Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition

Abstract

BackgroundTo develop targeted antifibrotic therapy for glaucoma filtration surgery; this study determines the effectiveness of small interfering RNA (siRNA) to reduce in vivo secreted protein acidic and rich in cysteine (SPARC) expression using the mouse model of conjunctival scarring.MethodsExperimental surgery was performed as described for the mouse model of conjunctival scarring. Scrambled (siScram) or Sparc (siSparc) siRNAs, loaded on layer-by-layer (LbL) nanoparticles, were injected into the conjunctiva immediately after surgery. Expression of Sparc, Col1a1, Fn1 and Mmp14 was measured by real-time PCR and immunoblotting on days 7 and 14 postsurgery. Live imaging of the operated eyes was performed using slit lamp, anterior segment-optical coherence tomography and confocal microscopy. Tissue pathology was evaluated by histochemical and immunofluorescent analyses of operated conjunctival cryosections. Tissue apoptosis was quantitated by annexin V assay.ResultssiSparc, delivered via expanded LbL nanoparticles, significantly inhibited Sparc transcription in both day 7 (2.04-fold) and day 14 (1.39-fold) treated tissues. Sparc suppression on day 7 was associated with a significant reduction of Col1a1 (2.52-fold), Fn1 (2.89-fold) and Mmp14 (2.23-fold) mRNAs. At the protein level, both SPARC and collagen 1A1 (COL1A1) were significantly reduced at both time points with siSparc treatment. Nanoparticles were visualised within cell-like structures by confocal microscopy, while overt tissue response or apoptosis was not observed.ConclusionsSPARC targeted therapy effectively reduced both SPARC and collagen production in the operated mouse conjunctiva. This proof-of-concept study suggests that targeted treatment of fibrosis in glaucoma surgery is safe and feasible, with the potential to extend to a range of potential genes associated with fibrosis.

Breaking Barriers in Eye Treatment: Polymeric Nano-Based Drug-Delivery System for Anterior Segment Diseases and Glaucoma

“…Further optimizing the dosage to ensure the sustained release of siSPARC is required. siSPARC was also delivered with similar anti-fibrotic characteristics using LbL (layer-by-layer) nanoparticles (Seet et al, 2018) [ 141 ].…”

Looking into the future: Gene and cell therapies for glaucoma

“…Gene therapy has a great potential to specifically target and modify these pathways for safe and long‐term prevention of bleb fibrosis. For example, small interfering RNA (siRNA) was developed to silence transcription factors involved in conjunctival tissue fibrosis: the myocardin‐related transcription factor/serum response factor (MRTF/SRF) pathway or secreted protein acidic and rich in cysteine (SPARC) 143‐145 . Intravitreal antisense oligonucleotides against transforming growth factor‐ beta2 (TGF‐ beta2 ) showed promising results in human subjects with OAG undergoing trabeculectomy 146 .…”

Is the Conjunctiva a Potential Target for Advanced Therapy Medicinal Products?

“…There have been several successful examples in experimental models, but few techniques have reached the clinical trial stage in humans. An example is the development of a small interfering RNA to silence transcription factors involved in conjunctival tissue fibrosis, such as the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway or secreted protein acidic and rich in cysteine (SPARC) [ 88 , 89 , 90 ]. Another example is the presurgical subconjunctival injection or topical administration onto the surgical field of recombinant adenovirus with the human p21 transgene (encoding the CDKN1A protein) in rabbits [ 91 ].…”