Preclinical development of genes therapies in a genetic eye disease

  • Amanda Kowalczyk

Student thesis: Doctoral Thesis


Using small interfering RNA (siRNA) as a therapeutic has been proven to be successful in a clinical setting. However, treating TGFBI corneal dystrophies can be a unique challenge as the treatment requires allele-specificity which has not been achieved clinically before. The unique qualities of the cornea offer both challenges and advantages when it comes to delivery of the siRNA, therefore an efficient delivery mechanism is essential.

Allele-specific siRNA designs to silence R124H-TGFBI were tested with dual-luciferase reporter assays. The best performing candidate siRNA4 was validated in vitro in a combination of cell lines. The siRNA was confirmed to be allele-selective. Safety of the siRNA was assessed with in silico off-target analysis, and absence of silencing of a panel of potential off-target genes was confirmed by RT-qPCR and RNA-seq.

After confirming that the delivery system works in vitro, in vivo experiments on an R124H-TGFBI mouse model were performed. Stemloop-qPCR was used to quantify the siRNA4 delivered to the cornea, while RT-qPCR was used to quantify TGFBI silencing. Fluorescent microscopy assessed the penetration to the cornea by using a Cy5.5-tagged siRNA4. No significant silencing or siRNA4 presence was observed in the treated corneas. Methods of tracking siRNA biodistribution were also developed. Tested chemical modifications did not improve efficacy of siRNA in vivo or in vitro.

As the proof of concept in a mouse model was unsuccessful, further work is required. This includes improved intrastromal injections acting as a positive control against the delivery system by bypassing corneal barriers. Improvements to the topical delivery can also be investigated by adding compounds that improve bioavailability of the siRNA to the cornea. An alternative proof of concept can be also used and developed by using patient-derived keratocytes that contain TGFBI with R124H mutation in conjunction with extensive in vivo work proving safety of the drug product ahead of clinical trials in humans. Lastly, to precisely measure the penetration to the cornea, confocal laser microscopy can be used.
Date of AwardJun 2023
Original languageEnglish
SupervisorAndrew Nesbit (Supervisor), Tara Moore (Supervisor) & Paulina Baran-Rachwalska (Supervisor)


  • siRNA
  • Topical application
  • Corneal dystrophy
  • Allele-specific siRNA

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