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. 2021 Jun;16(13):1081-1095.
doi: 10.2217/nnm-2020-0477. Epub 2021 May 7.

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

Hari Priya Vemana  1 , Aishwarya Saraswat  1 , Shraddha Bhutkar  1 , Ketan Patel  1 , Vikas V Dukhande  1
Affiliations

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

Hari Priya Vemana et al. Nanomedicine (Lond). 2021 Jun.

Abstract

Aim: To develop novel cationic liposomes as a nonviral gene delivery vector for the treatment of rare diseases, such as Lafora disease - a neurodegenerative epilepsy. Materials & methods: DLinDMA and DOTAP liposomes were formulated and characterized for the delivery of gene encoding laforin and expression of functional protein in HEK293 and neuroblastoma cells. Results: Liposomes with cationic lipids DLinDMA and DOTAP showed good physicochemical characteristics. Nanosized DLinDMA liposomes demonstrated desired transfection efficiency, negligible hemolysis and minimal cytotoxicity. Western blotting confirmed successful expression and glucan phosphatase assay demonstrated the biological activity of laforin. Conclusion: Our study is a novel preclinical effort in formulating cationic lipoplexes containing plasmid DNA for the therapy of rare genetic diseases such as Lafora disease.

Keywords: DLinDMA liposomes; Lafora disease; gene delivery; gene therapy; laforin; neurodegenerative disorders; rare diseases.

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Conflict of interest statement

Financial & competing interests disclosure

This research was supported by an award from the National Institute of General Medical Sciences of the NIH under Award Number SC2GM125550 to VV Dukhande and by funds from the College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA to VV Dukhande and K Patel. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1.
Figure 1.. Physicochemical characterization of DLinDMA-containing liposomal formulation.
(A) Electrostatic interaction between highly positive charge of cationic liposomes and negatively charged plasmid DNA to form lipoplexes. (B) Dynamic light scattering graphs illustrating unimodal particle size distribution and positive zeta potential of the liposomes.
Figure 2.
Figure 2.. Cytotoxicity studies of DLinDMA liposomes in HEK293 and SK-N-SH cell lines.
Blank liposomes cytotoxicity evaluated in HEK293 cells after 48 h by (A) MTT assay and (B) cell proliferation (Cyquant) assay with indicated concentrations (μM) of DOTAP and DLinDMA. (C) Blank DLinDMA liposomes cytotoxicity evaluated in SK-N-SH cells by MTT assay after 48 h with indicated concentrations (μM) of DLinDMA. Data represent mean ± standard error of mean of three individual experiments with n = 4 for each trial in HEK293 cell line and two individual experiments with n = 8 in SK-N-SH cells. Two-way ANOVA analysis in HEK293; One-way ANOVA analysis in SK-N-SH cell line; *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 3.
Figure 3.. DLinDMA liposomes show higher transfection efficiency than DOTAP liposomes.
Representative fluorescent images demonstrating transfection efficiency with (A) DOTAP liposomes and (B) DLinDMA liposomes at 1:5, 1:10, 1:20, 1:40 and 1:60 mass ratio in HEK293 cells after 48 h of transfection. n = 6; Scale bar: 200 μm. (C) Representative fluorescent images demonstrating transfection efficiency with DLinDMA liposomes at 1:5, 1:10, 1:20, 1:40 and 1:60 mass ratio after 48 h of transfection in SK-N-SH cells. n = 4; Scale bar: 200 μm. (D) Quantification of transfection efficiency of DLinDMA and DOTAP liposomes in HEK293 cells shown above. (E) Quantification of transfection efficiency of DLinDMA liposomes in SK-N-SH cells shown above. Scale bar: 200 μm. Graphical data represent mean ± standard error of mean of three individual experiments with n = 3 for each trial. One-way analysis of variance with post hoc Tukey; *p < 0.05, **p < 0.01 and ***p < 0.001.
Figure 4.
Figure 4.. Electrophoretic mobility shift assay gel image for indicated ratios of plasmid DNA:DLinDMA containing cationic liposomes.
Representative image from two independent experiments.
Figure 5.
Figure 5.. DLinDMA liposome-mediated protein expression is stable in cells.
(A) Representative images of HEK293 cells transfected with pEGFP-N3 and FLAG laforin with DLinDMA liposomes (1:20 ratio) obtained by florescence microscopy. n = 3; Scale bar: 100 μm. (B) Representative fluorescence images of stable EGFP expression in HEK293 cells transfected with pEGFP-N3:DLinDMA liposomes (1:20 ratio) for 5 and 10 days. n = 3; Scale bar: 200 μm. (C) Representative western blots to observe the stable expression of GFP (27 kDa) or laforin (40 kDa) after 5- and 10-day transfections using DLinDMA liposomes (1:20 ratio) in HEK293 cells. β-actin (42 kDa) was used as loading control. n = 3.
Figure 6.
Figure 6.. DLinDMA lipoplexes deliver functional laforin.
(A) SK-N-SH cells were transfected with FLAG laforin and mutant FLAG C266S laforin using DLinDMA liposomes (1:20 ratio). Representative western blots for protein expression of laforin (40 kDa) and β-actin (42 kDa) in whole cell lysate are shown. n = 4. (B) Representative western blot for expression of FLAG-laforin (40 kDa) in IP samples. n = 5. (C) Glucan phosphatase activity of functionally active laforin transfected using DLinDMA liposomes. Graphical data represent mean ± standard error of mean of three individual experiments with n = 5 for each trial. One-way analysis of variance; ***p < 0.001. IP: Immunoprecipitated.
Figure 7.
Figure 7.. In vitro hemolysis study results of indicated DLinDMA concentrations of cationic liposomes.
PBS: Phosphate-buffered saline; SLS: Sodium lauryl sulfate.
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