Recombinant elastin-based nanoparticles for targeted gene therapy

Abstract

Among viruses, lentiviral vectors have been popular vectors for gene delivery due to their efficient mode of gene delivery. However, the nonspecific delivery of genes associated with lentiviral vectors may result in undesirable side effects. Here we propose a heterogeneous nanoparticle (NP) delivery system for targeted delivery of lentiviral particles containing a therapeutic gene. The heterogeneous NPs consist of the low-density lipoprotein receptor repeat 3 (LDLR3) and the keratinocyte growth factor (KGF), each fused to elastin-like polypeptides (ELPs), LDLR3-ELP and KGF-ELP, respectively. Our results show that although homogeneous NPs comprising of LDLR3-ELP alone blocked viral transduction, heterogeneous NPs comprising of KGF-ELP and LDLR3-ELP enhanced viral transduction in cells expressing high levels of the KGF receptors compared with cells expressing low levels of KGF receptors. Overall, this novel design may help with the targeting of specific cells that overexpress growth factor receptors such as KGF receptors.

Figure 1. Fusion protein comprising of the viral envelope binding domain (VBD) and elastin like polypeptide (ELP) was successfully expressed and purified using inverse temperature cycling (ITC)

A)Sequence of LDLR3-ELP and ELP. B) The bacterial lysate and purified LDLR3-ELP fusion protein was run on a SDS-PAGE gel and stained with simply safe blue stain for total protein Lanes: L=ladder, lanes 1 and 2, 10 µM and 2 µM of purified LDLR3-ELP fusion respectively, and lanes 3 and 4 bacterial lysates. SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. Red color represents guest residues.

Figure 2. LDLR3-ELP maintains the phase transition property and self assembles into nanoparticles

A) LDLR3-ELP transitions around 30°C. The transition temperature of 2 µM LDLR3-ELP was taken using dynamic light scattering (DLS). B) LDLR3-ELP size is in the nanometer range at physiological temperature. The size distribution of LDLR3-ELP by intensity is 204 nm at 37°C. Samples were dissolved in 1X sterile PBS.

Figure 3. LDLR3-ELP inhibits VSV-G pseudotyped lentiviral vector infectivity

A) GFP expression in A549 cells, 20 µM ELP and 20 µM LDLR3-ELP, n=3. B) Average ± SD of the normalized GFP expression. A549 cells were starved for 24 hours. The lentiviral vectors were incubated with the NPs prior to cell treatment. Then the cells were incubated for 24 hours with the lentiviral vectors containing NPs. After 24 hours, the old media was discarded and fresh serum free 37°C media was added to each well. Then, 72 hours later, the transduction efficiency was quantified using flow cytometry. Treatments were normalized to control (virus). These experiments were repeated two more times with triplicates. ***indicates P<0.001 when compared to control (virus). Error bars represent ± SD.

Figure 4. LDLR3-ELP binds to the lentiviral vector and inhibits the VSV-G pseudotyped lentiviral vector infectivity in A549 cells at different concentrations

A) GFP expression in A549 cells, LDLR3 at 0.50 µM, 1 µM, 2 µM, and 10 µM B) Average ± SD of the normalized GFP expression of supernatant. A549 cells were starved for 24 hours and cells were treated for 24 hours with the lentiviral vector containing NPs as described in materials and methods. After 24 hours, the old media was discarded, and fresh 37°C serum free media was added to each well. Then, 72 hours later, the transduction efficiency was quantified using flow cytometry. Treatments were normalized to control (virus). All the treatments except with “virus no centrifuge” went through the centrifugation process. (C) LDLR-3-ELP has three times the binding affinity at 37°C compared to 4°C. 1 µM of biotinylated LDLR3-ELP was incubated with lentiviral vectors either at 4°C or 37°C for 30 minutes, followed by a 15 minute incubation at 4°C. Binding of the lentiviral vector to LDLR-3-ELP was assessed by immunoprecipitation assay as described in materials and methods. The viral binding was assessed by western blot for the VSV-G protein. Since the lentiviral vectors were not biotinylated, there is no band in lane three of LDLR3-ELP. Image Studio Lite was used to quantify the western blot bands. All experiments were repeated two more times with triplicates. ***indicates P<0.001 when compared to control (virus). Error bars represent ± SD.

Figure 5. LDLR3-ELP does not induce cell death

A549, H1650, H292, and H23 cells were starved for 24 hours then were treated in serum free media with the indicated treatments 0.2 µM, 2 µM, 10 µM and 20 µM LDLR3-ELP for 48 hours. After 48 hours, a MTT assay was performed and a spectrophotometer was used to read the absorbance at 570 nm. Treatments were normalized to control (no treatment). These experiments were repeated two more times with triplicates. ***indicates P<0.001, **indicates P<0.05, and *indicates P<0.1 when compared to control (no treatment). Error bars represent ± SD.

Figure 6. Targeted internalization of LDLR3-ELP using growth factors in high growth factor receptor expressing cells

A) Schematic of heterogeneous nanoparticle. B) Average ± SD of the normalized fold increase of cell uptake. A549 cells were starved for 24 hours then were treated in serum free media with the indicated treatments fluorescein-labeled 2 µM LDLR3-ELP and combination of fluorescein-labeled 2 µM LDLR3-ELP and ELP and KGF-ELP, and LDLR3-ELP for 48 hours. Lentiviral vectors were not added in this experiment. After 48 hours, flow cytometry was used to quantify the data. Before analysis, trypan blue was used in each sample suspension to capture only the internalized labeled fusion proteins and not the ones that are bound to the periphery of the cells. These experiments were repeated two more times with triplicates. ***indicates P<0.001when compared to control (2 µM LDLR3-ELP). Error bars represent ± SD.

Figure 7. Heterogeneous nanoparticles comprising of LDLR-3-ELP and KGF-ELP result in targeted delivery of the gene in high growth factor receptor expressing cells

A) GFP expression in A549 cells and H293 cells. B) Average ± SD of the normalized GFP expression of pellet. C) Average ± SD of the normalized geometric mean of cells. Cells were starved for 24 hours and treatment started when their confluency reached 30%. The cells were treated with the indicated NP formulations as described in materials and methods. 72 hours after treatment the transduction efficiency was quantified using flow cytometry. Treatments were normalized to control (1µM LDLR3-ELP + 1µM LDLR3-ELP +Virus) for each cell type to account for differences in transduction that may arise due to different cell types. These experiments were repeated two more times with triplicates. ***indicates P<0.001 and **indicates P<0.05 when compared to control (1 µM LDLR3-ELP + 1 µM LDLR3-ELP + Virus). Error bars represent ± SD.

Figure 8. Recombinant KGF increased the transduction efficiency blocked by LDLR3-ELP

A549 cells were starved for 24 hours and then treated with lentiviral vector bound to either LDLR-3-ELP homogeneous or KGF-ELP LDLR3-ELP heterogeneous nanoparticles in the presence or absence of recombinant KGF (rKGF). After 24 hours, the old media was discarded, and fresh 37°C serum free media was added to each well. 72 hours later, the transduction efficiency was quantified using flow cytometry. Treatments were normalized to control (1µM LDLR3-ELP + 1µM LDLR3-ELP +Virus). These experiments were repeated two more times with triplicates. ***indicates P<0.001 when compared to control (1 µM LDLR3-ELP + 1 µM LDLR3-ELP). Error bars represent ± SD.

Figure 9. ELP and KGF-ELP do not bind to the VSV-G pseudotyped lentiviral vector

A) GFP expression in A549 cells. B) Average ± SD of the normalized GFP expression of supernatant. A549 cells were starved for 24 hours. A binding assay was performed and cells were treated with the supernatant. Cells were treated for 24 hours with the lentiviral vector containing 12 µM ELP, 12 µM KGF-ELP, and 2 µM LDLR3-ELP were added respectively After 24 hours, the old media was discarded, and fresh 37°C serum free media was added to each well. Then, 72 hours later, the transduction efficiency was quantified using flow cytometry. Treatments were normalized to control (virus). These experiments were repeated two more times with triplicates. ***indicates P<0.001 when compared to control (virus). Error bars represent ± SD.

Figure 10. Lentiviral vector is released from LDLR3-ELP after internalization by cells

A549 were starved for 24 hours then were treated in serum free media with the indicated treatments. Cells were treated for 24 hours with the lentiviral vector containing nanoparticles that consisted of 1 µM of biotinylated LDLR3-ELP and 1 µM KGF-ELP. At the indicated time points, cells were put on ice for five minutes followed by three ice cold washed in PBS. The cells were lysed with radio immune protection assay (RIPA) buffer containing 2X halt protease and phosphatase inhibitors cocktail with 1X ethylene diamine triacetic acid (EDTA). The lysates were immunoprecipitated using streptavidin conjugated magnetic beads (dynabeads). Then a magnetic bar was used to separate the beads from the supernatant. Following a few washes, the immunoprecipitated complexes (dynabeads) and the supernatant were analyzed for the presence of virus using western blots. Anti VSV-G antibody was used to detect the lentiviral vector.