Antisense locked nucleic acid gapmers to control Candida albicans filamentation

Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNAASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2′-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field. © 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

The antisense technology uses antisense oligonucleotides (ASOs) to target a specific gene involved in diseases by means of RNA-targeting, which can lead to inhibition of gene expression and thus to a blockage in the transfer of genetic information from DNA to protein. 1 ASOs are short oligonucleotides designed to bind target RNA through standard Watson-Crick base pairing. 1 ASOs are often chemically modified in order to protect them against the action of nucleases, thus improving its biodistribution, RNA-affinity and potency. 2 The phosphorothioate (PS) backbone was one of the early developed chemical modifications, and it is characterized by the substitution of one of the nonbridging phosphate oxygen atoms by a sulfur atom ( Figure  S1). 1 , 3 , 4 The locked nucleic acid (LNA) is a sugar modification having, relative to native RNA, a methylene bridge between the 2′-oxygen and 4′-carbon atoms of the ribose sugar ( Figure  S1). 5 , 6 A derivative of LNA, named palmitoyl-2′-amino-LNA resulting from the inclusion of an N-palmitoylated nitrogen atom in the 2′-position of the ribose ring, has been developed 7 , 8 and shown to display similarly high-affinity binding as LNA to complementary RNA and DNA ( Figure S1). 2 , 7 , 9 Candidiasis is the primary fungal infection, with a mortality rate of about 40% 10 and high costs associated in hospitalized patients. 10 , 11 Candida albicans remains the most prevalent of all Candida species in the world, with a range of incidence of approximately 50%. 10 C. albicans pathogenicity is attributed to certain virulence factors, such as its ability to switch from yeast Nanomedicine: Nanotechnology, Biology, and Medicine 39 (2022) 102469 nanomedjournal.com to filamentous forms. [12][13][14][15] In addition, EFG1 is an important regulator gene of C. albicans filamentation, [16][17][18][19] and we recently proved that it is an important target to control C. albicans filamentation. 20 As a consequence of the rising levels of C. albicans resistance to the traditional antifungal therapies (e.g. fluconazole), 13 , 21 new alternative therapies, with novel mechanisms of action, enhanced therapeutic potential, improved pharmacokinetics, and lower toxicity, are urgently needed to reach the marketplace to control C. albicans infections. Despite the use of nucleic acid mimics to detect Candida species, [22][23][24] the application of ASOs to treat Candida infections is still scarce and it is limited to the first (PS) 25 and the second (2'OMe) 20 generation of ASOs. Thus, the main goal of this work was to evaluate the ability of a set of LNA-ASOs, in the so-called gapmer constitution, to control EFG1 gene expression and to reduce C. albicans filamentation (in vitro). The ability of the LNA-ASOs to control C. albicans virulence in an in vivo model (G. mellonella) was also assessed.

Design and synthesis of anti-EFG1 LNA-gapmers
Five LNA-gapmers were designed against the C. albicans EFG1 (gene orf19.610) target using the sequence 5′-AATAACGGTATGCC-3′ as starting point 20 for the inclusion of LNA nucleotide modifications. LNA-gapmer1 was used as the standard ASO during this study, and four additional LNAgapmers were subsequently designed by shortening the central DNA-nucleotide gap (LNA-gapmer2), adding PS-linkages (LNA-gapmer3 and LNA-gapmer5), and adding a palmitoyl-2′-amino-LNA modification (LNA-gapmer4 and LNA-gap-mer5) ( Table 1). In all LNA-gapmer designs, three LNA nucleotides were introduced consecutively at both 3′ and 5′ ends to increase the ASOs stability. Furthermore, in order to ensure compatibility with RNase H-mediated cleavage of the target RNA upon binding, the central regions (gap) were constituted of DNA nucleotides. 26 Finally, a scrambled PS LNA-gapmer control was synthesized with three mismatches within the gap ( Table 1).
The LNA-gapmers were synthesized using the standard phosphoramidite method on an automated nucleic acid synthesizer (PerSpective Biosystems Expedite 8909 instrument). All standard oligonucleotides were purchased from Integrated DNA Technologies (Leuven, Belgium). LNA phosphoramidites were purchased from Qiagen, and the synthesis was performed in 1.0 μmol scale using an LNA T 40 custom primer support (GE Healthcare). LNA phosphoramidites were incorporated according to the following conditions: trichloroacetic acid in CH 2 Cl 2 as detritylation reagent; 0.25 M 4,5-dicyanoimidazole (DCI) in CH 3 CN as an activator; acetic anhydride in THF (9:91; v/v) as capA solution; N-methylimidazole in THF (1:9; v/v) as capB solution; and a thiolation solution containing 0.2 M phenylacetyl disulfide (PADS) in 3-picoline/CH 3 CN (1:1, v/v) for 180 s. The coupling yields were based on the absorbance of the dimethoxytrityl cation (DMT + ) released after each coupling step. Palmitoyl-2′-amino-LNA phosphoramidite monomer was incorporated by manual-coupling 7 using 5-[3,5-bis(trifluoromethyl) phenyl]-H-tetrazole (0.25 M, in anhydrous acetonitrile) as an activator and extended coupling time (20 min). After the synthesis process, the LNA-gapmers were cleaved from the solid support and the protecting groups removed by treatment with a 1:1 mixture (v/v) of 98% aqueous methanol (v/v) and a 7 M solution of ammonia in methanol for 2 h at room temperature followed by treatment with 32% aqueous ammonia (w/w) at 55°C for 12 h. The ASOs were characterized by ion-exchange HPLC (IE-HPLC, Lachrom) ( Figure S2) and matrix-assisted laser desorption ionization time-to-flight mass spectrometry (MALDI-TOF, Microflex LT, Bruker, Daltonies) ( Figure S3). The purified ASOs were detritylated by treatment with an 80% (w/w) aqueous solution of acetic acid for 20 min at room temperature, precipitated by the addition of ice-cold acetone, and characterized by IE-HPLC (purity >90%) and MALDI-TOF mass spectrometry (confirmation of composition).

Melting temperatures (T m values)
The T m values determined for duplexes involving RNA complementary strands were measured on a PerkinElmer Lambda 35 UV/VIS spectrometer equipment with Peltier Temperature Programmer (PTP6). The concentration of each oligonucleotide was determined optically at 260 nm using their molar extinction coefficients (134,000 L per M −1 cm −1 of strands). All measurements were performed in medium salt buffer with 220 mM Na + (composition: 200 mM NaCl, 20 mM NaH 2 PO 4 and 0.1 mM EDTA, pH 7.0). The LNA-gapmer ASOs were mixed with the corresponding unmodified complementary strand at a 1:1 ratio (2.5 nmol of each strand). All melting curves for duplex denaturation were collected at a 260 nm wavelength as a function of temperature in the range from 8 to 80°C (heating rate of 1°C per min) ( Figure S4). The values of T m were determined as an average of two individual measurements.

Secondary structure
The secondary structure of the LNA-gapmers was determined by circular dichroism (CD) studies. The spectra were recovered on a JASCO DC 1500 spectrophotometer using cuvettes with 0.1 cm path length and averaged over three scans (320-200 nm, 50 nm min −1 intervals, 1 nm bandwidth, and 1 s response time) and with a background corrected using the buffer applied (5 mM MgCl 2 , 10 mM NaCl and 1 mM sodium phosphate). The LNAgapmer ASOs were used in the following constitution, i.e., 50 μM RNA, 5 mM MgCl 2 , 10 mM NaCl, and 1 mM sodium phosphate-pH 7.2. All other strands were prepared using 25 μM of each RNA using the same buffer. Hybridization step was performed by heating to 90°C for 5 min followed by slow cooling to room temperature followed by spectral recording.

Anti-EFG1 LNA-gapmers cytotoxicity
The cytotoxicity of the LNA-gapmers was determined using a 3T3 cell line (Fibroblasts, Embryonic tissue, Mouse from CCL3, American Type Culture Collection) according to the ISO10993:5. 27 For that, 3T3 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Biochrom, Germain) supplied by 10% of fetal bovine serum (FBS, Sigma Aldrich) and 1% of antibiotic-containing penicillin and streptomycin (P/ S) (Biochrom, Germain). After detachment, a suspension with 1 × 10 5 cells ml −1 was added to a 96-well plate and cells were allowed to grow until attaining 80% confluence. Different concentrations of each LNA-gapmer (10, 40 and 100 nM) were prepared in DMEM medium and 50 μL of each concentration was added to each well. The positive control was prepared by adding 50 μL of DMEM medium and the negative control by adding 50 μL of DMSO to the cells. The plates were incubated for 24 h at 37°C and 5% CO 2.

Microorganisms and growth conditions
The Candida strain used in this study was C. albicans SC5314, a Candida collection reference strain from the Biofilm group of the Centre of Biological Engineering (Braga, Portugal). The strain identification was confirmed using a chromogenic medium, CHROMagar™ Candida, through the distinction of colony colors and PCR-based sequencing with primers for TS1 and ITS4. 28 For all experiments, the yeast strain was subcultured on Sabouraud dextrose agar (SDA; Merck, Germany) and incubated for 24 h at 37°C. Cells were then inoculated in Sabouraud dextrose broth (SDB; Merck, Germany) and incubated overnight at 37°C, 120 rpm. After incubation, the cells' suspensions were centrifuged for 10 min, at 3000 ×g and 4°C, and washed twice with phosphate-buffered saline solution (PBS; pH 7, 0.1 M). Pellets were suspended in 5 ml of Roswell Park Memorial Institute 1640 medium (RPMI; pH 7; Sigma, St Louis, USA), and the cellular density was adjusted for each experiment using a Neubauer chamber (Marienfild, Land-Konicshofem, Germany) to 1 × 10 6 cells ml −1 . All experiments were performed in triplicate, and at least three independent assays were run.

Effect on filamentation
To evaluate the effect of the LNA-gapmers on C. albicans filamentation, yeast cells were incubated with each ASO for 24 h in an Erlenmeyer flask. For that, 5 mL of each LNA-gapmer at 40 nM (prepared on RPMI) was added to 5 mL of C. albicans suspension at 1 × 10 6 cells ml −1 (prepared on RPMI). The suspensions were incubated at 37°C under gentle agitation (120 rpm). The positive control was prepared with 10 mL of the same yeast cell concentration on RPMI. In addition, the LNAscrambled gapmer was also incubated with C. albicans for 24 h (as negative control). After 24 h, aliquots were recovered, and filaments were counted using a Neubauer chamber. The results were presented as percentage (%) of filamentation reduction through the following formula: In parallel, the levels of filamentation and the length of the filaments were confirmed through fluorescence microscopy (Olympus BX51) coupled with a DP71 digital camera and the filter used was DAPI 360-370/420 (Olympus Portugal SA, Porto, Portugal). C. albicans cells were stained with 1% (v/v) of calcofluor (Sigma-Aldrich, St. Louis, MO, USA) for 15 min in dark conditions, centrifuged for 5 min, and washed twice with ultra-pure water. Images were acquired with the program FluoView FV100 (Olympus). The filaments' length was determined using ImageJ plug-in software (Maryland, USA) software.

Effect on EFG1 gene expression
Reverse transcription-qPCR (qRT-PCR) was used to determine the effect of LNA-gapmers on EFG1 gene expression. After 24 h of incubation, 1 mL of each suspension was collected, subjected to centrifugation for 5 min at 6000 ×g and 4°C, and then washed once with PBS. RNA extraction was performed using the PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA). 20 To avoid potential DNA contamination, samples were treated with DNase I (Deoxyribonuclease I, Amplification Grade, Invitrogen) and the RNA concentration was determined by optical density measurement (NanoDrop 1000 Spectrophotometer Thermo Scientific®). The complementary DNA (cDNA) was synthesized using the Xpert cDNA Synthesis Mastermix (Grisp, Porto, Portugal) in accordance with the manufacturer's instructions, and qRT-PCR (CFX96, Biorad) was performed on a 96-well microtiter plate using Eva Green Supermix (Biorad, Berkeley, USA). Each reaction was performed in triplicate and mean values of relative expression were determined by the 2 −ΔΔCq method. The expression of the EFG1 gene was normalized using the ACT1 Candida reference gene. 29 Non-transcriptase reverse (NRT) controls were included in each run. The primers were designed using the Primer 3 web-based (Table S1).

Anti-EFG1 LNA-gapmers performance in vivo
The Galleria mellonella caterpillar infection model was used for the in vivo studies as previously described by Mil-Homens et al. 30 , 31 G. mellonella larvae were reared on a pollen grain and bee wax diet at 25°C in the darkness, and a micro syringe was adapted into a micrometer range so as to control the injection volume into hemolymph of larvae.

Toxicity evaluation
To test toxicity of the anti-EFG1 LNA-gapmers in vivo, 10 G. mellonella larvae were injected, via the hindmost left proleg previously sanitized with 70% (v/v) ethanol, with 5 μL of two distinct concentrations (40 and 100 nM both prepared with PBS) of each LNA-gapmer. As control, a set of larvae was injected with the same volume of PBS. Larvae were placed in Petri dishes and stored in the dark at 37°C. Larvae survival was recorded over 4 days, and survival curves were constructed.

Galleria mellonella survival
To study the effect of the LNA-gapmers on G. mellonella survival rate, larvae were injected with C. albicans and each LNA-gapmer. The concentration of C. albicans to be injected (7 × 10 7 cells mL −1 ) was selected on the basis of the G. mellonella lethality results after injection with different concentrations of yeast cells (between 7 × 10 7 cells mL −1 and 2 × 10 8 cells mL −1 ) (data not shown). Next, 10 larvae were injected with 5 μL of a suspension of C. albicans at 7 × 10 7 cells mL −1 mixed with 40 nM of each LNA-gapmer. Larvae were placed in Petri dishes and stored in the dark at 37°C for 3 days, whereupon survival curves were constructed. Caterpillars were considered dead when they displayed no movement in response to a touch with tweezers.
Histological analysis of G. mellonella fat bodies was also performed to evaluate the effects of LNA-gapmers on C.
albicans filamentation. For each condition, two larvae were recovered after pre-determined times (24 h and 48 h) and their fat bodies were removed through an incision in the midline of the ventral with a scalpel blade. The fat bodies were stored in 4% (v/ v) paraformaldehyde at 4°C to prepare for histological processing. The tissue was mounted in paraffin blocks and cut in sections of 4-5 μm, which were stained with hematoxylineosin (HE) and periodic acid Schiff (PAS). 32 , 33 The yeast and hyphae of C. albicans were observed under a light microscope. For analysis of filamentation, all areas of the histological section stained with PAS that contained hyphae and yeast cells were photographed with an OLYMPUS BX51 microscope coupled with a DP71 digital camera (Olympus Portugal SA, Porto, Portugal).

Statistical analysis
Data are expressed as the mean ± standard deviation (SD) of a least three independent experiments. Results were compared using two-way analysis of variance (ANOVA). All tests were performed with a confidence level of 95%. Kaplan-Meier survival curves were plotted and differences in survival were calculated by using log-rank Mantel-Cox statistical test. All performed with GraphPad Prism 6® (CA, USA). Figure 1 represents the results of the secondary structure for single-stranded (ss) and double-stranded(ds)-LNA:RNA compounds. All duplexes presented a similar overall conformation, thus indicating no major alterations in secondary structure. For single-strands, all LNA-gapmers present bands as expected from literature observations, 34 , 35 i.e. low positive bands around 270 nm and 220 nm and low negative bands around 240 nm and 210 nm. The dsRNA, defined as A-duplex, is characterized by a strong positive band at 270 nm coupled with a strong negative band at 210 nm, 34 , 36 , 37 as it can be seen for dsLNA:RNA in all LNA-gapmers. Table 2 and Figure S4 show the values of T m measured for all LNA-gapmers with RNA complement. The LNA-gapmer1 presented a T m of 70°C and similarly, the LNA-gapmer2 (exclusion of one nt) presented a T m of 72°C. In contrast, LNA-gapmer3 (addition of PS-linkages), LNA-gapmer4 (addition of the palmitoyl-2′-amino-LNA modification) and LNA-gapmer5 (addition of PS-linkages and the palmitoyl-2′-amino-LNA modification) showed a significant decrease in T m for 63°C, 65°C and 56°C, respectively.

Cytotoxicity of anti-EFG1 LNA-gapmers
The in vitro cytotoxicity was assessed on 3T3 cells and using different concentrations of LNA-gapmers (10, 40 and 100 nM) ( Figure 2). These results show that all LNA-gapmers are noncytotoxic in concentrations up to 40 nM, as the relative 3T3 cells viability was >70% under these conditions. 27

Anti-EFG1 LNA-gapmer ASO performance in vitro
Based on the cytotoxicity results, the ASO concentration of 40 nM was selected for the in vitro experiments. The effect of all LNA-gapmers on C. albicans filamentation and on the EFG1 gene expression was evaluated after 24 h of treatment. To note, the C. albicans strain used under this study presents a high filamentation capacity ( Figure S5, A) together with high levels of EFG1 gene expression ( Figure S5, B). Figure 3, A shows that all LNA-gapmers were able to control C. albicans filamentation with values of inhibition greater than 40%. The LNA-gapmer1 was capable to reduce around 45% of C. albicans filamentation, and this reduction increased for all other LNA-gapmers, however without statistically significant differences (P value > 0.05). The LNA-gapmer2 was able to reduce into 50% C. albicans filamentation and the LNA-gapmer3-5 into 55% (Figure 3, A). The results revealed that the LNA-gapmers modified with PSlinkages and the palmitoyl-2′-amino-LNA display a similar in vitro performance. To note, the LNA-scrambled ASO tested was unable to reduce C. albicans filamentation ( Figure S6). Figure 3, B shows that all LNA-gapmers were able to reduce the levels of EFG1 gene expression with values also greater than 40%. LNA-gapmer1 was able to reduce the levels of EFG1 expression by approximately 74%. Moreover, the LNA-gapmer2 and LNA-gapmer3 had a similar performance to LNA-gapmer1, with reductions of approximately 78% and 71%, respectively. Concerning the LNA-gapmer4 and LNA-gapmer5, the levels of gene inhibition were slightly reduced, reaching around 44% and 61%, respectively. It is important to note that the results were statistically significant when comparing the performance of all LNA-gapmers with the untreated cells (P value < 0.05), despite not presenting statistically significant differences among the LNA-gapmers (P value > 0.05).
The epifluorescence microscopy images (Figure 3, C) corroborate the results in the number of C. albicans' filaments decrease (Figure 3, A), also demonstrating its ability to reduce the filament length. Concerning this issue, the highest impact was observed with the LNA-gapmer3 and LNA-gapmer5, achieving values of reduction of around 65% and 56%, respectively.

Anti-EFG1 LNA-gapmers performance in vivo
The in vivo effects of the anti-EFG1 LNA-gapmer ASOs were evaluated using the G. mellonella model 38 (Figure 4). Notably, experiments showed the absence of in vivo toxicity for all the LNA-gapmers (40 nM) (Figure 4, A).
To evaluate the performance of the LNA-gapmers in vivo, G. mellonella larvae were injected with a lethal dose of C. albicans (7 × 10 7 cells/mL) together with 40 nM of each LNA-gapmer. As Table 2 Characterization of anti-EFG1 LNA-gapmer ASOs. The thermal denaturation temperatures (T m values,°C) of the LNA-gapmer ASOs were determined in medium salt buffer against its RNA complement. All melting curves were collected at a 260 nm wavelength as a function of temperature in the range from 8 to 80°C (heating rate of 1°C per min).

LNA-gapmer
Melting  illustrated in Figure 4, B, from the set of larvae injected with C. albicans cells (control) only 57% at 24 h, 33% at 48 h and 20% at 72 h of larvae were alive. It is important to underline that the treatment with all LNA-gapmers increased the G. mellonella survival rate. The larvae injection with the LNA-gapmer1 and LNA-gapmer2 increased the survival, respectively, to 80% and 67% at 24 h and to 53% and 47% at 48 h, with no effect observed at 72 h (with 20% of survival in control and with both LNAgapmers). The treatment of larvae with the LNA-gapmer3 and LNA-gapmer4 displayed an increase in the survival rate of approximately 90% and 83% at 24 h, 60% at 48 h and 30% and 33% at 72 h, respectively. The most promising of all LNA-gapmers was the LNA-gapmer5, which induced a survival rate of around 93% at 24 h, 77% at 48 h and 60% at 72 h. In parallel, the fat body of larvae ( Figure S7) was recovered and processed for histological analysis (Figure 4, C). As illustrated, yeast's cells are organized into clusters located around the organs or scattered around the adipose tissue. Sections of larvae infected only with C. albicans (control) showed intense C. albicans clusters composed mostly of filamentous forms, with a significant increase in tissue area engaged by hyphae from 24 h to 48 h post infection. Larvae sections treated with LNA-gapmer4 and LNA-gapmer5 revealed a notable decrease in the number of C. albicans as filamentous and consequently led to a significant reduction in the area of fat body invaded by C. albicans.

Discussion
In the last decade, the number of successful applications of ASOs for the treatment of human diseases [39][40][41][42][43][44][45][46] and to manage virus and bacterial infections [47][48][49] has increased. However, the ASO technology has been poorly exploited to control Candida virulence determinants, and to date there are no reports of applications of LNA chemical modification to control Candida virulence determinants. Through this work, the efficacy of LNAgapmer ASOs, designed to hybridize specifically to an EFG1 target, was compared, concerning its ability to control EFG1 gene expression and to reduce C. albicans filamentation, in order to control in vivo C. albicans virulence. Therefore, a set of five LNA-gapmers was designed with different lengths and different chemical modifications, such as the PS-linkages or/and the palmitoyl-2′-amino-LNA chemical modification/s. Initially, it was important to confirm the composition and the purity of all LNA-gapmers ( Figures S2 and S3) and to verify if the inclusion of LNA chemical modifications to the sequence influences its secondary structure ( Figure 1) and its hybridization stability (Table 2). In many aspects the LNA gapmer ASOs behaved quite similarly. However, the inclusion of PS-linkages (LNA-gapmer3) led to a decrease in duplex thermal denaturation temperature (T m value) against RNA complement. As described in the literature, 50 , 51 the inclusion of the PS modification and the palmitoyl-2′-amino-LNA (LNA-gap-mer4 and LNA-gapmer5) also reduced the thermal denaturation temperature of the corresponding duplexes when compared to the thermal denaturation temperatures of the duplexes involving the reference LNA-gapmer1. Of all LNAgapmers characterized, LNA-gapmer5 displayed the lowest affinity. The decrease in thermal denaturation temperature upon incorporation of the palmitoyl-2′-amino-LNA monomers corresponds with results reported earlier. 8 The in vitro and in vivo efficacy of each LNA-gapmer was evaluated at 40 nM since none of them presented in vitro ( Figure  2) or in vivo toxicity (Figure 4, A). The in vitro results demonstrate the capacity of the LNA-gapmers to control EFG1 gene expression by 40%-60% (Figure 3, B) and C. albicans' filamentation around 50% (Figure 3, A), as well as the filaments length on 60% (Figure 3, C). The inclusion of PS-linkages (LNA-gapmer3) enhanced filamentation reduction, however, without any additional effect on EFG1 gene silencing. On the other hand, the inclusion of palmitoyl-2′-amino-LNA monomers on LNA-gapmer4 and LNA-gapmer5 did not significantly enhance the filamentation control or the gene silencing when compared to the performance of the other LNA-gapmers. It should be pointed out that the ability of LNA-gapmer ASOs to control C. albicans' filamentation and to reduce the filaments' length is crucial to reduce C. albicans pathogenicity, once it decreases C. albicans' ability to invade the human body tissues and to escape of host's immune system. [52][53][54] The G. mellonella caterpillar infection model was used to evaluate the effects of the LNA-gapmer ASOs on C. albicans virulence ( Figure 4). Interestingly, in contrast to the in vitro experiments, in vivo results highlight pronounced differences among the individual LNA-gapmers. Thus, although the LNA-gapmer5 did not show the best performance in vitro (Figure 3), this gapmer was the most efficient with respect to the control of in vivo C. albicans' virulence ( Figure 4, B and C). In fact, larvae treated with LNA-gapmer5 significantly increased G. mellonella survival from 20% to 60% (at 72 h of treatment). The histological images corroborated these findings with a notable reduction in the number of C. albicans filamentous cells and a consequent reduction in area of G. mellonella invasion (Figure 4, C).
This work revealed that it is possible to design nucleic acid mimics to control important virulence factors behind the Candida infections. The application of LNA-gapmer ASO confirms the possibility to control virulence determinants of C. albicans and thus to control its pathogenicity. Moreover, the LNA-type gapmer ASOs modifications with PS-linkages and palmitoyl-2′amino-LNA monomers are favorable for in vivo performance and constitute promising lead structures for development of drugs against Candida species.
However, considering any possible future clinical applications in the control of local candidiasis (oral, vaginal, and urinary), as well as of systemic infections (blood), additional studies are imperative. It will be necessary to investigate strategies for LNA-gapmer ASO delivery and to extend the in vivo studies for a more complex and robust model, as a mouse model.

Credit Author Statement
Daniela Araújo, Sónia Silva, Per Trolle Jørgensen and Jesper Wengel conceived and designed the study. Daniela Araujo, Dalila Mil-Homens and Maria Elisa Rodrigues conducted the experiments. Daniela Araujo and Sónia Silva wrote the manuscript. Mariana Henriques, Sónia Silva and Jesper Wengel performed the analysis and read the paper. All authors read and approved the manuscript.