Character Bio at ARVO 2025
Poster A0074: Optimization of lipofection-based RNA interference in mature human induced pluripotent stem cell-derived retinal pigmented epithelium
David E. Buchholz, Adria R. Frazier, Maria Avrutsky, Jonathan Gumucio, Laura Lee Carter, Erik Karrer
Purpose
Cation lipid-based commercial transfection reagents pose a simple and economical delivery option for small interfering RNA (siRNA), however mature, postmitotic cells are notoriously difficult to transfect. In particular, this has proven true for cultures of the retinal pigmented epithelium (RPE). We screened and optimized transfection conditions using eleven lipofection-based commercial reagents to enable RNA interference (RNAi) in mature RPE derived from human induced pluripotent stem cells (iPSC-RPE).
Methods
Human iPSC-RPE were purchased from Fujifilm Cellular Dynamics (#R1101) and grown on 96-well transwell permeable supports for >42 days, reaching transepithelial electrical resistance (TEER) of ≥400 Ω*cm2. Commercial lipofection reagents Lipofectamine RNAiMAX, Lipofectamine Stem, Lipofectamine LTX (ThermoFisher), Metafectene PRO (Biontex), TransIT-TKO, TransIT-X2, TransIT-siQUEST (Mirus Bio), and DharmaFECT 1–4 (Dharmacon) and siRNAs (ThermoFisher) were screened using 1.6 μL of transfection reagent and 4.3 pmol siRNA directed to a specific target. Further optimization was performed on the best performing reagents. Reagents were assessed for their ability to not interfere with cell physiology (transepithelial electrical resistance, TEER) and to reduce mRNA levels of the specific target. TEER was recorded pre-transfection and 48 h post-transfection. RNA knockdown efficiency was measured by qRT-PCR at 48 h post-transfection.
Results
Lipofectamine RNAiMAX, Lipofectamine Stem, Lipofectamine LTX, TransIT-TKO, TransIT-X2, and Metafectene Pro had no deleterious effect on TEER. RNA knockdown efficiency ranged from 0–60%, with DharmaFECT 3 and Metafectene PRO producing the highest knockdown efficiency of ~60%.
Conclusions
Of the 11 transfection reagents tested, Metafectene PRO had the greatest RNA knockdown efficiency without affecting TEER. This transfection approach could prove useful for rapid interrogation of gene function by RNAi in fully mature iPSC-RPE cultures.
Poster B0510: Association between drusen volume and progression to advanced dry AMD in a large longitudinal study of intermediate AMD
Joe Davis, Benjamin S. Glicksberg, Zehua Wei, Yunju Yang, Andrea Neeranjan, Laura Lee Carter, Dinah Chen
Purpose
To investigate the relationship between drusen volume and risk of conversion to advanced dry age-related macular degeneration (AMD) in patients with intermediate AMD (iAMD).
Methods
This retrospective, real-world observational study consists of a platform containing multi-modal data, e.g., imaging, electronic health records, from consented participants from over 120+ clinics across the United States. Imaging data included full volumetric spectral-domain optical coherence tomography (SD-OCT). We analyzed a baseline population of 1306 study eyes (more advanced eye, earliest date of first visit) that started with iAMD and at least one supporting SD-OCT. Drusen volume was quantified using a segmentation machine learning model at each visit on SD-OCT. Development of advanced dry AMD, as identified by large hypertransmission defect (defined as bright regions having a greatest linear dimension that is ≥ 250 μm on an enface sub-RPE generated slab), was assessed by an automated segmentation algorithm. To evaluate the association between baseline drusen volume and progression to advanced dry AMD, we used a repeated measures logistic regression model with a random intercept for study eye and fixed effects for gender, years from baseline, and baseline drusen volume and age.
Results
Patients were followed for a median of 4.0 years, with a median of 2.8 visits per year. The baseline characteristics of our population (N = 1306 study eyes) were: median age of 74.8 years, 503 males versus 803 females, median baseline drusen volume of 0.041 mm3. 4.5% and 23.2% of patients converted to advanced dry disease within 3 and 5 years, respectively. Patients with baseline <0.2 mm3 convert within 5 years at a significantly lower rate than those with baseline >0.2 mm3 (19.4% and 41.8%, respectively; p-value 2.3x10–4). Controlling for baseline age, follow-up time, and sex, we observe a highly significant effect for baseline drusen volume as a predictor of conversion (log odds ratio 8.5; p-value 8.3x10–21).
Conclusions
We found that baseline drusen volume is a significant predictor of progression to advanced dry AMD in patients with intermediate AMD. These findings highlight the potential utility of drusen volume as a biomarker for stratifying patients at higher risk of disease progression, and support using drusen volume as a target for future therapies or to optimize clinical intervention.
Poster A0502: Enhanced surface binding activity of CTX114 augments multiple physiological functions of Complement Factor H.
Maria Avrutsky, Nate Mooi, Nadkarni Tejasveeta, Laura Lee Carter, Erik Karrer
Purpose
CTX114 is a novel complement inhibitor designed to reduce the rate of retinal cell death and vision loss in geographic atrophy associated with age-related macular degeneration (AMD). Loss of retinal surface binding activity by the Y402H variant of Complement Factor H (CFH) and its alternative splicing isoform, FHL-1, is one of the strongest genetic risk factors for AMD. CTX114 was designed via protein engineering as an enhanced version of FHL-1 to confer increased retinal surface binding and complement regulatory activity. Here we characterize modeled retinal surface binding activity and biomolecule displacement activity of CTX114 in comparison with CFH and FHL-1.
Methods
Competitive binding assays were performed by ELISA using microplates functionalized with heparin (surrogate for retinal surfaces) or MDA (malondialdehyde, surrogate for oxidized retinal surfaces). Complement inhibitory activity was measured by WIESLAB Complement assay. Concentration response was quantified by nonlinear regression averaged across multiple experiments to calculate standard deviation and mean IC50 values.
Results
CTX114 (IC50 = 18.9 ± 8.1 nM), CFH (IC50 = 179.4 ± 12.7 nM), and FHL-1 (IC50 = 324.2 ± 37.1 nM) inhibited ApoB binding to heparin. CTX114 inhibited heparin binding by ApoA-I (IC50 = 168.7 ± 188.2 nM) and ApoE (IC50 = 1.6 ± 0.5 nM), while CFH and FHL-1 did not have activity in those assays. Commercial C3/C5 inhibitors had no inhibitory activity towards lipoprotein binding to heparin. MDA-coated ELISA plates potentiated deposition of C1q, which was suppressed by CTX114 (IC50 = 48.1 ± 5.5 nM) and anti-C1q antibody ANX005 (IC50 = 0.6 ± 0.2 nM), but not by CFH/FHL-1 or commercial complement C3/C5 inhibitors. In a C1q-dependent complement activation assay (WIESLAB Classical Complement Assay), CTX114 potently inhibited complement activity (IC50 = 12.6 ± 1.8 nM).
Conclusions
CTX114 impacts multiple nodes in the complement activation and amplification cascade to protect modeled retinal surfaces from AMD-relevant stressors. In addition to regulating complement alternative pathway, CTX114 protects modeled retinal surfaces from lipoprotein deposition, inhibits C1q deposition on MDA epitopes, and inhibits classical complement activity. These preclinical studies further support advancement of CTX114 as a novel, best-in-class therapeutic candidate for treatment of AMD.
Layman Abstract:
Age-related macular degeneration (AMD) is a chronic, progressive disorder which damages retinal cells and is a major cause of vision loss in older adults. Evidence from human genetics implicates deficient retinal surface binding activity of an endogenous complement regulator (CFH/FHL-1) in multiple aspects of AMD disease progression. CTX114 is a novel complement inhibitor designed to augment the retinal surface binding activity of FHL-1, to reduce the rate of retinal cell death and vision loss in geographic atrophy, an advanced form of AMD. Here, we present preclinical data evaluating how CTX114 protects surfaces from challenges relevant to retinal damage in AMD.
