OPTIDICER REDUCES LONG CUG RNA ACCUMULATION IN 
CORNEAL ENDOTHELIAL CELLS FROM PATIENTS WITH 
FUCHS’ DYSTROPHY 
 
 
 
 
 
 
 
by 
SANJANA CAROLINE BASAK 
 
 
 
 
 
 
 
 
 
 
A THESIS 
 
 
 
Presented to the Department of Biology  
and the Robert D. Clark Honors College  
in partial fulfillment of the requirements for the degree of  
Bachelor of Science 
 
June 2024 
 
 
 
 
An Abstract of the Thesis of 
Sanjana C. Basak for the degree of Bachelor of Science 
in the Department of Biology to be taken June 2024 
 
 
Title: Optidicer Reduces Long CUG RNA Accumulation in Corneal Endothelial Cells From 
Patients With Fuchs’ Dystrophy 
 
 
 
 
Approved:  Balamurali K. Ambati, M.D., Ph.D.  
Primary Thesis Advisor 
 
This study will examine the viability of the modified endonuclease, known as OptiDicer, 
in preventing corneal endothelial cell loss and halting the progression of late-onset Fuchs’ 
Endothelial Corneal dystrophy (FECD). FECD is a debilitating, heritable disease that could 
impact 415 million people by the year 2050 (Aiello et al., 2022). The consequences of this 
disease are significant and include loss of vision, painful corneal edema, and collagen 
excrescences. The current treatment for FECD involves surgical transplantation of donor 
corneas. This treatment is limited in its capability due to high costs, high transplant rejection and 
infection rates, and painful long recuperation periods. Developing a targeted gene therapy such 
as OptiDicer could provide a watershed medical care advancement for patients suffering from 
late-onset FECD.   
This disease manifests from a progressive decrease in corneal endothelial cell density. 
Prior research indicates that a trinucleotide expanded repeat mutation in the TCF4 gene affects 
diseased corneal endothelial cells. The excessive repetition of the CTG nucleotide in this gene 
leads to the overproduction of CUG RNA in the cell nucleus. The overabundance of RNA forms 
distinct structures, or foci, which are toxic to the corneal endothelial cells. Our treatment, 
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OptiDicer, is a modified form of the endogenous protein DICER1 that cleaves CUG RNA in cell 
nuclei via RNase III activity. Unlike DICER1, OptiDicer is miRNA-resistant and can 
continuously cleave accumulated CUG RNA foci. We expect that OptiDicer will significantly 
reduce CUG RNA foci in corneal endothelial cell nuclei and prevent endothelial cell death in the 
cornea. 
  
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Acknowledgements 
 
I’d like to thank the members of the Ambati Lab: Dr. Hiro Uehara, Dr. Sangeetha Ravi-
Kumar, Bonnie Archer, Kieley Trempy, Baila Shakaib, Saumya Keremane, and Dr. Bala 
Ambati. Working and learning from this team for over 3 years has been the highlight of my 
college experience. I’d like to give special thanks to Hiro for being a wonderful mentor; he 
believed in my potential and gave me a chance to prove myself as a researcher in training. 
Interacting with Hiro, Sangeetha, and the rest of the Ambati lab inspired me to pursue a 
biomedical PhD and dedicate my career to research. I’m incredibly lucky to have found such a 
welcoming, kind, supportive, and talented group of scientists in my time at UO.  
I am dedicating this thesis to my parents, Gloria and Indranil Basak. Their loving support 
has allowed me the freedom in college to explore my interests and pursue my ambitions. I’m so 
grateful to have them as my family and to celebrate this moment in my life with them.  
This thesis would not be possible without the snuggles provided by my best buddy and 
darling shih tzu, Opus Basak.  
  
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Table of Contents 
INTRODUCTION 7 
Cornea Physiology and FECD Pathology 7 
FECD Genetic Markers and Underlying Mechanisms 10 
Current and Future Treatments for FECD 16 
METHODS 17 
OptiDicer Design and Plasmid Construction 17 
Immunofluorescence 18 
Western blot 19 
Fluorescent in situ hybridization (FISH) 20 
RT-PCR for RNA splicing analysis 21 
RESULTS 22 
Development of nuclear OptiDicer 22 
NLS-OptiDicer reduced CUG-RNA accumulations in the nucleus 24 
NLS-OptiDicer shifts RNA splicing 26 
DISCUSSION 27 
Bibliography 28 
 
  
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List of Figures  
Fig 1: Human Eye and Cornea Physiologies. 8 
Fig 2: Patient with advanced Fuchs’ dystrophy. 10 
Fig. 3: Long (CTG)n repeat (n>30-40) in TCF4 is associated with FECD. 13 
Fig 4: Comparison of OptiDicer modifications and endogenous DICER1. 17 
Fig. 5: 40x immunofluorescence and western blot gel images showing DICER1, OptiDicer, and 
NLS-OptiDicer expression in F35T cells and control Hela cells. 23 
Fig. 6: FISH visualization of CUG RNA foci in F35T cells. A, (CAG)6- CA-5′ Texas red-labeled 
2-Omethyl RNA 20-mers probe concentration assay. B,  NLS-OptiDicer and D2A control 
transfection and untreated control with 0.08 ng/uL RNA probe. 25 
Fig. 7: RT-PCR gel images showing splicing of MBNL1 and ADD3 with NLS-OptiDicer 
transfection in F35T cells and control corneal endothelial cells. 26 
 
 
 
 
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INTRODUCTION  
Late-onset Fuchs’ endothelial corneal dystrophy (FECD) is a heritable degenerative 
disorder that is characterized by progressive vision loss and corneal edema. FECD has been 
demonstrated to be prevalent in Caucasian and Asian populations in adults aged sixty years or 
older (Mootha et al., 2015; Jurkunas et al., 2010). The disorder has been documented in 11% of 
females and 7% of males in Reykjavik, Iceland, 8.5% of Chinese Singaporeans, 5.5% of 
Japanese citizens, and 5% of Caucasians older than 40 years in the United States (Mootha et al., 
2015). In the U.S., 14,153 keratoplasties out of 72,736 total corneal transplants in 2013 were 
performed to treat Fuchs’ dystrophy (Xing et al., 2014). In 2022, Fuchs’ dystrophy and 
endothelial dysfunction accounted for 28,143, or 59.7%, of the keratoplasty procedures 
performed in the U.S. (Mathews et al., 2023). With FECD projected to impact 415 million 
people globally by 2050, patients and practitioners would greatly benefit from novel treatments 
for this disorder (Aiello et al., 2022).   
 
Cornea Physiology and FECD Pathology 
Fuchs’ dystrophy is a corneal disease that manifests from a dysfunctional corneal 
endothelium. Composed of three cellular layers (epithelium, stroma, and endothelium) and two 
acellular layers (Bowman’s and Descemet’s membranes), the transparent cornea regulates vision 
quality by focusing images on the retina (Faye et al., 2021). The outermost cellular layer, the 
epithelium, is constructed as a multilayered epithelial sheet of self-renewing cells that continue 
to achieve mitosis (Sheerin et al., 2012). The stroma is a cellular layer below the epithelium, 
structured as a lattice of collagen and proteoglycans maintained by keratocytes (Sheerin et al., 
2012). The innermost cellular layer is the endothelium, a terminally differentiated cell monolayer 
7 
 
 
that separates the cornea from aqueous humour (aqueous fluid) (Sheerin et al., 2012). Due to the 
absence of growth factors in the aqueous humor, corneal endothelial cells are unlikely to enter 
mitosis and initiate self-regeneration (Sheerin et al., 2012). 
 
 
 
 Fig 1: Human Eye and Cornea Physiologies.  
 
A functional corneal endothelium maintains a cell density of 400-500 cells per square 
millimeter (Sarnciola et al. 2019). The cells are connected at their apical surfaces by tight 
junctions and joined to adjacent cells by gap junctions (Haschek et al. 2002). The endothelial 
cells regulate corneal hydration, thickness, and transparency by pumping aqueous fluid between 
the stroma and the anterior chamber of the cornea (Nanda et al., 2019). Aqueous fluid is pumped 
into the stroma to facilitate the active transport of nutrients (Sheerin et al., 2012). In contrast, 
aqueous fluid is pumped out of the stroma so the structure can attain a state of dehydration or 
deturgescence to maintain corneal transparency (Nanda et al., 2019). The movement of aqueous 
fluid between the stroma and anterior chamber is mediated by sodium-potassium (Na+/K+) and 
bicarbonate anion-activated ATPase pumps located in the outer endothelial cell membrane 
(Haschek et al. 2002). 
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The progressive loss of corneal endothelial cell density and alterations in underlying 
cellular mechanisms inform the pathology of Fuchs’s dystrophy. An FECD patient’s corneal 
endothelium experiences dysregulated senescence (cell cycle arrest), which expedites apoptosis 
or programmed cell death (Xing et al., 2014). As increased cell death occurs, the unaffected 
endothelial cells cannot enter mitosis and self-renew due to their terminally differentiated state. 
Instead, the remaining cells migrate to cover low-density areas in the endothelium (Mootha et al. 
2015). The remaining corneal endothelial cells also experience morphological changes such as 
thinning, stretching, and enlargement (Jurkunas et al., 2010). Cell size and shape changes, or 
polymegethism and pleomorphism, disrupt endothelial cells’ wild-type hexagonal morphology 
(Jurkunas et al., 2010). When the cell density becomes too low to be corrected by malformed 
migratory cells, the corneal endothelium loses its pumping function, and excess aqueous fluid 
overhydrates the stroma (Hamill et al., 2013). Dysregulated stromal hydration results in corneal 
opacity, edema, scarring, and reduced vision (Hamill et al., 2013). 
An additional pathological marker of FECD is guttae formation near the endothelium. 
Descemet’s membrane, located directly below the endothelium, becomes diffusely thickened 
with FECD progression (Mootha et al., 2015). The thickened membrane induces the formation of 
focal collagenous excrescences, known as guttae (Mootha et al., 2013). Guttae formation and 
corneal endothelial cell loss are initiated in the central cornea and progress to the periphery of the 
tissue (Jurkunas et al., 2010). Through visualizing the mound-shaped collagenous excrescences 
with slit lamp biomicroscopy, studies suggest the number of endothelial cells remaining in the 
cornea is inversely proportional to the number of guttae (Jurkunas et al., 2010). 
 
 
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                   Fig 2: Patient with advanced Fuchs’ dystrophy.  
 
FECD Genetic Markers and Underlying Mechanisms 
Late-onset FECD is a disorder with locus heterogeneity and multiple genetic markers not 
integrated into a cohesive pathology (Mootha et al., 2015). While smoking and low body mass 
index have been implicated as potential FECD risk factors, environmental risk factors have not 
been found to inform FECD pathogenesis (Wieben et al., 2012). Prior studies describe the 
disorder as an autosomal-dominant trait transmitted through generations (Hamill et al., 2013). 
However, late-onset Fuchs’ dystrophy has been demonstrated to exhibit incomplete penetrance 
and phenocopies in large pedigrees (Mootha et al., 2015). In other words, individuals who carry 
the FECD variant gene may not phenotypically exhibit the disorder, while individuals with the 
disorder may not carry the FECD gene variant. The gene variants discussed in this paper include 
TCF8, LOXHD1, and TCF4, in addition to current data on RNA-mediated toxicity, aberrant 
splicing, differential mitochondrial activity, and oxidative stress implicated in late-onset FECD 
pathogenesis.  
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In a minority of late-onset FECD cases, the development of the disorder is attributed to 
mutations in the TCF8 and LOXHD1 loci. TCF8 encodes ZEB1, a zinc-finger transcription 
factor family protein with gene-repressive and enhancive capabilities (Hamill et al. 2013). TCF8 
expression in the corneal endothelium is presumed to regulate epithelial-mesenchymal transition 
or the process of epithelial cells obtaining migratory properties and becoming mesenchymal stem 
cells (Hamill et al. 2013). Five loss-of-function mutations in the TCF8 gene on chromosome 10 
may contribute to the development of FECD (Hamill et al., 2013). Another genetic marker 
documented in a minority of late-onset FECD cases is a mutation in the LOXHD1 gene on 
chromosome 18 (Hamill et al., 2013). LOXHD1 encodes a protein consisting of PLAT domains 
and is suspected of regulating protein interactions in the plasma membrane (National Library of 
Medicine). FECD patients have exhibited LOXHD1 mutations, which overexpress the LOXHD1 
protein in the cornea (Hamill et al., 2013). High levels of LOXHD1 have been observed to result 
in cytotoxic aggregations, which could contribute to corneal endothelial cell loss and FECD 
pathogenesis (Hamill et al., 2013). 
The gene variant found in 70% of FECD cases in the United States is a repeat-expansion 
mutation in the transcription factor 4 (TCF4) locus on chromosome 18 (Hu et al., 2018). In 2010, 
Baratz et al. conducted a genome-wide association study (GWAS) that indicated a correlation 
between the single nucleotide polymorphism (SNP) rs613872 in the TCF4 gene and late-onset 
FECD. Subsequently, in 2012, Wieben et al. demonstrated that a cytosine-thymine-guanine 
(CTG) repeat in intron 3 of TCF4 had greater specificity as a FECD genetic marker. The Wieben 
et al. study reported that 52 of 66 FECD subjects had the expanded CTG alleles, while only 2 of 
63 controls displayed this genotype (Mootha et al., 2013). These findings agreed with a 1997 
study by Breschel et al., which identified the intronic trinucleotide repeat locus, termed 
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CTG18.1, as a statistically significant genetic marker in a Caucasian cohort (Mootha et al., 
2013). The TCF4 gene extends over 437 kb on chromosome 18 and includes 20 exons (Du et al., 
2015; Mootha et al., 2013). The gene gives rise to 48 different transcripts from more than 20 
mutually exclusive transcription start sites (Du et al., 2015). TCF4 encodes for E2-2, a 667-
amino acid, class I, basic helix-loop-helix transcription factor that binds to the canonical E-box 
sequence CANNTG of promoters of target genes (Mootha et al., 2013; Mootha et al., 2015).  
Studies conducted by Breschel et al., Wieben et al., Du et al., and Mootha et al. have 
implicated expanded CTG18.1 alleles of n>30-40 repeats to be present in late-onset FECD 
patients. A copy of the expanded CTG18.1 allele confers a more than 32-fold increased risk of 
developing late-onset FECD (Mootha et al., 2013). Despite the evidence identifying the 
expanded CTG18.1 allele as a risk factor, the mechanisms contributing to corneal endothelial 
degeneration and FECD development are unknown. Potential mechanisms of the CTG repeat 
mutation in FECD may include RNA-mediated toxicity, haploinsufficiency of TCF4, or an 
unsuspected mechanism (Mootha et al., 2015). RNA-mediated toxicity is a strong contender in 
FECD pathogenesis since the expanded CTG18.1 allele is located in an intron; no protein 
product is ultimately translated from this non-coding region (Du et al., 2015). In other repeat-
expansion diseases such as myotonic dystrophy 1 and 2 (DM1 and DM2), expanded 
microsatellite DNA sequences are also located in non-coding intronic regions (Du et al., 2015). 
In DM1 pathogenesis, CTG repeats are transcribed into poly(CUG)n mRNA (Du et al., 2015). 
The DM1 RNA transcripts with expanded CUG repeats form stable hairpin structures, which 
interact and sequester RNA binding proteins and induce degenerative molecular pathways 
(Mootha et al., 2015).  The hairpin RNA structures, denoted as foci, further exhibit cytotoxic, 
gain-of-function activity by stimulating misplicing events and apoptosis (Mootha et al., 2015).  
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Fig. 3: Long (CTG)n repeat (n>30-40) in TCF4 is associated with FECD. 
 
Mootha et al. and Du et al. demonstrate that, similarly to DM1, ribonuclear foci can be 
detected in late-onset FECD corneal endothelial cells. A 2015 study identified discrete nuclear 
RNA foci of variable size in all eight endothelial samples of FECD subjects with CTG18.1 
expansion (Mootha et al., 2015). Nuclear RNA foci were undetectable in five control endothelial 
samples without the expansion, while two control samples with ribonuclear foci were found to 
have mutant CTG18.1 alleles (Mootha et al., 2015). The average percentage of cells with nuclear 
RNA foci in the eight CTG18.1 expansion-positive FECD endothelial samples ranged from 33% 
to 88% (Mootha et al., 2015). The observed morphology of the foci was spheroidal, and foci size 
ranged from 0.43 to 2.59 μm2 (Mootha et al., 2015). While Mootha et al. observed a range of 
0.39 to 1.55 average foci per cell nucleus, Du et al. reported 2.35 ± 1.14 foci per cell nucleus.  
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When the pathology of late-onset FECD is compared to DM1 again, poly(CUG)n RNA in 
DM1 co-localizes with and sequesters the mRNA-splicing factor MBNL1 (Du et al., 2015). 
Sequestration of MBNL1 disrupts the splicing of MBNL1-regulated mRNAs, such as the 
chloride channel ClC-1 and the insulin receptor (Du et al., 2015). Du et al. demonstrate in their 
2015 study that two FECD patients with CTG18.1 expansion exhibited co-localization of CUG 
RNA foci and MBNL1 protein, while CUG RNA foci and MBNL1 aggregation were not 
detected in a CTG18.1 expansion-negative FECD patient. Further investigation by Du et al. on 
potential CUG RNA foci interference with splicing factors revealed that 342 genes with robust 
expression in the corneal endothelium had differential expression of at least one isoform in 
FECD samples. Top differential splicing events identified by Du et al. involved MBNL1 and the 
MBNL1-regulated genes ADD3 and INF2. Implementation of  MISO (Mixture of Isoforms) 
software by Du et al. provided estimates of isoform expression at the exon level. MISO analysis 
of MBNL1 splicing in CTG18.1 expansion-positive FECD samples revealed that the inclusion of 
exon 6 (352-bp band) of MBNL1 is more highly favored in FECD patients than in the control 
(Du et al., 2015). MBNL1 splicing in a control sample resulted in two strong PCR products with 
sizes of 352-bp and 298-bp, while an FECD sample resulted in a strong 352-bp product and very 
little of the 298-bp product (Du et al., 2015). Similarly, analysis of splicing of the gene product 
ADD3 indicated that the inclusion of exon 14 (380-bp band) is favored in CTG18.1 expansion-
positive FECD samples (Du et al., 2015). In contrast, control samples for ADD3 splicing exhibit 
a robust 298-bp PCR product and little of the 380-bp PCR product (Du et al., 2015). Lastly, 
splicing analysis for the INF2 gene product demonstrates preferential exclusion of exon 22 (352-
bp band) (Du et al., 2015). The 352-bp PCR product shows up strongly in control samples, but 
only CTG18.1 expansion-positive samples exhibit a 295-bp PCR product (Du et al., 2023). The 
14 
 
 
differential splicing events in MBNL1 and MBNL1-regulated ADD3 and INF2 transcripts 
support previous findings that CUG RNA foci in CTG18.1 expansion-positive FECD cells 
sequester MBNL1 and induce aberrant splicing. Furthermore, ADD3 and INF2 proteins are 
suspected to be involved in actin dynamics within cells and cell-cell adhesion (Du et al., 2015). 
Dysregulated splicing of these proteins involved in mechanical support and movement of cells 
may contribute to corneal endothelial cell death and late-onset FECD pathogenesis.    
Current literature emphasizes the role of CTG18.1 expansion, RNA-mediated toxicity, 
and differential splicing in FECD pathogenesis. However, multiple studies demonstrate that 
dysregulated mitochondrial activity and oxidative stress may be critical in FECD development. 
Corneal endothelial cells are highly populated with mitochondria, which are cell organelles that 
generate chemical energy via oxidative phosphorylation (Haschek et al. 2002). A robust 
mitochondrial presence is necessary for endothelial cells to maintain corneal hydration, 
thickness, and transparency (Nanda et al., 2019). The bicarbonate anion-activated ATPase 
located within mitochondria allows endothelial cells to perform the metabolically costly activity 
of pumping stromal fluid into the anterior chamber of the cornea (Haschek et al. 2002). The 
progression of FECD may be correlated with increased oxidative stress in corneal endothelial 
cells due to an inefficient mitochondrial system (Nanda et al. 2019). FECD cells have been found 
to have increased mitochondrial DNA damage, decreased mitochondrial membrane potential, 
and mitochondrial fragmentation (Nanda et al. 2019). Jurkunas et al. further report that a marker 
of oxidative DNA damage, 8-hydroxy-2’-deoxyguanosine, co-localizes to mitochondria in FECD 
samples, demonstrating that the mitochondrial genome is the specific target of oxidative stress in 
FECD. A promising avenue for FECD gene therapy could be preventing the decline of 
15 
 
 
mitochondrial mass and correcting the oxidant-antioxidant imbalance in corneal endothelial 
cells. 
Current and Future Treatments for FECD  
Current treatments for late-onset Fuchs’ dystrophy are solely surgical. Penetrating 
Keratoplasty (PKP) has become outdated in FECD treatment practice due to its extensive 
recovery period, permanent weakening of tectonic eye strength, high rates of transplant rejection, 
and serious risk of suture infection (Sarnicola et al. 2019). Endothelial Keratoplasty, the current 
standard for late-onset FECD treatment, has improved upon the failures of PKP and results in 
faster recovery, rescued vision loss, and low transplantation rejection (Sarnicola et al. 2019). 
However, with FECD cases on the rise, the need for quality corneal transplant tissue has 
outpaced the supply chain.  
The global cornea donor shortage has provided a prime opportunity to position a genetic 
therapy in FECD care. Our treatment, OptiDicer, is a modified endonuclease that aims to treat 
FECD by continuously cleaving CUG RNA accumulations and preventing the formation of 
cytotoxic RNA foci. OptiDicer is a modified form of the endogenous protein DICER1 
responsible for degrading miRNA and mRNA via  RNase III activity. In contrast to DICER1, 
OptiDicer does not respond to negative feedback and can cleave accumulated CUG RNA 
continuously. Removal of the helicase domain in OptiDicer allows RNase III activity to occur 
unchecked. We expect that OptiDicer will significantly reduce CUG RNA accumulations in 
corneal endothelial cell nuclei and rescue corneal endothelial cell death by preventing RNA-
mediated toxicity, sequestration of MBNL1, and dysregulated splicing.  
 
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METHODS 
OptiDicer Design and Plasmid Construction 
 
Fig 4: Comparison of OptiDicer modifications and endogenous DICER1. 
 
OptiDicer was initially developed by Dr. Hironori Uehara to prevent retinal pigmented epithelial 
cell loss in Geographic Atrophy. The original sequence of OptiDicer was posted in GenBank as 
MN910264. Endogenous DICER1consists of a helicase domain, PAZ domain, two RNase III 
domains, and a DRBM domain and has a full length of 5.8 kb. Previous studies demonstrate that 
the N-terminus helicase domain has an inhibitory effect on DICER1 double-strand RNA 
cleavage (Ma et al., 2008). The N-terminus helicase domain was removed to optimize RNA 
cleavage efficiency and reduce dicer’s size. To package DICER1 into an adeno-associated virus 
(AAV) for transfection, the promoter and polyadenylation signal were modified to reduce the 
dicer packaging size to ~5.0kb. In addition to the N-terminus helicase domain, miRNA 
production via DICER1 cleavage is also suspected to have an inhibitory function by inducing a 
negative feedback loop. OptiDicer was modified to be resistant to let-7 miRNA with silent 
mutations on let-7 target regions (DNA sequence changes, but not protein sequence). The 
17 
 
 
negative feedback loop stimulated by miRNA production was further corrected by removal of all 
potential miRNA targeting sites. Since mature miRNA is ~22 nt, the seed sequence was reduced 
to ~7 nt to only recognize target mRNA. miRDB (http://mirdb.org/miRDB/index.html) was used 
to alter the seed miRNA sequence of OptiDicer with silent mutations. Lastly, NLS-OptiDicer 
was given a nuclear localization signal (NLS) for expression in cell nuclei only. We introduced 
NLS at the N-terminus, C-terminus, and both termini. We found C-terminus NLS decreased 
OptiDicer expression. Therefore, NLS-OptiDicer was modified to contain c-myc derived NLS 
(PAAKRVKLD) at the N-terminus. 
 
Cell culture  
The patient derived FECD corneal endothelial cells F35T (>1500 CTG repeats) were a generous 
gift from Dr. Albert Jun. Cells were cultured in “Joyce” medium designed by Dr. Nancy C. 
Joyce. The Joyce medium consisted of 2% fetal bovine serum, 8% chondroitin sulfate (C9819), 
P/S/A (anti-anti), BPE (pituitary extract) (Gibco, 13028-014), 100mg/mL CaCl2, 10mg/mL 
ascorbic acid (A4403), and 100μg/mL EGF, and OptiMEM (1x)/GlutaMAX (Gibco). When cells 
achieved confluence in Joyce media, the media was changed to human corneal endothelial 
maturation medium (Gibco).   
 
Immunofluorescence  
Cells were plated in an 8-well chamber slide with FNC coating at 0.05 x 106 cells/well. The cells 
were incubated overnight at 37°C, and transfection was performed with Lipofectamine3000 
Transfection Reagent (Thermofisher). The protocol involved diluting 0.25ug AAV plasmid, 
0.375 μL Lipofectamine3000, and 0.5μL P3000 with 25μL OptiMEM (1X)/GlutaMAX. After 10 
18 
 
 
minutes of incubation, 25μL of diluted transfection solution was added dropwise to a well 
containing 250μL of Joyce medium.  Joyce medium was changed after 24 hours of incubation.   
After washing cells twice with 1X PBS, 500μL of 4% PFA was used to fix cells for 15 
minutes. Following a triple-wash with 70% RNase-free ethanol, 500μL of 70% EtOH was 
permeabilized in each well for 30 minutes at 4°C. Cells were washed twice with 1x PBS and 
blocked with 5% FBS, 0.02% triton, and 1x PBS for 60 minutes at room temperature. The 
meniscus of the 8-well chamber slide was removed, and wells were washed with a blocking 
buffer. The cells were incubated with 50μL of Dicer rabbit monoclonal primary antibody 
(D38E7, 1:100) dispersed dropwise. Cells were placed overnight in a humidified incubation tray 
at 4°C.   
Cells were washed with 1X PBST (0.01% T detergent) 6 times for 10 minutes. 
Afterward, they were incubated with 100μL Alexa Fluor goat anti-rabbit IgG HRP secondary 
antibody (1:500, Thermofisher) for 1-2 hours at room temperature. The PBST wash regimen was 
repeated, and cells were stained with mounting media with DAPI. The cells were imaged with an 
EVOS fluorescence microscope at 10X and 40X magnification.   
 
Western blot  
Cells were plated in a 24-well plate with FNC coating at 0.1 x 106 cells/well. The cells were 
incubated overnight at 37°C, and transfection was performed with Lipofectamine3000 
Transfection Reagent (Thermofisher). The protocol involved dilution of 0.5μg AAV plasmid, 
0.75μL Lipofectamine3000, and 1μL P3000 with 50μL OptiMEM (1X)/GlutaMAX. After 10 
minutes of incubation, 50μL of diluted transfection solution was added dropwise to a well 
containing 500μL of Joyce medium. Joyce medium was changed after 24 hours of incubation, 
and the cells were incubated for 48 hours.   
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Following a double wash with cold PBS, 200μL of RIPA buffer and protease inhibitor 
was dispensed to each well and incubated for 30 minutes on ice. The cells were collected with a 
cell scraper and lysed by centrifugation at 12,000g for 5-10 minutes. The supernatant was 
collected, and protein concentration was ascertained with a BCA protein assay kit. A buffer 
comprised of 10% BME and 4X LDS (Invitrogen NuPAGE) was added to each sample and the 
samples were incubated for 10-15 minutes at 70°C. Equal amounts of protein were loaded onto 
4-12% Bis-Tris 10-well polyacrylamide gels (Invitrogen NuPAGE) and transferred onto PVDF 
membranes. Westerns were performed with 1X MOPS and 10% MeOH 1X transfer buffer (20X 
transfer buffer, RO water, 100% MeOH). Protein samples were incubated with Dicer rabbit 
monoclonal primary antibody (D38E7, 1:500) and Alexa Fluor goat anti-rabbit IgG HRP 
secondary antibody (1:2000, Thermofisher) in 3% blocking buffer (milk powder, 1X PBST). The 
gel images were obtained by Azure Biosystems™ c280 (Azure Biosystems).  
 
Fluorescent in situ hybridization (FISH) 
Cell plating and transfection occurred as previously described in the immunofluorescence 
protocol. After washing with 1X PBS, cells were fixed with 500 μL 4% PFA for 30 minutes at 
room temperature. Cells were washed twice with 70% EtOH and permeabilized with 500 μL 
70% EtOH overnight at 4°C. The following day, cells were washed with wash buffer (10% 
formamide in 0.2X SSC) for 15 minutes and incubated with prehybridization buffer (40% 
formamide in 0.2X SSC) for 20 minutes at 43°C in a humified incubation tray. Following 
replacement with (CAG)6CA-5' Texas red-labeled 2-Omethyl RNA 20-mers probe in 
hybridization buffer (1:1000, 10% dextran sulfate and 40% formamide in 0.2X SSC), cells were 
incubated at 37°C overnight. FISH performed with ecc43 cells involved differently formulated 
wash buffer (10% formamide in 2X SSC), prehybridization buffer (40% formamide in 2X SSC), 
20 
 
 
and hybridization buffer (10% dextran sulfate and 40% formamide in 2X SSC). The cells were 
washed twice with wash buffer for 15 minutes at 37°C and stained with mounting media with 
DAPI. The cells were imaged with an EVOS fluorescence microscope at 10X and 40X 
magnification.  
 
RT-PCR for RNA splicing analysis 
RNA splicing variants were determined by RT-PCR. Briefly, total RNAs were purified from 
cells using Direct-zol RNA Miniprep Kits (Zymo Research) with DNaseI treatment. 
Complementary DNA was synthesized with iScript™ cDNA Synthesis Kit (Biorad). Taq DNA 
Polymerase with Standard Taq Buffer (NEB) was used for PCR. After agarose electrophoresis 
with ethidium bromide, the gel images were obtained by Azure Biosystems™ c280 (Azure 
Biosystems). The intensity of the bands was measured with Image J (reference, 
https://pubmed.ncbi.nlm.nih.gov/22930834/).  We used the following primers:  
MBNL1 forward; 5’- TCAAGGCTGCCCAATACCAG-3’,  
Reverse; 5’-TGTTGGCTAGAGCCTGTTGG-3’.  
ADD3 forward; 5’- ACCAGCTCCTCCTAACCCAT-3’,  
Reverse; 5’-GCCTTCAGGTGACAGGACTT-3’.  
21 
 
 
RESULTS 
Development of nuclear OptiDicer 
 
 
22 
 
 
Fig. 5: 40x immunofluorescence and western blot gel images showing DICER1, OptiDicer, and 
NLS-OptiDicer expression in F35T cells and control Hela cells.  
 
As demonstrated in Fig. 5A, OptiDicer (yellow fluorescence) is mainly expressed in the cell 
cytosol. In the F35T cells, we were unable to detect endogenous DICER1 by immunofluorescence. 
DICER1 is generally localized to the cytosol but is also present in the nucleus. To target nuclear 
CUG RNA, we introduced a nuclear localization signal (NLS, SV40) at the N-terminus (NLS-
OptiDicer, Fig. 5B). Although NLS-OptiDicer is still expressed in the cytosol, it is expressed 
efficiently in the nucleus as demonstrated by the colocalization of DAPI staining (blue) and NLS-
OptiDicer expression (yellow fluorescence). In Fig. 5C, we confirmed the protein level expression 
of DICER1, OptiDicer, and NLS-OptiDicer in F35T cells by western blot with GFP and EFS-
OptiDicer controls. NLS-OptiDicer showed efficient protein expression greater than DICER1 
expression, and equivalent or more than OptiDicer1 expression.  
  
 
 
 
 
 
 
 
 
 
 
 
23 
 
 
NLS-OptiDicer reduced CUG-RNA accumulations in the nucleus 
A 
 
 
 
 
 
24 
 
 
B 
 
Fig. 6: FISH visualization of CUG RNA foci in F35T cells. A, (CAG)6- CA-5′ Texas red-labeled 2-
Omethyl RNA 20-mers probe concentration assay. B,  NLS-OptiDicer and D2A control transfection and 
untreated control with 0.08 ng/uL RNA probe. 
 
Next, we examined whether OptiDicer overexpression can reduce CUG-RNA 
accumulation (red fluorescence) in F35T cells (nuclei stained DAPI blue). We performed a 
concentration assay and determined an optimized RNA 20-mers probe concentration of 0.08 
ng/uL (Fig. 6A). 0.08 ng/uL of probe was dispensed in FISH experiments with OptiDicer 
transfected, D2A-OptiDicer transfected, and untreated F35T cells (Fig. 6B).  In this experiment, 
we used D2A-OptiDicer as a control and NLS-OptiDicer experimentally (Fig. 6B). The 
transfection efficiency was estimated as 50%. We did not find significant cell loss by pCMV-
NLS-OptiDicer or pCMV-D2A-OptiDicer transfection. Accumulation of CUG RNA was 
counted in 276 OptiDicer transfected and 222 D2A-OptiDicer transfected F35T cells. The 
average number of CUG RNA accumulation was 1.9 +/- 1.4 in OptiDicer-F35T and 2.9 +/- 1.7 
in D2A-OptiDicer control F35T (p = 1.28E-11), respectively. In comparison, the average number 
of CUG RNA accumulations counted in 17 untreated F35T cells was 2.5 +/- 1.1 (p = 0.045).  
25 
 
 
NLS-OptiDicer shifts RNA splicing 
 
Fig. 7: RT-PCR gel images showing splicing of MBNL1 and ADD3 with NLS-OptiDicer 
transfection in F35T cells and control corneal endothelial cells.  
 Finally, we investigated whether NLS-OptiDicer shifts RNA splicing of MBNL1 
and ADD3 in F35T cells. MBNL1 and ADD3 are genes previously identified as being influenced 
by mutant TCF4. We observed alteration of splicing in the genes with transfection of NLS-
OptiDicer. When compared to RT-PCR gels of untreated F35T cells, we found that NLS-
OptiDicer expression modified the band intensity of exons included in F35T cells. The band 
intensity of exons qualified in the RT-PCR gel images was measured with ImageJ. The ratio of 
the upper band intensity to the lower band for MBNL1 was calculated to be 0.90 +/ 0.06 for the 
control and 0.63 +/- 0.03 for NLS-OptiDicer. For ADD3, the ratio of the upper band intensity to 
the lower band for ADD3 was calculated to be 0.23 +/- 0.03 for the control and 0.17 +/- 0.02 for 
NLS-OptiDicer.   
26 
 
 
DISCUSSION 
We found that OptiDicer significantly decreased CUG-RNA accumulation in late-onset 
FECD patient derived corneal endothelial cells (F35T) from 2.9+/-1.7 foci per cell to 1.9+/-1.4 
foci per cell. Our results also indicated that NLS-OptiDicer expression shifts alternative splicing 
of MBNL1 and ADD3 in F35T cells. While OptiDicer had robust expression in 
immunofluorescent images and western blot gel images, transfection efficiency was 
approximated to be only 50% (i.e., only 50% of cells expressed the GFP tag). This low 
transfection efficiency may underestimate the therapeutic effect of OptiDicer.   
We did not include our experimentation with the FECD cell lines ecc20 and ecc43 in this 
report. In contrast to F35T, transfection efficiency for ecc20 and ecc43 was approximated to be 
30-40%. The low transfection efficiency made it impossible to yield significant results with 
immunofluorescence, western blot, and RT-PCR data. All three cell lines mentioned are derived 
from late-onset Fuchs’ dystrophy patients. The age of the donor renders the cell line more 
susceptible to mutation. In addition, corneal endothelial cells are difficult to culture in regard to 
maintaining their phenotypes. These factors may contribute to low transfection efficiency and 
failure to replicate the results found in F35T with other FECD cell lines.  
Future studies will explore OptiDicer in other cell lines from FECD patients (e.g., ecc20 
and ecc43) with improved transfection methods. I began the process of optimizing OptiDicer 
transfection by performing AAV incubation and electroporation with a NucleofectorTM machine. 
Further experimentation with these methods may increase OptiDicer transfection efficiency to 
our target of 80-100%. However, our current results preliminarily suggest that OptiDicer can be 
a potential treatment for long CUG RNA repeat FECD.    
27 
 
 
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