01 Introduction
Vitreous floaters are visual phenomena resulting from floating opacities inside the eye, primarily consisting of aggregated collagen fibers that cast shadows on the retina as light enters the eye. With an aging population and increasing prevalence of myopia, the incidence of floaters continues to rise.
Although often considered benign, recent studies confirm that floaters significantly impact quality of life. Patients with symptomatic floaters may experience decreased contrast sensitivity function, leading to impaired visual quality. One study demonstrated that symptomatic degenerative vitreous floaters negatively affect health-related quality of life, with younger patients (≤55 years) more willing to risk blindness to eliminate floater symptoms. The degree of distress is difficult to quantify with routine visual acuity tests but undeniably affects daily visual activities.
Current clinical treatments include YAG laser vitreolysis and pars plana vitrectomy. An estimated 40,000 vitrectomies for symptomatic vitreous opacities are performed annually in the United States. However, these invasive procedures carry risks including retinal detachment and infection. Thus, a non-invasive therapeutic strategy targeting the underlying pathology is urgently needed.
02 Design of Modules (please click the text box)
Module ① Promoter & Translation Enhancement
Elements: CMV enhancer promoter
Function: Drives high-efficiency transcription and translation of downstream genes（like FOXOX3A-TM & HAS2）
Highlight: CMV promoter ensures long-term expression in ocular cells (e.g.HVC, ARPE-19).
Module ② Core Effector – FOXO3A-TM
Elements: Constitutively active FOXO3A mutant (T32A/S257A/S315A)
Function: Sustained nuclear localization; activates antioxidant enzymes (MnSOD, Catalase); inhibits matrix metalloproteinases (MMP-2/9)
Highlight: Triple mutation of phosphorylation sites to alanine resists oxidative stress-induced inactivation, mimicking persistent FOXO3A activation.
Module ③ Co-expression Linker
Elements: GSG Linker + P2A self-cleaving peptide
Function: Enables 1:1 stoichiometric expression of FOXO3A-TM and HAS2 as independent proteins
Highlight: High-efficiency P2A cleavage with GSG optimization ensures functional separation without interference.
Module ④ Matrix Repair Effector – HAS2
Elements: Hyaluronan Synthase 2
Function: Catalyzes hyaluronic acid (HA) synthesis to restore vitreous matrix stability
Highlight: Synergizes with FOXO3A to double-guarantee HA production, reversing vitreous liquefaction.
Module ⑤ Leakage-preventing part
Elements: 4×miR-124
Function: If our plasmid "escape" into retinal cells which have high-abundance miR-124, miR-124 will act as a guide, binding to the 4×miR-124 targets of the mRNA. This recruits the AGO2-RISC complex, which functions as molecular scissors to cleave and degrade the transcript, effectively silencing off-target protein expression to protect retinal neurons while allowing treatment in the vitreous.
Module ⑥ Smart Negative Feedback
Elements: minTK promoter + 4×NF-κB response elements (reverse orientation)
Function: Upon HA overproduction, NF-κB activation drives reverse transcription, generating antisense RNA that pairs with forward mRNA to form dsRNA and block translation
Highlight: Auto-titration mechanism prevents excessive HA synthesis, enhancing therapeutic safety via “on-demand” regulation.
Module ⑦ Termination & Backbone
Elements: bcr poly(A) signal + pVXA1 backbone
Function: mRNA polyadenylation for stability; backbone with ampicillin resistance for bacterial amplification
Highlight: Commercially proven vector ensures stable expression and facilitates downstream cell line generation.
03 Genetic Circuit & Parts
Mechanism of Engineered Exosome Targeting
Functional Plasmid:pVXA1-FOXO3A-TM-HAS2-4×miR-124-NFκB (6977 bp) – HindIII/ApaI insertion – full vector map
Targeting Plasmid
All modules designed as BioBrick compatible parts with reference support.
04 Experimental Design
Overall Experimental Design
1. Genetic Circuit Construction
Vector pVXA1 (6977 bp) with HindIII/ApaI insertion. Modules: CMV promoter + FOXO3A-TM (T32A/S257A/S315A), GSG-P2A, HAS2, -4×miR-124, minTK+4×NF-κB (reverse), bcr poly(A). Exosome targeting module (CD44-HCPSYGRS fused to LAMP2B) constructed separately.
2. Stable Cell Line Generation
Co-transfect HEK-293FT with recombinant plasmid + psPAX2 + pMD2.G. Harvest virus, transduce human vitreous cells (HVC) or ARPE-19. Select with kalamycin (2 μg/mL) for 2 weeks.
3. Exosome Production & Purification
Culture stable cells in exosome-depleted medium 48-72 h. Purify: 0.22 μm filtration → TFF (100 kDa) → SEC (qEV). QC: NTA (~120 nm), Western blot (CD9/CD63⁺, Calnexin⁻), detect FLAG and LAMP2B.
4. In Vitro Functional Validation
Targeting: Cy5-exosomes + confocal.
Autophagy: LC3-II/LC3-I, p62, LysoTracker.
Anti-fibrosis: COL1, FAP, FSP-1.
HA synthesis: ELISA.
Pathway: p-mTOR/mTOR, p-Smad2/3, FOXO3A nuclear localization.
5. Disease Model Validation
In vitro fibrosis model: TGF-β1 (10 ng/mL, 72 h) in vitreous cells + exosome treatment. High-content imaging (Calcein-AM/PI, LysoTracker, JC-1). RNA-seq for KEGG/GO (autophagy, ECM-receptor, TGF-β).
PCR Detection of Exosomal Plasmid
Exosomes are treated with DNase I, lysed, and internal DNA extracted.PCR targeting plasmid-specific sequences confirms encapsulation.Controls: non-DNase-treated exosomes, medium, non-engineered exosomes, and plasmid positive control. DNase-treated (+) and medium (-) confirm packaging.
05 Team & Contributions
Xu Houdian
Genetic circuit design, experimental design
Zhan Yuzhi
Web development, PPT creation
Lin Ziqing
Artwork & illustrations
06 References
- Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol. 2010 Apr;22(2):124-31. doi: 10.1016/j.ceb.2009.11.014
- SGK1 is necessary to FoxO3a negative regulation, oxidative stress and cardiac fibroblast activation induced by TGF-β1. Cell Signal. 2023 Sep;109:110778. doi: 10.1016/j.cellsig.2023.110778
- The miR-15b-5p/miR-379-3p-FOXO axis regulates cell cycle and apoptosis in scleral remodeling during experimental myopia. J Transl Med. 2024 Jul 30;22(1):710. doi: 10.1186/s12967-024-05523-x
- Retinal cell-targeted liposomal ginsenoside Rg3 attenuates retinal ischemia-reperfusion injury via alleviating oxidative stress and promoting microglia/macrophage M2 polarization. Free Radic Biol Med. 2023 Sep;206:162-179. doi: 10.1016/j.freeradbiomed.2023.06.024
- An anti-FAP-scFv-functionalized exosome-carrying hydrogel delivers SKI mRNA to fibrotic nucleus pulposus cells to alleviate intervertebral disc degeneration by regulating FOXO3. Theranostics. 2025 Mar 3;15(9):3877-3899. doi: 10.7150/thno.107776
- Exosome-loaded degradable polymeric microcapsules for the treatment of vitreoretinal diseases. Nat Biomed Eng. 2024;8(11):1436-1452. doi:10.1038/s41551-023-01112-3