Targeted downregulation of VEGF-A to inhibit angiogenesis in psoriasis using CRISPR/Cas9 complexed lipopolymeric nanoplexes

Psoriasis, an incurable chronic inflammatory skin disease, is characterised by enhanced angiogenesis, abnormal keratinocyte differentiation, and immune cell invasion. Angiogenesis, a critical factor in endothelial cell proliferation, adhesion, and migration, is driven by the overexpression of the VEGF-A gene. Downregulating VEGF- A offers a potential therapeutic strategy for angiogenesis-related disorders. This study investigates the application of CRISPR/Cas9 gene editing to target and downregulate VEGF-A expression in psoriasis. However, their large size and negative charge hinder the delivery of CRISPR/Cas9 components in vitro and in vivo. To address these challenges, we developed lipopolymeric nanoplexes to deliver Cas9 RNPs specifically to target cells. The nanoplexes were characterised by a particle size of 142.2 nm, a PDI of 0.144, and a zeta potential of +4.27 mV. In vitro studies demonstrated over 70% transfection efficiency in HaCaT cells, with an Indel frequency of approximately 30% for the VEGF-A gene. The nanoplexes were further characterised using XPS, confirming their stability and low toxicity. Hemocompatibility assays indicated non-toxicity. Ex vivo skin permeation analysis showed a 66% permeation rate after 24 hours. The optimised nanoplex formulation was incorporated into a carbopol-based gel with non-Newtonian flow characteristics, exhibiting shear-thinning behaviour and variable thixotropy. This gel-delivered nanoplex achieved a 48% skin permeation rate after 24 hours in ex vivo mouse skin. In vivo skin toxicity testing confirmed a low toxicity profile. Efficacy assessment in a psoriasis-like inflammation model in Swiss albino mice demonstrated significantly improved PASI scores, reduced skin damage, and decreased proliferation compared to the naked RNP and Blank gel. The enhanced cellular uptake, high skin penetration with increased skin retention, and improved efficacy collectively highlight the potential for future clinical applications in treating psoriasis.

Audience Take Away Notes:

Advanced Delivery Techniques:

• How we've developed lipopolymeric nanoplexes to deliver CRISPR/Cas9 components precisely to target cells, overcoming traditional delivery challenges\
• The detailed characterization process, including particle size, PDI, and zeta potential, and how these parameters influence delivery efficiency

Gene Editing for Psoriasis:

• The crucial role of VEGF-A in psoriasis and the therapeutic potential of its downregulation using CRISPR/Cas9
• The significant in vitro, ex vivo, and in vivo results demonstrate this approach's potential to alleviate psoriasis symptoms

Formulating Topical Treatments:

• The development of a carpool-based gel that effectively delivers our nanoplexes, featuring desirable properties like shear-thinning behavior
• Insights into how these properties enhance the gel's performance and application in a clinical setting

Safety and Efficacy:

• Comprehensive methods for evaluating hemocompatibility, toxicity, and skin permeation, ensuring the treatment's safety
• Robust in vivo efficacy assessments, showing marked improvements in psoriasis models

Future Clinical Potential:

• The implications of this research for future clinical applications, including potential adaptations for other angiogenesis-related conditions
• Steps towards translating this innovative approach into human treatments, paving the way for new therapies

Research Applications:

• Researchers can apply our delivery techniques and characterization methods to other gene-editing projects, potentially expanding the scope of their work
• Insights from our study can inspire new research directions, particularly in targeting gene expression for various diseases

Clinical Practice:

• Clinicians will gain an understanding of emerging treatments for psoriasis, which could inform future therapeutic strategies and patient care
• Safety and efficacy assessment techniques can be adapted for evaluating other topical treatments

Pharmaceutical Development:

• Pharmaceutical scientists can use our findings to design more efficient delivery systems for gene therapies and other treatments
• Our methodologies for optimizing nanocarrier formulations can streamline the drug development process, making it more efficient and effective

Benefits to the Audience:

Expanding Research and Teaching:

• Faculty can incorporate these advanced techniques and findings into their teaching, enriching the education of students in the fields of gene therapy and nanotechnology
• Researchers can build on our work to explore new treatment avenues, potentially leading to breakthroughs in other areas

Practical Solutions:

• The study provides practical solutions for delivering large and negatively charged gene-editing components, addressing a significant challenge in the field
• Our optimized delivery system simplifies the development process for other gene therapies, making them more accessible

Improved Accuracy and Information:

• Enhanced delivery and targeting mechanisms improve the accuracy and efficacy of gene-editing therapies
• Detailed characterization and assessment techniques offer valuable information for solving design problems in drug delivery systems

Overall Benefits:

• The potential for improved therapeutic outcomes for patients with psoriasis and possibly other conditions
• A solid foundation for future innovations in nanomedicine and gene therapy, driving the field forward