Institute of Biomedical Engineering, University of Toronto, 164 College Street, M5S 3G9 Toronto, Ontario, Canada; Donnelly Centre, University of Toronto, 160 College Street, M5S3E1 Toronto, Ontario, Canada.
Donnelly Centre, University of Toronto, 160 College Street, M5S3E1 Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, 1 King's College Circle, M5S 1A8 Toronto, Ontario, Canada.
Acta Biomater. 2023 Apr 15;161:37-49. doi: 10.1016/j.actbio.2023.03.003. Epub 2023 Mar 8.
Retinitis pigmentosa (RP) is a group of genetic diseases that results in rod photoreceptor cell degeneration, which subsequently leads to cone photoreceptor cell death, impaired vision and eventual blindness. Rod-derived cone viability factor (RdCVF) is a protein which has two isoforms: a short form (RdCVF) and a long form (RdCVFL) which act on cone photoreceptors in the retina. RdCVFL protects photoreceptors by reducing hyperoxia in the retina; however, sustained delivery of RdCVFL remains challenging. We developed an affinity-controlled release strategy for RdCVFL. An injectable physical blend of hyaluronan and methylcellulose (HAMC) was covalently modified with a peptide binding partner of the Src homology 3 (SH3) domain. This domain was expressed as a fusion protein with RdCVFL, thereby enabling its controlled release from HAMC-binding peptide. Sustained release of RdCVFL was demonstrated for the first time as RdCVFL-SH3 from HAMC-binding peptide for 7 d in vitro. To assess bioactivity, chick retinal dissociates were harvested and treated with the affinity-released recombinant protein from the HAMC-binding peptide vehicle. After 6 d in culture, cone cell viability was greater when cultured with released RdCVFL-SH3 relative to controls. We utilized computational fluid dynamics to model release of RdCVFL-SH3 from our delivery vehicle in the vitreous of the human eye. We demonstrate that our delivery vehicle can prolong the bioavailability of RdCVFL-SH3 in the retina, potentially enhancing its therapeutic effects. Our affinity-based system constitutes a versatile delivery platform for ultimate intraocular injection in the treatment of retinal degenerative diseases. STATEMENT OF SIGNIFICANCE: Retinitis pigmentosa (RP) is the leading cause of inherited blindness in the world. Rod-derived cone viability factor (RdCVF), a novel protein paracrine factor, is effective in preclinical models of RP. To extend its therapeutic effects, we developed an affinity-controlled release strategy for the long form of RdCVF, RdCVFL. We expressed RdCVFL as a fusion protein with an Src homology 3 domain (SH3). We then utilized a hydrogel composed of hyaluronan and methylcellulose (HAMC) and modified it with SH3 binding peptides to investigate its release in vitro. Furthermore, we designed a mathematical model of the human eye to investigate delivery of the protein from the delivery vehicle. This work paves the way for future investigation of controlled release RdCVF.
色素性视网膜炎（RP）是一组遗传性疾病，导致视杆细胞变性，随后导致视锥细胞死亡、视力受损，最终导致失明。视杆细胞源性视锥细胞生存因子（RdCVF）是一种蛋白质，有两种亚型：短型（RdCVF）和长型（RdCVFL），作用于视网膜中的视锥细胞。RdCVFL 通过减少视网膜中的高氧来保护感光细胞;然而，持续输送 RdCVFL 仍然具有挑战性。我们开发了一种用于 RdCVFL 的亲和控制释放策略。透明质酸和甲基纤维素（HAMC）的可注射物理混合物通过Src 同源 3（SH3）结构域的肽结合伴侣进行共价修饰。该结构域被表达为与 RdCVFL 的融合蛋白，从而能够从 HAMC 结合肽中控制其释放。首次证明 RdCVFL-SH3 从 HAMC 结合肽体外释放 7 天。为了评估生物活性，从鸡视网膜分离物中收获并用 HAMC 结合肽载体中的亲和释放重组蛋白处理。在培养 6 天后，与对照相比，用释放的 RdCVFL-SH3 培养的锥体细胞活力更高。我们利用计算流体动力学来模拟我们的输送载体在人眼玻璃体内释放 RdCVFL-SH3。我们证明我们的输送载体可以延长 RdCVFL-SH3 在视网膜中的生物利用度，从而增强其治疗效果。我们的基于亲和性的系统构成了一种多功能的输送平台，可用于最终在眼内注射治疗视网膜退行性疾病。意义声明：色素性视网膜炎（RP）是世界上导致遗传性失明的主要原因。视杆细胞源性视锥细胞生存因子（RdCVF）是一种新型的旁分泌因子，在 RP 的临床前模型中有效。为了延长其治疗效果，我们开发了一种用于 RdCVF 长型（RdCVFL）的亲和控制释放策略。我们将 RdCVFL 表达为与Src 同源 3 结构域（SH3）融合蛋白。然后，我们利用由透明质酸和甲基纤维素（HAMC）组成的水凝胶，并对其进行 SH3 结合肽修饰，以研究其体外释放情况。此外，我们设计了一个人类眼睛的数学模型，以研究从输送载体输送蛋白质。这项工作为进一步研究控制释放的 RdCVF 铺平了道路。
