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Design of Liposome Formulations for CRISPR/Cas9 Enzyme Immobilization: Evaluation of 5-Alpha-Reductase Enzyme
Hasan Akbaba1, Gülşah Erel-Akbaba2, Yücel Başpınar1
1Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Abstract:
The enzyme steroid type II 5-alpha-reductase (SRD5α2) is responsible for the conversion of testosterone to dihydrotestosterone (DHT), which is involved in prostate cancer, benign prostatic hyperplasia, and androgenic alopecia. Inhibition of SRD5α2 activity has been explored and presented as a potential treatment for these conditions, but current drugs have side effects and alternative treatment approaches are needed. The CRISPR/Cas9 system, an innovative gene-editing tool, shows potential for targeting the SRD5α2 gene knockout as a therapeutic approach. Liposomes have been used for the immobilization and delivery of different proteins, and studies have shown that liposomes can enhance the stability and activity of enzymes. In this study, we provided the immobilization of Cas9 protein by encapsulating it in a novel cationic liposome formulation that carries sgRNA on its outer surface for gene delivery approaches. This novel delivery system has shown promising results in terms of physicochemical properties, stability, cytotoxicity, in vitro cellular uptake, and gene knockout efficiency, together with providing flexibility in sgRNA selection. The optimized final formulations showed an average diameter of 229.1 ± 3.66 nm, a polydispersity index of 0.089 ± 0.013, and a zeta potential value of 25.7 ± 0.87 mV. The encapsulation efficiency of the developed formulations has been revealed as 80.60%. The cellular uptake efficiency was evaluated and measured as 45.6% for the final formulation. Furthermore, the Lipo/Cas9:sgRNA (1.5:1) formulation decreased the relative SRD5α2 mRNA expression by 29.7% compared to the control group. The results of this study reveal that the liposomal formulation based on enzyme immobilization of Cas9 protein using CRISPR technology, an innovative gene-editing tool for SRD5α2 suppression, might be an alternative treatment option for prostate cancer or BPH treatment without current drug side effects.
Insights
This study developed a novel liposomal delivery system for CRISPR/Cas9 gene editing to target the SRD5α2 gene. This approach shows potential as a new treatment for prostate cancer and benign prostatic hyperplasia, reducing side effects.
Area of Science:
- Biotechnology
- Gene Therapy
- Nanomedicine
Background:
- Steroid type II 5-alpha-reductase (SRD5α2) is crucial in conditions like prostate cancer and benign prostatic hyperplasia (BPH).
- Current SRD5α2 inhibitors cause side effects, necessitating alternative therapeutic strategies.
- CRISPR/Cas9 gene editing offers a promising tool for targeting SRD5α2, but effective delivery is key.
Purpose of the Study:
- To develop and characterize a novel liposomal formulation for CRISPR/Cas9 delivery targeting the SRD5α2 gene.
- To evaluate the physicochemical properties, stability, cytotoxicity, cellular uptake, and gene knockout efficiency of the liposomal system.
- To assess the potential of this system as an alternative treatment for SRD5α2-related conditions.
Main Methods:
- Cas9 protein was immobilized within cationic liposomes, with sgRNA attached to the outer surface.
- Liposomal formulations were characterized for size, polydispersity index, and zeta potential.
- Encapsulation efficiency, cellular uptake, and SRD5α2 gene expression knockdown were evaluated in vitro.
Main Results:
- The optimized liposomal formulation exhibited favorable physicochemical properties (229.1 nm diameter, 0.089 PDI, 25.7 mV zeta potential) and high encapsulation efficiency (80.60%).
- The system demonstrated significant in vitro cellular uptake (45.6%) and effectively reduced SRD5α2 mRNA expression by 29.7%.
- The liposomal CRISPR/Cas9 system showed no significant cytotoxicity, indicating a good safety profile.
Conclusions:
- The developed liposomal formulation provides a stable and efficient delivery system for CRISPR/Cas9 targeting SRD5α2.
- This approach holds promise as a novel therapeutic strategy for prostate cancer and BPH, potentially overcoming the limitations of current treatments.
- Further research into this gene-editing delivery system could lead to improved treatment options with fewer side effects.
