Suppression of Keratoepithelin and Myocilin by Small Interfering Rnas (Sirna) in Vitro Ching Yuan University of Minnesota - Twin Cities

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2007 Suppression of keratoepithelin and myocilin by small interfering RNAs (siRNA) in vitro Ching Yuan University of Minnesota - Twin Cities

Emily J. Zins University of Minnesota - Twin Cities

Abbott .F Clark Alcon Research Ltd.

Andrew J.W. Huang Washington University School of Medicine in St. Louis

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Recommended Citation Yuan, Ching; Zins, Emily J.; Clark, Abbott .;F and Huang, Andrew J.W., ,"Suppression of keratoepithelin and myocilin by small interfering RNAs (siRNA) in vitro." Molecular Vision.13,. 2083-2095. (2007). https://digitalcommons.wustl.edu/open_access_pubs/1812

This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision Received 25 July 2007 | Accepted 24 October 2007 | Published 7 November 2007

Suppression of keratoepithelin and myocilin by small interfering RNAs (siRNA) in vitro

Ching Yuan,1 Emily J. Zins,1 Abbott F. Clark,2 Andrew J.W. Huang1,3

1Department of Ophthalmology, University of Minnesota Minneapolis, MN; 2Alcon Research Ltd., Fort Worth, TX; 3Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, MO

Purpose: Mutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. We investigated the potential use of small interfering RNAs (siRNAs) to suppress the expression of KE and MYOC and the related cytotoxicity of mutant myocilins in vitro. Methods: cDNAs of the human keratoepithelin (KE) gene and myocilin (MYOC) gene were amplified by polymerase chain reaction and subcloned into pEGFP-N1 to construct respective plasmids, KEpEGFP and MYOCpEGFP, to produce fluorescence-generating fusion proteins. Short hairpin RNAs (shRNAs) were generated from an RNA polymerase III promoter-driven vector (pH1-RNA). Transformed HEK293 and trabecular meshwork (TM) cells were cotransfected via liposomes with either KEpEGFP or MYOCpEGFP and respective shRNA-generating plasmids to evaluate the suppression efficacy of shRNAs. Suppression of KE-EGFP fusion protein by KE-specific shRNAs was evaluated by fluorescence microscopy and western blotting. Suppression of MYOC-EGFP fusion protein by myocilin-specific shRNAs was quantified with UN-SCAN-IT software on digitized protein bands of western blots. The cellular stress response of TM cells induced by misfolded mutant myocilins was evaluated with a BiP promoter-driven luciferase reporter assay. Results: One shRNA (targeting the coding sequence starting at 1,528 bp of KE) reduced the expression of KE-EGFP in HEK293 cells approximately by 50% whereas the other shRNA (targeting the 3'-UTR region of KE) suppressed more than 80% of the expression of fusion protein. Cotransfection of MYOCpEGFP and various shRNA-generating plasmids targeting different regions of MYOC (containing amino acid residues R76, E352, K423, or N480 associated with inherited glaucoma) showed effective reduction of MYOC-EGFP fusion protein, ranged from 78% to 90% on average. The activation of the BiP gene (a cellular stress response induced by mutant myocilins) in transformed TM cells was significantly reduced when mutant myocilin proteins were suppressed by myocilin-specific shRNAs. Conclusions: KE-specific or MYOC-specific shRNAs effectively suppressed the expression of recombinant KE or myocilin proteins and the related cytotoxicity of mutant myocilins in vitro. RNA interference may have future therapeutic implications in suppressing these genes.

Keratoepithelin (KE) or transforming growth factor-β in- autosomal dominant corneal dystrophies. In humans, KE is ducible (TGFβI) protein is an essential constituent of the ex- located at chromosome 5q3l. Several 5q3l-linked corneal dys- tracellular matrix responsible for cell adhesion and cell-ma- trophies such as lattice type I, Avellino, granular type I, and trix interactions. The encoding gene of KE was first discov- Reis-Bückler are correlated with permutations of KE [5-7]. ered from a subtraction library screening in human adenocar- To date, at least 13 different types of KE-related corneal dys- cinoma cell line A549 treated with transforming growth fac- trophies attributed to at least 30 missense mutations of KE tor-β1 (TGFβ-1) [1]. Two other groups later independently have been reported. These corneal dystrophies are typically isolated the KE protein from pig cartilages [2] and rabbit cor- presented with untoward subepithelial or stromal opacities with neas [3] as a collagen fiber-associated protein. KE has various reduced vision and often painful recurrent erosions due to poor other names such as TGFBI (TGFβ1-induced protein), BIGH3, epithelial adhesions. Research evidence indicate that these βIGH3, βig-h3, beta ig-h3, keratoepithelin, or RGD-CAP (in corneal opacities are caused by amyloid or non-amyloid pro- chicken and pig) [2,4]. KE is composed of 683 amino acids tein aggregates secondary to the accumulation of KE and re- and is highly conserved among species (human, mouse, lated mutant proteins. Perturbation of mutant KE to reduce chicken, and pig). It is widely distributed in human tissues the production and/or accumulation of those undesirable mu- such as the cornea, skin, lung, bone, bladder, and kidney. Dur- tant proteins may potentially mitigate the aggregation of ab- ing corneal wound healing, upregulation of KE is associated normal corneal deposits and associated corneal opacities. with an increase of TGFβ-1 [3]. Myocilin (MYOC) is a secretory glycoprotein of 55 kDa In addition to its role in corneal wound healing [3], KE with myosin-like and olfactomedin-like domains, it was first also plays an important role in the pathogenesis of several identified in cultured human trabecular meshwork (TM) cells treated with dexamethasone [8]. The actual functions of Correspondence to: Andrew J.W. Huang, MD, Washington Univer- myocilin remain to be elucidated. Recent myocilin researches sity School of Medicine, 660 S. Euclid Avenue, Campus Box 8096, have implied its roles in the regeneration in glial cells and in St. Louis, MO, 63110-1093; Phone: (314) 362-0403; FAX: (314) 747- 2851; email: [email protected] the central nerve system [9,10]. Myocilin may also contribute 2083 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision to the structural integrity of the myelin sheath of peripheral silencing that was first discovered in C. elegans and plants nerves [11,12]. In situ hybridization revealed that myocilin is [20,21]. RNAi can be found in eukaryotes as diverse as yeast present in many ocular tissues including sclera, TM, and cor- and mammals and likely plays a crucial role in regulating gene nea and in non-ocular tissues such as smooth muscle [13]. expression in all eukaryotes [22,23]. Currently, there are two Although the actual functions of myocilin in the eye have yet commonly employed methods in applying RNAi for gene sup- to be delineated, it is believed that the intracellular accumula- pression: synthetic small interfering RNAs (siRNAs), which tion of misfolded mutant myocilins induces apoptosis of TM usually are short double-stranded RNAs of 21-23 nucleotide cells with subsequent obstruction of TM and increased resis- pairs [22,24-26], and short hairpin siRNAs (shRNAs) gener- tance of aqueous outflow [14-16]. The resultant elevation of ated by RNA polymerase III promoters such as human H1- intraocular pressure eventually leads to axon degeneration of RNA or murine U6 RNA promoters (Figure 1) [24,25]. The the optic nerve and loss of visual fields. Mutations of MYOC promoter-mediated shRNA technology has recently gained have been implicated in at least two types of inherited open- popularity due to advantages such as low production costs and angle glaucoma (OAG) like primary open-angle glaucoma prolonged durations of gene suppression. This recent advance (POAG) and juvenile open-angle glaucoma (JOAG) [17]. In in RNAi technologies would make shRNA-mediated gene humans, MYOC is located in chromosome 1 (1q21-q31) and therapies possible if it could be further combined with effica- was initially named the TIGR (TM-inducible-glucocorticoid- cious nucleotide delivery vehicles such as viral vectors or response protein) gene. Currently, at least 3%-4% of POAG nanoparticles with high efficiency and low toxicity. and JOAG patients have been associated with 43 myocilin Since mutations of KE and MYOC have been implicated mutations via genetic linkage analysis [18,19]. in autosomal dominant corneal dystrophies and inherited Human genes can be manipulated by many mechanisms. OAGs, respectively, we surmise that siRNA-mediated sup- RNA interference (RNAi) is a powerful technique for gene pression of mutant KE and MYOC may potentially mitigate

Figure 1. Gene silencing by short hairpin RNA. RNA interference (RNAi)-mediated gene silencing is a posttranscriptional mechanism targeting a specific mRNA in which a short double-stranded RNA of 21-23 nucleotide pairs (known as “small interfering RNA”, siRNA) induces a sequence-specific knock-down of its complementary gene. To prevent degradation of RNA fragments and to achieve a stable expression of siRNA in cultured cells, self- looped short hairpin RNAs (shRNAs) as siRNA precursors can be designed by linking the sense and antisense strands of siRNA with an oligonucleotide linker (spacer) and a poly-T as a terminator. The sequences can then be subcloned into a plasmid, containing RNA polymerase III promoters, such as human H1-RNA promoter to generate shRNAs. These plasmids can transfect target cells via liposomes or viral vectors. Once inside the cell, the shRNAs generated by the plasmids are cleaved by a Dicer (an RNase) to form siRNAs. The double stranded siRNA is unwound to form a single- stranded ribonucleoprotein complex known as RNA-induced silencing complex (RISC), which mediates a sequence-specific degradation of the mRNAs involved in coding a target protein. After pairing with a siRNA strand, the target mRNA is cleaved and further degraded, leading to an inter- ruption in synthesis of the disease-causing protein such as myocilin or keratoepithelin (KE). The RISC complex is naturally stable thus enabling siRNAs to interact consecutively with multiple mRNAs with a potent suppression of protein synthesis. With suppression of mutant KE and myocilin, the amyloidogenic response from aggregations of abnormal KE and the cytotoxic response of trabecular meshwork caused by misfolded myocilin can be mitigated, respectively. 2084 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision the protein aggregations of abnormal KE in cornea or of ab- Plasmid construction to generate short hairpin RNA: normal myocilin in TM and alleviate the untoward corneal Oligonucleotides, containing the sequence of human H1 RNA opacities or glaucoma, respectively. The anterior segment of promoter, were synthesized by the Microchemical facilities at the eye is readily accessible for topical delivery of therapeu- the University of Minnesota. To generate short hairpin RNAs tics to the target tissues such as cornea or trabecular mesh- (shRNAs) to interfere with the expression of fusion proteins, work. To investigate the feasibility of shRNA-mediated gene pH1 plasmids were first produced by subcloning the synthe- suppression as a new potential therapeutic strategy for inher- sized human H1-RNA promoter into the pBluescript KS(+) II ited ocular conditions, we produced several shRNAs from the (Stratagene, La Jolla, CA) via BamHI and EcoRI sites. The RNA polymerase III promoter-containing plasmids to evalu- pH1 plasmids were further digested by BglII and HindIII and ate their efficacy in suppressing the expression of KE and then gel purified. For comparison, we also obtained a mU6P MYOC in vitro. plasmid from Dr. David Turner at the University of Michigan, Ann Arbor, MI to generate shRNAs [25]. The mU6P plasmid METHODS contains the murine U6 RNA promoter and has been used in Plasmid construction to generate fusion proteins: Amplifica- murine tissues and several other cell lines to generate shRNAs. tion of KE cDNA and construction of expression plasmids were We found that both pH1 and mU6P plasmids successfully gen- performed as in our previous report [27]. For experiments re- erated shRNAs capable of suppressing target genes (data not garding shRNAs targeting the coding region of KE, cDNA of shown). human KE was amplified by polymerase chain reaction (PCR) The candidate siRNA sequences specific for human KE from an I.M.A.G.E. (Integrated Molecular Analysis of Ge- and MYOC were selected and designed by using online tools nomes and Expression Consortium) clone (clone ID 4837646; from various vendors (such as programs from Ambion or GenBank BE206112) and ligated into a green fluorescent pro- Oligoengines; see Table 1 for siRNA sequences). The selected tein-producing pEGFP-N1 vector to construct KEpEGFP plas- candidate siRNA sequences were also checked to avoid any mids to generate KE-EGFP fusion proteins. The KE gene, possible match with other genes or polymorphism of the tar- containing the coding region after the signal peptide and addi- get gene by Blast search. To ensure stable expression of tional downstream 454 bp of the 3'-untranslated region (3'- shRNAs in HEK293 cells, TTCAAGAGA was used as a de- UTR), was amplified and subcloned into pEGFP-C3 to evalu- fault spacer (hairpin loop) sequence for pH1-RNA to generate ate the suppression efficiency of shRNAs targeting the 3'-UTR shRNAs. The sense and antisense strands of each shRNA con- region. taining the selected siRNA sequence, hairpin loop, and The I.M.A.G.E. clone of human MYOC was purchased pentathymidine terminator were synthesized and cloned into from ResGen (clone ID: 5179076; Huntsville, AL). The PCR- the prepared vector arm of pH1 plasmids (named as “KEpH1- amplified full-length MYOC cDNA was subcloned into shRNA” or “MYOCpH1-shRNA,”, respectively). For specific pEGFP-N1 (Clontech, Palo Alto, CA) via BamHI and EcoRI suppression of MYOC, we constructed pH1 plasmids to pro- sites to construct MYOCpEGFP plasmids to produce the duce MYOC-specific shRNAs that were complementary to the myocilin-EGFP fusion proteins in HEK293 cells (primer se- mutated amino acid sequences associated with primary or ju- quences: HMYOC-BamHI.3: 5'-GGC TGG ATC CAT CTT venile OAGs (R76K, E352K, K423E, and N480K) as previ- GGA GAG CTT GAT G-3', and HMYOC-EcoRI.5: 5'-GAA ously reported by other authors [16,17,28]. A control plasmid GAA TTC ATG AGG TTC TTC TGT GCA C-3'). The trun- generating shRNA with no sequence similarity to any known cated myocilin mutant, Q368X, was generated by PCR-am- mammalian genes (sequence of the sense strand: 5'-CAG TCG plification of a cDNA fragment containing amino acid resi- CGT TTG CGA CTG G-3') was also constructed to serve as dues 1-367 and subsequently fused with EGFP. The specific our negative control. The sequences of all our clones were sequences were further confirmed by automated sequencing further confirmed by a standard automated sequencing method at the Microchemical Facilities at the University of Minne- at the Microchemical Facilities at the University of Minne- sota. sota. For comparison, we also used a commercial plasmid, pSuper (Oligoengine, Seattle, WA) to generate similar MYOC- TABLE 1. PLASMID SEQUENCES FOR SMALL INTERFERING RNAS specific shRNAs. The results were comparable between the pH1 and pSuper (data not shown). Name Sequence (sense strand) ------Suppression of KE or MYOC by short hairpin RNA: The 1. control 5'-AACAGTCGCGTTTGCGACTGG-3' siRNA-mediated gene suppression experiments were con- 2. siKE-1528 5'-AAGGGAGACAATCGCTTTAGC-3' 1,528 bp ducted in transformed HEK293 or TM5 cells. HEK293 cells 3. siKE-3’UTR 5'-AACTTGCCCGTCGGTCGCTAG-3' 3'-UTR 4. siMYOC-A 5'-AACTTACAGAGAGACAGCAGC-3' R76 were purchased from American Tissue Culture Collection 5. siMYOC-B 5'-AATACCGAGACAGTGAAGGCT-3' E352 (ATCC, Manassas, VA) and maintained in DMEM/F12 cul- 6. siMYOC-C 5'-AACATCCGTAAGCAGTCAGTC-3' K423 ture medium (Invitrogen, Carlsbad, CA) with the addition of 7. siMYOC-D 5'-AACCCCCTGGAGAAGAAGCTC-3' N480 10% fetal bovine serum (FBS, HyClone Laboratories, Logan,

The oligonucleotide sequences of the sense strands inserted into pH1- UT) and antibiotics at 5% CO2/humid atmosphere. TM5 cells shRNA to generate KE- or MYOC-specific shRNAs for this study. A (obtained from Dr. A. F. Clark of Alcon Research Ltd., Fort Blast search of the control sequence did not find a similarity to any Worth, TX) were maintained in DMEM medium, 10% FBS mammalian genes or to EGFP cDNA. without sodium pyruvate as reported previously [29-31]. The 2085 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision media were replenished every other day, and cells were split son to cotransfection with control plasmid was used as an in- twice weekly. dicator for the suppression efficiency for each shRNA. To KE-EGFP or MYOC-EGFP plasmids were cotransfected ensure consistent transfection efficiency in cultured cells with the control plasmid or previously selected shRNA-gen- among experiments, a pCMV-βgal (Invitrogn) plasmid that erating plasmids into HEK293 cells. HEK293 cells were produces β-galactosidase was included in each transfection seeded into 60 mm culture dishes and were grown to a experiment as an internal control. The activity of β-galactosi- confluency between 70% and 90%. Transfections of HEK293 dase was measured with the Luminescent β-gal detection kit cells were performed with lipofectamine (Invitrogen) accord- (Clontech) in a Lumat LB9507 luminometer (Berthold Tech- ing to the manufacturer’s instructions. For each 60 mm dish, nologies USA, Oak Ridge, TN) according to the 0.05 µg KEpEGFP was cotransfected with 0.1 µg pCMV-βgal manufacturer’s instructions. Only transfection experiments (Invitrogen) and 1.0 µg of each specific KEpH1-shRNA in with a variation of less than 10% of β-galactosidase activity 0.5 ml of Opti-MEM. After 4 h of incubation at 37 °C, 1.5 ml were included for analysis. Five transfections were performed of serum-containing growth medium was added to each plate. to evaluate the suppression efficiency of each shRNA. The medium was completely replaced with fresh, serum-con- As lipofectamine caused significant cell death in the TM5 taining growth medium at 24 h after transfection. HEK293 cell line, transfections of this cell line was performed with cells were harvested at 24 h or 48 h after transfection. The Fugene 6 (Roche, Applied Science, Indianapolis, IN). TM5 fluorescent signals of EGFP fusion proteins generated by KE- cells were seeded into six well plates at 50% confluency 24 h EGFP or MYOC-EGFP plasmids in cultured HEK293 cells before transfection. Ninety-seven microliters of OptiMem con- were evaluated with an Axiovert 200 fluorescence microscope taining 0.5 µg of MYOCpH1-shRNA, 0.025 µg of (Zeiss, Thornwood, NY) at 48 h after transfection. The reduc- MYOCpEGFP, and 0.05 µg of pCMV-βgal were mixed with tion of EGFP-fusion protein fluorescence signal in compari- 3 µl of Fugene 6. After incubation at room temperature for 20

Figure 2. Suppression of KE-EGFP in HEK293 cells by a short hairpin RNA targeting the coding region of the KE gene. Cells were transfected with KEpEGFP along with the control plasmid or shRNA-generating plasmid, KE-1528pH1-shRNA. The fluorescence photographs were taken at the same exposure times (140 msec) 48 h after cotransfections. A: HEK293 cells were cotransfected with KEpEGFP plasmids and control plasmids as a baseline. B: Fluorescent signals of KE-EGFP in cultured HEK293 cells were significantly reduced by cotransfection with KE-1528pH1-shRNA. C: Western blot of protein lysates of HEK293 cells from A (lane 1) and from B (lane 2) to demonstrate the reduction of KE by KE-1528pH1-shRNA is shown. Protein bands (10 µg/lane) were probed with a custom-made anti-KE antibody. Presence of KE-EGFP fusion proteins in HEK293 cells was noted after cotransfection of KEpEGFP with control plasmid in lane 1 (as indicated by KE). Significant reduction of KE-EGFP fusion proteins (as indicated by KE) was noted after cotransfection of KEpEGFP with KE-1528pH1- shRNA in lane 2. D: Northern hybridization of mRNAs from A (left lane) and B (right lane), using IR analysis by Li-Cor, is shown. Equal amounts of mRNAs were loaded in each lane as judged by the intensities of 28S RNA (as indicated by 28S). Significant reduction of IR signals of KE-EGFP mRNA (as indicated by KE) was noted after cotransfection of KEpEGFP with KE-1528pH1-shRNA in right lane. 2086 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision min, the mixtures were added directly onto cultured cells and cific oligonucleotide probes (PCR-amplified coding sequences incubated for 24 h. The medium was completely replaced with including exons 12-13). The hybridizations were performed fresh medium 24 h after transfection. Cells were harvested with the ULTRAhyb-OS Northern kit (Ambion, Austin, TX). and evaluated similarly to transfected HEK293 cells. The spe- The membranes were further incubated with streptavidin cific cell confluency and seeding density for HEK293 and TM5 IRDye 800CW conjugate (Rockland Immunochemicals, cells (at 70%-90% and 50%, respectively) was determined by Gilbertsville, PA) to detect biotin-labeled probes and imaged pilot studies to determine the optimal transfection efficiency on the Odyssey IR image system (Li-Cor). (data not shown). Cell viability after transfection by After being rinsed with 1X PBS and trypsinized, a frac- lipofectamine varied among different cell lines but remained tion of the cells was removed to determine the activity of β- consistent for the each cell line. For HEK293 cells, as much galactosidase, and the remaining cells were extracted with ly- as 80% of cells could be transfected by lipofectamine with the sis buffer (1% SDS/1xPBS) to prepare lysates for western blots. above amount of DNA (1.15 µg per 60 mm plate) with negli- Protein concentrations of cell lysates were determined using a gible cell death. We noted that the transfection efficiency could BCA Protein Assay Kit (Pierce, Rockford, IL). Equal amounts be boosted to 90%-95% by increasing the amounts of DNA of protein (10-20 µg/lane) from each cell lysate were subjected (to more than 2 mg) for transfection. However, transfecting to electropheresis on 12% SDS-PAGE gels. The gels were blot- higher amounts of DNA also led to significant cell death. The ted onto nitrocellulose membranes at 350 mA for 1 h in the transfection efficiency of TM5 cells by Fugene 6 was only 1X TG buffer (BioRad, Carlsbad, CA)/20% methanol. KE- 15% and cell death was less than 10%. EGFP fusion proteins were detected with our custom-made HEK293 or TM5 cells transfected as described above were rabbit anti-KE antibody raised against E.coli-expressed recom- harvested at 48 h and subjected to northern hybridization and binant KE [32]. MYOC-EGFP fusion proteins were detected western blotting. For northern blot experiments, the RNeasy with a mouse anti-EGFP monoclonal antibody (Clontech) at mini kit (Qiagen, Valencia, CA) was used for the total RNA 1:1,000 dilution followed by a goat anti-mouse secondary an- extraction. After the samples were separated on a denaturing tibody conjugated with alkaline phosphatase (Sigma, St. Louis, formaldehyde-agarose gel (1.2%) and transferred onto Odys- MO) at 1:1,000 dilution. The same membranes were also sey nylon membrane for Li-Cor Odyssey system (Li-Cor, Lin- probed with a mouse anti-β-actin antibody (1:5,000; Sigma) coln, NE), they were hybridized with biotin-labeled KE-spe- to determine the amount of β-actin, which would act as an

Figure 3. Suppression of KE-EGFP in HEK293 cells by a short hairpin RNA targeting the 3'-UTR region of KE. A: HEK293 cells were cotransfected with KEpEGFP plasmids and control plasmids as a baseline. B: Fluorescent signals of KE-EGFP in cultured HEK293 cells were significantly reduced by cotransfection with KE- 3’UTRpH1-shRNA. C: Western blot of protein lysates of HEK293 cells from A (lane 1) and from B (lane 2) to demonstrate the reduction of KE by KE-3’UTRpH1-shRNA is shown. Protein bands (10 µg/lane) were probed with a custom-made anti-KE antibody. Pres- ence of KE-EGFP fusion proteins in HEK293 cells was noted after cotransfection of KEpEGFP with control plasmid in lane 1 (as indicated by KE). Significant reduction of KE-EGFP fusion proteins (as indicated by KE) was noted after cotransfection of KEpEGFP with KE-3’UTRpH1-shRNA in lane 2. 2087 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision internal standard to ensure that equal amounts of protein were Statistical analysis: One-way t-tests were used to deter- loaded in each lane for electrophoresis. The BCIP/NBT-blue mine the difference in the intensity ratios between myocilin substrate system (Sigma) was used to visualize the antigen- and β-actin and the luciferase ratios between control and antibody complexes, and the colored protein bands were then myocilin-specific shRNAs. At least three sets of transfection scanned and digitized with a flatbed scanner. Quantification were performed for each experiment with shRNAs unless oth- of the digitized bands and β-actin was performed with UN- erwise stated, and the mean values with standard deviation SCAN-IT software (Silk Scientific, Orem, UT). The pixel in- were reported. A p<0.05 was used to determine significant tensities from the bands detected by the anti-EGFP antibody differences between the groups. were normalized to the pixel intensities from the bands detected by anti-β-actin. The ratio of intensities between the con- RESULTS trol shRNA and a selected shRNA was used to determine the Identification and evaluation of keratoepithelin-specific short suppression efficiency for each shRNA. hairpin RNAs: Using several online tools, possible shRNA Luciferase assay: To evaluate the protein misfolding re- candidates for those targeted gene sequences based on algo- sponse and stress of endoplasmic reticulum (ER) caused by rithms proposed by several groups were searched [22,25]. The the accumulation of mutant myocilins, we also investigated selection of shRNA candidate sequences was based on the GC the activation of BiP by mutant myocilins in TM5 cells using content of the gene sequence, the optimal sequences for hair- luciferase reporter assays (Dual Luciferase Reporter System, pin siRNAs, and screening by Blast search. Specific sequences Promega, Madison, WI). A plasmid, BiPpGL3 (a gift from of each siRNA were are listed in Table 1. Dr. C.D. Chen, Boston University, Boston, MA), that contains Several KEpH1-shRNA plasmids were cotransfected with rat grp78 (BiP) promoter region -457 to -39 bp and generates KEpEGFP plasmids into cultured HEK293 cells to evaluate firefly luciferases, was constructed as previously described their potency in suppressing the expression of KE-EGFP. Two [33]. TM5 cells were harvested at 48 h after cotransfection KE-specific shRNAs, which significantly reduced the expres- with MYOCpH1-shRNA (0.1 µg), MYOCpEGFP (0.005 µg), sion of KE genes in HEK293 cells, were identified. As indi- BiPpGL3 (0.1 µg), and pRL (0.025 µg, generating Renilla cated by the EGFP signals, KE fusion proteins in control cells luciferases as internal control). After aspirating the media and (Figure 2A) were reduced to approximately 50% by KE- washing cells with 1 ml of 1X PBS, cells were lysed by add- 1528pH1-shRNA plasmids (Figure 2B), which generated ing 100 µl of 1X passive lysis buffer (Promega) to each well shRNAs targeting the coding sequence including 1,528-1,548 of the 24-well plates, and the culture plates were gently shaken bp. The percentage of fluorescence-positive cells was approxi- on a rotating platform for 15 min at room temperature. Twenty mately 80% and 60% for Figure 2A,B, respectively. In our microliters of the above lysate was used to measure luciferase pilot experiments for quality controls, the viability was con- activities with a luminometer as mentioned above. The ex- sistently around 90% after each transfection (data not shown). pression of firefly luciferases and Renilla luciferases was As shown in these photographs, not all cells were transfected measured sequentially for each sample, and the BiP promoter with equal amounts of plasmids or generated similar amounts activity was derived from the ratio of firefly luciferase to of fusion proteins. Therefore, the reduction of fluorescence Renilla luciferase. could only be deemed as supporting evidence since the detec-

Figure 4. Myocilin-specifc hairpin siRNAs targeting various mutation regions. Myocilin-specific shRNAs were generated from shMYOCpH1 plasmids that targeted amino acids including R76 (A), E352 (B), K423 (C), and N480 (D) of myocilin protein. Mutations of these residues have been associated with POAG and JOAG. R76 is located in the myosin-like domain of myocilin whereas the other three amino acids are located in the olfactomedin-like domain of myocilin. sp: signal peptide; myosin: myosin-like domain; olfactomedin: olfactomedin-like domain. 2088 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision tion of fluorescence could be affected by various empirical control shRNA (Figure 2C, lane 1), the expression of KE fu- conditions such as detection threshold and exposure time. The sion protein was reduced to approximately 50% by this shRNA most conclusive evidence for the efficacy of shRNAs should (Figure 2C, lane 2, arrowhead). Northern hybridization fur- be the final protein reduction in a standardized pool of cells. ther confirmed the reduction of KE-EGFP mRNA by KE- Consistent with the results observed in tissue culture (Fig- 1528pH1-shRNAs in HEK293 cells (Figure 2D, right lane) ure 2A,B), a representative western blot with a custom-made when compared with the control shRNA (Figure 2D, left lane). anti-KE antibody confirmed that when compared with the Another KE-3’UTR-5pH1-shRNA plasmid (generating

Figure 5. Suppression of MYOC-EGFP by short hairpin RNAs specific to various coding regions of MYOC in cultured HEK293 cells. A: Representative photographs of HEK293 cells from fluorescence microscopy after cotransfection of MYOCpEGFP with various shMYOCs are shown. The fluorescence photographs were taken with the same exposure times at 48 h after cotransfection. C: Cotransfection of MYOCpEGFP with a control plasmid; 1: Cotransfection of MYOCpEGFP with shMYOC-A; 2: Cotransfection of MYOCpEGFP with shMYOC- B; 3: Cotransfection of MYOCpEGFP with shMYOC-C; and 4: Cotransfection of MYOCpEGFP with shMYOC-D. Significant reduction of MYOC-EGFP was noted in HEK293 cells cotransfected with MYOCpEGFP and shMYOC-A, -B, -C, and -D, respectively (A 1-4). B: Western blot of protein lysates from cultured HEK293 cells after cotransfection of MYOCpEGFP with various shMYOCs as in A is shown. Equal amounts of protein lysates from HEK 293 cells (20 µg/lane) were loaded for each lane as indicated by similar intensities of β-actin in each lane. The MYOC-EGFP fusion protein and β-actin were detected with anti-EGFP and anti β-actin antibodies, respectively. Consistent with the findings of fluorescence microscopy in A, significant reduction of MYOC-EGFP was noted in HEK293 cells cotransfected with MYOCpEGFP and shMYOC-A, -B, -C, and -D, respectively (lanes 1-4). These protein bands were then digitized to quantify the suppression efficiency of each shRNA (see the Results section for average suppression efficiency of each shMYOC). 2089 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision siRNAs targeting the 3'-UTR region) suppressed greater than than 80% as shown in Figure 3C, lane 2, arrowhead) by this 80% of the KE-EGFP in HEK293 cells (Figure 3A,B). West- shRNA. These results indicated that these two shRNAs could ern blot of protein lysates from HEK293 cells further con- effectively suppress the expression of KE gene. Similar sup- firmed a significant suppression of KE fusion proteins (greater pression of KE-EGFP mRNA was also confirmed by North- ern hybridization (data not shown). In summary, by transfecting HEK293 cells at 70%-90% confluency, we identified two shRNAs that could effectively suppress the expression of KE fusion proteins by approximately 50% and 80%, respectively. Identification of myocilin-specific short hairpin RNAs: From the published coding sequence of human MYOC, 102 candidate siRNAs were initially identified. Candidate siRNAs with sequences covering amino acids R76, E352, K423, or N480 were chosen to test their suppression efficiencies as mutations of these residues have been reported to be associated with POAG (Table 1 and Figure 4). The Blast search indicated that these siRNAs were specific for human MYOC.

Figure 6. Suppression of wild-type (MYOC-WT) and mutant Q368X (MYOC-Q368X) myocilin proteins by shMYOC-A (siMYOC-A) in TM5 cells. A: Western blot of protein lysates from cultured HEK293 cells after cotransfections of MYOCpEGFP or MYOCQ368XpEGFP with control pH1-RNA or shMYOC-A plasmids is shown. The Figure 7. BiP activation as a stress response to myocilins in TM5 myocilin fusion protein and β-actin were detected with anti-EGFP cells by luciferase reporter assays. BiPpGL3 vector was cotransfected and anti β-actin antibodies, respectively. After cotransfection of in TM5 cells with either MYOCpEGFP (MYOC-WT) or MYOCpEGFP with control pH1-RNA, abundant wild-type MYOC- MYOCQ368XpEGFP (MYOC-Q368X) along with control pH1- EGFP was noted in HEK 293 cell lysates. Moderate suppression of RNA or shMYOC-A. Dual luciferase assays were performed at 48 h wild-type MYOC-EGFP by shMYOC-A was noted after after transfections. The results were from three independent trans- cotransfection of MYOCpEGFP with shMYOC-A (MYOC-WT). fection experiments and each experiment was tested in triplicates Similarly, abundant mutant Q368X-EGFP was noted in HEK 293 (n=9, bars=SD). The activity of BiP in TM5 cells after the cell lysates after cotransfection of MYOCQ368XpEGFP with con- cotransfection of MYOCpEGFP (MYOC-WT) and control pH1-RNA trol pH1-RNA. Moderate reduction of mutant Q368X-EGFP was was used as a baseline control (100%, as seen in the second bar). noted after cotransfection of MYOCQ368XpEGFP with shMYOC- Compared with the baseline, cotransfection of pEGFP (without A (MYOC-Q368X). B: Suppression efficiency of wild-type and MYOC) and the control, pH1-RNA, showed less activation of BiP in mutant myocilins by shMYOC-A is shown in a graph. The protein TM5 cells (as shown in the first bar), indicating that the presence of bands from A were digitized to quantify the suppression efficiency MYOC-WT could induce moderate stress response in TM5 cells. of shMYOC-A on wild-type MYOC-EGFP and mutant Q368X-EGFP Significant activation of BiP was noted in TM5 cells after using UN-SCAN-IT software. The pixel intensities from the myocilin cotransfection of MYOCQ368XpEGFP (MYOC-Q368X) and con- fusion proteins were normalized to the pixel intensities from the β- trol pH1-RNA plasmids, indicating that mutant MYOC-Q368X in- actin bands. The ratio of intensities between the control shRNA and duced a more pronounced stress response than MYOC-WT (The as- shMYOC-A was used to determine the suppression efficiency. Com- terisk indicates that p<0.001, as seen in the third bar). Cotransfection pared with their respective control of wild-type (MYOC-WT) and of shMYOC-A significantly reduced the activation of BiP induced mutant Q368X (MYOC-Q368X) myocilins, shMYOC-A reduced the by mutant MYOC-Q368X (The double asterisk indicates that expression of MYOC-WT and MYOC-Q368X to 58.9%±10.6% (the p<0.001, as seen in the fourth bar), indicating shMYOC-A could re- asterisk indicates a p<0.02) and 60.8%±6.4% (the double asterisk duce the stress response induced by the mutant myocilin to a baseline indicates a p<0.03), respectively (n=3, bars=SD). level similar to that induced by wild-type myocilin. 2090 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision

These candidate siRNAs had neither a sequence similarity to 58.9%±10.6% and 60.8%±6.4% of the control level, respec- EGFP nor the capability of suppressing EGFP expression when tively (Figure 6B). Under these experimental conditions, tested in cultured HEK293 cells (data not shown). The tar- shMYOC-A on average suppressed the expression of both wild geted myocilin domains of these shRNAs were shown in Fig- type and mutant myocilins by 40% in TM5 cells when trans- ure 4. One shRNA was targeted at the myosin-like domain fected at 50% confluency. (R76, shMYOC-A), and the other three were targeted at the Activation of BiP by BiP promoter-driven luciferase as- olfactomedin region (E352, shMYOC-B; K423, shMYOC-C; say: To further study the capability of MYOC-specific shRNAs and N480, shMYOC-D). in reducing the cytotoxic effects induced by mutant myocilins, Plasmids generating MYOC-specific shRNAs were the activation of BiP, one of the stress-response elements in cotransfected with MYOCpEGFP to evaluate their efficiency endoplasmic reticulum (ER) was investigated in TM5 cells. of suppressing myocilin expression in cultured HEK293 cells. Using a BiP promoter-driven luciferase assay, cotransfection As shown in the representative photographs of Figure 5A 1-4, of TM5 cells with MYCOpEGFP plasmids generating wild- evident reduction of MYOC-EGFP fluorescence was noted in type myocilin (MYOC-WT) and the control shRNA plasmids HEK293 cells cotransfected by MYOCpEGFP with shMYOC- along with BiPpGL3 (Figure 7, bar 2) showed a mild increase A, -B, -C, or-D, respectively, when compared with control of BiP activation when compared with the pEGFP (contain- shRNA (Figure 5A,C). These results suggested that success- ing no myocilin as a baseline control, Figure 7, bar 1). On the ful suppression of fusion proteins was achieved by these other hand, cotransfection of mutant Q368XpEGFP generat- MYOC-specific shRNAs. ing truncated Q368X mutant myocilin (MYOC-Q368X) with The suppression of MYOC-EGFP proteins by these control shRNA plasmids and BiPpGL3 (Figure 7, bar 3) re- MYOC-specific shRNAs was further confirmed by western sulted in statistically significant activation of BiP in TM5 cells blots of HEK293 cell lysates after each shRNA was when compared to transfection with MYCO-WT (with an av- cotransfected with MYOCpEGFP. As shown in one of the rep- erage increase of 214% BiP activation) or EGFP (with an av- resentative blots (Figure 5B), shRNAs targeting regions sur- erage increase of 300% BiP activation). Most importantly, rounding amino acids R76, E352, K423, and N480, respec- cotransfection of shMYOC-A with MYOC-Q368X (Figure 7, tively, were effective in suppressing the expression of MYOC- bar 4) significantly reduced the activation of BiP to 50% when EGFP fusion proteins (lanes 1-4) when compared with a con- compared to cotransfection of MYOC-Q368X with the control shRNA (lane C). The staining intensity of β-actin indi- trol sh-RNA plasmids (Figure 7, bar 3). Furthermore, our re- cated comparable protein loadings in each sample. The reduc- sults showed that shMYOC-A could effectively mitigate the tion of MYOC-EGFP intensity was further quantified with stress response of TM5 cells induced by mutant MYOC- UN-SCAN-IT software on digitized protein bands of the west- Q368X (as indicated by BiP activation) to a level comparable ern blots. When compared with the control siRNA (Figure 5B, to that induced by wild-type myocilin (MYOC-WT; Figure 7, lane C), the expression of MYOC-EGFP was reduced to cf. bars 2 and 4). 13.3%±10.9%, 10.2%±15.3%, 10.5%±14.2%, and 11.3%±9.9% (mean±SD, n=5) by shMYOC-A, -B, -C, and - DISCUSSION D, respectively. When compared with the control shRNA, all The pathogenesis for genetic diseases could be due to one of of these four MYOC-specific shRNAs showed statistically sig- two mechanisms: (1) gain-of-function (for example, mutant nificant suppression of myocilin in HEK293 cells (Student’s proteins generate cytotoxic or pathogenic effects) or (2) loss- t-test, p<0.05). Reduction of MYOC-EGFP mRNAs by these of-function or haloinsufficiency (such as the failure of pro- shRNAs was also noted, but we did not specifically quantify ducing sufficient amount of proteins to achieve or maintain the percentage of reduction (data not shown). As shown by proper cellular functions). RNA interference (RNAi) is a use- analyses with western blots, these MYOC-specific shRNAs ful tool to silence those untoward gene mutations, especially on average achieved between 80%-90% reduction of myocilin the ones associated with abnormal protein production known fusion protein in HEK293 cells when transfected at 70%-90% as gain-of-function. Accumulation of abnormal KE and cell confluency. myocilin proteins as a result of gene mutations has been linked Suppression of mutant myocilin by short hairpin RNAs in to autosomal dominant corneal stromal dystrophies and cer- cultured TM5 cells: In addition to suppressing wild type tain types of inherited open-angle glaucoma, respectively. myocilin, the suppression of mutant myocilins in TM5 cells, a There is strong evidence suggesting that KE-related corneal transformed cell line of trabecular meshwork, by those dystrophies and myocilin-related OAGs are due to the gain- myocilin-specific shRNAs was further evaluated. Such experi- of-function mechanism rather than the loss-of-function mecha- ments were conducted in TM5 cells rather than the routine nism. For KE-related corneal dystrophies, it is self-evident HEK293 cells to simulate glaucoma in vitro. As shown in Fig- that accumulation of mutant proteins leads to corneal opaci- ure 6A, when compared with the control pH1-RNA plasmid, ties and poor epithelial adhesion to the corneal stroma. The shMYOC-A effectively reduced the expression of both severity of corneal dystrophies correlates well with the extent MYOC-EGFP (wild type) and the Q368X-EGFP mutant in of mutant KE aggregations. TM5 cells. Using UN-SCAN-IT software to digitize the pro- There is evidence indicating the presence of misfolded tein bands of western blots, the expression of MYOC-EGFP proteins in glaucoma patients such as overexpression of αB- and Q368X-EGFP mutant was reduced by shMYOC-A to crystallin along with myocilin [34] and colocalization of pro- 2091 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision tein disulfide isomerase (PDI) with aggregated mutant pressed MYOC-EGFP fusion proteins up to 90% in HEK293 myocilin in the endoplasmic reticulum (ER) [16]. When cells and around 40% in TM5 cells (Figure 6B, bar 2) with a cDNAs, encoding mutant myocilin, were transfected into cul- linear dose-response of MYOC suppression by MYOC- tured TM cells, the expressed mutant myocilins failed to be shRNAs in the cotransfection experiments (data not shown). secreted extracellularly and formed aggregates of misfolded Increases in dosages of MYOC-shRNAs rendered more sup- proteins inside the TM cells. Mutant myocilins also prevented pression of MYOC fusion proteins. The dose or molar ratio of the secretion of wild-type protein when cDNAs of both wild- siRNA to target gene will be an important factor to consider type and mutant myocilins were cotransfected into cultured when applying RNAi technology for potential clinical use. TM cells [14,16]. Since myocilin forms dimers or even oligo- Although the BiP activation by Q368X mutant myocilins was mers in vivo [35], it is likely that misfolded mutant myocilins effectively reduced by shMYOC-A to a baseline level induced bind and “trap” wild-type proteins inside the cells. Further- by wild type myocilin (MYOC-WT in Figure 7) in TM5 cells, more, it has been demonstrated that the expression of mutant the correlation between the suppression of mutant myocilins myocilin in cultured TM cells led to cellular deformity, de- and the increased survival of TM5 cells has not yet been es- creased cell proliferation, increased ER stress, and significant tablished. cell death of TM cells [16,28]. Taken together, these data sug- While being able to devise shRNAs with successful sup- gest that glaucoma associated with abnormal secretion of pression of wild type KE and myocilin in vitro as demon- mutant and/or wild-type myocilins in humans are mediated strated herein, we intend to further investigate the feasibility through a gain-of-function mechanism with untoward cyto- of using mutation-specific siRNAs to selectively suppress the toxicity and cell death of TM induced by mutant myocilins. mutant genes. Since the physiological functions of KE and To treat these conditions associated with mutant proteins, myocilin proteins still remain unclear, the nondiscriminating it is logical to suppress the expression of mutant genes to ame- nature of those shRNAs used in this study certainly raises the liorate the accumulation of untoward disease-causing proteins. concerns of safety regarding their clinical applications. Their In this study, the feasibility of using RNAi to suppress the safety and efficacy profiles should be further evaluated in vivo, expression of KE and MYOC in vitro was explored. Our re- preferably using pertinent animal models. To the best of our sults demonstrated that KE-specific and MYOC-specific knowledge, multiple attempts to produce transgenic animals shRNAs could effectively suppress the expression of recom- with genetic knock-in or knockout of KE have failed to show binant KE and myocilin proteins, respectively, in HEK293 cells any corneal phenotype consistent with corneal stromal dys- and TM5 cells. trophies or other systemic pathology (personal communica- Since our working hypotheses was that myocilin-related tions with Dr. Gordon Klintworth, Duke University, Durham, glaucoma is induced by the misfolding of mutant myocilins NC, December, 2006). Similarly, MYOC-knockout mice did and related cytotoxicity of TM, shRNAs that suppress the ac- not reveal any discernable phenotype with normal IOP being cumulation of mutant myocilins may potentially be used to noted in MYOC-null animals [36]. Furthermore, patients with mitigate the adverse consequences in TM cells. Many myocilin the deletion of MYOC did not develop glaucoma or other ocu- mutations linked to POAG are missense mutations within the lar abnormalities [37]. It was estimated in a recent study that olfactomedin domain. Among them, the myocilin Q368X non- 3% of the Asian control subjects carrying an Arg46Stop mu- sense mutation is one of the most common mutations found in tation, which results in a severely truncated form of myocilin, POAG patients. The experiments of transfecting MYOC- missing more than 90% of amino acids of the wild-type pro- Q368X into TM5 cells (simulating an empirical glaucoma tein, were not affected by glaucoma [38]. Among many indi- model) with subsequent upregulation of BiP reconfirmed the viduals carrying this Arg46Stop mutation, only a few were notion that mutant myocilins can cause an untoward stress found to have evidence of glaucoma [38,39]. The fact that response in TM cells. As shown in Figure 7, suppression of patients with a deleted MYOC gene or with the Arg46Stop MYOC-Q368X by myocilin-specific shRNAs indeed reduced mutation remain asymptomatic and that no phenotype has been the activation BiP (indicative of reduced ER stress) in TM5 observed in KE-knockout animals further supports the notion cells. These MYOC-specific shRNAs were capable of suppress- that loss of wild-type myocilin or keratoepithelin as a result ing the expression of wild type and mutant myocilins in cul- of RNAi should not result in significant ocular pathology. Con- tured HEK293 and TM5 cells. More importantly, they could sequently, these findings may also argue in favor of using the mitigate the stress response induced by mutant myocilins such non-selective suppression of KE-related or myocilin-related as MYOC-Q368X. Their high suppression efficiency and their mutations for clinical applications. capabilities of reversing the cytotoxicity induced by mutant Although several reports have indicated that mutation- myocilins should render them as a potential for future studies specific suppression can be achieved in various genes [40,41], on the functions of myocilin. others have questioned whether such specificity could actu- It remains unclear whether cell death of TM is linearly ally be achieved or would be necessary due to the RNAi mecha- correlated with the accumulation of mutant myocilins. In other nism. It has been suggested that during the course of RNAi, words, what is the relationship between the reduction of mu- the target mRNA is converted to long dsRNA and enzymati- tant proteins and the improved survival of TM cells? In this cally digested by Dicer into siRNAs [42]. These newly gener- study, 100% suppression of myocilin could not be achieved ated siRNAs will in turn “amplify” and drive gene silencing by any individual shRNA of ours. These four shRNAs sup- further, resulting in a chain reaction of mRNA degradation. In 2092 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision other words, although the initiation of RNAi can be directed efficacy of our shRNAs may be affected by the local tissue to the mutant mRNA, the mutant-specific siRNA may eventu- temperature in vivo studies especially the anterior segment of ally suppress wild-type mRNAs in trans via siRNA amplifi- the eye with the surrounding temperature being cooler than cation. the core temperature of the body. Compared with synthetic While our MYOC-specific shRNAs could effectively sup- siRNAs, shRNAs are likely to be more thermally stable and press the expression of myocilin, it remains possible that com- less susceptible to RNA degradation, given its looped hair-pin plete removal of mutant proteins is necessary to abolish the configuration. Regardless, we believe that our study is the first cytotoxic effect exerted by mutant myocilins. Our MYOC- in vitro investigation on exploring the use of shRNAs to in- shRNAs were originally designed based on their potential hibit the synthesis of myocilin and keratoepithelin. ability to inhibit a target mutation selectively. However, to Protein misfolding and conformational changes are the our pleasant surprise, these shRNAs showed potent suppres- main causes of many diseases such as cystic fibrosis and sion of both wild-type and mutant myocilins under our ex- Alzheimer disease [45]. Therefore, these diseases are known perimental conditions. Since there is no adverse effect observed as “conformational diseases”. In each condition, the mutated in MYOC-knockout animals, these nonselective shRNAs may or degraded form of disease-associated proteins together with potentially be used for myocilin-related glaucoma. As such, other nonprotein components show a tendency to form fibril they may be used as an individual agent or as a mixture of or plaque (amyloid-like) structures. Fibril or amyloid forma- shRNAs to alleviate nonselectively the aggregations of mu- tion of the mutated proteins may be the underlying cause of tant and/or wild type myocilins. We intend to further explore these conformational diseases. KE-related corneal dystrophies the synergistic effects of these shRNAs by combining all or have characteristic stromal deposits of rod-shaped amyloid or some of them to achieve maximal or complete myocilin sup- curly, non-amyloid fiber aggregates. We therefore surmise that pression. Instead of using mutation-specific shRNAs to selec- those abnormal stromal deposits are caused by a misfolding tively suppress both copies of the mutant gene in homozy- or conformational change of mutant KEs and may contribute gous cells, such a “shotgun” approach by combining nonse- to the observed clinical morbidities. Interestingly, even though lective shRNAs targeting the nonmutated sequence to sup- mutations are present systemically in affected individuals with press both wild type and mutant myocilins may more effec- KE-related corneal dystrophies, the cornea seems to be the tively reduce myocilin-related cytotoxicity in heterozygous only tissue known to be affected. Current therapies are lim- cells (containing one copy of the wild type gene and one copy ited to supportive treatment for corneal erosions at the early of the mutant gene). Such an approach is advantageous in that stages and invasive superficial keratectomy or penetrating shRNA treatment does not need to be tailored to each glau- keratoplasty for corneal opacities at the advanced stages. Such coma-related mutation individually. If needed, a synthetic a unique tissue-specific expression of gene mutation makes myocilin gene that has silent mutations to escape from the KE-related corneal dystrophies ideal candidates for a tissue- suppression of shRNA could then be introduced into cells to specific gene manipulation instead of difficult systemic gene restore the expression of myocilin. Such a strategy known as therapy with stem cells. Although we are confident that RNAi “gene switching” has been proposed as a potential therapy for can suppress the expression of mutant KEs or myocilins, al- gain-of-function genetic diseases [43]. Conversely, those two ternative strategies to suppress the aggregation of disease-caus- KE-specific shRNAs were not designed to specifically target ing proteins do exist. Recently, we have reported on the suc- any point mutation of KE. They most likely suppress the ag- cessful use of several short synthetic KE peptides with N- gregation of both mutant and wild-type KEs thereby offering methylation of selective amino acids (known as meptides) to the potential advantage for being universal agents to suppress reduce the amyloid aggregates of recombinant KE in vitro [46]. any given KE mutation. Nonetheless, all these shRNAs will Despite the ongoing enthusiasm about the potential ocu- have to be thoroughly evaluated in animal models or other lar applications of RNAi, several recent studies using highly relevant in vitro systems before any conclusion regarding their sensitive microarray analyses have shown that siRNAs can potential clinical application can be drawn. have off-target effects by silencing unintended genes [47,48]. As shown in our results, the applicability of RNAi can be These unwanted off-target effects can be potentially minimized affected by cell type, cell density, and various experimental by modifying the siRNAs to prevent incorporation of the sense conditions. Interestingly, a temperature-dependent gene silenc- strand into RNA-induced silencing complex (RISC) or by se- ing was noted among several commercially available synthetic lecting sequences with minimal complementarities to other myocilin-specific siRNAs in a recent report [44]. The myocilin known genes in the database [49]. Choosing siRNAs that are suppression efficiency by these synthetic siRNAs ranged from effective at low concentrations may also help abrogate or side- 30%-70% at temperatures from 33 °C to 37 °C. Such an effect step some of these problems. At present, the lack of animal is likely related to the thermally-modulated folding status of models for KE-related corneal dystrophies or myocilin-related mRNA and its propensity of a particular region to be base glaucoma precludes us from investigating any unintended off- paired or single-stranded. The suppression efficiency of those target effects of our shRNAs. In summary, this report demon- synthetic siRNAs is lower than what has been observed in our strated the feasibility of using shRNAs as effective “molecu- shRNAs, albeit those synthetic siRNAs and our shRNAs tar- lar silencers” to suppress the expression of KE and myocilin get different regions of the MYOC gene. Since our in vitro in vitro. studies were done with cultured HEK293 cells at 37 °C, the 2093 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision

ACKNOWLEDGEMENTS ization of subtracted cDNAs from a human ciliary body library This work was supported in part by grants from the National encoding TIGR, a protein involved in juvenile open angle glau- Eye Institute (EY016088 for A.J.W.H.), The Glaucoma Foun- coma with homology to myosin and olfactomedin. FEBS Lett dation, Fight for Sight, Eye Bank Association of America, 1997; 413:349-53. 14. Caballero M, Borras T. Inefficient processing of an olfactomedin- Minnesota Medical Foundation, and Research to Prevent deficient myocilin mutant: potential physiological relevance to Blindness. This manuscript is an abridgement of a thesis sub- glaucoma. Biochem Biophys Res Commun 2001; 282:662-70. mitted in partial fulfillment of requirements for membership 15. Russell P, Tamm ER, Grehn FJ, Picht G, Johnson M. The pres- in the American Ophthalmological Society, May 2007. ence and properties of myocilin in the aqueous humor. Invest Ophthalmol Vis Sci 2001; 42:983-6. REFERENCES 16. Sohn S, Hur W, Joe MK, Kim JH, Lee ZW, Ha KS, Kee C. Ex- 1. Skonier J, Neubauer M, Madisen L, Bennett K, Plowman GD, pression of wild-type and truncated myocilins in trabecular Purchio AF. cDNA cloning and sequence analysis of beta ig-h3, meshwork cells: their subcellular localizations and cytotoxici- a novel gene induced in a human adenocarcinoma cell line after ties. Invest Ophthalmol Vis Sci 2002; 43:3680-5. treatment with transforming growth factor-beta. DNA Cell Biol 17. Stone EM, Fingert JH, Alward WL, Nguyen TD, Polansky JR, 1992; 11:511-22. Sunden SL, Nishimura D, Clark AF, Nystuen A, Nichols BE, 2. Hashimoto K, Noshiro M, Ohno S, Kawamoto T, Satakeda H, Mackey DA, Ritch R, Kalenak JW, Craven ER, Sheffield VC. Akagawa Y, Nakashima K, Okimura A, Ishida H, Okamoto T, Identification of a gene that causes primary open angle glau- Pan H, Shen M, Yan W, Kato Y. Characterization of a cartilage- coma. Science 1997; 275:668-70. derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to 18. Fingert JH, Stone EM, Sheffield VC, Alward WL. Myocilin glau- collagen. Biochim Biophys Acta 1997; 1355:303-14. coma. Surv Ophthalmol 2002; 47:547-61. 3. Rawe IM, Zhan Q, Burrows R, Bennett K, Cintron C. Beta-ig. 19. Tamm ER. Myocilin and glaucoma: facts and ideas. Prog Retin Molecular cloning and in situ hybridization in corneal tissues. Eye Res 2002; 21:395-428. Invest Ophthalmol Vis Sci 1997; 38:893-900. 20. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello 4. Kawamoto T, Noshiro M, Shen M, Nakamasu K, Hashimoto K, CC. Potent and specific genetic interference by double-stranded Kawashima-Ohya Y, Gotoh O, Kato Y. Structural and phyloge- RNA in Caenorhabditis elegans. Nature 1998; 391:806-11. netic analyses of RGD-CAP/beta ig-h3, a fasciclin-like adhe- 21. Hamilton AJ, Baulcombe DC. A species of small antisense RNA sion protein expressed in chick chondrocytes. Biochim Biophys in posttranscriptional gene silencing in plants. Science 1999; Acta 1998; 1395:288-92. 286:950-2. 5. Munier FL, Korvatska E, Djemai A, Le Paslier D, Zografos L, 22. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl Pescia G, Schorderet DF. Kerato-epithelin mutations in four T. Duplexes of 21-nucleotide RNAs mediate RNA interference 5q31-linked corneal dystrophies. Nat Genet 1997; 15:247-51. in cultured mammalian cells. Nature 2001; 411:494-8. 6. Klintworth GK. Advances in the molecular genetics of corneal 23. Tijsterman M, Ketting RF, Plasterk RH. The genetics of RNA dystrophies. Am J Ophthalmol 1999; 128:747-54. silencing. Annu Rev Genet 2002; 36:489-519. 7. Munier FL, Frueh BE, Othenin-Girard P, Uffer S, Cousin P, Wang 24. Brummelkamp TR, Bernards R, Agami R. A system for stable MX, Heon E, Black GC, Blasi MA, Balestrazzi E, Lorenz B, expression of short interfering RNAs in mammalian cells. Sci- Escoto R, Barraquer R, Hoeltzenbein M, Gloor B, Fossarello ence 2002; 296:550-3. M, Singh AD, Arsenijevic Y, Zografos L, Schorderet DF. BIGH3 25. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression mutation spectrum in corneal dystrophies. Invest Ophthalmol of short-interfering RNAs and hairpin RNAs in mammalian cells. Vis Sci 2002; 43:949-54. Proc Natl Acad Sci U S A 2002; 99:6047-52. 8. Fauss DJ, Bloom E, Lui GM, Kurtz RM, Polansky JR. Glucocor- 26. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. ticoid (GC) effects on HTM cells: biochemical approaches and Short hairpin RNAs (shRNAs) induce sequence-specific silenc- growth factor responses. In: Lutjen-Drecoll E, editor. Basic ing in mammalian cells. Genes Dev 2002; 16:948-58. Aspects of Glaucoma Research III. Stuttgart: Schattauer-Verlag; 27. Yuan C, Yang MC, Zins EJ, Boehlke CS, Huang AJ. Identifica- 1993. p. 319-330. tion of the promoter region of the human betaIGH3 gene. Mol 9. Jurynec MJ, Riley CP, Gupta DK, Nguyen TD, McKeon RJ, Buck Vis 2004; 10:351-60. CR. TIGR is upregulated in the chronic glial scar in response to 28. Joe MK, Sohn S, Hur W, Moon Y, Choi YR, Kee C. Accumula- central nervous system injury and inhibits neurite outgrowth. tion of mutant myocilins in ER leads to ER stress and potential Mol Cell Neurosci 2003; 23:69-80. cytotoxicity in human trabecular meshwork cells. Biochem 10. Ohlmann A, Goldwich A, Flugel-Koch C, Fuchs AV, Schwager Biophys Res Commun 2003; 312:592-600. K, Tamm ER. Secreted glycoprotein myocilin is a component 29. Jacobson N, Andrews M, Shepard AR, Nishimura D, Searby C, of the myelin sheath in peripheral nerves. Glia 2003; 43:128- Fingert JH, Hageman G, Mullins R, Davidson BL, Kwon YH, 40. Alward WL, Stone EM, Clark AF, Sheffield VC. Non-secretion 11. Ricard CS, Agapova OA, Salvador-Silva M, Kaufman PL, of mutant proteins of the glaucoma gene myocilin in cultured Hernandez MR. Expression of myocilin/TIGR in normal and trabecular meshwork cells and in aqueous humor. Hum Mol glaucomatous primate optic nerves. Exp Eye Res 2001; 73:433- Genet 2001; 10:117-25. 47. 30. Pang IH, Shade DL, Clark AF, Steely HT, DeSantis L. Prelimi- 12. Borras T, Morozova TV, Heinsohn SL, Lyman RF, Mackay TF, nary characterization of a transformed cell strain derived from Anholt RR. Transcription profiling in Drosophila eyes that human trabecular meshwork. Curr Eye Res 1994; 13:51-63. overexpress the human glaucoma-associated trabecular mesh- 31. Wordinger RJ, Lambert W, Agarwal R, Talati M, Clark AF. Hu- work-inducible glucocorticoid response protein/myocilin (TIGR/ man trabecular meshwork cells secrete neurotrophins and ex- MYOC). Genetics 2003; 163:637-45. press neurotrophin receptors (Trk). Invest Ophthalmol Vis Sci 13. Ortego J, Escribano J, Coca-Prados M. Cloning and character- 2000; 41:3833-41. 2094 Molecular Vision 2007; 13:2083-95 ©2007 Molecular Vision

32. Yuan C, Reuland JM, Lee L, Huang AJ. Optimized expression efficient tools to inactivate oncogenic mutations and restore p53 and refolding of human keratoepithelin in BL21 (DE3). Protein pathways. Proc Natl Acad Sci U S A 2002; 99:14849-54. Expr Purif 2004; 35:39-45. 41. Miller VM, Xia H, Marrs GL, Gouvion CM, Lee G, Davidson 33. Szczesna-Skorupa E, Chen CD, Liu H, Kemper B. Gene expres- BL, Paulson HL. Allele-specific silencing of dominant disease sion changes associated with the endoplasmic reticulum stress genes. Proc Natl Acad Sci U S A 2003; 100:7195-200. response induced by microsomal cytochrome p450 overproduc- 42. Lipardi C, Wei Q, Paterson BM. RNAi as random degradative tion. J Biol Chem 2004; 279:13953-61. PCR: siRNA primers convert mRNA into dsRNAs that are de- 34. Lutjen-Drecoll E, May CA, Polansky JR, Johnson DH, graded to generate new siRNAs. Cell 2001; 107:297-307. Bloemendal H, Nguyen TD. Localization of the stress proteins 43. Morcos PA. Achieving efficient delivery of morpholino oligos in alpha B-crystallin and trabecular meshwork inducible gluco- cultured cells. Genesis 2001; 30:94-102. corticoid response protein in normal and glaucomatous trabe- 44. Russell P, Walsh E, Chen W, Goldwich A, Tamm ER. The effect cular meshwork. Invest Ophthalmol Vis Sci 1998; 39:517-25. of temperature on gene silencing by siRNAs: implications for 35. Fautsch MP, Johnson DH. Characterization of myocilin-myocilin silencing in the anterior chamber of the eye. Exp Eye Res 2006; interactions. Invest Ophthalmol Vis Sci 2001; 42:2324-31. 82:1011-6. 36. Kim BS, Savinova OV, Reedy MV, Martin J, Lun Y, Gan L, Smith 45. Zerovnik E. Amyloid-fibril formation. Proposed mechanisms and RS, Tomarev SI, John SW, Johnson RL. Targeted Disruption of relevance to conformational disease. Eur J Biochem 2002; the Myocilin Gene (Myoc) Suggests that Human Glaucoma- 269:3362-71. Causing Mutations Are Gain of Function. Mol Cell Biol 2001; 46. Yuan C, Berscheit HL, Huang AJ. Identification of an 21:7707-13. amyloidogenic region on keratoepithelin via synthetic peptides. 37. Wiggs JL, Vollrath D. Molecular and clinical evaluation of a pa- FEBS Lett 2007; 581:241-7. tient hemizygous for TIGR/MYOC. Arch Ophthalmol 2001; 47. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, 119:1674-8. Mao M, Li B, Cavet G, Linsley PS. Expression profiling re- 38. Lam DS, Leung YF, Chua JK, Baum L, Fan DS, Choy KW, Pang veals off-target gene regulation by RNAi. Nat Biotechnol 2003; CP. Truncations in the TIGR gene in individuals with and with- 21:635-7. out primary open-angle glaucoma. Invest Ophthalmol Vis Sci 48. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, 2000; 41:1386-91. Umayam L, Lee JC, Hughes CM, Shanmugam KS, 39. Yoon SJ, Kim HS, Moon JI, Lim JM, Joo CK. Mutations of the Bhattacharjee A, Meyerson M, Collins FS. Short interfering TIGR/MYOC gene in primary open-angle glaucoma in Korea. RNAs can induce unexpected and divergent changes in the lev- Am J Hum Genet 1999; 64:1775-8. els of untargeted proteins in mammalian cells. Proc Natl Acad 40. Martinez LA, Naguibneva I, Lehrmann H, Vervisch A, Tchenio Sci U S A 2004; 101:1892-7. T, Lozano G, Harel-Bellan A. Synthetic small inhibiting RNAs: 49. Pebernard S, Iggo RD. Determinants of interferon-stimulated gene induction by RNAi vectors. Differentiation 2004; 72:103-11.

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