension was then probe sonicated at 30 amplitudes for 120 s to obtain nanoliposomes. The liposomes were stirred at room temperature to remove the traces of ethanol.

Preparation of liposome–DNA complex

The DNA/liposome complexes were prepared by mixing the plasmid DNA with different ratios of the liposomal formulation to identify the DNA-binding affinity of the cationic liposomes. Briefly, different volumes of the formulation were mixed with 1 μg of DNA and incubated for 30 min at room temperature (RT) to form the DNA/liposome complex.

Physicochemical characterization of cationic liposomes

Nanocarriers were diluted with Milli-Q water. Average particle size, size distribution and zeta potential analysis were performed by dynamic light scattering particle size analyzer (Malvern Zetasizer Nano ZS). Samples were analyzed using folded capillary cells at 25°C with a scattering angle of 173°.

Cell viability determination

Cytotoxicity of liposomes was measured in HEK293 cells and SK-N-SH cells by MTT cell viability assay. The cells were seeded in 96-well plates at density of 5000 cells per well. After 16 h incubation, cells were treated with DOTAP or DLinDMA liposomes at concentrations ranging from 1.56 to 200 μM. Transit LT1 (Mirus Bio) and PEI max were used as positive controls. After 48 h treatment, MTT dye (0.05% w/v final concentration) was added and cells were incubated at 37°C for 3 h. Formazan crystals formed from the reduction of MTT dye were dissolved in DMSO and the absorbance was measured at 570 nm using the SpectraMax M5e plate reader (Molecular Devices).

Cell proliferation assay

Cell proliferation was determined using the CyQuant Direct Proliferation Assay kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. Briefly, at the end of DLinDMA liposome treatments (ranging from 1.56 to 200 μM) of HEK293 cells, medium was carefully aspirated from each well without disturbing the cells. 50 μl of 2X detection reagent was added to each well containing 50 μl media with cells and incubated for 30 min at 37°C. Fluorescence was measured for each well at wavelengths 480/535 nm with a Spark 10 M plate reader (Tecan Life Sciences).

Transfection efficiency

The transfection efficiency of DLinDMA liposomes was evaluated in HEK 293 and SK-N-SH cells. The cells were seeded in 96-well plates at density of 5000 cells per well and maintained in complete culture medium overnight prior to transfection. Lipoplexes were formed at 1:5, 1:10, 1:20, 1:40 and 1:60 mass ratio of pEGFP-N3 DNA:DLinDMA/DOTAP and incubated for 30 min. Cells were incubated with these lipoplexes at 37°C in 5% CO₂ for 3 h in opti-MEM medium. The transfection medium was replaced with fresh complete culture medium and the cells were incubated for 48 h post-transfection. Commercial transfection reagents Transit LT1 and PEI max were used as positive controls at 1:3 ratio of pEGFP-N3 DNA:Transit LT1/PEI Max (3 μl reagent per 1 μg DNA). The transfection efficiency of pEGFP-N3 was monitored by fluorescence microscopy using EVOS microscope. Fluorescence images of transfected cells were taken after 48 h at 200X magnification.

Electrophoretic mobility shift assay

DLinDMA liposomes are capable of forming electrostatic complexes with DNA and this complex formation was measured by electrophoretic mobility shift assay (EMSA). The DLinDMA:DNA complexes at different charge ratios ranging from 1:5, 1:10, 1:20, 1:40, 1:60 mass ratio of DNA:DLinDMA were incubated at room temperature for 30 min and then analyzed by electrophoresis on a 1% (w/v) agarose gel in tris-acetate-EDTA (TAE) buffer containing SYBR green dye (Life Technologies). EDTA-free loading dye was used in order to curb the interaction of EDTA with the cationic lipids used in the liposomes. The gel was run for 1 h at 100 V and visualized by ultraviolet illumination using Azure biosystems c500 imager and electrophoretic mobility was investigated to evaluate the quantity of DNA entrapment. Naked plasmid DNA was used as control.

Fluorescence microscopy

SK-N-SH cells were grown on poly-D-lysine-coated glass coverslips in 24-well cell culture plates. Next day, cells were treated with DLinDMA lipoplexes containing pEGFP-N3 or FLAG-laforin. After 48 h of transfection, cells were washed with phosphate-buffered saline (PBS) and fixed using formalin (3.7% v/v in PBS). Cells were permeabilized using cold Triton X-100 (0.25% v/v in PBS) for 5 min, washed with PBS and incubated in FBS solution (10% v/v in PBS) for 1 h. Next, samples were incubated with anti-FLAG M2 antibody (Sigma-Aldrich) for 2 h followed by washes and incubation with Alexa Fluor 594-conjugated secondary antibody. Coverslips were mounted on slides using DAPI Fluoromount, and images were taken using EVOS FL Auto Imaging System.

Immunoprecipitation

SK-N-SH cells were transfected with FLAG laforin or FLAG laforin C266S using DLinDMA liposomes in 100-mm cell culture dishes. Cells were lysed using modified RIPA buffer (Tris pH 8.0 50 mM, NaCl 150 mM, NP40 1%, glycerol 10%, NaF 10 mM and EDTA 0.4 mM) with protease inhibitors. Lysates were incubated with M2 FLAG-agarose beads (Millipore Sigma) for 2 h at 4°C. The samples with the beads were washed with modified RIPA buffer three-times and resuspended in 100 μl of modified RIPA buffer. 5 μl of sample was used for each replicate of the phosphatase assay (n = 5). 2X SDS PAGE dye was added to the remaining sample and heated for 10 min. Heated samples were centrifuged at 4°C at 1100 g for 3 min and the supernatant was collected. Samples were analyzed by western blotting with 1:1000 laforin antibody.

Western blot analysis

SK-N-SH cells were lysed after transfection for 48 h using DLinDMA liposomes. The cell lysates were centrifuged at 4°C for 10 min at 10,000 g and the supernatant was collected. 4X SDS PAGE dye was added to the samples and were heated at 95°C for 12 min. The samples were loaded onto a 4–20% SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane. The blots were blocked with 5% milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h at RT and then incubated at 4°C overnight with primary antibodies for anti-FLAG M2 (Sigma-Aldrich) and β-actin (Proteintech). The membranes were washed three-times with TBST and incubated with secondary antibody (Bio-Rad) for 1 h at RT. The proteins were visualized by chemiluminescent substrate (Thermo Fisher Scientific) on Azure biosystems c500 imager.

Glucan phosphatase assay

Molybdate-based malachite green assay is widely used for quantifying picomolar quantities of inorganic-free phosphate and was employed to assess laforin’s glucan phosphtase activity [38,39]. Malachite green reagent was made by mixing 1 volume of 4.2% (w/v) ammonium molybdate tetrahydrate in 4 M HCl and three volumes of 0.045% (w/v) malachite green carbinol hydrochloride and filtered through 0.45-μm filter. Tween-20 was added at last to the required amount of malachite green reagent to make the final concentration of Tween-20 to 0.01% v/v. A mastermix of the assay components consisting of 5X buffer (0.5 M sodium acetate, 0.25 M Bis-Tris, 0.25 M Tris-HCl, pH 5), 100 mM dithiothreitol, 5 mg/ml amylopectin was prepared and immunoprecipitated (IP) laforin sample was added to this aliquot of mastermix in a microcentrifuge tube to initiate the reaction. All reactions were incubated for 30 min at 37°C. The enzymatic activity of laforin was stopped by adding 0.1 M NEM to the reaction mixture and the tube was vortexed for 10 s. Malachite green reagent with 0.01% Tween-20 was added to each of the reaction tubes and vortexed for 10 s. All reactions were incubated for 90 min at room temperature before measuring the absorbance at 620 nm using a spectrophotometer.

In vitro hemolysis study

Experimental protocol was approved by the St. John’s University Institutional Animal Care and Use Committee for collection of blood from mice for laboratory use. Red blood cells (RBCs) from mice were used to carry out the in vitro hemolysis study of cationic liposomes containing DLinDMA. C57BL/6 mice (5–6 weeks old) were received from Jackson laboratories (CT, USA). Briefly, mice were anesthetized by 2.5% isoflurane followed by a one-time blood collection using cardiac puncture technique. Then, the animals were immediately euthanized by carbon dioxide. Initially, the collected blood was centrifuged at 450 g for 10 min to separate the RBCs from plasma. The cell pellet was washed with PBS and redispersed into an appropriate volume of PBS to achieve the same hematocrit. Then, cationic liposomes were added to the RBC dispersion to achieve various cationic lipid (DLinDMA) concentrations. Following this, all the samples were incubated for 30 min at 37°C and then centrifuged at 450 g for 10 min. The supernatant was diluted with PBS and analyzed for hemoglobin release using a UV spectrophotometer at 550 nm. PBS was used as the negative control and 5% sodium lauryl sulfate (SLS) solution was used as the positive control (100% hemoglobin release). Percentage hemolysis was calculated by following formula:

Statistical analyses

The data presented are a mean of three independent experiments ± SEM unless noted otherwise. The statistical significance of difference was assessed with one-way analysis of variance, followed by Dunnett’s or Tukey’s post hoc analysis using GraphPad Prism to determine statistical significance as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Development & characterization of cationic liposomes for gene delivery

Liposomes containing cationic lipids DLinDMA or DOTAP were prepared by the ethanol injection method. The average size, size distribution, and zeta potential of both the formulations are shown in Table 2. Overall, particle size of both the liposomal formulations was below 200 nm, indicating the formation of nanosized liposomes with a narrow size distribution and positive zeta potential to facilitate the complexation with negatively charged plasmid DNA via electrostatic interaction. The dynamic light scattering illustrated unimodal particle size distribution and positive zeta potential of liposomes containing DLinDMA as the cationic lipid (Figure 1).

Safety of cationic liposomal formulations

Cell viability (MTT) and cell proliferation (Cyquant) assays were performed to assess the cytotoxicity of DLinDMA- and DOTAP-containing liposomal formulations in HEK293 cells. Cells not treated with liposomes in their regular media and OptiMEM media were used as controls as the transfections were performed in serum-free media. Commercially available transfection reagents PEI Max and Transit LT1 were used as positive controls. The toxicity of DLinDMA liposomal formulation was observed at the concentrations ranging from 6.25 to 200 μM, whereas for DOTAP liposomal formulations, the toxicity was observed at the concentrations ranging from 12.5 to 200 μM (Figure 2A). Interestingly, the toxicity of commercially available transfection reagents such as PEI Max and Transit LT-1 at recommended 1:3 ratio of DNA:Transit LT1/PEI Max (3 μl reagent per 1 μg DNA) was similar compared with that of our liposomal formulations with higher ratio (Figure 2A). Similar to the cell viability studies, cell proliferation assay results also showed that these liposomal formulations are nontoxic at lower concentrations. A significant decrease in cell proliferation was only observed at the highest concentration 200 μM for DOTAP and at concentrations ranging from 25 to 200 μM for DLinDMA liposomal formulations (Figure 2B). As DLinDMA liposomes were less toxic compared with DOTAP, their cytotoxicity was also evaluated in SK-N-SH, a neuroblastoma cell line, at concentration range of 1.56–200 μM. Transit LT1 and PEI max were used as positive controls. The toxicity was observed at concentrations of 12.5 μM onward, which was similar to the effect observed in HEK293 cells (Figure 2C). These results indicate that liposomes with DOTAP and DLinDMA are relatively safe when compared with commercially available transfection reagents such as PEI Max and Transit LT1.

Transfection efficiency of DLinDMA liposomes is higher than DOTAP liposomes

We used fluorescence microscopy to determine the transfection efficiency of DOTAP- and DLinDMA liposomes in HEK293 and SK-N-SH cells. The transfection efficiency of liposomes in both the cell lines was studied at different ratio of DNA to DLinDMA/DOTAP liposomes at 1:5, 1:10, 1:20, 1:40 and 1:60 (Figure 3A–C). The number of EGFP-expressing cells transfected with different ratios of liposomes was quantified (Figure 3D & E). The results show that transfection efficiency was higher with DLinDMA liposomes compared with DOTAP liposomes in HEK293 cells (Figure 3D). In addition, prominent transfection was observed with DLinDMA liposomes in SK-N-SH cells, a neuroblastoma cell line that is difficult to transfect (Figure 3E). Due to the higher transfection efficiency and lower cytotoxicity of DLinDMA liposomes compared with DOTAP liposomes, liposomes containing DLinDMA as the cationic lipid were selected to perform further experiments.

DLinDMA formed functional lipoplexes

The efficiency of the liposomes to form a complex binding with DNA was evaluated using electrophoretic mobility shift assay (EMSA). Naked pEGFP-N3 was used as a control and DNA to DLinDMA liposomes forming a complex with the at 1:1, 1:5, 1:10, 1:20, 1:40 and 1:60 mass ratio were studied. The liposomal formulations at 1:20, 1:40 and 1:60 mass ratio of DNA:DLinDMA showed the plasmid DNA complexation with no DNA migration as a result of binding and neutralization of the negatively charged DNA by the cationic liposomes (Figure 4). Based on the data from cytotoxicity assay, cellular transfections and EMSA, we decided to use DLinDMA liposomes at the ratio of 1:20 that corresponds to DLinDMA liposomes at 32 μM for immunofluorescence microscopy or 16 μM for immunoprecipitation experiments.

DLinDMA liposomes efficiently deliver laforin in cells

After studying the transfection efficiency of pEGFP-N3 in HEK293 and SK-N-SH cells, we evaluated the transfection of FLAG laforin in SK-N-SH neuroblastoma cells using cationic liposomes at DNA:DLinDMA mass ratio of 1:20. FLAG laforin-transfected cells were immunolabeled using fluorescent-dye conjugated secondary antibody to anti-FLAG antibody. As seen in Figure 5, fluorescence exhibited by cells transfected with pEGFP-N3 as well as FLAG-laforin indicated that cells were effectively transfected with DLinDMA cationic liposomes. In addition, cells transfected with pEGFP-N3 and FLAG-laforin showed similar cell density and cellular morphology when compared with control. As compared with HEK293 cells, SK-N-SH cells are difficult to transfect which implies that 1:20 mass ratio of DNA:liposomes are safe and effective vehicle for gene delivery. Furthermore, in order to assess intracellular protein persistence and length of protein expression, we performed DLinDMA liposomal transfection and studied the protein expression after 5- and 10-day periods in HEK293 cells by fluorescence microscopy and western blotting. The EGFP expression in HEK293 cells increased from days 5 to 10 (Figure 5B). The western blotting data showed that the EGFP or FLAG-laforin protein expression was stable after 5- and 10-days of transfection using DLinDMA liposomes in HEK293 cells (Figure 5C).

DLinDMA lipoplexes deliver functional laforin with intact glucan phosphatase activity

Protein expression studies were performed using western blotting to confirm the presence of laforin in SK-N-SH cells transfected using the cationic liposomes. The cellular expression of laforin was confirmed in whole cell lysates by employing FLAG antibody that showed the presence of laforin in the samples transfected with FLAG laforin and mutant FLAG C266S laforin whereas, as expected, no bands were observed in control and blank liposome lanes (Figure 6A). Furthermore, IP laforin was assessed by western blot using laforin antibody. The results show the expression of laforin in the cells transfected with FLAG laforin and mutant FLAG C266S laforin (Figure 6B).

Malachite green-based glucan phosphatase assay was used to determine whether the lipoplex-mediated transfected laforin is functionally active. The assay measures glucan phosphatase activity of laforin as it uses phosphorylated glucan polymer amylopectin as a substrate that is similar to laforin’s endogenous substrate glycogen [5,39]. Phosphate is released in the presence of laforin and forms a complex with ammonium molybdate in malachite green reagent and the resulting color can be measured at 620 nm. A standard curve was generated before the experiment and picomol of phosphate released was calculated using the standard curve. Glucan phosphatase activity was significant in FLAG laforin-transfected samples compared with control, blank liposomes and the cells transfected with mutant FLAG C266S laforin that is catalytically inactive (Figure 6C). In addition, we performed silencing of endogenous laforin in HEK293 cells and then introduced DLinDMA liposomal FLAG-laforin to confirm that the expression and activity observed is from laforin delivered through liposomes. Our data showed robust FLAG-laforin in siRNA-treated cells (Supplementary Figure 1) confirming data from Figure 6A and C. These results show that the DLinDMA cationic liposomes are not only efficient in delivering laforin DNA into the cells but also indicate that the expression and functional activity of laforin is intact.

Biocompatibility of DLinDMA liposomes analyzed by hemolytic potential

The effect of varying concentrations of DLinDMA in developed cationic liposomes was evaluated on mice RBCs by performing the in vitro hemolysis study. As shown in Figure 7, cationic liposomes exhibited negligible hemolysis (<5%) at all the tested concentrations (25, 50 and 100 μM) of cationic lipid (DLinDMA), in comparison to the positive control (SLS), which demonstrated 100% hemolysis. Notably, rapid and complete redispersion of RBCs following centrifugation implied that the DLinDMA cationic liposomes did not change the surface characteristics of RBCs at any of the tested concentrations.