Duchenne Muscular Dystrophy (DMD) is a rare genetic disease that prevents muscle regeneration, which occurs at the early childhood stage: 3-5 years old. Over time, children with DMD will develop difficulties in walking and breathing due to the muscle breakdown. This disease is caused by a genetic mutation that prevents the body from producing dystrophin, a protein that muscles need in order to work properly. A potential treatment for most DMD patients is gene therapy for the delivery of a therapeutic gene to skeletal and cardiac muscle, in order to restore the dystrophin protein. To increase the stability of the gene against degradation, the use of particulate carriers may be considered as the more realistic approach of the gene delivery [1]. Hydrogels are three-dimensional, cross-linked networks that are used to provide a fundamental tool for a variety of clinical applications including gene therapy for inherited disorders and drug delivery [2]. PEG fibrinogen (PF) hydrogel, which was formulated in Seliktar lab (figure.1A) with adjustable physical and mechanical properties can be used as a suitable delivery platform for the therapeutic gene in order to restore the dystrophin protein expression. For this purpose, biocompatible, biodegradable and semi-permeable PEG-Fibrinogen hydrogel microspheres (figure.1B) were designed for the encapsulation of the gene with the purpose of facilitating delivery to the local muscle tissue and providing protection against the gene degradation and clearance.

INTRODUCTION

EXPERIMENTAL

RESULTS

Gene Therapy using PEG-fibrinogen Hydrogel Controlled Release System for the treatment of Duchenne Muscular Dystrophy.

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Summary

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Joleen Tanous and Nathan Slotnik

This project was conducted at Prof. Dror Seliktar lab, Faculty of Biomedical Engineering, Technion

Mentor: Ph.D Candidate Shani Cohen

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Encapsulation Technique

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Size Distribution of PF Microspheres Microspheres Images by using Light Microscopy

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Figure 5: Light microscopy images of PF microspheres with addition of 1% (A), 2%(B), 3%(C) of PEG-DA.

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Figure 4: Size distribution of PF microspheres using laser diffraction method usingMastersizer 3000, n=3.

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Figure 3: peak value of storage modulus G' during cross-linking of PF with addition of 1%, 2%, 3% of PEG-DA.

Table 1: Average of size distributionof PF microspheres using laserdiffraction method using Mastersizer,n=3.

Figure 2: Evolution in time of storage modulus G' measured by rheometer during polymerization of PF with addition of 1%(A), 2%(B), 3%(C) PEG-DA . The higher the G’ value, the higher the cross-linking density and mechanical stiffness of the hydrogel.

Fluorescent RNA Loaded Microspheres

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Figure 7: Confocal Images of PF microspheres loaded with (6-FAM) fluorescentRNA with addition of 1% (A), 2% (B) and 3% (C) of PEG-DA.

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Acknowledgments

References[1] Lim KR, Maruyama R, Yokota T. Drug Des Devel Ther. (2017) Feb 28;11:533-545.[2] Almany, L., & Seliktar, D. (2005). Biomaterials, 26(15), 2467–2477.

• Addition of PEG-DA to the hydrogel precursor results with higher G’ value (figures 2 and 3)• PF microspheres were not toxic to C-57 mouse satellite cell culture after 24 hours of

incubation (figure 6)• RNA polyplexes were successfully encapsulated within PEG-Fibrinogen microspheres by dual

photo initiator emulsion technique (figure 7)• Characterization of microspheres result with spherical microspheres with uniform size and

with homogenic RNA polyplexes distribution (figures 4,5 and 7)

Live/Dead Toxicity Experiment -24H

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B CAFigure 6: Live-Dead experiment 24h after treatment with PF microspheres.Live/Dead staining with Calcein (green) and Ethidium (red) show a relatively high number of viable cells.

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We would like to thank PhD candidate Shani Cohen and Prof. Dror Seliktar for hostingand guiding us through our research in his laboratory. We would also like to thank thefoundations and donors for their generous support of the SciTech Program.

PEG-Fibrinogen Hydrogel AssemblyPEGylated Denatured Fibrinogen

UV Photo-initiator

A. Assembly of PF hydrogel by UV photo-polymerization [1]B. PF microspheres fabrication using emulsion based dual photo initiator techniqueC. PF microspheres characterization by mastersizer and by light and confocal microscopy

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