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ELEONORA MAURIZI

Ricercatore t.d. art. 24 c. 3 lett. A
Dipartimento di Scienze della Vita sede Centro di Medicina Rigenerativa

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Pubblicazioni

2023 - GSK-3 inhibition reverts mesenchymal transition in primary human corneal endothelial cells [Articolo su rivista]
Maurizi, E.; Merra, A.; Macaluso, C.; Schiroli, D.; Pellegrini, G.
abstract

Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK-3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of α-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed that CHIR99021 induced downregulation of EnMT markers (α-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the β-catenin and TGFβ pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT, providing a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy.

2023 - Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro [Articolo su rivista]
Parisi, L.; Bianchi, M. G.; Ghezzi, B.; Maurizi, E.; Macaluso, G. M.; Bussolati, O.; Lumetti, S.
abstract

Background/purpose: Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper in vitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype. Materials and methods: We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates. Results: Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. Conclusion: These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application.

2022 - Fluctuations in Corneal Endothelial LAP2 Expression Levels Correlate with Passage Dependent Declines in Their Cell Proliferative Activity [Articolo su rivista]
Maurizi, E.; Merra, A.; Schiroli, D.; Ghezzi, B.; Macaluso, C.; Pellegrini, G.
abstract

The corneal endothelium is the inner corneal mono‐layered epithelium, fundamental for preserving corneal hydration and transparency. However, molecular mechanisms that regulate corneal endothelial cells (CEnCs), in particular regarding their proliferative capacity, have been only partially elucidated. CEnCs are quiescent in vivo and they easily undergo endothelial to mesenchymal transition (EnMT) in vitro. This study aims to analyze CEnCs behavior and expression in vitro, either in sub‐confluent growing (S) or confluent (C) CEnCs cultures. Primary rabbit and human CEnCs were cultured and used for RT‐PCR, immunofluorescence or western blot analysis. These methods allowed identifying a novel molecular marker, LAP2, that is upregulated in S while downregulated in C human or rabbit CEnCs. Those results were observed for several subsequent passages in culture and this, together with the correlation between ki67 and LAP2 expression, suggested LAP2 as a novel possible indicator for culture ageing. Finally, treatment with FGF and TGFβ in rCEnCs highlighted how LAP2 can vary as the cells regulate their proliferative state. In conclusion, we have identified a novel marker for CEnCs, LAP2, that regulates its expression depending on the cells sub/confluent state and that correlates with CEnCs proliferation.

2022 - Mother-newborn ABO group discrepancy caused by a rare BW.17 variant [Articolo su rivista]
Marraccini, C.; Iotti, B.; Vanzanelli, P.; Bedeschi, E.; Farnetti, E.; Nicoli, D.; Berni, P.; Razzoli, A.; Maurizi, E.; Gavioli, G.; Mazzi, A.; Baricchi, R.; Merolle, L.; Schiroli, D.
abstract

Several ABO gene mutations are known to determine rare subgroups: these ABO variants are often responsible for weak or null phenotypes and may cause an incorrect determination of the serotype. Here we describe for the first time the phenotypic discrepancy of a rare B allele within the same Caucasian family that depends on the co-inheritance with A or H antigen. Blood samples from newborns, mothers, and grandmothers were analysed through routine serotype and genotype testing. Blood compatibility test was performed for red blood cells or serum of the grandmother. ABO exons were investigated through PCR and sanger sequencing. According to serology, the phenotype of the mother was AB, while it was O for the newborn. Genotype analysis confirmed that the mother was AB, while the newborn was found to be B. Sanger sequencing revealed the presence of a rare mutation in both individuals (784 G>A, D262N), corresponding to the ABO*BW.17 allele. The grandmother was found to have the same genotype/serotype of the newborn. Crossmatch testing suggested that subjects with this genotype/serotype might be considered O donors and recipients.

2022 - Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium [Articolo su rivista]
Maurizi, E.; Martella, D. A.; Schiroli, D.; Merra, A.; Mustfa, S. A.; Pellegrini, G.; Macaluso, C.; Chiappini, C.
abstract

Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction.

2022 - Nanoneedles for targeted {siRNA} silencing of p16 in the Human Corneal Endothelium [Articolo su rivista]
Maurizi, Eleonora; Alessandro Martella, Davide; Schiroli, Davide; Merra, Alessia; Ahmad Mustfa, Salman; Pellegrini, Graziella; Macaluso, Claudio; Chiappini, Ciro
abstract

2022 - Platelet-rich Plasma Lysate for Treatment of Eye Surface Diseases [Articolo su rivista]
Merolle, Lucia; Iotti, Barbara; Berni, Pamela; Bedeschi, Elisa; Boito, Katya; Maurizi, Eleonora; Gavioli, Gaia; Razzoli, Agnese; Baricchi, Roberto; Marraccini, Chiara; Schiroli, Davide
abstract

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite and growth factor content, which promotes eye surface regeneration. Blood-based eye drops can be prepared from different sources (i.e., whole blood or platelet apheresis donation), as well as with different protocols (e.g., different dilutions and freeze/thaw cycles). This variability hampers the standardization of clinical protocols and, consequently, the evaluation of their clinical efficacy. Detailing and sharing the methodological procedures may contribute to defining common guidelines. Over the last years, allogenic products have been diffusing as an alternative to the autologous treatments since they guarantee higher efficacy standards; among them, the platelet-rich plasma lysate (PRP-L) eye drops are prepared with simple manufacturing procedures. In the transfusion medicine unit at AUSL-IRCCS di Reggio Emilia, Italy, PRP-L is obtained from platelet-apheresis donation. This product is initially diluted to 0.3 x 109 platelets/mL (starting from an average concentration of 1 x 109 platelets/mL) in 0.9% NaCl. Diluted platelets are frozen/thawed and, subsequently, centrifuged to eliminate debris. The final volume is split into 1.45 mL aliquots and stored at -80 °C. Before being dispensed to patients, eye drops are tested for sterility. Patients may store platelet lysates at -15 °C for up to 1 month. The growth factor composition is also assessed from randomly selected aliquots, and the mean values are reported here.

2022 - The cell as a tool to understand and repair urethra [Capitolo/Saggio]
Sceberras, Virginia; Maria Magrelli, Federica; Adamo, Davide; Maurizi, Eleonora; Attico, Eustachio; Giuseppe Genna, Vincenzo; Lazzeri, Massimo; Barbagli, Guido; Pellegrini, Graziella
abstract

2021 - Nanomedicine Approaches to Negotiate Local Biobarriers for Topical Drug Delivery [Articolo su rivista]
Mustfa, S. A.; Maurizi, E.; Mcgrath, J.; Chiappini, C.
abstract

Topical treatments have been widely adopted to address a broad range of conditions across multiple sites thanks to their convenience, versatility, and effectiveness. While bypassing systemic biobarriers and avoiding systemic side effects by delivering directly to the target tissue, topical treatments still face significant local biobarriers that limit their efficacy. The toolset available for nanodelivery systems and their inherent multifunctionality can contribute to simultaneously address otherwise intractable challenges related to barrier function evasion, drug solubility, bioavailability, pharmacokinetics, smart and sustained release, quantitative co-delivery, and local targeting which are key to successful topical treatments. This review summarizes the outstanding challenges associated with the topical treatments of key diseases of the skin, mucosae, eyes, and ears, and highlights how nanodelivery systems are being developed to address them effectively.

2021 - Regenerative Medicine of Epithelia: Lessons From the Past and Future Goals [Articolo su rivista]
Maurizi, Eleonora; Adamo, Davide; Magrelli, FEDERICA MARIA; Galaverni, Giulia; Attico, Eustachio; Merra, Alessia; Maffezzoni, MARIA BENEDETTA RIZZARDA; Losi, Lorena; Genna, VINCENZO GIUSEPPE; Sceberras, Virginia; Pellegrini, Graziella
abstract

This article explores examples of successful and unsuccessful regenerative medicine on human epithelia. To evaluate the applications of the first regenerated tissues, the analysis of the past successes and failures addresses some pending issues and lay the groundwork for developing new therapies. Research should still be encouraged to fill the gap between pathologies, clinical applications and what regenerative medicine can attain with current knowledge.

2020 - A fine-tuned β-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis [Articolo su rivista]
Maurizi, E.; Schiroli, D.; Zini, R.; Limongelli, A.; Misto, R.; Macaluso, C.; Pellegrini, G.
abstract

Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to re-activate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo—G0/G1) and proliferating (in vitro—G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed β-catenin and transforming growth factor (TGF-β) pathways to be correlated with the identified proteins. Treatment of rCEnC with a β-catenin activator and inhibitor showed that β-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-β were regulated through β-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC re-activate in vitro, consolidating the role of β-catenin and TGF-β.

2019 - A novel role for CRIM1 in the corneal response to UV and pterygium development [Articolo su rivista]
Maurizi, E.; Schiroli, D.; Atkinson, S. D.; Mairs, L.; Courtney, D. G.; O'Hagan, B.; Mcgilligan, V. E.; Pagnamenta, A. T.; Taylor, J. C.; Vasquez, J. J. D.; Illanes-Velarde, D. E.; Goldsmith, D.; Gouws, P.; Moore, J. E.; Nesbit, M. A.; Moore, C. B. T.
abstract

Pterygium is a pathological proliferative condition of the ocular surface, characterised by formation of a highly vascularised, fibrous tissue arising from the limbus that invades the central cornea leading to visual disturbance and, if untreated, blindness. Whilst chronic ultraviolet (UV) light exposure plays a major role in its pathogenesis, higher susceptibility to pterygium is observed in some families, suggesting a genetic component. In this study, a Northern Irish family affected by pterygium but reporting little direct exposure to UV was identified carrying a missense variant in CRIM1 NM_016441.2: c.1235 A > C (H412P) through whole-exome sequencing and subsequent analysis. CRIM1 is expressed in the developing eye, adult cornea and conjunctiva, having a role in cell differentiation and migration but also in angiogenesis, all processes involved in pterygium formation. We demonstrate elevated CRIM1 expression in pterygium tissue from additional individual Northern Irish patients compared to unaffected conjunctival controls. UV irradiation of HCE-S cells resulted in an increase in ERK phosphorylation and CRIM1 expression, the latter further elevated by the addition of the MEK1/2 inhibitor, U0126. Conversely, siRNA knockdown of CRIM1 led to decreased UV-induced ERK phosphorylation and increased BCL2 expression. Transient expression of the mutant H412P CRIM1 in corneal epithelial HCE-S cells showed that, unlike wild-type CRIM1, it was unable to reduce the cell proliferation, increased ERK phosphorylation and apoptosis induced through a decrease of BCL2 expression levels. We propose here a series of intracellular events where CRIM1 regulation of the ERK pathway prevents UV-induced cell proliferation and may play an important role in the in the pathogenesis of pterygium.

2019 - Effective In Vivo Topical Delivery of siRNA and Gene Silencing in Intact Corneal Epithelium Using a Modified Cell-Penetrating Peptide [Articolo su rivista]
Schiroli, D.; Gomara, M. J.; Maurizi, E.; Atkinson, S. D.; Mairs, L.; Christie, K. A.; Cobice, D. F.; Mccrudden, C. M.; Nesbit, M. A.; Haro, I.; Moore, T.
abstract

Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations.

2016 - CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting [Articolo su rivista]
Courtney, D. G.; Moore, J. E.; Atkinson, S. D.; Maurizi, E.; Allen, E. H. A.; Pedrioli, D. M. L.; Mclean, W. H. I.; Nesbit, M. A.; Moore, C. B. T.
abstract

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

2015 - Genetic modification possibilities in treating corneal diseases [Articolo su rivista]
Moore, T.; Courtney, D.; Atkinson, S.; Maurizi, E.; Allen, E.; Mclean, I.; Pedrioli, D. Leslie; Moore, J.; Nesbit, A.
abstract

2015 - In vivo gene silencing by siRNA delivery to the corneal epithelium in a keratin-12-bioluminescence mouse model [Relazione in Atti di Convegno]
Moore, Tara; Atkinson, Sarah; Allen, Edwin; Moore, Jonathan; Courtney, David; Maurizi, Eleonora; Nesbit, Andrew; Mclean, Irwin; Pedrioli, Deena Leslie
abstract

2015 - Keratin 12 missense mutation induces the unfolded protein response and apoptosis in meesmann epithelial corneal dystrophy [Articolo su rivista]
Allen, E. H. A.; Courtney, D. G.; Atkinson, S. D.; Moore, J. E.; Mairs, L.; Poulsen, E. T.; Schiroli, D.; Maurizi, E.; Cole, C.; Hickerson, R. P.; James, J.; Murgatroyd, H.; Smith, F. J. D.; Macewen, C.; Enghild, J. J.; Nesbit, M. A.; Leslie Pedrioli, D. M.; Mclean, W. H. I.; Moore, C. B. T.
abstract

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.

2015 - Protein composition of TGFBI-R124C- and TGFBI-R555W-associated aggregates suggests multiple mechanisms leading to lattice and granular corneal dystrophy [Articolo su rivista]
Courtney, D. G.; Poulsen, E. T.; Kennedy, S.; Moore, J. E.; Atkinson, S. D.; Maurizi, E.; Andrew Nesbit, M.; Tara Moore, C. B.; Enghild, J. J.
abstract

PURPOSE. Transforming growth factor beta-induced (TGFBI)–related dystrophies constitute the most common heritable forms of corneal dystrophy worldwide. However, other than the underlying genotypes of these conditions, a limited knowledge exists of the exact pathomechanisms of these disorders. This study expands on our previous research investigating dystrophic stromal aggregates, with the aim of better elucidating the pathomechanism of two conditions arising from the most common TGFBI mutations: granular corneal dystrophy type 1 (GCD1; R555W) and lattice corneal dystrophy type 1 (LCD1; R124C). METHODS. Patient corneas with GCD1 and LCD1 were stained with hematoxylin and eosin and Congo red to visualize stromal nonamyloid and amyloid deposits, respectively. Laser capture microdissection was used to isolate aggregates and extracted protein was analyzed by mass spectrometry. Proteins were identified and their approximate abundances were determined. Spectra of TGFBIp peptides were also recorded and quantified. RESULTS. In total, three proteins were found within GCD1 aggregates that were absent in the healthy control corneal tissue. In comparison, an additional 18 and 24 proteins within stromal LCD1 and Bowman’s LCD1 deposits, respectively, were identified. Variances surrounding the endogenous cleavage sites of TGFBIp were also noted. An increase in the number of residues experiencing cleavage was observed in both GCD1 aggregates and LCD1 deposits. CONCLUSIONS. The study reveals previously unknown differences between the protein composition of GCD1 and LCD1 aggregates, and confirms the presence of the HtrA1 protease in LCD1-amyloid aggregates. In addition, we find mutation-specific differences in the processing of mutant TGFBIp species, which may contribute to the variable phenotypes noted in TGFBI-related dystrophies.

2014 - Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I. [Articolo su rivista]
Courtney, Dg; Atkinson, Sd; Moore, Je; Maurizi, E; Serafini, Chiara; Pellegrini, Graziella; Black, Gc; Manson, Fd; Yam, Gh; Macewen, Cj; Allen, Eh; Mclean, Wh; Moore, Cb
abstract

PURPOSE: This study aimed to investigate the potency and specificity of short-interfering RNA (siRNA) treatment for TGFBI-Arg124Cys lattice corneal dystrophy type I (LCDI) using exogenous expression constructs in model systems and endogenous gene targeting in an ex vivo model using corneal epithelial cell cultures. METHODS: A panel of 19 TGFBI-Arg124Cys-specific siRNAs were assessed by a dual-luciferase reporter assay. Further assessment using pyrosequencing and qPCR was used to identify the lead siRNA; suppression of mutant TGFBIp expression was confirmed by Western blot and Congo red aggregation assays. An ex vivo model of LCDI was established using limbal biopsies from corneal dystrophy patients harboring the Arg124Cys mutation. Treatment efficiency of the siRNA was assessed for the inhibition of the mutant allele in the primary patient's corneal epithelial cells using pyrosequencing, quantitative PCR (qPCR), and an ELISA. RESULTS: A lead siRNA was identified, and demonstrated to be potent and specific in inhibiting the TGFBI-Arg124Cys mutant allele at the mRNA and protein levels. Besides high allele specificity, siRNA treatment achieved a 44% reduction of the endogenous Arg124Cys allele in an ex vivo model of LCDI. CONCLUSIONS: We have identified a lead siRNA specific to the TGFBI-Arg124Cys mutant allele associated with LCDI. Silencing of exogenous TGFBI was observed at mRNA and protein levels, and in an ex vivo model of LCDI with an efficient suppression of the endogenous mutant allele. This result indicates the potential of siRNA treatment as a personalized medicine approach for the management of heritable TGFBI-associated corneal dystrophies.

2014 - Identifying the role of matrix metalloproteinases in the pathomechanism of TGFBI Arg124Cys related Lattice Corneal Dystrophy Type I [Relazione in Atti di Convegno]
Moore, Johnny E.; Courtney, David G.; Atkinson, Sarah D.; Maurizi, Eleonora; Nesbit, Andrew M.; Pellegrini, Graziella; Azar, Dimitri T.; Mclean, Irwin W.; Moore, Tara C. B.
abstract

2014 - siRNA silencing of the mutant keratin 12 allele in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy. [Articolo su rivista]
Courtney, Dg; Atkinson, Sd; Allen, Eh; Moore, Je; Walsh, Cp; Pedrioli, Dm; Macewen, Cj; Pellegrini, Graziella; Maurizi, E; Serafini, Chiara; Fantacci, Monica; Liao, H; Irvine, Ad; Mclean, Wh; Moore, Cb
abstract

PURPOSE: The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles. METHODS: Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC). RESULTS: The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression. CONCLUSIONS: Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.