Application of antisense oligonucleotides to prevent Candida albicans infections
Abstract
Candida albicans is the main cause of candidiasis and its pathogenicity is supported by a series of virulence factors, including the switch from yeast to filamentous forms. The transcription factor EFG1 plays a central role in C. albicans regulation morphology. In addition, there is rise of C. albicans multidrug resistance accompanied by the scarcity of new classes of fungal drugs. Antisense oligonucleotides (ASOs) can hold great promise as a therapeutic agent, however yet they are poorly explored for controlling Candida infections. Therefore, the main goal of this work was to develop a therapeutic approach based on antisense therapy (AST) to track C. albicans filamentation. To this end, two major objectives were addressed: I) Exploitation and application of ASOs to control C. albicans filamentation; II) Creation of strategies for ASOs cargo and delivery. The first goal of this research was the exploitation of the second and third generations of ASOs to control EFG1 gene and C. albicans filamentation. To this end, the anti-EFG1 2’OMethylRNA was firstly projected based on second generation of ASOs and was proved to have an impact on the reduction of EFG1 gene expression (by 60 %) and on Efg1p protein translation (by 60 %). Interestingly, the ASO was able to inhibit filamentation and to attenuate the virulence of C. albicans on in vivo Galleria mellonella (increasing its survival on 30 % at 72h with a double-dose administration). Based on the third generation of ASOs, a set of anti-EFG1 LNA (Locked nucleic acids) ASOs were developed, and their performance was tested in vitro and in vivo. All LNA-ASOs were able to reduce EFG1 gene expression (by 40-80 %) and consequently filamentation (by 45-55 %) in vitro. The in vivo results revealed that the LNA-ASO modified with PS-linkages and palmitoyl-2’-amino-LNA as the most favorable for an in vivo application (increasing G. mellonella survival by 40 % only with a single-dose administration). The second goal of this research was the development of strategies for anti-EFG1 ASO cargo and delivery. Two distinct approaches were tested: (i) development of polyplexes based on polyamide 4 and 6, that revealed to be feasible carriers, once the ASO maintains its activity against C. albicans cells; (ii) application of lipid-based formulations, where the DOTAP/DOPC 80/20 ρ=3 revealed to be the most promising when tested in a G. mellonella model (25 % of increase on larvae survival in comparison to ASO-free at 72 h). In summary, this research underlines the therapeutic potential of ASOs for controlling C. albicans virulence determinants, as is the case of EFG1 gene, as well as the importance of developing carriers’ systems for ASOs cargo. Considering the future research into the management of C. albicans infections, this research provides valuable information to the development of a credible and alternative method to control C. albicans infections, based on AST.
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Universidade do Minho Escola de Engenharia Daniela Eira Araújo Application of antisense oligonucleotides to prevent infections october 2021 UMinho | 2021 Daniela Araújo Application of antisense oligonucleotides to prevent infections Candida albicans Candida albicans
This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the PhD grant SFRH/BD/121417/2016 and the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This study was also supported by the project funding by the “02/SAICT/2017 – Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT) – POCI-01-0145-FEDER-028893”, designated as Antisen4CandiB - Application of antisense oligomers for controlling Candida species biofilm formation on medical surfaces”.
Daniela Eira Araújo Application of antisense oligonucleotides to prevent infections Doctoral Thesis Chemical and Biological Engineering Work supervised by Dr. Sónia Silva Dr. Mariana Henriques Dr. Jesper Wengel Universidade do Minho Escola de Engenharia october 2021 Candida albicans
ii COPYRIGHT AND CONDITIONS FOR THE USE OF WORK BY THIRD PARTIES This is an academic work that can be used by third parties in accordance with internationally accepted rules and good practice in respect of copyright and related rights. Thus, the present work can be used under the terms of the license indicated below. In case the user needs permission to be able to make use of the work under conditions not foreseen in the indicated license, he should contact the author through the RepositóriUM of the University of Minho. License granted to users of this work Atribuição-CompartilhaIgual CC BY-SA https://creativecommons.org/licenses/by-sa/4.0/
iii Acknowledgments Somos feitos do amor das pessoas que nos acompanham diariamente. Aliado a todo este trabalho, estão as pessoas que me acompanharam, e que me ajudaram a tornar tudo isto possível. Desde os momentos de laboratório, nas trocas de ideias até aos momentos de lazer, de sorrisos e risos. Sou imensamente grata por esta experiência e por este trabalho. A ti Sónia, quero agradecer-te com o meu coração todo a orientação neste meu percurso. Foi incrível toda a tua disponibilidade e ajuda incondicional que nos levou a todo este trabalho que tantos desafios nos tem dado e nos alicia dia após dia a continuar. Adorei partilhar todo este trabalho contigo e sou muito grata pela oportunidade e pela amizade. À professora Mariana, obrigado pela orientação e por todas as oportunidades ao longo deste longo percurso. É uma gratidão enorme todo o conhecimento partilhado comigo e por toda a sua disponibilidade e atenção para com o meu trabalho. To Per and Jesper for your supervision, knowledge, and help during my stay in Denmark. I would like to also thank Joan Hansen and Tina Hansen for their valuable technical assistance and knowledge. Thank you, Joan, to turn this an easy journey, and for all the conversations and all the tours around Denmark. I would like to also thank all people from Department of Physics, Chemistry and Pharmacy. It was a lovely journey in this beautiful and peaceful city. Ao grupo MH, Elisa, Isabel, Fernanda, Liliana, Ana, Nuno, que diretamente fizeram parte deste trabalho, desde as reuniões aos dias passados no laboratório. Foram muitas as ideias trocadas entre todos, as risadas que fizeram acompanhar este trabalho e tanto fazem tornar a vida especial. Ao grupos dos Biofilmes e a pessoas tão especiais que fizeram parte desta etapa da minha vida, Vânia e a Daniela (Trio maravilha da minha vida), Tânia, Susanas, Paula, Diana, Joana, Andreia (Gang das Marmitas), à Graça (a minha companheira do doutoramento), ao Rodrigo, ao Henrique, à Priscila, ao Luís, e a todos aqueles que estiveram presentes direta e indiretamente. Ao prof. Zlatan, à prof Nadya, à Joana, À Dalila, ao Bruno e ao Ricardo, pelas colaborações estabelecidas que fortaleceram o trabalho desenvolvido e por toda a aprendizagem em cada uma das áreas. A vocês meus amigos que estiverem sempre presentes e que me acompanham tanto nas vitórias como nas derrotas. Em especial um muito obrigado, Catarina Simões e Diana Castro pela vossa amorosa amizade e é verdadeiramente bom ter-vos na minha vida. Catarina, és como uma minha irmã, a minha companheira de tudo e aquela que está sempre por perto. À minha família, que fazem parte de mim e que estiveram lá para me propocionar momentos de pura diversão e que tanta força e amor me dão para que isto seja possível. A vocês, José Araújo, Clementina Eira e Milinho porque são o meu Mundo. Por toda a confiança depositada em mim ao longo da minha vida e pela partilha de conhecimento. Fazem parte de mim todos os dias e vocês merecem ser tão mas tão felizes. Obrigado por todos os momentos partilhados, por todas as dificuldades ultrapassadas e por todas as vitórias alcançadas. A ti, Fábio Reina, por todo o teu amor e companhia. Esta jornada tornou-se ainda mais fácil quando tu te juntaste a mim e desde aí tudo parece mais bonito todos os dias. Que toda esta luta das nossas vidas se torne em algo tão incrível quanto aquilo que partilhamos todos os dias. O teu sorriso tranquiliza-me e sou muito grata por fazeres parte de mim e por teres feito parte deste meu percurso. “Tenho em mim todos os sonhos do mundo” Fernando Pessoa
iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho.
v Application of antisense oligonucleotides to prevent Candida albicans infections Abstract Candida albicans is the main cause of candidiasis and its pathogenicity is supported by a series of virulence factors, including the switch from yeast to filamentous forms. The transcription factor EFG1 plays a central role in C. albicans regulation morphology. In addition, there is rise of C. albicans multidrug resistance accompanied by the scarcity of new classes of fungal drugs. Antisense oligonucleotides (ASOs) can hold great promise as a therapeutic agent, however yet they are poorly explored for controlling Candida infections. Therefore, the main goal of this work was to develop a therapeutic approach based on antisense therapy (AST) to track C. albicans filamentation. To this end, two major objectives were addressed: I) Exploitation and application of ASOs to control C. albicans filamentation; II) Creation of strategies for ASOs cargo and delivery. The first goal of this research was the exploitation of the second and third generations of ASOs to control EFG1 gene and C. albicans filamentation. To this end, the antiEFG1 2’OMethylRNA was firstly projected based on second generation of ASOs and was proved to have an impact on the reduction of EFG1 gene expression (by 60 %) and on Efg1p protein translation (by 60 %). Interestingly, the ASO was able to inhibit filamentation and to attenuate the virulence of C. albicans on in vivo Galleria mellonella (increasing its survival on 30 % at 72h with a double-dose administration). Based on the third generation of ASOs, a set of antiEFG1 LNA (Locked nucleic acids) ASOs were developed, and their performance was tested in vitro and in vivo . All LNA-ASOs were able to reduce EFG1 gene expression (by 40-80 %) and consequently filamentation (by 45-55 %) in vitro . The in vivo results revealed that the LNA-ASO modified with PS-linkages and palmitoyl-2’-aminoLNA as the most favorable for an in vivo application (increasing G. mellonella survival by 40 % only with a single-dose administration). The second goal of this research was the development of strategies for antiEFG1 ASO cargo and delivery. Two distinct approaches were tested: (i) development of polyplexes based on polyamide 4 and 6, that revealed to be feasible carriers, once the ASO maintains its activity against C. albicans cells; (ii) application of lipid-based formulations, where the DOTAP/DOPC 80/20 ρ=3 revealed to be the most promising when tested in a G. mellonella model (25 % of increase on larvae survival in comparison to ASO-free at 72 h). In summary, this research underlines the therapeutic potential of ASOs for controlling C. albicans virulence determinants, as is the case of EFG1 gene, as well as the importance of developing carriers’ systems for ASOs cargo. Considering the future research into the management of C. albicans infections, this research provides valuable information to the development of a credible and alternative method to control C. albicans infections, based on AST. Keywords: antisense oligonucleotides, Candida albicans, EFG1 gene, filamentation, non-viral vectors
vi Aplicação de oligómeros de antisense para prevenir infeções por Candida albicans Sumário Candida albicans é uma das principais causas de candidíase e a sua patogenicidade é suportada por um conjunto de fatores de virulência, incluindo a capacidade de filamentar. EFG1 é um fator de transcrição com papel fundamental na filamentação. O aumento da multirresistência de C. albicans é acompanhado pela escassez de novos antifúngicos. Os oligonucleotídeos de antisense (OAS) têm revelado potencial como agentes terapêuticos, contudo ainda pouco explorados no controlo da candidíase. Assim, o principal objetivo deste trabalho foi desenvolver uma abordagem terapêutica baseada na Terapia Antisense (TA) para controlar a filamentação de C. albicans . Para tal, dois grandes objectivos complementares foram definidos: I) Exploração e aplicação de OAS para controlar a filamentação e II) Criação de estratégias para o transporte e entrega dos OASs. Como primeiro objetivo explorou-se a segunda e terceira gerações de OASs para controlar o gene EFG1 . O antiEFG1 2’ O MethylRNA foi projetado com base na segunda geração, e provou ter um impacto na redução da expressão do gene EFG1 (em 60 %) e na tradução da proteína Efg1p (em 60 %). O OAS foi capaz de inibir a filamentação e atenuar in vivo a virulência de C. albicans em Galleria mellonella (30 % de aumento de sobrevivência às 72 h após dupla administração). Por outro lado, um conjunto de OAS modificados com LNA (terceira geração) foram desenvolvidos e testados in vitro e in vivo . In vitro , os OAS-LNA inibiram a expressão do gene de EFG1 (em 40-80 %) e a filamentação (em 45-55 %). In vivo , o OAS-LNA com as modificações de PS e palmitoyl-2’amino-LNA revelou ser o mais favorável (40 % de aumento de sobrevivência da G. mellonella com uma administração). Como segundo objetivo exploraram-se poliplexos e lipoplexos como estratégias para o transporte e entrega do antiEFG1 2’OMe. Como primeira abordagem, desenvolveram-se poliplexos à base de poliamida 4 e 6, que revelaram ser veículos viáveis, uma vez que o OAS manteve a sua atividade contra as células de C. albicans . Em seguida, foram exploradas formulações lipídicas, sendo que o DOTAP/DOPC 80/20 ρ=3 revelou ser a mais promissora quando testada in vivo (25 % de aumento de sobrevivência da G. mellonella em comparação com o OAS livre às 72 h). Em suma, esta investigação destaca o potencial terapêutico de OAS para o controlo de determinantes de virulência de C. albicans , como o caso do gene EFG1 , bem como a importância do desenvolvimento de sistemas de veículos para o seu transporte. Considerando futuros trabalhos sobre o controlo de infeções por C. albicans , esta investigação fornece informação valiosa para o desenvolvimento de um método credível e alternativo para o controlo destas infeções, com base na TA. Palavras-chave: Candida albicans, filamentação, gene EFG1, oligonucleotídeos antisense, vetores nãovíricos
vii Index List of figures…………………………………………………………………………………………………………..………..xii List of tables……………………………………………………………………………………………………….…………....xx Symbols……………………………………………………………………………………………………….…………………xxii Nomenclature……………………………………………………………………………………………………….………....xxii Scientific Outputs……………………………………………………………………………………………..……………..xxvii Objectives and structure of the thesis…………………………………………………………….……………..……..xxix Chapter I. Literature review………………………………………………………..……….…..………….1 I.1 Candida and Candidiasis……………………………………………………………………………………….2 I.1.1 Epidemiology and risk factors of candidiasis……………………………………………………..2 I.1.2 Candida albicans …………………………………………………………………………………………4 a. Biology of Candida albicans ……………………………………………………………………….4 b. Candida albicans virulence factors………………………………………………………………4 Hydrolytic enzymes production………………………………………………………………5 Adhesion…………………………………………………………………………………………..5 Biofilm formation………………………………………………………………………………..7 Dimorphic ability……………………………………………………………………………….10 c. Candida albicans antifungal resistance……………………………………….…..………….13 I.2 Antisense Therapy…………………………………………………………………………….……….….......14 I.2.1 ASOs chemical modifications……………………………………………………………………14 a. First-generation of ASOs………………………………………………………………..15 b. Second-generation of ASOs……………………………………………………………16 c. Third-generation of ASOs……………………………………………………...……….16 I.2.2 ASO mechanism of action…………………………………………………………….…..……..17 I.2.3 ASO cargo and delivery strategies………………………………………………………………19 a. Polymeric complexes: Polyplexes……………………………………………….......20 b. Liposomes complexes: Lipoplexes…………………………………………………….21 I.2.4 ASO pharmacokinetic properties……………………………………………………….……..22 I.2.5 ASO toxicology……………………………………………………………………………….…….23 I.2.6 Antisense Drugs approved by FDA and EMA……………………………………………….25 References………………………………………………………………………………………………………………..26
xiv human body fluids (AS and AU at 24 h and horse blood at 48 h). Error bars represent standard deviation. * Significantly differences between 6 h and the other times tested (P-value<0.05). + Significantly differences between untreated and treated cells (P-value<0.05).……………..…………………………………..52 Chapter II.2 AntiEFG1 2’- O MethylRNA antisense oligonucleotide inhibits Candida albicans filamentation and attenuates the candidiasis in Galleria mellonella Figure II.2.1 AntiEFG1 2’ O Me ASO toxicity evaluation in Galleria mellonella model. For each condition, 10 larvae were injected with 40 nM and 100 nM of ASO and their survival was monitored over 96 h. As control larvae were injected only with PBS……………………………………………………………………60 Figure II.2.2 AntiEFG1 2’ O Me ASO effect on the survival of Galleria mellonella larvae infected with Candida albicans . Survival curves of infected larvae were treated with: (A) a singledose of antiEFG1 2’ O Me ASO (0 h post infection) and (B) a double-dose of antiEFG1 2’ O Me ASO (0 h and 12 h post infection). Larvae infected with C. albicans cells were treated with 40 nM and 100 nM of antiEFG1 2’ O Me ASO. As control larvae infected were injected only with PBS. + Significant difference among control and a single-dose of 100 nM of antiEFG1 2’ O Me ASO at 24 h (P-value < 0.05). * Significant difference among control and a double-dose of 40 nM of antiEFG1 2’ O Me ASO for all times (P-value < 0.05). *** Significant difference among control and a double-dose of 100 nM of antiEFG1 2’ O Me ASO for all times (P-value < 0.001).………………………………………………………………………………………………61 Figure II.2.3 AntiEFG1 2’ O Me ASO effect on Candida albicans cells morphology and progression into fat body of Galleria mellonella. Histological images of larvae infected (A) with C. albicans (at 24 h and 48 h) and treated (B) with a single-dose (0 h post infection) and (C) with a doubledose (0 h and 12 h post infection) of 40 nM and 100 nM of anti -EFG1 2’ O Me ASO . The larvae sections were labelled with periodic acid Schiff (PAS) coloration. The magnification images were at 400x.………..62 Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation Figure II.3.1 Characterization of AntiEFG1 LNA-gapmer ASOs. (A) Thermal denaturation temperatures (T values, °C) determined in medium salt buffer with RNA complement, and evaluation of superficial charge by zeta potential determination. (B) Evaluation of overall conformation of the secondary
xv structure by CD spectral analysis of single-strands (ssLNA) and double-stranded complexes (dsLNA:RNA)..………………………………….………………………................................................................72 Figure II.3.2 Cytotoxicity of AntiEFG1 LNA-gapmer ASOs . Relative cell viability (%) determined by the absorbance (Abs (490 nm) cm-2) of formazan product obtained from 3T3 cells, treated with different concentrations of LNA-gapmers (10, 40, 100 nM). The control is compared to cells without ASO treatment.. … …………………………………………………………………………….………………………………………73 Figure II.3.3 AntiEFG1 LNA-gapmer ASOs in vitro . Effect of treatment of Candida albicans with LNA-gapmers (40 nM) during 24 h. (A) Filamentous inhibition (%). (B) Levels of EFG1 gene expression evaluated by qRT-PCR. (C) Length of filaments determined by epifluorescence microscopy image analysis of yeast cells stained with calcofluor. Untreated represents an experiment prepared only with cells on RPMI (without ASOs). Error bars represent standard deviation. *Significant differences between the untreated cells and the LNA-gapmers tested (P-value<0.05)..………………………………………………………74 Figure II.3.4 AntiEFG1 LNA-gapmer ASOs in vivo . (A) Toxicity evaluation at 40 nM and 100 nM in the Galleria mellonella model evaluated (96 h post treatment); (B) Survival curves for G. mellonella larvae infected with C. albicans SC5314 (7 x 107 cells mL-1 injected per larva) and LNA-gapmers (40 nM) evaluated 72 h post treatment. Larvae were infected with a single dose of each LNA-gapmer at the same time of C. albicans cells. (C) Histological sections of the fat bodies of G. mellonella infected with C. albicans and treated with LNA-gapmers (40 nM) at 24 h and 48 h. The larvae sections were labelled with PAS coloration. The magnification was 400x. Results represent means of three independent assays for 10 larvae per treatment. *** Significant difference between the control ( Candida + PBS) and the LNAgapmer5 ( p -value < 0.001)..…………………..…………………………………………………………………………….76 Chapter III. Creation of strategies for C. albicans antisense oligonucleotides cargo and delivery Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’- O MethylRNA EFG1 ASO Figure III.1.1 Scanning electron microscopy of neat carriers and polyplex samples based on (A) PA6 microparticle and (B) PA4 microparticle: (a-c) neat microparticle; (d-f) Entrapped sample; (g-i) Immobilized sample. For sample designation see Table III.1.1.………………………………89
xvi Figure III.1.2 Comparison of Fourier-transform infra-red spectroscopy with attenuated total reflection spectra of polyplexes based on (a) PA6 microparticle and (b) PA4 microparticle. The inset displays the area 1650-1800 cm-1 where the terminal carboxyl groups appear. Thermogravimetric analysis traces of polyamide polyplexes (c) integral curves; (d) first derivative curves: (1) PA6; (2) PA6-Ent-ON; (3) PA6-Imm-ON; (4) PA4; (5) PA4-Ent-ON; (6) PA4-Imm-ON.………………………………….90 Figure III.1.3 Linear synchrotron wide-angle X-ray scattering patterns of: (a) PA6-based polyplexes; (b) PA4-based polyplexes. (1) neat polyamide carriers; (2) PA-Imm-ON samples; (3) PA-EntON samples.……………………………………………….…………………………………………………………………….92 Figure III.1.4 (A) Synchrotron wide-angle X-ray scattering analysis of neat MP and polyplexes. (B) Deconvolution of polyamide-based polyplexes: (a) PA4; (b) PA4-Ent-ON; (c) PA4-Imm-ON and PA6-based polyplexes: (d) PA6; (e) PA6-Ent-ON; (f) PA6-Imm-ON.……………………………………………………………..93 Figure III.1.5 Linear small-angle X-ray scattering patterns of (a) PA4-based polyplexes; (b) PA6based polyplexes. (1) neat polyamide carriers; (2) PA-Imm-ON samples; (3) PA-Ent-ON samples.……..95 Figure III.1.6 Relative cell viability of (A) PA4-based polyplexes and (B) PA6-based polyplexes microparticles determined by the absorbance values (Abs (490 nm) cm-2) of formazan product obtained from 3T3 cells in contact with 6 and 10 mg mL-1 microparticles. The control is related to the cells without any treatment. Error bars represent standard deviation………………………………………………………………96 Figure III.1.7 Cumulative controlled release profiles of antiEFG1 2’OMe ASO from immobilized (Imm) and entrapped (Ent) into (A) PA4-based polyplexes and (B) PA6-based polyplexes over the time.……………………………………………………………………………………………………………………………….96 Figure III.1.8 Effect of antiEFG1 2’OMe ASO released from immobilized (Imm) and entrapped (Ent) polyplexes microparticles on C. albicans cells filamentation. (A) Number of non-filamentous C. albicans cells normalized by the quantity of ASO released at each time; (B) Epifluorescence microscopy images of C. albicans stained with Calcofluor after 24 h and 48 h in contact with the polyplex microparticles and the average of the hyphae size for each condition. The assays were performed for C. albicans SC5314. Error bars represent SD. *Significant differences between samples of the same polymer at each time (P-value<0.05). +Significant differences between both times analysed for the samples of the same polyplex microparticle (P-value<0.05). Arrows highlight yeast cells.……………..98
xvii Chapter III.2 Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’- O MethylRNA antisense oligonucleotide Figure III.2.1 In vitro effect of antiEFG1 2’OMe ASO lipid-based formulations on C. albicans filamentation. Levels of C. albicans filamentation reduction (%) of (A) DOTAP/DOPC; (B) DOTAP/DOPE and (C) DOTAP/MONO after 24, 48 and 72 h of treatment with each lipoplex. As positive control, C. albicans cells were incubated in same conditions with the antiEFG1 2’OMe ASO-free. Error bars represent standard deviation. *Significant differences between antiEFG1 2’OMe ASO lipid-based formulations and ASO-free at each time of incubation (P-value<0.05). +Significant differences between DOTAP/DOPE 80/20 ρ=10 and DOTAP/DOPE 30/70 ρ=10 at 24 h of incubation (P-value<0.05)……………………….107 Figure III.2.2 In vitro effect of antiEFG1 2’OMe ASO lipid-based formulations on C. albicans filament’s length. (A) Filament’s length inhibition (%) determined by using ImageJ software and (B) epifluorescence microscopy images of C. albicans cells stained with calcofluor after 72 h of incubation with lipoplexes. Control represents an experiment prepared only with cells on RPMI. AntiEFG1 2’OMe free represents the cells treated with free ASO. *Significant differences between antiEFG1 2’OMe ASO lipid-based formulations and ASO-free (P-value<0.05)…………............................................................109 Figure III.2.3 (A) AntiEFG1 2’OMe ASO lipid-based formulations toxicity evaluation on Galleria mellonella model. For each condition, 10 larvae were injected with each lipoplex and their survival was monitored over 72 h. As control larvae were injected only with PBS. (B) Effect of Lipid-based formulations empty of ASO on the Galleria mellonella survival infected with Candida albicans… ………………………….110 Figure III.2.4 Single dose antiEFG1 2’OMe ASO lipid-based formulations effect on the survival of Galleria mellonella survival infected with Candida albicans. Survival curves of infected larvae treated with a single-dose (0 h post infection) of (A) DOTAP/DOPC lipoplexes; (B) DOTAP/DOPE lipoplexes and (C) DOTAP/MONO lipoplexes. As control larvae infected were injected with antiEFG1 2’OMe ASO-free and only with PBS…………………………………………………………………………111 Figure III.2.5 Double-dose antiEFG1 2’OMe ASO lipid-based formulations effect on Galleria mellonella survival infected with Candida albicans. Survival curves of infected larvae treated with a double-dose (0 h and 12 h post infection) of DOTAP/DOPC 80/20 ρ=3 lipoplex. As control larvae infected were injected only with antiEFG1 2’OMe ASO-free and only with PBS……………………………….112
xviii Annex I Figure AI.1 AntiEFG1 2’OMe Candida albicans strains sensitivity determined by fluorescence in situ hybridization (FISH). The images were obtained by epifluorescence microscopy with the same exposure time for cells incubated in absence and presence of ASO. The values of exposure time used varied between 32.05 ms and 340.7 ms. Negative controls were prepared only with 20 µL of hybridization solution without probe……………………………………………………………………………………..120 Annex AI.2 Evaluation of C. albicans filamentation and EFG1 gene expression in the untreated cells. (A) Percentage of filamentous forms (%) of Candida albicans SC5314 at different time points (4, 6 and 8 h) in RPMI (Without ASO); (B) Percentage of EFG1 gene expression obtained from Candida albicans SC5314 at different time points (4, 6 and 8 h). Error bars represent standard deviation. *Significantly differences between 4 h and the other times tested (P-value<0.05)…………………………..121 Annex AI.3 Scrambled ASO effect on Candida albicans filamentation. Percentage of inhibition of filamentous forms (%) at different time points (4, 6 and 8 h) with 40 nM of scrambled ASO in RPMI……………………………………………………………………………………………………………………………..121 Annex AI.4 Candida albicans behaviour on different simulated human body fluids. (A) Number of cultivable cells (log CFUs cm-2) at different time points (0, 6, 8, 10 and 24 h); (B) Percentage of filamentous forms (%) at different time points (6, 8, 10, 24 and 48 h) and (C) Percentage of EFG1 gene expression in different simulated human body fluids (AS, AU at 24 h and horse blood at 48 h); without ASO obtained from Candida albicans SC5314. Error bars represent standard deviation. *Significantly differences between 0 h and the other times tested (p<0.05). + Significantly differences between 6 h and the other times tested (P-value<0.05)…………………………………………………………….122 Annex II Figure AII.1 Study of Galleria mellonella lethality. For each control, 10 larvae were injected with two different concentrations of Candida albicans cells (2x108 and 7x107 cells mL-1 cells mL-1)…………….123
xix Annex III Figure AIII.1 Evaluation of C. albicans filamentation and EFG1 gene expression in the untreated cells. Percentage of (A) Candida albicans filamentation and (B) EFG1 gene expression after 24 h of incubation on RPMI (without ASO)……………………………………………………………………………..124 Figure AIII.2 Histological sections of the fat body of Galleria mellonella (control, without Candida albicans cells). The larvae sections were labelled with haematoxylin-eosin (HE) coloration. The magnification images were at 100x…………………………………………………………………………………124 Annex IV Figure AIV.1 UV-VIS analysis for ASO before and after adsorption immobilization on PA4 and PA6 carriers…………………………………………………………………………………………………………..125 Figure AIV.2 Circular dichroism of antiEFG1 2’- O MethylRNA.The spectra were recovered on a JASCO DC 1500 spectrophotometer using cuvettes with a 0.1 cm path length. Spectra were averaged over three scans (320-200 nm, 50nm/min intervals, 1 nm bandwidth, and 1 s response time) and background corrected with the solvent (5 mM MgCl2, 10 mM NaCl and 1 mM sodium phosphate)……..125 Figure AIV.3 Chemical reactions occurring during AAROP of GBL (R1 = (CH2)3) or ECL (R1 = (CH2)5) to PA4 or PA6 MPs. The active substance of the AAROP activator is designated as C20; The chemical structure of the AAROP initiator dicaprolactamato-bis-(2-methoxyethoxo)-aluminate wherein R = OCH2CH2OCH3 is designated by DL………………………………………………………………………………………126 Figure AIV.4 Size distribution curves in neat PA6 and PA4 MP supports and in the respective polyplexes: a – PA6; b – PA6-Ent-ON; c – PA6-Imm-ON; d – PA4; e – PA4-Ent-ON; f – PA4-Imm-ON. Bright field optical microscopy of all samples was performed evaluating the particles' sizes, roundness, and their distributions were performed in an Olympus BH-2 microscope (Japan) equipped with the Leica Application Suite 4.4 software for image processing…………………………………127
xx List of Tables Chapter I. Literature review Table I.1 Incidence and mortality rate of population-based studies and hospital based-studies in European countries based on the report by Koehler et al. [4]………………………..………………………..…….3 Table I.2 Antisense drugs that received market authorization by FDA and/or EMA. Adapted from [227]………………………………………………………………………………………………………………………………26 Chapter II. Exploitation of the antisense oligonucleotides to control Candida albicans filamentation Chapter II.1 Application of 2’- O MethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant Table II.1.1 Primers used for qRT-PCR, with the respective theoretical Tm obtained from the calculator from IDT and amplification product……………………………………………………………………………………….43 Table II.1.2 Sequence of antiEFG1 2’OMe (m) and scramble ASO, with the respective size, theoretical melting temperature (Tm) and GC content…………………………………………….………….…………………….47 Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation Table II.3.1 Sequence of antiEFG1 LNA-gapmer ASOs, with the respective size and GC content………67 Table II.3.2 Primers used for real time PCR, with the respective melting temperature (Tm) and amplification product (AP)…………………………………………………………………………………………………..70
xxi Chapter III. Creation of strategies for C. albicans antisense oligonucleotides cargo and delivery Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’- O MethylRNA EFG1 ASO Table III.1.1 Designation and some characteristics of PA6 and PA4 MP and polyplexes.…………..…….87 Chapter III.2 Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’OMethylRNA ASO Table III.2.1 Different liposomes prepared and its molar ratios, cationic-to-anionic charge ratio (ρchg) and type of lipoplexes structure…………………………………………………………………………………………..103 Annex IV Table AIV.1 Z-potential of neat polyamide MPs and their polyplexes. Amount of particles: 0.5 mg/mL…………………………………………………………………………………………………………………….......128
xxii Symbols σM charge density ρchg charge ratio P Significance value % Percent °C Degrees Nomenclature 2’OMe 2’- O -Methyl 2’-MOE 2’- O -methoxyethyl 2’-F 2’-fluoro AAROP Activated anionic ring opening polymerization ALS Agglutinin-like sequence ANOVA Analysis of variance AP Amplification product ARP Arginine-rich peptide AS Artificial saliva ASO Antisense oligonucleotides AST Antisense Therapy ATCC American type culture collection AU Artificial urine bHLH Basic helix-loop-helix-type BLAST Basic local alignment search tool bp Base pairs cAMP Cyclic adenosine monophosphate CAN acetonitrile cDNA Complementary DNA CFU Colony forming units CGD Candida genome database
xxiii CHROMagarrTM Chromogenic media agar DCI Dicyanoimidazole DDS Drug delivery system DL Dilactamate DMEM Dulbecco’s modified eagle medium DMSO Dimethyl-sulfoxide DMT Dimethoxytrityl cation DNA Deoxyribonucleic acid DOPC 1,2-dioleoylsn-glycero-3-phosphatidylcholine DOPE Di-Oleoyl-Phosphatidyl-Ethanoalamine DOTAP 1,2-DiOleoyl-3-TrimethylAmmonium Propane DOX Doxycycline ds double-stranded DSC Differential scanning calorimetry DSPC 1,2-DiStearoyl-sn-glycero-3PhosphoCholine DTT Dithiothreitol ECL ε-caprolactam EDT1 Efg1-dependent transcript 1 EDTA Ethylenediamine tetraacetic acid EED1 Epithelial Escape and Dissemination 1 EMA European Medicines Agency FA Formic acid FASP Filter-aided sample preparation FBS Fetal Bovine Serum FDA Food and Drug Administration FISH Fluorescence in situ hybridization FTIR-ATR Fourier-transform infra-red spectroscopy with attenuated total reflection GBL γbutyrolactam GC Guanine-cytosine
xxx described the effect of antiEFG1 2’OMe ASO on EFG1 gene expression, on Efg1p protein translation and on C. albicans cell filamentation in simulated human body fluids. To complement the in vitro results presented in Chapter II.1, into Chapter II.2 – “AntiEFG1 2’- O MethylRNA antisense oligonucleotide inhibits Candida albicans filamentation and attenuates candidiasis in Galleria mellonella ”, the antiEFG1 2’OMe ASO performance was validated in vivo using the Galleria mellonella model. The Chapter II.3 – “Antisense locked nucleic acid gapmers to control Candida albicans filamentation” describes the application of a set of Locked nucleic acid gapmers to control in vitro EFG1 gene expression, and C. albicans filamentation and the in vivo performance on the G. mellonella survival. Afterwards, Chapter III – “Creation of strategies for C. albicans antisense oligonucleotides cargo and delivery” is focused on the development of different vectors for antiEFG1 2’OMe ASO cargo and delivery. The Chapter III is sub-divided into two complementary sub-chapters. Chapter III.1 – “Polyamide microsized particulate polyplex carriers for 2’- O MethylRNA EFG1 ASO” describes the application of anionic and cationic polyplexes based on poly(γ-butyrolactam) (PA4) or poly(ε-caprolactam) (PA6) respectively as a valid strategy to antiEFG1 2’OMethyl ASO cargo. In Chapter III.2 – “Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’- O Methyl antisense oligonucleotide” is described the application of cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’OMethyl ASO. Finally, Chapter IV - “General conclusions and work perspectives” comprehends the major conclusions, obtained in this work, concerning the application of ASOs to control EFG1 gene expression and C. albicans filamentation and proposes suggestions for future research that can contribute for enhanced knowledge about applicability of AST to control Candida infections.
1 Chapter I Literature review
Chapter I Literature review 2 I.1 Candida and Candidiasis The members of the genus Candida are the most frequently recovered from fungal infections and there is an extremely heterogeneous group of over 200 species. However, it is well established that only 20 have been associated with human infectious disease [1,2]. These Candida infections are collectively referred to as candidiasis and may either be superficial, affecting the skin or mucosal membranes, or systemic, which involves the major body organs. The increase of candidiasis has been associated to many factors, in which the increase of immunocompromised patients and the use of broad-spectrum antibiotics are the most reported. In this sense, fungal infections have emerged as an important public health challenge with high economic and medical relevance due to the increased costs of care, time of hospitalization and high levels of mortality, with rates of about 30-50 % in the last years [3,4]. Candida species normally exist as commensals; therefore, this status depends on the host microbiome as a whole and the immune condition of the host. Obviously, if some perturbation of mucosal microbiota or weakening of host immunity happens, Candida species switch from commensal to pathogen microorganism, increasing its ability to cause superficial and systemic infections [5]. Most of candidiasis cases are attributed to Candida albicans , however, in the recent decades, the improved diagnostic methods and the multi-drug resistance to certain antifungals have led to the emergence of nonCandida albicans Candida (NCAC) species, particularly Candida glabrata, Candida parapsilosis , Candida tropicalis, Candida Krusei and more recently Candida auris [6–8]. I.1.1 Epidemiology and risk factors of candidiasis Fungal infections have become increasingly important as a public health problem since invasive candidiasis is a severe infection associated with high morbidity and mortality rates [3,4,9–11]. Normally, the most common presentation of invasive candidiasis is the development of a candidemia [11]. In fact, Candida species are in the first three pathogens causing health-care associated bloodstream infections, and approximately 50 % of candidemia episodes occurs in the Intensive Care Unit (ICU) [4]. There are many risk factors associated with candidiasis, however the most common include exposure to broadspectrum antibacterial agents, long-term ICU stay, a recent major surgery and the presence of an indwelling central venous catheter [7,9,12]. In fact, catheters could be a problem in ICU that required an important attention since candidemia associated with intravenous lines can act as substrates for biofilm production [3,10,13].
Chapter I Literature review 3 Candida species can also colonize several human body sites as skin, oropharynx, lower respiratory tract, and gastrointestinal and genitourinary tract [1,13]. It is important to refer that Candida species are commensal yeasts and there are part of the normal human skin and gut microbiota, so normally they are found in up to 60 % of healthy individuals [13,14]. Candida albicans remains the predominant species recovered from infections counting with 7090 % cases among all candidiasis-causing fungi [13]. In a report of Montagna et al. [15] in 2014, C. albicans represented 50-70 % of all Candida infections in European countries. In the last decades, it has been confirmed that in 95 % of the Candida infection’s cases, the most common species involved are C. albicans , C. glabrata , C. tropicalis , C. parapsilosis and Candida krusei [1,13]. However, the distribution of C. albicans varies from region to region and depend upon the study and the time of surveillance. In addition, other reasons can explain the differences from region to region, such as, differences in medical practices, a reduced number of health care workers and difficulties in the implementation of infection control programs in hospitals and the excessive use of antifungal agents. Indeed, Koehler et al. [4] reported recently that the incidence rate (IR) for candidemia has been higher in Southern Europe than in Northern or Western Europe (Table I.1). In that work the differences in the regions are explained by factors as climatic differences, the methods used for antibiotic prescription and demographic development [4]. However, the local health care systems have significant impact in candidemia incidence. In terms of a pooled crude mortality rate (MR) was about of 40 % in population-based studies [16,17], and in the case of ICU-only studies, the report of crude MR was approximately 49 %. In the population-based studies reported in Koehler et al. [4] , C. albicans remained the most prevalent species involved in candidemia, even though cases with nonCandida species were increasing. Table I.1 Incidence and mortality rate of population-based studies and hospital based-studies in European countries for candidemia based on the report by Koehler et al. [4] European Countries Population-based studies (per 100.000 inhabitants) Hospital-based studies (per 100.000 inhabitants) Mortality rate (%) Southern 5.29 1.13 37 Northern 3.77 0.31 35 Western 2.5 0.47 37
Chapter I Literature review 4 I.1.2 Candida albicans a. Biology of Candida albicans Candida albicans is a diploid polymorphic yeast that is part of the human microflora and is found in the human gastrointestinal (GI), respiratory and genitourinary tracts [7,18]. Normally, C. albicans is present as blastoconidia, which is a harmless commensal fungus, however, in immunocompromised patients, this fungal species can become into an opportunistic pathogen causing superficial, as well as systemic infections [7,19–21]. Candida albicans cells present a range of size between 4-6 µm in the blastoconidia form, reaching higher sizes when grow to a filamentous form [22]. Polymorphism ability of C. albicans corresponds to a reversible morphologic transition between yeast and filamentous forms, which can be a pseudohyphae or true hyphae (Figure I.1 A) [2,7,23,24]. In addition to microscopic differences, it is possible to distinguish from other Candida species by colony colour on through CHROMagarTM Candida , based on a set of reactions of species-specific enzymes with a proprietary chromogenic substrate [25]. In the case of C. albicans , the colonies produce a light green colour (Figure I.1 B) [25]. Figure I.1 (A) Candida albicans cells stained with calcofluor obtained by microscopic epifluorescence and (B) macroscopic colonies on CHROMagarTM of Candida albicans . b. Candida albicans virulence factors As mentioned before, C. albicans can become pathogenic invading host tissues and leading to superficial and systemic infections. The pathogenicity of C. albicans is supported by several virulence factors, including hydrolytic enzyme secretion, the ability to adhere to medical devices and/or on host cells and to form biofilms, and the morphological transition from yeast to hyphal forms.
Chapter I Literature review 5 Hydrolytic enzymes production Candida albicans cells have the ability to produce and secrete some enzymes that facilitate penetration into the host cells. These enzymes are involved in adhesion, cell damage and tissue invasion and there are three different classes of secreted hydrolases expressed by C. albicans : proteinases, phospholipases and lipases. The secreted aspartic proteinases ( SAP genes) are involved in the invasion and colonization of host tissues, promoting the disruption of host mucosal membranes and degradation of defence proteins [6,26,27]. The family of SAP genes consists a group of 10 members ( SAP1 to SAP10 ), which eight of them ( SAP1 to SAP8 ) are secreted into the extracellular space and SAP9 and SAP10 are part of membrane-anchored GPI proteins [6,28]. According to the some studies, the regulation of SAP genes is depending on the transition from yeast to hyphal form, changes in growth environment, and expression of alternative switch phenotypes [28–30]. In the case of SAP1 and SAP3 , they are involved in the regulation of phenotypic switching, being expressed in the ‘opaque’, but it is not expressed during ‘white’ form [28,31]. The genes SAP4-SAP6 are mostly expressed during the development of hyphal forms at neutral pH [28,32,33]. In contrast, it has been described that SAP9 and SAP10 are expressed in all growth conditions, so these genes are not regulated by environmental conditions [28,34,35]. Phospholipases (PL) are responsible for the hydrolyse one or more ester linkages in glycerophospholipids, leading to the host cell membrane damage and adhesion of yeasts to host tissues [36]. The ability of C. albicans to produce these extracellular phospholipases is significantly higher [36] and these enzymes have been differentiated according to its mode of action and the target within the phospholipid molecule. There are four subclasses of PL genes identified in C. albicans , which are classified as A, B, C and D [28]. In C. albicans , seven phospholipases were reported to be involved in the pathogenesis, namely, PLA , PLB1-2 , PLC1-3 and PLD1 [37]. The secreted hydrolytic lipases are involved in the hydrolysis of ester bonds of monodiand triacylglycerols. So far, 10 genes have been identified in C. albicans called as LIP1-10 and the first extracellular lipase identified was LIP1 . In contrast to other enzymes, these enzymes are less studied. It was known that deletion of LIP8 in C. albicans produced significant effects on its virulence [38]. Adhesion Adherence of C. albicans to mucosal surfaces and/or synthetic materials is the early step leading to proliferation and consequently biofilm formation and infection (Figure I.2) [27]. The adhesion
Chapter I Literature review 6 mechanism is based on interaction between the C. albicans cell wall and the host cells’ surface and is ruled by environmental conditions [39]. The adhesion process can be modulated by microbial adhesins or host cell receptors, or by physical and chemical manipulations [40]. Figure I.2 presents the gene cascades that are involved in Candida adhesion and biofilm formation. One of those genes is BCR1 , a C2H2 zinc finger protein essential for biofilm formation in C. albicans and which is essential for the expression of several cell wall proteins in C. albicans [41], namely Als1, Als3 and Hwp1 [42,43]. Nobile et al. [41] described that the expression of ALS3 and HWP1 is reduced in a ∆ bcr1/bcr1 strain [41,44]. The CFEM (common in fungal extracellular membranes) family of proteins are targets of BCR1 and can act as cell surface receptors or as adhesins [45]. However, the CFEM family, namely RBT5 , PGA10 and CSA1 has just been described in C. albicans as having a role in biofilm development [46,47]. The presence of specific cell-wall proteins, designated normally as adhesins, is a trigger in the modulation of the adhesion process [6]. In C. albicans , the adhesion is mediated by the family of agglutinin-like sequence (Als) proteins, which comprises eight members, Als1-7 and 9, with a similar structure containing an Nterminal secretory signal sequence [48,49]. In the literature, it is reported that ALS1 and ALS3 are involved in biofilm surface attachment, however their expression is depending on C. albicans cell morphology (Figure I.2) [50]. ALS1 expression is detectable in both growth forms, yeast, and hyphal cell [51], in contrast to ALS3 which is only expressed in the hyphal lifestyle [52]. The hyphal forms of C. albicans that are deficient to ALS3 exhibit defective adhesion to endothelial cells [49,53]. Figure I.2 Regulatory genes involved in (A) initial adhesion process and (B) in the basal layers of biofilm formation. Eap1 is a glycosylphosphatidylinositol-dependent (GPI-linked) cell wall protein, which is also involved in the cell-cell adhesion in C. albicans and it is expressed in both yeast and hyphal cells (Figure I.2) [54]. This protein mediates surface binding, affecting cell adhesion and biofilm formation in vitro and in vivo and its expression is regulated by the transcription factor EFG1 [51] . The EAP1 mutants showed
Chapter I Literature review 7 reduced adhesion to plastic surfaces and epithelial cells and it was demonstrated that are involved in the control of adhesion to yeast cells [51,54]. Hyphal wall protein I ( HWP1 ) is a fungal cell wall mannoprotein that promotes attachment of Candida cells to the host surface (Figure I.2) [43]. In the literature, this gene is described as involved in the formation of germ-tubes and the hyphal forms of C. albicans , promoting physical contact between epithelial cells and the fungi, concluding that HWP1 is an important effector of C. albicans pathogenicity [24,55,56]. Biofilm formation Biofilms are communities of microorganisms properly organized and embedded in an extracellular matrix [57]. Candida albicans isolates are good biofilms formers, and their presence during infection has been related to higher morbidity and mortality rates compared to isolates incapable of forming biofilms [58]. It is important to address that biofilms have variability in their structure and matrix composition differing between species and strains [59]. The biofilm structure of C. albicans normally consists of two layers, a basal deposit of blastospores covered by a thick matrix film with hyphal forms (Figure I.3). The matrix of C. albicans biofilm is composed mainly by carbohydrates, proteins, phosphorus and hexosamines [23,60,61]. Figure I.3 Candida albicans biofilm structure. (a) Filamentous forms (hyphae and/or pseudohyphae). Images obtained with scanning electron microscopy after 24 h of biofilm growth. Biofilm formation is a sequential phenomenon, which involves attachment, maturation and detachment [62]. Attachment and colonisation of yeast cells to an abiotic or/and biotic surface is the first step of biofilm development, as described in the previous section (Figure I.2 A). Initial attachment of C. albicans cells is followed by cells division, leading to the formation of a basal layer of anchoring microcolonies [62,63] (Figure I.2 B), and then subsequent biofilm maturation (Figure I.4 A). The biofilm
Chapter I Literature review 8 maturation is, generally, characterized by the presence of filamentous forms, pseudohyphae and/or hyphae, and by the production of extracellular matrix [63,64]. The matrix role is to protect Candida cells from phagocytic cells and to act as a barrier to drugs and toxic substances [65,66]. Moreover, this matrix allows the maintenance of nutrients within reach of biofilm cells [65,66]. Finally, mature biofilms have the ability to initiate detachment and dispersion (Figure I.4 B) on their own and this cells release from the original biofilm community is a step forward in generating novel communities at new locations [67]. Figure I.4 Final stages of Candida albicans biofilm formation and its corresponding networks genes. (A) Mature biofilm constituted by cells with diverse morphologies and extracellular matrix; (B) Biofilm detachment and dispersion. The biofilm formation in C. albicans is regulated by several transcription factors that play a fundamental role in various pathways and they have an important potential in the regulation of other genes involved in biofilm formation [24,41]. Nobile et al. [62] investigated the transcriptional network and identified a set of six transcription factors in C. albicans that play an important role in the regulation of biofilm formation, namely BCR1 , EFG1, TEC1, NDT80, ROB1 and BRG1 (Figure I.2 and I.4) [62]. Furthermore, C. albicans biofilms are defective when any of these regulators are deleted [62]. EFG1 is one transcription factor required for biofilm formation that regulates the cell surface and hyphal formation [68]. Holland et al. [69] demonstrated a reduction in C. albicans biofilm when EFG1 is eliminated. Moreover, ∆ efg1/efg1 strains were unable to form hyphae in C. albicans , even grown under hypha-
Chapter I Literature review 9 inducing conditions [70]. TEC1 is a gene required for hyphal formation and virulence in C. albicans [42] and is a member of TEA/ATTS transcription factor family [71]. The production of extracellular matrix is another important feature in biofilm maturation [63]. One of the carbohydrates present in C. albicans matrix is β-1,3 glucan [72]. However, recent study by Zarnowski et al. [73] demonstrated that β-1,6 glucan is also an important matrix component, that it is highly dependent on the environmental conditions used. The gene responsible for glucan synthase is FKS1 (Figure I.4), more commonly designated by GSC1 , and it has been implicated in C. albicans biofilm resistance to fluconazole [72]. The susceptibility to fluconazole is the result of FKS1 disruption which reduces the deposition of β-1,3 glucan in the biofilm matrix [74]. Furthermore, the increase in FKS1 transcript is due to the reduction of the delivery of glucan to matrix [75]. In C. albicans , RLM1 and ZAP1 are consider two other biofilm regulators involved in matrix production in biofilms (Figure I.4). The transcription factor ZAP1 is predominantly a negative regulator of biofilm matrix production, ∆ zap1/zap1 produces a biofilm with high levels of β-1,3 glucan both in vitro as in vivo [76]. Some targets genes of ZAP1 are CSH1, IFD6, GCA1, GCA2 and ADH5 , which modulate levels of β-1,3 glucan in the biofilm matrix (Figure I.4) [76]. In the case of CSH1 and IFD6 , when ZAP1 activates the expression of these genes, the production of β-1,3 glucan decreases and therefore these genes are considered as negative regulators of matrix production [76]. However, GCA1 , GCA2 and ADH5 are positive regulators, since there is an increase of β-1,3 glucan when these genes are activated by the ZAP1 gene [76]. Another regulator of matrix production is RLM1 , a positive regulator, which deletion promoted a reduction in its matrix levels [77]. Taff et al. [75] described a role for BGL2 , PHR1 and XOG1 (Figure I.4) as glucan modifying genes involved in glucan delivery and matrix incorporation. The BGL2 and PHR1 genes encode glucanosyltransferases and XOG1 is a β-1,3 exoglucanase [78–80]. The last step of biofilm formation is characterized by the dispersion of yeast cells and/or pieces of the biofilm from its mature form which allows the organism to colonize new sites for further adherence and colonization [62], completing the biofilm life-cycle (Figure I.4). Biofilm dispersion occurs in response to environmental changes, such as a decrease or lack of nutrients or other modifications in the growth media composition [81]. Furthermore, the dispersion of biofilm cells can lead to a development of infections in deep organs due to the ability to invade the blood stream [81]. In the past decade, early events associated with Candida biofilm formation have received considerable attention. Recent studies in C. albicans biofilms have reported that the majority of dispersed cells are yeast cells and PES1, UME6 and NRG1 are three main regulators genes in this step (Figure I.4) [63]. Uppuluri et al. [81] demonstrated that the major yeast cells dispersed from biofilm were released from the upper hyphal layers.
Chapter I Literature review 16 b. Second-generation of ASOs The alkyl modifications are characterized by modifying the 2’ position of the ribose sugar, which belongs to the second-generation of ASOs. These chemical modifications were developed to enhance nuclease resistance and increase binding affinity for target mRNA [126] and also reduces immune stimulation [138]. The second generation is formed by 2’- O -Methyl (2’-OMe) and 2’- O -Methoxyethyl (2’- MOE), characterized by the introduction of an oxygenated group (Figure I.8) [139]. This generation is less toxic than PS, described in first generation, and increases the nuclease resistance. Moreover, there is a jump in hybridization affinity [140–142]. For ASO with 2’-OMe-PS, the toxicity presented was very mild, which in fact has been shown to not interfere, after delivery in rodent brain, with their desired effects [143,144]. The ASO sequence can be modified also with 2’-fluoro (2’-F) modification, which increase the ASO affinity. In terms of mechanism of action, the second generation does not allow the recruitment of RNase H [139]. To overcome this limitation, the RNase H activity can be restored through the insertion of a central unmodified or PS-DNA sequence, called as gapmer, which consist of regions of 2’-modified residues that flank a central DNA region of the oligonucleotide. These “wings” increase affinity and nuclease resistance, and additionally allows RNase H to join in the central gap and activate the degradation of target mRNA [126,130]. c. Third-generation of ASOs In addition to promoting target affinity and nuclease resistance, third-generation is created to further enhance biostability and pharmacokinetics and it is the most heterogeneous generation [145]. The most common modification include locked nucleic acid (LNA), peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomer (PMO) (Figure I.8) [145]. LNA modification is a sugar modification, in which nucleotides are chemically modified with a ribose containing a methylene bridge between the 2’-oxygen and the 4’-carbon of the ribose [146]. LNA substitution improve the binding affinity with mRNA target through the increase of thermal stability between DNA and RNA heteroduplexes [147]. Moreover, the LNA-modified sequence show low toxicity in biological systems [148], which explains the fact of LNAs being used as an ASO molecule, both in vitro and in vivo [149,150] . Tricyclo-DNA (tc-DNA) is a sugar modification that belongs to the conformational constraint of the LNA modification. It is characterized by the addition of an ethylene bridge merged with a cyclopropane unit that lead to a decrease of flexibility around the C3’-C4’ and C4’-C5’ bounds [143].
Chapter I Literature review 17 This modification is not compatible with RNase H and the binding affinity of tc-DNA is lower than in the case of LNA [151]. However, the studies in cells has shown more potent splice-switching applications compared to 2’-OMe-PS oligonucleotides [132,152]. PNA modification is an uncharged synthetic DNA and was firstly described by Nielsen et al. [153] . This modification is characterized by the substitution of sugar phosphate backbone by N- (3-aminoethyl) glycine units [154]. PNA is not degraded by nucleases or peptidases, showing high biostability in biological fluids [126]. PMO modification is a non-charged agent that binds and sterically blocks translation machinery or alters splicing of pre-mRNA, since PMO does not support RNase H activity [155]. This modification is characterized by the replacement of ribose sugar by morpholino ring and phosphodiester bond by phosphoroamidate linkage [155]. It has been known that PMO ASOs have a great resistance to nucleases and proteases in biological fluid [126]. Recent studies have conjugated PMO modification with argininerich peptide (ARP) in order to increase the thermal stability of heteroduplex cellular uptake, leading to an increase of cellular uptake and ASO potency [156]. I.2.2 ASO mechanism of action In absence of ASO, the normal gene expression leads to a protein expression (Figure I.9 A). According to Stanley Crooke [157], the activity of ASOs can be divided into three phases, namely prehybridization, hybridization and posthybridization. After the ASO enters and distributes in the cell, it must recognize the nucleic acid sequence space to hybridize to its cognate site, which is a complex process involving interactions within proteins [157]. The events before hybridization between ASO and target sequence need to be better understood. It is important to determine the effective concentrations in the region and identify the major intracellular proteins that bind ASOs which allow the hybridization between ASO and the “receptor” [157,158]. However, proteins or lipids involved in the hybridization step have not been yet identified and for the researchers this step is considered a “black box”. Crooke et al. [159] demonstrated that the RNA structure is a major factor on potency and specificity of the potential hybridization. On the other hand, they shown that the number of copies of target RNA and the RNA halflife is irrelevant for this step. After hybridization, the target RNA can be degraded, disabled, or modified by several mechanisms which is dependent on the ASO chemistry and design, the position of the RNA where ASO will bind and the function of the target RNA.
Chapter I Literature review 18 Figure I.9 Antisense oligonucleotides modes of action. A) normal gene protein expression in absence of ASO. B) formation of an ASO-mRNA heteroduplex capable to induce RNase H activation, leading to selective degradation of bound mRNA or C) steric interference of ribosomal assembly into cell cytoplasm. Alternatively, the ASO can enter the nucleus and regulate mRNA maturation D) inhibition of 5´cap formation, E) inhibition of mRNA splicing and F) activation of RNase H. Basically, the mechanism of action are divided into two broad categories: the ASO which activate RNase H (Figure I.9 B, F), and those that do not (Figure I.9 C, D, E) [145]. The first ASOs activate the RNase H, which is a ubiquitous enzyme that hydrolyses the RNA strand of an RNA/DNA duplex, which means that recognizes RNA-DNA heteroduplexes, and induce the degradation of mRNA, releasing the intact DNA [160]. The kinetics associated to molecular pharmacology of RNase H1 activating ASOs is extremely slow [161] and it was demonstrated that the onset of action occurs about two hours after ASO enters the cell [157]. It was necessary 60 min for intracellular distribution, 20 min for ASO search the RNA sequence and hybridize to the site and 40 min for RNase H1 recruitment and cleavage [157]. It has been shown that RNase H1, specifically, mediates the RNA cleavage and the intact ASO released from RNase H1 can be used in cleavage of many RNA molecules, which increase ASO potency [130,160]. The ideal ASO should be designed with perfect match to the target sequence and more than 3 base pair mismatches to all other genes [162]. Thus, the inactivation of RNase H is very dependent of sequence specificity because RNase H loses the degrading activity when 3 or more mismatches exist [130,162].
Chapter I Literature review 19 The ASOs that do not activate RNase H, prevent or inhibit the progression of translation by sterically blocking the ribosomal subunits or modulating alternative splicing. In the first case, ASOs can be involved in the prevention of the movement of ribosomes down the transcript and/or inhibit the construction of ribosomal subunits (40S and 60S) [130]. On the other hand, several studies demonstrated that ASOs can also bind to pre-mRNA structure and modulate splicing, both in vitro as in vivo , leading to exons inclusion or exclusion [163,164]. MicroRNAs are sequences of RNAs with approximately 21 to 23 nucleotides that inhibit translation of several mRNAs targets, leading to the control of the regulation of around 200-300 genes [130,165,166]. Thus, ASOs can be designed to bind microRNAs, blocking the possible linkage with RNA sequences and one miRNA could be an effective strategy once microRNAs block translation of multiple targets [167,168]. The first miRNA which inhibits miR-122 in hepatitis C infection show therapeutic promise in vivo [169] , since decreased the hepatitis C in monkeys as well as human patients in phase II human trial [170,171]. I.2.3 ASO cargo and delivery strategies The major problem for ASO-based therapeutics is the cargo and the delivery of the molecule to its site of action, since this delivery shall be in the tissue of therapeutic interest and in the right intracellular compartment [172]. It is very difficult to administer systematically the ASO in its naked form, due to its negative charge that cause a repulsion with the cell membrane, leading to a low uptake by target cells. Moreover, naked ASOs present more toxicity and are easily eliminated and degraded by serum nucleases [126]. The precise mechanisms involved in ASO penetration into the targeted cells is not clear, so it is important to understand how these ASOs enter the cell. The uptake of the ASO depends on temperature, the structure and the concentration of the ASO and the cell line [173–175]. The mechanisms of internalization are dependent on ASO concentration and the two major mechanisms are adsorptive endocytosis and fluid phase pinocytosis. For a low concentration, the membrane-bound receptors are enough, so the internalization occurs mainly by endocytosis [173,174]. However, for the high concentration, the pinocytosis process occurs since these receptors are saturated. It is known that ASOs tend to localize in endosomes/lysosomes and become unavailable for antisense technology. In order to improve the cellular uptake and ASO activity, in addition to the variety of chemical modifications developed, it has also been introduced Drug Delivery System (DDS): techniques and transporters, such as vectors [128]. So, it is important to develop vectors for efficient and stable delivery of ASO to the target cells and these vectors could be a viral or non-viral system. The most common
Chapter I Literature review 20 vector to deliver the ASO is the incorporation into a non-viral system, which determines the tissue distribution and cellular interactions of the oligonucleotide [172]. These non-viral vectors present many advantages to viral vectors, in terms of the relative safety, lack of immunogenicity, ease large-scale production and their surface is easy to modify for tissue-specific targeting [176–180]. As mentioned above, a single piece of ASO (Figure I.10 A) present a rapid renal clearance and can easily activate the immune response. The strategy to deliver the ASO can be divided into two broad strategies, molecular and nanoscale [172]. It is possible to conjugate the ASO with a ligand (Figure I.10 B) or a polymer (Figure I.10 C). The association of the ASO to a ligand allows to improve the half-life of the ASO in blood, providing time to transit to the targeted organ or cell [181]. The conjugation of ASO with a ligand allows a selective delivery to cells or tissue through receptor mediated mechanisms, which can be an advantage in relation to naked ASO [181]. Alternatively, the ASO can be conjugated with a polymer, as described in Figure I.10 C and in this case, the retention in blood circulation is improved [182]. Through the biodegradable bonds that can be readily dissociated, the ASO can be released intact into the cytoplasm [181,182]. Figure I.10 Delivery systems of oligonucleotides. A) single piece of oligonucleotide, B) ligandoligonucleotide conjugate, C) polymer-oligonucleotide conjugate and D) lipid or polymer nanoparticle. Because of the negative charge of ASO, the non-viral vectors more commonly used are the cationic liposomes and polymers (Figure I.10 D), forming a complex more easily [177,183]. a. Polymeric complexes: Polyplexes An example of ASO delivery strategy is the use of polymeric particles, which have a shell of polymer with the ASO inside (Figure I.10 D). Cationic polymers have been an alternative class of non-viral vectors and poly(L-lysine) (PLL) and polyethylenimine (PEI), being the earlier polymeric vectors used [178]. Very recently, polyamide porous microparticles (MP) were developed by activated anionic ring
Chapter I Literature review 21 opening polymerization (AAROP) of lactams [184,185] and proven useful for protein recognition [186] or enzyme carriers [187]. The interaction between the vectors and ASO is via electrostatic interactions [128] and normally, the ASO is internalized via an endocytosis mechanism. The first application of PLL polymer was in the late 1980s for non-viral liver-targeted gene delivery, where PLL was conjugated with a glycoprotein [188,189]. However, to reduce the in vitro cytotoxicity of unmodified PLL, some variants have been reported. One example is the use of the hydrophilic polymer PEG to cover the PLL, since PEG helps to minimize nonspecific interactions with serum components, increasing the circulation time of nanoparticles [190,191]. Another polymeric material most studied for gene delivery was PEI and its variants, and the first demonstration of its application for transfection in vitro and in vivo was in 1995 [192]. In this case, the transfection efficiency and cytotoxicity depend on its structural properties [178]. To overcome the issues associated with the toxicity, other polymeric non-viral vectors have been developed, with poly[(2-dimethylamino) ethyl methacrylate] (pDMAEMA) as an example. b. Liposomes complexes: Lipoplexes Another strategy is to use the lipid based nanoparticles (LNPs), which are the most widely used and clinically advanced non-viral vectors and the first generation of vectors developed (Figure I.10 D) [178,179]. Liposomes are spherical particles composed of phospholipids that present different structures, dimensions, lipid composition and surface charge [193]. In relation to the structure, liposomes can be composed by several concentric bilayers separated by aqueous compartments or only one phospholipid bilayer surrounding an aqueous compartment [194]. In the first case, liposomes are called Multilamellar Vesicles (MLVs), with a size range between 500 nm and 5 µm [194,195]. In the second case, liposomes are called as Small Unilamellar Vesicles (SUVs), Large Unilamellar Vesicles (LUVs) and Giant Unilamellar Vesicles (GUVs), with a size range of 20 to 200 nm, 200 to 1 µm and larger than 1 µm, respectively [194]. Moreover, they can be formed by vesicles surrounded by other vesicles, which form a multi-compartmental structures, called Multi Vesicular Vesicles (MVVs) [194]. The LNP consist of vesicles composed of bilayers of phospholipids and the nature of the phospholipids determines the liposome charge, which can be neutral or cationic [128]. The positive charge of these vectors promote a high affinity with cell membrane, because of their negative charge under physiological conditions [179,195,196]. It can be used as a lipid with positive charge, i.e., the 1,2-DiOleoyl-3-TrimethylAmmonium Propane (DOTAP) [177–180,195,197] and include a “helper” lipid to promote a more efficiently intracellular uptake of ASO and their endosomal escape [179,180,195]. These “helper” lipids are
Chapter I Literature review 22 normally neutral lipids and the most common used are 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC) [177,180,197,198], Di-Oleoyl-Phosphatidyl-Ethanoalamine (DOPE) [178,180,197,198] and 1,2DiStearoyl-sn-glycero-3PhosphoCholine (DSPC) [178,180]. I.2.4 ASO pharmacokinetic properties The pharmacokinetic properties of ASOs depend on its structure, if it is a single or double stranded, the charge, if it is negatively charged or neutral, and how it is the formulation (into a nanoparticle or not) [159,199]. As mentioned before, an unmodified ASO is generally rapidly filtered out of blood by the kidney and excreted in urine because there are normally small molecules. There are several routes of administration, namely intravenous administration, subcutaneous route, local administration, or inhalation. The most favourable route of administration depends on the site to reach its target. For systemic applications, the intraperitoneal, subcutaneous, or intravenous (IV) delivery of ASOs are the primary route of administration. An ASO modified by PS chemical modification, is enough to prolong the time of circulation and to distribute to tissue, due to the increased binding to serum proteins. The administration of PS-modified ASO by subcutaneous or intravenous route allows a high biodistribution mainly in the peripheral tissues, taking liver and kidney the highest concentrations [199,200]. The dominant phase of distribution happens immediately after the administration when ASO are moved to tissues in minutes to hours, declining rapidly in the plasma after this initial distribution phase [201]. This initial clearance is dependent on protein binding, which it is saturable and the distribution kinetics is dose-dependent [159]. The cell uptake is predominantly facilitated by endocytosis, which ASOs transit into cells by moving down concentration gradients from extracellular to intracellular compartments [159,201]. The slower terminal elimination from tissues is the last phase, whose can be extended up to several weeks, leading to a balance between the post-distribution phase plasma concentrations and tissue concentrations. The ultimate clearance and elimination is facilitated by endoand exonuclease metabolism, since the resultant small-molecular-weight fragments lose the ability to bind plasma proteins and they are easily eliminated in urine [201]. The second generation of ASOs is composed by chemical modifications that allows to improve pharmacokinetic and pharmacodynamic properties when compared to first generation (PS-ASOs). The pharmacokinetic properties of the second generation of ASOs after parenteral administration are very similar for both 2’- O Me and 2’-MOE modifications. The drugs are initially adsorptive from the plasma to the tissues, quickly and extensively, presenting a rapid distribution half-lives of plasma profiles, of about hours. The peak plasma concentrations when second generation of ASOs are injected intravenously, is
Chapter I Literature review 23 reached at the end of infusion, in contrast to subcutaneous injection where the ASOs are rapidly absorbed into the systemic circulation and the maximum concentration is reached on 3 to 5 h after injection [202,203]. After reaching the peak of concentration, the ASOs concentration decrease in a multiphasic decline, which is defined by a fast-initial distribution phase (intravenous distribution with a half-life of about 0.5 to 3 h and subcutaneous distribution of about 3 to 5 h). Consequently, the ASOs elimination is a slower phase and is characterized by very low plasma concentrations with elimination half-lives of about 2 to 4 weeks. The ASOs modified by the second generation are, in general, more stable against endoand exonucleases than PS-ASOs, mainly due to the ‘wings’ at the 3’ and 5’ ends, which protect them from being metabolized, justifying the metabolization over 2 to 4 weeks. These oligonucleotides are highly bound to plasma proteins [201,204], which rates are above 90 % and have a higher bioavailability following subcutaneous injection [203]. Furthermore, these compounds are extremely excreted by urinary tract mainly in metabolites produced slowly within tissues [202,204]. When LNA ASOs are transfected without any delivery agent, called as ‘Gymnosis’, any cellular response is directly related with the administration of the ASO. It was shown that ‘naked’ LNAs present a higher uptake and potency in contact with cell cultures [205,206]. Normally, the cellular uptake of LNA ASOs is predominantly carried out by endocytosis, as happens with other chemical modification [206]. Recently, it was reported that after subcutaneous administration, LNA ASOs are highly bound to plasma proteins, however, the extension is dependent on species. After 24 h of the first dose, the plasma concentration decays for at least half of the concentration, since the oligonucleotides are rapid distributed to different tissues, firstly in the liver and kidney [206]. Following intravenous administration of the ASO, the concentration in the plasma declined rapidly in a multi-exponential phase. As in the case of secondgeneration of ASO, the initial phase is characterized by initial rapid distribution, wherein the ASO is distributed from circulation in the tissues, of about hours [207,208]. I.2.5 ASO toxicology The pharmacological effect of any class of drug can result on one potential mechanism of toxicity and like any drug, the ASOs exhibit dose-dependent toxicities. These ASOs toxicities can be classified as hybridization dependent or hybridization independent [199]. The hybridization with nearly homologous sequences on nontarget mRNA, alterations of endogenous metabolic pathways and nonspecific interactions with proteins can induce toxicity. Exaggerated pharmacological effects and the hybridization to non-target RNAs are the most common processes associated to the hybridization-dependent toxicities [209]. These toxicities can be avoided through a proper and careful selection of the sequence with perfect
Chapter I Literature review 24 matches or a few mismatches by bioinformatic analysis. The shorter oligonucleotides can be a problem because of the interaction with non-targeted transcripts which are degraded if RNase is active [210]. Moreover, it is important to perform preclinical models in order to characterize the pharmacology and toxicology of the ASOs [199]. The interaction between oligonucleotides and proteins could be a second mechanism of ASO toxicity which can be sequence dependent or independent. Typically, there are two main factors associated to the sequence independent toxicity, which are the chemistry of the oligonucleotides and the chemical class of the proteins that ASO interact [199]. The most serious toxicities related to the systemic administration of ASOs are the coagulation activation and the complement and hypotension [211–213]. In the case of the first generation of ASOs, it is described in the literature that the toxicity is due to the combination of some factors, such as, the dose, extent of PS modification, sequence, and route and duration of administration [199,213,214]. The sequence that are fully modified by PS, ASO exhibit nonspecific effects, in contrast to partially modified sequence [214]. The first side effects associated with PS chemistry was reported in human trials of Bcl-2 PS ASOs which were increased blood glucose levels, dose-dependent thrombocytopenia, and mild hyperglycaemia [213,215]. The second generation of ASOs has been demonstrated to be better accepted than the PSmodified ASOs.[209,216]. For example, thrombocytopenia is a side effect with ASO treatment, and it was reported that in mice the administration of a first-generation ASO resulted frequently in a reduction in platelet counts [217–219]. In contrast, this incidence decreases with the administration of a 2’-MOE ASO. Moreover, in cancer studies which investigated the first-generation of ASO, the thrombocytopenia has been frequently reported [220,221], in contrast with the second-generation [217,219,222]. The LNA modified ASOs have the potential to increase the potency of the ASOs [223], however, comparing to the second generation of the ASO, LNA-modified ASOs provoke a risk of hepatotoxicity that was chemistry-, sequenceand design-dependent [147]. For example, it has also been shown that LNA ASOs with a size between 14 and 20 nucleotides significantly reduced the hepatotoxicity. Moreover, sequences with different positions of LNA modifications can present different hepatotoxic profiles [206,224].
Chapter I Literature review 25 I.2.6 Antisense Drugs approved by FDA and EMA Numerous studies have documented the use of AST as biochemical tools for studying human target diseases. Up to now, there are ten antisense drugs that have been approved by Food and Drug Administration (FDA), as well as, by European Medicines Agency (EMA) [225–227]. These antisense drugs are presented in Table I.2. However, there are around 187 antisense drugs that are under different stages of clinical trials, and only 17 in the third phase [227,228]. The first antisense drug that received market authorization was formivirsen, that was a drug developed in a collaboration between Isis Pharmaceuticals with Novartis Ophthalmics approved by FDA in 1998 and by EMA in 1999 [229,230]. However, Novartis interrupted the drug marketing in 2002 in Europe and in 2006 in the United States [226]. In the last two years, three antisense drugs were approved, namely, Volanesorsen, Givosiran and Golodirsen [226,227]. The potential antisense therapeutics have been studied for the treatment of some human diseases, such as, cancer (e.g., leukemia, lung cancer, prostate cancer) [131,231–234] and, Huntington’s disease [131,143,235]. In recent years, the AST has been applied to bind specific targets to treat infections, for example as targeting specific antibiotic resistance determinants. The first application of AST to bacteria was demonstrated through a PNA-modified ASO, which demonstrated an antisense inhibition of the Escherichia coli beta-lactamase gene [236]. More recently, a PMO-modified ASO was synthesized and conjugated with cell-penetrating peptides to enhance cellular delivery. An CPPPMO was designed to target E. coli gyrA that is a conserved gene in several bacteria species, and it was demonstrated that the ASO has reduced the expression of gyrA mRNA and reduced the viability of Enterococcus faecalis and Staphylococcus aureus [237,238] . Another application was described by Liang et al. [239] that have designed a peptide-conjugated PNA (PPNA) to target the ftsZ gene, essential for cell division in S. aureus and it was demonstrated that PPNA ASO inhibited growth and expression of ftsZ mRNA [239–241]. However, application of AST as antiCandida agents are still scarce and usually are limited to the first and the second generation of ASOs. Do authors knowledge there is only one study using the AST to interrupt and efficiently inhibit C. albicans in vivo splicing using a PS-modified ASO [242]. The most exciting long-term possibility is to develop antisense molecules for clinical application; however, this naturally requires much more research to develop a credible and alternative approach to control Candida infections.
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37 Chapter II Exploitation of the antisense oligonucleotides to control Candida albicans filamentation Main goal To explore the second (2’- O Methyl) and third (LNA) generation of antisense oligonucleotides modifications to control Candida albicans switch from yeast to filamentous forms.
38 Chapter II.1 Application of 2’- O MethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant Main goal To design an antisense oligonucleotide based on the second generation of antisense oligonucleotides targeting the EFG1 mRNA of Candida albicans and to validate in vitro its applicability through filamentation enumeration, EFG1 gene expression and Efg1p protein translation. Conclusions This study validated the possibility to use antisense oligonucleotides with 2’OMe chemical modifications to control C. albicans virulence determinants . The anti -EFG1 2’OMe ASO was able to significantly reduce EFG1 gene expression and Efg1p protein translation, and effectively prevent C. albicans cell filamentation, even in different simulated human body fluids. This chapter is based on the following publications: Araújo, D.; Azevedo, N.M.; Barbosa, A.; Almeida, C.; Rodrigues, M.E.; Henriques, M. and Silva, S. (2019) Application of 2’- O MethylRNA’ Antisense Oligomer to Control Candida albicans EFG1 Virulence Determinant. Molecular Therapy Nucleic Acids. 18, 508-517 Silva, S.; Araújo, D.; Azevedo, N.; Azeredo, J.; Henriques, M. (2020). Antisense oligomers for controlling Candida albicans infections. WO 2020/174366 A1
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 39 II.1.1 Introduction As referred previously (Chapter I), candidiasis is the primary fungal disease, with a mortality rate of about 30-50 % and with costs associated with hospitalized patients that range from €5,700 to €85,000 (in U.S. dollars, approximately $6,286 to $93,752) per episode [1,2]. This important clinical, social and economic problem is due to the recognized phenomenon of Candida species antifungal resistance, associated with the indiscriminate use of traditional antifungal agents [1–3]. Candida albicans remains the most prevalent of all Candida species in Europe, with a range of incidence of around 40 % [2,4,5]. The pathogenicity of C. albicans is supported by a series of virulence factors, one of the most alarming being its ability to switch from yeast to filamentous forms, a tightly regulated process by a network of genes known as dimorphic switching [6]. This virulence factor requires C. albicans to sense and respond to the host environment and is essential for its pathogenicity [7–9]. EFG1 is one of the most important and well-studied regulator genes involved in C. albicans filamentation [10–15]. As a consequence of the rising levels of C. albicans multi-resistance to the traditional antifungal treatments, new alternative therapies, with novel mechanisms of action, enhanced therapeutic potential, improved pharmacokinetics, and less toxicity, are urgently needed [16,17]. Antisense therapy (AST) holds great promise for the treatment of many human chronic non-infectious diseases; [18–24] however, for controlling Candida species growth, the knowledge is scarce [23,25]. Moreover, the control of yeast virulence determinants has never been exploited before with AST. The concept underlying AST is relatively straightforward: the use of a complementary sequence to a specific mRNA that can inhibit gene expression, inducing a blockage in the transfer of genetic information from DNA to protein [26]. Antisense oligonucleotides (ASOs) are simply short strands of nucleic acids that have a sequence that is complementary to the target mRNA, and that bind to this target by means of standard WatsonCrick base pairing [26]. Up to now, there have been three generations of ASOs [24-26] with several chemical modifications in order to increase its nuclease resistance, reduce its toxicity, and enhance its affinity and half-life [22]. The 2’- O MethylRNA (2’OMe) sugar modification belongs to the second generation of acid mimics; however, these do not support RNase H activity (a specific degradation mechanism cleaving the target mRNA) [27,28]. An insertion of a longer central unmodified region, known as gapmers , has been used as a popular strategy to allow that RNase to join and activate the degradation of the mRNA target [29,30]. Thus, this work is based on that if a pathogen’s genetic sequence of a specific gene is a determinant of virulence, as is the case with the EFG1 gene, it will be possible to synthesize a nucleic
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 40 acid mimic that will bind to the mRNA produced and degrade it, blocking its translation into protein and, consequently, reducing its virulent phenotype (which, in this case, would be the filaments development). II.1.2 Materials and Methods a. Microorganisms A total of 11 clinical strains (Figure II.1.1 A), including Candida albicans (n=10) and Saccharomyces cerevisiae (n=1), recovered from different body sites, were used during this study. All isolates were recovered from vaginal, urinary, and oral tracts and were obtained from Candida collection of the Biofilm group of the Centre of Biological Engineering, University of Minho, Braga, Portugal. Four reference strains - Candida albicans (SC5314), Candida parapsilosis (ATCC 22019) , Candida tropicalis (ATCC 750) and Candida glabrata (ATCC 2001)- were included in this study. The mutant strain C. albicans ΔΔefg1 (HLC52) was also tested [31]. b. Growth conditions For all experiments, yeast strains were subcultured on sabouraud dextrose agar (SDA; Merck, Darmstadt, Germany) and incubated for 24 h at 37 °C. Cells were then inoculated in sabouraud dextrose broth (SDB; Merck, Darmstadt, Germany) and incubated overnight at 37 °C, 120 rpm. After incubation, the cells’ suspensions were centrifuged for 10 min at 3000 g at 4 °C and washed twice with phosphatebuffered saline (PBS, pH 7, 0.1 M). Pellets were suspended in 5 mL of Roswell Park memorial institute 1640 medium (RPMI, pH 7, Sigma-Aldrich, St Louis, USA), and the cellular density was adjusted for each experiment using a Neubauer chamber (Paul Marienfild, Lauda-Königshofen, Germany) to 1 x 105 or 1 x 106 cells mL-1. All experiments of this work were performed in triplicate and in a minimum of three independent assays. c. Design and synthesis To design a specific ASO for C. albicans EFG1 , the target region of the gene was selected based on a search conducted at the Candida Genome Database (CGD) (http://www.candidagenome.org/cgibin/compute/blast_clade.pl). Several EFG1 gene sequences were aligned to make sure that conserved regions were used for the design. Also, a BLAST search was performed to ensure that the sequences were not targeting any sequence of the human genome or a similar region in another C. albicans gene. The EFG1 sequence 5’-ACAATAACGGTATGCC-3’ was selected as the target, taking into account its
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 41 high specificity to the C. albicans genome, its non-binding against the Homo sapiens genome, and the number of nucleotides [26]. Specific ASOs were then designed for the use of 2’ ribose modification. 2’OMe was selected, since it is one of the most used for antisense applications [28–30,32]. A gapmer was introduced to increase the odds of activating RNase H activity [33]. The calculator from Integrated DNA Technologies (IDT: http://eu.idtdna.com/calc/analyzer) was used to determine the theoretical Tm and the GC content of the possible ASO for that target region. The selected ASO was produced according to the user’s own specifications at EXIQON and purified by high-pressure liquid chromatography (HPLC). The same ASO was synthetized with an orange-fluorescent fluorophore (TYE563). A scrambled ASO, similar to the EFG1 ASO, was also synthesized to be used as negative control. d. Sensitivity and specificity tests The sensitivity and specificity of antiEFG1 2’OMe ASO was determined against different yeast strains (Figure II.1.1 A) by fluorescence in situ hybridization (FISH) [34]. For that, 20 µL of an inoculum of Candida cells adjusted to 1 x 106 cells mL-1 were transferred to a slide and fixated with 30 µL 4 % (v/v) paraformaldehyde (Sigma-Aldrich) for 10 min, and the excess was removed. After that, cells were permeabilized with 30 µL 50 % (v/v) ethanol for an additional 10 min and allowed to air dry. The hybridization step was performed with 20 µL ASO (200 nM) coupled with orange-fluorescent fluorophore diluted in hybridization solution (900 nM NaCl [Panreac Applichem, Barcelona, Spain], 30 % formamide [Sigma-Aldrich, Sintra, Portugal], 20 mM Tris-HCl [Sigma-Aldrich, Sintra, Portugal], and 0.01 % SDS (Sigma-Aldrich, Sintra, Portugal]). Negative controls were prepared only with 20 µL hybridization solution without probe. Samples were then covered with coverslips and incubated at 37 °C for 3 h in dark conditions. After hybridization, slides were submerged in wash solution (20 mM Tris-HCl, 0.01 % SDS, and 900 mM NaCl) and incubated for 30 min at the same temperature. The images from cells were acquired with an epifluorescence microscope (Olympus Portugal, Porto, Portugal). Cells were observed using a 40x objective. The exposure time, gain, and saturation values were fixed for each sample. The TRITC filter (530-550/591) was used for images acquisition. e. Cytotoxicity In order to select the concentration of anti -EFG1 2’OMe without cytotoxicity to be used during this study, the ASO cytotoxicity was determined against 3T3 cell line (fibroblast cells, embryonic tissue, mice from the CCL 163 line, American Type Culture Collection). For that, 3T3 cells were grown in Dulbecco’s modified eagle medium (DMEM, Biochrom, Berlin, Germany) supplied by 10 % fetal bovine serum (FBS;
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 48 b. AntiEFG1 2’OMe cellular uptake, sensitivity and specificity Sensitivity and specificity of the nucleic acid mimics are two important factors to the success of the ASO applicability [54–56]. In this study it was used the FISH test, a standard methodology used to identify microorganisms that makes use of nucleic acid coupled with fluorochromes [57–60], and epifluorescence analysis to evaluate the anti -EFG1 2’OMe cellular uptake, sensitivity and specificity against C. albicans cells. The anti -EFG1 2’OMe specificity was tested against 10 strains of C. albicans and other 4 strains of other fungi (Figure II.1.1). AntiEFG1 2’OMe binding in C. albicans was confirmed by the positive signal (presence of fluorescence) observed for all C. albicans strains tested (n = 10) (Figure II.1.1 A and II.1.1 B; Figure AI.1). The negative signal (absence of fluorescence) obtained for the other fungi tested and for C. albicans ΔΔ efg1 reinforces ASO specificity for C. albicans cells (Figure II.1.1). Figure II.1.1 AntiEFG1 2’OMe sensitivity and specificity obtained by FISH. (A) List of strains and species used and their origin, as well as the respective results obtained by FISH at 37 °C, during 3 h. (B) Illustrative images obtained by epifluorescence microscopy. The exposure time was the same for each strain: Candida albicans SC5314 were obtained with 218.7 ms; Candida albicans HLC52 (ΔΔefg1 mutant strain) with 713.2 ms, and Candida tropicalis ATCC750 with 293.9 ms of exposure. Negative controls were prepared only with 20 µL of hybridization solution without probe.
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 49 These studies demonstrate the anti -EFG1 2’OMe Candida cellular uptake without carriers or transfection agents for instance, by adsorptive endocytosis as in other microorganisms [61–63], and its ability to hybridize with the respective target with high specificity for C. albicans cells. c. AntiEFG1 2’OMe ASO behaviour In order to determine the concentration of anti -EFG1 2’OMe to be used in vitro validation studies, C. albicans SC5314 was incubated with different concentrations of ASO (10-60 nM) (Figure II.1.2). Additionally, the same was applied to investigate the cytotoxic effect of the ASO on 3T3 cell line (Figure II.1.2 A). Figure II.1.2 Anti -EFG1 2’OMe effect on Candida albicans filamentation. (A) Relative cell viability (%) determined by the absorbance values (Abs; 490 nm cm-2) of formazan product obtained from 3T3 cells treated with different concentrations of ASO (10, 20, 40, and 60 nM). The control is related to the cells without ASO treatment.
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 50 (B) Percentage of inhibition (%) of filamentous forms, after treatment with different concentration of ASO (10, 20, and 40 nM). (C) Levels of EFG1 gene expression obtained by Pfaffl method, after application of 40 nM ASO, at different time points (4, 6 and 8 h) in RPMI. Error bars represent standard deviation. *Significant differences among 10 nM and the other concentrations of ASO tested (P-value< 0.05). + Significant differences between untreated and treated cells (P-value< 0.05). Cytotoxicity evaluation Figure II.1.2 A presents the results of ASO cytotoxicity on the 3T3 cell line for the determination of the minimal ASO concentration capable to inhibit C. albicans filamentation and EFG1 gene expression. MTS assays were performed to infer about the anti -EFG1 2’OMe cytotoxicity against 3T3 cells. The results demonstrated that the ASO concentrations of 10, 20, and 40 nM tested were not cytotoxic, since the relative cell viability is higher than 70 % of the control (absence of ASO) (Figure II.1.2 A) [64]. However, the relative cell viability for 60 nM is approximately 70 %, so it could be considered a cytotoxic concentration. Therefore, it was decided to use 40 nM of ASO for the next experimental assays. Effect on filamentation and gene expression Concerning the anti -EFG1 2’OMe effect on C. albicans filamentation, it was possible to verify a reduction for all the concentrations tested (Figure II.1.2 B). As expected, the percentage of filamentation of C. albicans without ASO increased from 4 h to 8 h, reaching 80% filamentation (Figure AI.2 A). In the presence of ASO, after 4 h of incubation, approximately 10% reduction was observed (Figure II.1.2 B) without statistically differences among the ASO concentrations tested (P-value>0.05). Additionally, the results revealed a more pronounced effected after 6 h, specifically with 40 nM ASO, with approximately 20 % reduction (P-value<0.05). After 8 h of incubation, a similar performance was observed with 15 % of reduction, even for the lower concentration (20 nM) of ASO. Additionally, the ASO scramble was unable to reduce C. albicans filamentation (Figure AI.3). The EFG1 expression levels were determined for C. albicans SC5314 cells growing in the presence and the absence of 40 nM of ASO in order to evaluate the effect of anti -EFG1 2’OMe in the blockage of the expression of the respective gene. As expected, this strain expresses the EFG1 gene and a 3-fold increase on its expression levels was noticed from 4 h to 8 h (Figure AI.2 B). Regarding ASO treatment, qRT-PCR studies revealed a decrease on the levels of EFG1 expression after 6 h and 8 h (Pvalue<0.05) (Figure II.1.2 C). Indeed, a reduction of 54 % at 6 h and 60 % at 8 h on the EFG1 levels of expression was demonstrated (P-value<0.05).
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 51 After defining the most appropriate concentration of antiEFG1 2’OMe to be used (40 nM), it was evaluated the performance of the ASO on longer periods (Figure II.1.3). In terms of C. albicans filamentation reduction (Figure II.1.3 A), the results showed an increase on inhibition over time, reaching 80 % after 24 h of treatment (P-value<0.05) compared to the absence of ASO. It is important to address that the dimorphic switching in C. albicans is dependent on a network of genes [12,14,65–68]. Thus, it was not expected a total reduction on C. albicans filamentation. Subsequent examination of epifluorescence microscopy images confirms these results and also revealed a significant and relevant decrease in terms of the filaments’ length (74 m to 34 m at 6 h, 81 m to 54 m at 8 h, 68 m to 37 m at 10 h and 143 m to 56 m at 24 h of treatment) (Figure II.1.3 D). This is an important result once C. albicans filamentation is considered one of the most problematic virulence factors, increasing its capability to invade human cells and causing tissue damage [69,70]. Figure II.1.3 Anti -EFG1 2’OMe effect on Efg1p translation . (A) Percentage inhibition (%) of filamentous forms at different time points (6, 8, 10, and 24 h). (B) Levels of EFG1 gene expression obtained by the Pfaffl method at 24 h. (C) Levels of Efg1p translation normalized with the translation of Act1p at 24 h. (D) Epifluorescence microscopy images of Candida cells stained with Calcofluor after treatment with 40 nM ASO (control was prepared only with cells in RPMI; without ASO). The assays were performed for C. albicans SC5314. Error bars represent standard deviation. *Significant differences between 6 h and the other times tested (Pvalue<0.05). + Significant differences between untreated and treated cells (P-value<0.05).
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 52 As mentioned earlier, ASOs affect cellular functions through transcription attenuation and protein translation inhibition [71–74]. The effect of antiEFG1 2’OMe on EFG1 gene expression and Efg1 protein translation were determined at 24 h of treatment (Figure II.1.3 B and II.1.3 C), as data from filamentation indicated this treatment time as quite effective. The results obtained showed a significant reduction in levels of EFG1 expression (around 59%) (Figure II.1.3 B) and in Efg1p protein translation (around 57 %) (Figure II.1.3 C), corroborating the morphological data (Figure II.1.3 A and II.1.3 D). Performance on simulated human body fluids To mimic human body environments, the performance of anti -EFG1 2’OMe was also evaluated on different simulated human body fluids (AS and AU) and horse blood (Figure II.1.4). It is important to highlight that C. albicans was able to grow and filament in all simulated human body fluids tested, but in a timeand fluid-dependent manner (Figure AI.4 A and AI.4 B). Figure II.1.4 Anti -EFG1 2’OMe effect on simulated human body fluids (AS, AU and horse blood). (A) Percentage of inhibition of filamentous forms (%) at different time points (6, 8, 10 and 24 h for AS and AU; 48 h for horse blood) and (B) Levels of EFG1 gene expression for C. albicans SC5314 obtained by Pfaffl method, after treatment with 40 nM of ASO in the presence of different simulated human body fluids (AS and AU at 24 h and horse blood at 48 h). Error bars represent standard deviation. * Significantly differences between 6 h and the other times tested (P-value<0.05). + Significantly differences between untreated and treated cells (P-value<0.05). Interestingly, it can be noticed that anti -EFG1 2’OMe maintains its performance in simulated human body fluids, reducing C. albicans filamentation and EFG1 gene expression. In fact, it was verified that the ASO was able to reduce 90 % and 80 % of C. albicans filamentation after 24 h of incubation in AS and AU (P-value<0.05) respectively, and 50 % after 48 h of incubation in horse blood (Figure II.1.4 A).
Chapter II.1 Application of 2’-OMethylRNA antisense oligonucleotide to control Candida albicans EFG1 virulence determinant 53 Figure II.1.4 B shows the levels of EFG1 gene expression and demonstrates a decrease in the levels of expression of 89 % in AS, 61 % in AU and, 74 % in horse blood (P-value<0.05). It is important to highlight that the levels of EFG1 expression in the absence of ASO were different in all simulated human body fluids tested (Figure AI.4 C), which justifies the different levels of reduction observed. Considering any possible future clinical applications of the anti -EFG1 2’OMe in the control of local candidiasis (oral and urinary), as well as of systemic infections (blood), these are important results once the ASO maintains its performance in human fluids, inhibiting C. albicans filamentation and the EFG1 gene expression. This data demonstrates, for the first time, that it is possible to use antisense oligonucleotides with 2’OMe chemical modifications to control virulence determinants of C. albicans. The anti -EFG1 2’OMe that it was projected has significantly reduced EFG1 gene expression and effectively prevented C. albicans cell filamentation in different simulated human body fluids. Undeniably, this work provides potentially valuable information for future research into the management of Candida infections. Thus, in the future, it will be possible to develop a credible and alternative method to control oral and urinary candidiasis, as well as systemic infections, based on AST methodology. References 1 Quindós G (2018) Epidemiology of invasive mycoses: A landscape in continuous change. Rev. Iberoam. Micol. 35, 171–178 2 Koehler P, Stecher M, Cornely O A et al (2019) Morbidity and mortality of candidaemia in Europe: an epidemiologic meta-analysis. Clin. Microbiol. Infect. 25(10), 1200-1212 3 Negri M, Henriques M, Svidzinski T I et al. (2009) Correlation between Etest®, disk diffusion, and microdilution methods for antifungal susceptibility testing of Candida species from infection and colonization. J. Clin. Lab. Anal. 23, 324–330 4 Yapar N (2014) Epidemiology and risk factors for invasive candidiasis. Ther. Clin. Risk Manag. 10, 95– 105 5 Gonçalves B, Ferreira C Alves C T et al. (2016) Vulvovaginal candidiasis: Epidemiology, microbiology and risk factors. Crit. Rev. Microbiol. 42, 905–927 6 Araújo D, Henriques M and Silva S (2017) Portrait of Candida species biofilm regulatory network genes. Trends Microbiol. 1, 62–75 7 Mayer F L Wilson D and Hube B (2013) Candida albicans pathogenicity mechanisms. Virulence 4, 119– 28 8 Casadevall A and Pirofski L (2001) Host-pathogen interactions: the attributes of virulence. J. Infect. Dis. 184, 337–344 9 Saville S P, Lazzell A L, Monteagudo C and Lopez-Ribot J L (2003) Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection. Eukaryot. Cell 2, 1053–60 10 Nobile C J, Fox E P, Nett J E et al. (2011) A recently evolved transcriptional network controls biofilm development in Candida albicans . Cell 148, 126–138 11 Nobile C J and Mitchell A P (2005) Regulation of cell-surface genes and biofilm formation by the C. albicans
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57 Chapter II.2 AntiEFG1 2’- O MethylRNA antisense oligonucleotide inhibits Candida albicans filamentation and attenuates candidiasis in Galleria mellonella Main goal To validate in vivo the applicability of antiEFG1 2’OMe antisense oligonucleotide for inhibiting Candida albicans filamentation and to attenuate candidiasis. Conclusions This work confirmed that the antiEFG1 2’OMe ASO was able to inhibit C. albicans filamentation and to attenuate the C. albicans virulence on a G. mellonella model. This chapter is based on the following article: Araújo D, Mil-Homens D, Henriques M, Silva S. AntiEFG1 2’- O MethylRNA oligomer inhibits Candida albicans filamentation and attenuates the candidiasis in Galleria mellonella . Molecular Therapy – Nucleic Acids, MTNA-D21-00236
Chapter II.2 AntiEFG1 2’- O MethylRNA antisense oligonucleotide inhibits Candida albicans filamentation and attenuates candidiasis in Galleria mellonella 64 References 1 Fedhila S, Buisson C, Dussurget O, et al. (2010) Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella . J Invertebr Pathol 103, 24–9. 2 Junqueira J C. (2012) Galleria mellonella as a model host for human pathogens. Virulence 3, 474–6. 3 Fuchs B B, O’Brien E, Khoury J B E and Mylonakis, E. (2010) Methods for using Galleria mellonella as a model host to study fungal pathogenesis. Virulence 1, 475–82. 4 Mil-Homens D, Ferreira-Dias S and Fialho A M. (2016) Fish oils against Burkholderia and Pseudomonas aeruginosa : In vitro efficacy and their therapeutic and prophylactic effects on infected Galleria mellonella larvae. J Appl Microbiol 120, 1509–19. 5 Vilela S F G, Barbosa J O, Rossoni R D, et al. (2015) Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C. albicans and attenuates the experimental candidiasis in Galleria mellonella . Virulence 6, 29–39. 6 Rossoni R D, dos Santos Velloso M, Figueiredo L M A, et al. (2018) Clinical strains of Lactobacillus reduce the filamentation of Candida albicans and protect Galleria mellonella against experimental candidiasis. Folia Microbiol (Praha) 63, 307–14. 7 Straarup E M, Fisker N, Hedtjärn M, et al. (2010) Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100–11. 8 Torres A, Kozak J, Korolczuk A, et al. (2016) Locked nucleic acid-inhibitor of miR-205 decreases endometrial cancer cells proliferation in vitro and in vivo . Oncotarget 7, 73651–63. 9 Macedo D, Leonardelli F, Dudiuk C, et al. (2019) In vitro and in vivo evaluation of voriconazole-containing antifungal combinations against Mucorales using a Galleria mellonella model of mucormycosis. J Fungi 5, 5. 10 Kloezen W, Parel F, Brüggemann R, et al. (2018) Amphotericin B and terbinafine but not the azoles prolong survival in Galleria mellonella larvae infected with Madurella mycetomatis . Med Mycol 56, 469–78.
65 Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation Main goal The main goal of this part of the work was to evaluate of a set of LNA-ASOs, in the so-called gapmer constitution, to control of EFG1 gene expression and reduction of in vitro filamentation, also in the control C. albicans virulence in an in vivo model. Conclusions This work showed that LNA-type gapmer ASOs modifications with PS-linkages and palmitoyl-2’-amino-LNA monomers are very favorable for an in vivo application and therefore constitute promising lead structures for development of drugs against Candida species. This chapter is based on the following article: Araújo D, Mil-Homens D, Rodrigues ME, Henriques M, Jørgensen P, Wengel J, Silva S. Antisense locked nucleic acid gapmers to control Candida albicans filamentation. Submitted to Nanomedicine – Nanotechnology, Biology and Medicine, JN202197
Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation 66 II.3.1 Introduction As described on Chapter I, antisense oligonucleotides (ASOs) are short oligonucleotide sequences which are complementary to the target RNA which bind through standard Watson-Crick base pairing [1]. Normally, ASOs are chemically modified in order to protect them against the action of nucleases, to improve delivery and biodistribution and 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 non-bridging phosphate oxygen atoms by sulphur [1,3,4]. To overcome some issues related to the chemical modifications, as in the case of PS and 2’OMethyl (as previously applied in Chapters II.1 and II.2), further chemical modifications were developed over the time. The third generation was, in fact, created to further enhance biostability and pharmacokinetics in addition to enhance nuclease resistance and the target affinity. The locked nucleic acid (LNA) is a sugar modification that belongs to the third generation, having, relative to native RNA, a methylene bridge between the 2’- oxygen and 4’-carbon atoms of the ribose sugar [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 [2,7,9]. The main goal of this part of the work was to evaluate of a set of LNA-ASO, in the so-called gapmer constitution, to control of EFG1 gene expression and reduction of in vitro filamentation, and also to control C. albicans virulence in an in vivo model of G. mellonella . II.3.2 Methods a. Design and synthesis of AntiEFG1 LNA-gapmer ASOs Five LNA-gapmers were designed against the C. albicans EFG1 (gene orf19.610) target using the sequence 5’-AATAACGGTATGCC-3’ as starting point for introduction of LNA nucleotide modifications. Further, the LNA-gapmers were designed based on previous results. LNA-gapmer1 was the standard LNAgapmer constitution one reference in this study, and four additional LNA-gapmers were subsequently designed by shortening the central DNA-nucleotide gap (LNA-gapmer2), adding PS-linkages (LNAgapmer3 and LNA-gapmer5), and adding a palmitoyl-2’-amino-LNA modification (LNA-gapmer4 and LNAgapmer5) (Table II.3.1). In all LNA-gapmers, chemical modifications were introduced distally within the sequences to increase the ASOs stability, whereas the central regions were constituted by DNA nucleotides in order to ensure compatibility with RNase H thus preserving a potential for RNA target cleavage upon hybridization between an ASO and its RNA target [10].
Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation 67 Table II.3.1 Sequence of antiEFG1 LNA-gapmer ASOs, with the respective size and GC content 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 IDT (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 following the following procedures: Trichloroacetic acid in CH2Cl2 as detritylation reagent; 0.25 M 4,5-dicyanoimidazole (DCI) in CH3CN 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 disulfid (PADS) in 3-picoline/CH3CN (1:1, v/v) for 180 sec. 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 7M 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) and matrix-assisted laser desorption ionization time-to-flight mass spectrometry (MALDI-TOF, Microflex LT, Bruker, Daltonies). The purified ASOs were detritylated by treatment with an 80 % (w/w) aqueous solution of acetic acid for 20
Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation 68 min at room temperature, precipitated by addition of ice-cold acetone, and characterized by IE-HPLC (purity >90 %) and MALDI-TOF mass spectrometry (confirmation of composition). b. Characterization of AntiEFG1 LNA-gapmer ASOs Melting temperatures (Tm values) The Tm values 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 (134000 L M-1 cm-1 of strands). All measurements were performed in medium salt buffer with 220 mM Na+ (composition: 200 mM NaCl, 20 mM NaH2PO4 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 min-1). The values for Tm were determined as an average of two individual measurements. Secondary structure The secondary structure of the LNA-gapmer ASOs 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 background corrected using the buffer applied (5 mM MgCl2, 10 mM NaCl and 1 mM sodium phosphate). The LNA-gapmer ASOs were used in the following constitution, i.e., 50 µM RNA, 5 mM MgCl2, 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. Surface charge The surface charge of the LNA-gapmer ASOs was measured using t Malvern Zetasizer ZS (Malvern, CA). A dispersion of each LNA-gapmer ASOs (25 nM) in ultra-pure water was placed in a disposable cuvette and the zeta potential was measured at room temperature in triplicate.
Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation 69 c. Cytotoxicity The cytotoxicity of the LNA-gapmer ASOs was determined using a 3T3 cell line (Fibroblasts, Embryonic tissue, Mouse from CCL3, American Type Culture Collection). For that, 3T3 cells were grown in DMEM (Biochrom, Germain) supplied by 10 % of FBS (Sigma Aldrich) and 1 % of antibiotic-containing P/S (Biochrom, Germain). After detachment, a suspension with 1 x 105 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 %CO2. The MTS procedure was carried out as described in the previous Chapter II.1. d. AntiEFG1 LNA-gapmer ASOs in vitro Microorganisms and growth conditions The Candida strain used in this study was C. albicans SC5314, which is 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, CHROMagarTM Candida , through the distinction of colony colours and by PCR-based sequencing with primers for ITS1 and ITS4 [11]. 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 (PBS; pH 7, 0.1 M). Pellets were suspended in 5 mL of RPMI (pH 7; Sigma, St Louis, USA), and the cellular density was adjusted for each experiment using a Neubauer chamber (Paul Marienfild, LaudaKönigshofen, Germany) to 1 x 106 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-gapmer ASOs on C. albicans filamentation, yeast cells were incubated with each ASO during 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 x 106 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. After 24 h, aliquots were
Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation 70 recovered, and filaments were counted using a Neubauer chamber. The results were presented as percentage (%) of filamentation reduction, as previously described in Chapter II.1. In addition to the filamentation inhibition studies, the length of the filaments was quantified through fluorescence microscopy analysis as previously described in Chapter II.1. 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), as in previous Chapter II.1. To avoid potential DNA contamination, samples were treated with DNase I (Amplification Grade, Invitrogen) and the RNA concentration was determined by optical density measurement (NanoDrop 1000 Spectrophotometer Thermo Scientific®). The 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 [12]. Non-transcriptase reverse (NRT) controls were included in each run. The primers were designed using the Primer 3 web-based (Table II.3.2). Table II.3.2 Primers used for real time PCR, with the respective melting temperature (Tm) and amplification product (AP) Candida albicans Gene Systematic Name Sequence (5’-3’) Primer Tm (°C) AP (BP) EFG1 CR_07890W_A / Orf19.610 5’-TTCTGGTGCAGGTTCCAC-3’ 5’-CCTGGTTGTGATGCAGGT-3’ Forward Reverse 57 168 ACT1 C1_13700W_A / Orf19.5007 5’-AATGGGTAGGGTGGGAAAAC-3’ 5’-AGCCATTTCCATTGATCGTC-3’ Forward Reverse 57 150 AP, amplification product; BP, base pairs.
Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation 71 e. AntiEFG1 LNA-gapmer ASOs in vivo The Galleria mellonella caterpillar infection model was used for the in vivo studies as previously described by Mil-Homens et al. [13,14]. Galleria 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 antiEFG1 LNA-gapmer ASOs 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 were 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-gapmer ASOs on G. mellonella survival rate, larvae were injected with C. albicans and each LNA-gapmer. The concentration of C. albicans to be injected (7 x 107 cells mL1) was selected based on the G. mellonella lethality, as described in previous Chapter II.2. Next, 10 larvae were injected with 5 µL of a suspension of C. albicans at 7 x 107 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 were also performed to evaluate the effects of LNA-gapmers on C. albicans filamentation. For each condition, two larvae were recovered after predetermined times (24 h, 48 h and 72 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 PAS and HE [15,16]. 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).
Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation 72 f. Statistical analysis Data are expressed as the mean ± standard deviation of a least three independent experiments. Results were compared using two-way ANOVA and Tukey’s multiple comparisons tests. 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® (GraphPad Software, San Diego, CA, USA). II.3.3 Results a. Characterization of Anti-EFG1 LNA-gapmer ASOs Figure II.3.1 A shows the values of Tm measured for all LNA-gapmer ASOs with RNA complement. The LNA-gapmer1 presented a Tm of 70 °C and similarly, the LNA-gapmer2 (exclusion of one nt) presented a Tm of 71.6 °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 Tm (Figure II.3.1 A). Figure II.3.1 Characterization of AntiEFG1 LNA-gapmer ASOs. (A) Thermal denaturation temperatures (T values, °C) determined in medium salt buffer with RNA complement, and evaluation of superficial charge by zeta potential determination. (B) Evaluation of overall conformation of the secondary structure by CD spectral analysis of single-stranded (ssLNA) and double-stranded complexes (dsLNA:RNA).
Chapter II.3 Antisense locked nucleic acid gapmers to control Candida albicans filamentation 73 As expected, all LNA-gapmers showed a negative charge with the LNA-gapmer1 surface charge of approximately -30 mV (Figure II.3.1 A). All other LNA-gapmers showed a decrease on surface charge, with LNA-gapmer2 and LNA-gapmer3 presenting a little increase in the negative charge of the ASOs, with values of -31.9 mV and -35.1 mV, respectively. In contrast, the LNA-gapmer4 and LNA-gapmer5 showed a slight increase in ASO charge with values of -44 mV and -54 mV, respectively. Figure II.3.1 B represents the results of the secondary structure for single-stranded (ss) and double-stranded(ds)-LNA:RNA compounds for all LNA-gapmers. It is that for all duplexes a similar overall conformation is observed thus indicating no major alteration in secondary structure. For ssLNA, all LNAgapmers present bands as expected based on literature observations, [17,18] 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 [17,19,20], as it can be seen for dsLNA:RNA for all LNA-gapmers. The in vitro cytotoxicity was assessed on 3T3 cells and using different concentrations of LNAgapmer ASOs (10, 40 and 100 nM) (Figure II.3.2). These studies verify that all LNA-gapmers are noncytotoxic in concentrations up to 40 nM, as the relative 3T3 cells viability was higher than 70 % under these conditions [21]. Figure II.3.2 Cytotoxicity of AntiEFG1 LNA-gapmer ASOs . Relative cell viability (%) determined by the absorbance (Abs (490 nm) cm-2) of formazan product obtained from 3T3 cells, treated with different concentrations of LNA-gapmers (10, 40, 100 nM). The control is compared to cells without ASO treatment. b. AntiEFG1 LNA-gapmer ASOs in vitro Based on the cytotoxicity results, an ASO concentration of 40 nM was selected for the in vitro experiments. The effect of all LNA-gapmer ASOs on C. albicans filamentation (Figure AIII.1 A) and on the R e la tiv e C e ll V ia b ility (% ) L N A -g a p m e r 1 L N A -g a p m e r2 LN A -g a p m e r3 L N A -g a p m e r4 LN A -g a p m e r5 0 10 20 30 40 50 60 70 80 90 100 110 C o n tro l 1 0 n M 4 0 n M 1 0 0 n M
80 Chapter III Creation of strategies for C. albicans antisense oligonucleotides cargo and delivery Main goal To create carrier’s systems based on polymers and liposomes for the antiEFG1 2’- O MethylRNA ASO cargo and delivery.
81 Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’- O MethylRNA EFG1 ASO Main goal To develop anionic and cationic polyplexes microparticles based on poly(γ-butyrolactam) (PA4) or poly(εcaprolactam) (PA6) respectively, for antiEFG1 2’- O MethylRNA ASO cargo and delivery. Conclusions This study showed that PA4 and PA6 polyplexes microparticles are feasible carriers for antiEFG1 2’ O Me ASO either using the entrapped and immobilized strategies, since the ASO released maintained its activity against C. albicans cells. This chapter is based on the following article: Araújo D, Braz J, Dencheva N, Carvalho I, Henriques M, Denchev Z, Malfois M, Silva S. Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 antisense oligonucleotide. ACS Applied Bio Materials. doi.org/10.1021/acsabm.1c00334
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 82 III.1.1 Introduction As described on Chapter I, delivery of ASOs to their site of action remains a challenge, and it appears that redesigning or finding new delivery vehicles is generally more problematic since there is no optimal delivery strategy [1]. As a result, development of such vehicles is necessary so as to ensure that ASOs are effectively protected from the environmental body conditions and to deliver them to their site of action. Polyplexes represent polymer materials and are mostly based on positively charged (cationic) polymers since the ASOs are negatively charged. The polymers can exhibit different polymeric architectures, such as linear, branched, hyperbranched, star-shaped, or dendritic structures [1–4]. Very recently, polyamide porous microparticles (MPs) were developed by activated anionic ring opening polymerization (AAROP) of lactams [5,6] and proven useful for protein recognition [7] or enzyme carriers [8]. To the best of the knowledge, so far, ASOs have not been introduced into porous microsized polyamide polyplexes. In this context, taking in account the promising results of the efficacy of antiEFG1 2’- O MethylRNA ASO (Chapters II.1 and II.2), the main goal of this part of the work was to develop anionic and cationic polyplexes MPs based on poly(γ-butyrolactam) (PA4) or poly(ε-caprolactam) (PA6) respectively, for ASO’s cargo and delivery. III.1.2 Materials and Methods a. Materials The antiEFG1 2’OMe ASO with the sequence 5’ mG mG mC mA TACCGTTA mU mU mG mU 3’ (m2’- O Me), was designed based on the second generation of nucleic acid mimics and synthesized according to the user’s own specifications at EXIQON, as described in Chapter II.1. A stock of ASO at 4 µM was prepared in sterile ultrapure water and stored at -20 °C for later use. The γbutyrolactam (GBL) monomer used in the PA4-based polyplex preparation and all solvents in this work are of analytical grade supplied by Merck/Sigma Aldrich, Portugal. The ε-caprolactam, (ECL) (special grade for anionic polymerization) used in the PA6 polyplex preparation and the activator of AAROP (Brüggolen C20, containing 80 wt.% of aliphatic diisocyanate blocked in -caprolactam) are products of Brüggemann Chemical, Germany, which are used as received. The initiator of AAROP sodium dicaprolactamato-bis-(2-methoxyethoxo)-aluminate (dilactamate, DL) was a commercial product purchased from Katchem, Czech Republic, which is used as received.
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 83 b. Synthesis of PA4 and PA6 polyplex MPs Two types of polyplex supports were prepared in this study wherein the ASO payload was either entrapped in the polyamide MPs during the AAROP or immobilized upon them by adsorption on prefabricated MPs. The AAROP of the respective lactams to PA4 and PA6 MPs was described in detail previously [6–8]. In a typical AAROP of GBL to PA4 MPs, 0.2 mol of the monomer was stirred for a period of 6 h with 1.5 mol. % of C20 and 3.0 mol % DL in an inert atmosphere at 40 C, fixing the residual pressure to 50 mbar. The resulting solid reaction product was dispersed in acetone and filtered, followed by a two-fold wash with methanol. The fine white powder so produced was extracted with methanol in a Soxhlet for 4 h to get the neat PA4 MPs to produce the adsorption-immobilized ASO polyplex (PA4-ImmON). For the preparation of the polyplex with ASO entrapment (PA4-Ent-ON), the above AAROP was performed with 66 mmol GBL adding 4.0 µM (0.024 mg) of lyophilized ASO, using the same reaction conditions and concentrations of the activator/initiator complex as for the neat PA4 MPs. In a typical AAROP of ECL to PA6 MPs, 50 mmol monomer dissolved in 90 mL of toluene/xylene mixture (1:1 by volume) were stirred for a period of 2 h with 1.5 mol. % of C20 and 3.0 mol % at 130 °C under reflux [5]. Then, the reaction mixture was vacuum-filtered, and the resulting fine white powder was washed and extracted with methanol, as in the case of PA4 MPs. Then the neat PA6 MPs were used to prepare the PA6-Imm-ON sample by physical adsorption of the ASO payload. For the preparation of the polyplex with ASO entrapment (PA6-Ent-ON), the above AAROP was performed with 50 mmol ECL adding 4.0 µM (0.024 mg) of lyophilized ASO at 90 °C using the same concentrations of the activator/initiator complex. For the preparation of the PA6-Imm-ON and PA4-Imm-ON samples, 100 mg of each neat MP type was added to an Eppendorf tube containing 1 mL of a 4 M aqueous solution of the ASO and incubated at 37 °C for 24 h using a laboratory orbital shaker. After centrifugation, the aqueous supernatant was decanted. The resulting PA6 or PA4 MPs with adsorption-immobilized ASO were washed with double distilled water and stored at 5 °C. Their immobilization efficiency was 100 % determined by the UV-vis absorbance at 260 nm before and after the immobilization (Figure AIV.1). c. Structural and morphological characterization of the samples Fourier-transform infra-red spectroscopy with attenuated total reflection (FTIR-ATR) was applied using a Perkin-Elmer Spectrum 100 apparatus with a horizontal ATR attachment with the ZnSe crystal. The spectra were acquired between 4000 and 600 cm-1 accumulating up to 16 spectra with a resolution of 2 cm-1. The samples were studied in the form of fine powders.
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 84 The scanning electron microscopy (SEM) studies were performed on a NanoSEM-200 apparatus of FEI Nova (USA) using mixed secondary electron/black scattered electron in-lens detection. The pulverulent samples were observed after sputter-coating with the Au/Pd alloy in the 208 HR equipment of Cressington Scientific Instruments (UK) with high-resolution thickness control. All the samples of this study were subjected to thermogravimetric analysis (TGA) in a Q500 gravimetric balance by TA Instruments, by heating the samples in the 40-600 °C range at a rate of 20 C min-1 in a nitrogen atmosphere. The differential scanning calorimetry was carried out in the 200 F3 equipment of Netzsch at a heating/cooling rate of 10 °C min-1 under nitrogen purge. The samples were heated to 290 °C, cooled down to 0 °C, and then heated back to 290 °C. The typical sample weights were in the 10-15 mg range. Synchrotron wide- (WAXS) and small-angle X-ray scattering (SAXS) measurements were performed in the NCD-SWEET beamline of the ALBA Synchrotron facility in Barcelona, Spain.[9] Twodimensional detectors were used, namely LH255-HS (Rayonix, USA) and Pilatus 1 M (Dectris, Switzerland) for registering the WAXS and SAXS patterns, respectively. The sample-to-detector distance was set to 131 mm for WAXS and 2690 mm for SAXS measurements, the λ of the incident beam being 0.1 nm and the beam size being 0.35 × 0.38 mm (h × v). The 2D data were reduced to 1D data using pyFAI software.[10] For processing of the WAXS and SAXS patterns the commercial packages Peakfit 4.12 by SeaSolve Software were implemented. d. Polyplexes MPs cytotoxicity assays The cytotoxicity side effects of the different polyplexes MPs were determined with MTS solution (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega) and 1 % of DMEM without phenol assays. For that, the 3T3 cell line (fibroblast cells, Embryonic tissues, Mouse from CCL3, American Type Culture Collection) was used and grown in DMEM (Biochrom, Germain) supplied with 10% FBS (Sigma Aldrich) and 1 % antibioticcontaining P/S (Biochrom, Germain). After cells detachment, 1 x 105 cells mL1 of cells suspension was added to a 96-well plate, and cells grew until achieving 80 % of confluence. Different concentrations of neat polymers and polyplexes of PA4 and PA6 (1 and 5 mg mL-1) were prepared in DMEM and 50 µL of each concentration was added each well. Negative control was prepared by adding 50 µL of DMSO to the cells and positive control by adding 50 µL of DMEM. The plates were incubated for 24 h at 37 °C and 5 % CO2. The MTS procedure was carried out as described in the previous Chapter II.1.
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 85 e. Controlled release of antiEFG1 2’OMe ASO from polyplexes MPs The controlled release of antiEFG1 2’OMe ASO from PA4 and PA6 polyplexes MPs was studied over 48 h. For that, approximately, 5-6 mg of each polyplex MP formulation [PA4 (PA4-Imm-ON and PA4Ent-ON) and PA6 (PA6-Imm-ON and PA6-Ent-ON)] was added to 1 mL of phosphate-buffered saline (PBS; pH 7, 0.1 M) and incubated in 24-well plates at 37 °C and 120 rpm. At each specific time point (2, 4, 6, 8, 24, 26, 28, 30 and 48 h), an aliquot of 50 µL was recovered, and the polymer was obtained by centrifugation at 1000 g during 10 s. The related supernatants were collected, and the amount of ASO released from polyplexes MPs was determined by measuring the values of UV absorption at 260 nm (Figure AIV.2). The results are presented as the cumulative release of ASO quantity over time (µM h-1). All experiments were performed in triplicate and in a minimum of three independent assays. f. Microorganism and growth conditions Candida used in this study was the reference strain C. albicans SC5314 belonging to Candida collection of the Biofilm group of the Centre of Biological Engineering and its identity was confirmed by PCR-based sequencing with specific primers (ITS1 and ITS4) [11]. The Candida cells were subcultured on Sabouraud dextrose agar (SDA; Merck, Germany) and incubated for 24 h at 37 ᵒC. An inoculum was prepared in Sabouraud dextrose broth (SDB; Merck, Germany) and incubated overnight at 37 °C, 120 rpm. After incubation, the cell suspensions were centrifuged for 10 min at 3000 g at 4 °C and washed twice with PBS (pH 7, 0.1 M). Pellets were resuspended in 5 mL of Roswell Park Memorial Institute cell culture medium (RPMI, pH 7; Sigma, St Louis, USA), and the cellular density was adjusted for each experiment using a Neubauer chamber (Paul Marienfild, Lauda-Königshofen, Germany) to 1 x 106 cells mL-1. All experiments were performed in triplicate and in a minimum of three independent assays. g. Effect on C. albicans filamentation The effect of antiEFG1 2’OMe ASO released from PA4 and PA6 polyplexes MPs was evaluated in terms of its ability to reduce C. albicans filamentation. For that, on 24-well polystyrene microtiter plates (Orange Scientific, Braine-l’Alleud, Belgium), approximately 5-6 mg mL-1 of each MP with ASO was resuspended in RPMI together with a suspension of C. albicans SC5314 at 1 x 106 cells mL-1. The suspensions were incubated at 37 °C at 120 rpm during 48 h. The positive control was prepared with 1 mL of C. albicans cells and the negative controls with each polyplex MPs without ASO together with C. albicans cells in RPMI. After 24 h and 48 h, the aliquots were recovered by centrifugation for 5 min at
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 86 6000 g and 4 °C, and in a total of 100 C. albicans cells, the number of cells as filaments was enumerated using a Neubauer chamber. The number of non-filamentous cells was normalized by the quantity of ASO released from each polyplex MPs at 24 h and 48 h. The results are presented as the number of nonfilamentous cells per µM of ASO. In parallel, epifluorescence microscopy images were obtained to confirm the levels of filamentation and to determine the filament length, as previously described in Chapter II.1. h. Statistical analysis Data are expressed as the mean ± standard deviation of a least three independent experiments. Results were compared using two-way ANOVA and Tukey’s multiple comparisons tests using GraphPad Prism® (GraphPad Software, San Diego, CA, USA). All tests were performed with a confidence level of 95 %. III.1.3 Results and Discussion Generally, ASOs represent short chains of certain nucleic acids that can bind to the target RNA by means of standard Watson-Crick base pairing through hydrogen bonds [12]. This capacity for H-bond formation can be used to attach the ASO to a suitable polymeric carrier. The polyplex so-formed will be expected to display better in vivo stability to the bioactive entities in the body fluids, improving the cellular uptake and the protection against the serum nucleases and other enzymes [4,13,14]. The purpose of this work was to use as polymeric carriers, the microsized porous PA4 or PA6 MPs, for ASO cargo and future delivery. Two ways to obtain the respective polyplexes were studied: by physical adsorption of ASO upon prefabricated polyamide microcapsules or by their entrapment into polyamide MPs in-situ forming via AAROP. Thus, AAROP of GBL or ECL was performed without or with ASO inclusion in the reaction mixture, respectively. A simplified scheme of AAROP of ECL is presented in Figure AIV.3. It is expected that the presence of secondary amide groups in both PA4 and PA6 carriers would enable intensive Hbond formation with the nitrogen nucleobases in ASO leading to immobilization of the oligonucleotides in the polyamide particles. In the case of physical adsorption of ASO, one may expect only H-bond formation. In the case of in-situ entrapment during AAROP that occurs at 40 °C (PA4 carrier) or 90 °C (PA6 carrier), some chemical reactions between ASOs and the forming polyamide MP are theoretically possible. This is possible since in each step of polyamide chain growth or in the termination of AAROP, the necessary extraction of proton from primary or secondary amine or amide groups can involve some of the ASO
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 87 pyrimidine or purine bases causing incorporation of the oligonucleotide into the polyamide chain. Hence, this work will present a comparative discussion on the morphology, structure, and biological activity of polyplexes obtained by adsorption or entrapment on PA4 or PA6 MP carriers against C. albicans cells. As seen from Table AIV.1, both types of empty MP carriers are negatively charged, the values varying between -23 (PA4 MP) and -12 eV (PA6 MP). At a pH slightly below the neutral and similar to the one at which the adsorption-immobilization process is carried out, the value of the negative charge will depend on the amount of terminal carboxyl groups in the PA6 or PA4 macromolecules, most probably being larger in the latter case. The second factor will be the protonation of the CONH group of the polyamide carrier that will depend on its crystalline structure. Having in mind that the ASO molecules are always negatively charged, it should be therefore concluded that the interaction between the ASO and the polyamide carrier in the adsorption immobilization is not based on electrostatic forces but on the formation of multiple hydrogen bonds that overcome the repulsion between the equally charged ASO and the polyamide carrier. a. Characterization of PA4 and PA6 MPs and polyplexes on their basis Microscopy studies As explained in the experimental part, for the adsorption-immobilization approach the purified PA4 and PA6 MPs were incubated in aqueous solutions of ASO. Then, PA4-Imm-ON and PA6-Imm-ON were obtained after decanting the water and drying. PA4-Ent-ON and PA6-Ent-ON were synthesized by adding ASO molecules to the reaction mixture of AAROP of ECL or GBL, respectively. All polyplex samples represent are fine powders. Some of their properties are presented in Table III.1.1 in comparison to neat PA4 and PA6 MPs. Table III.1.1 Designation and some characteristics of PA6 and PA4 MP and polyplexes Sample PA yield, %a) η, dL.g-1 dmax,b) µm dmax/dmin b) PA4 50.4 0.926 5-10 1.1-1.3 PA4-Imm-ON - - 5-15 1.1-1.6 PA4-Ent-ON 46.6 0.833 10-25 1.1-1.6 PA6 52.0 0.983 20-30 1.2-1.4 PA6-Imm-ON - - 15-35 1.1-1.5 PA6-Ent-ON 39.8 0.902 20-60 1.1-1.5
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 88 (a) relation to the lactam monomer; (b) Interval of values including >80 % of the particles number. The intrinsic viscosity [η] of 0.983 dL g-1 of the neat PA6 (Table III.1.1, Annex IV) corresponds to circa (ca.) 27.500 g/mol. The same viscosity average molecular weight Mv is ascribed to the PA6-ImmON sample. For the PA4 and PA4-Imm-ON samples Mv cannot be calculated since the Mark-Houwink parameters K and α are unavailable. However, the [η] value of 0.926 dL g-1 for PA4 and PA4-Imm-ON reached in this study is similar to that of PA4 microspheres obtained by a different method involving AAROP of GBL [15]. The polymerization yields of the neat polyamides are 50-52 %, close to those found in previous studies. The AAROP during the entrapment of ASO in PA4 and PA6 occurred normally with similar yields of PA4-Ent-ON and PA6-Ent-ON. Judging from the lower [η] values of the entrapped samples, they are of lower molecular weights compared to the neat PA6 and PA4 MPs. The average maximum size of the particles dmax in all neat polyamides and polyplex samples based on optical microscopy with imageprocessing was found to be between 5 and 60 μm (Table III.1.1, Figure AIV.4). The dmax , of neat PA4 MP is the smallest, ranging between 5 and 10 µm. Similar values of 5-15 µm are registered for the PA4-ImmON samples, while in the PA4-Ent-ON sample the upper limit of dmax grows above 25 µm. The neat PA6 produces MPs with dmax in the 20-30 µm range. The respective PA6-Imm-ON and PA6-Ent-ON polyplexes are characterized with very broad size distributions with upper limits in the latter case of 60 µm. As seen from Table III.1.1, the presence of ASO in both adsorption-immobilized or entrapped form leads to an increase of the roundness parameter dmax/dmin meaning less spherical MPs as compared to the neat PA4 and PA6 MPs. More details on the morphology of the empty supports and ASO-carrying particles can be obtained by SEM. Figure III.1.1 shows the micrographs of the neat PA6 (Figure III.1.1 A(a-c)) displaying spheroidal particles with sizes of the individual entity of 20-30 µm that can form also aggregates with average sizes close to 50 µm. At larger magnification, the PA6 particles display a highly porous, scaffoldlike topology with average visible pore diameters larger than of 500 nm. The pore sizes do not seem to change significantly in the PA6-Ent-ON (Figure III.1.1 A(d-f)) and PA6-Imm-ON samples (Figure III.1.1 A(gi)); however, both entrapment and adsorption of ASO results in more expressed agglomeration of MPs to aggregates with dmax of 60-100 µm, better expressed in the adsorption-immobilized polyplex.
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 89 Figure III.1.1 Scanning electron microscopy of neat carriers and polyplex samples based on (A) PA6 microparticle and (B) PA4 microparticle: (a-c) neat microparticle; (d-f) Entrapped sample; (g-i) Immobilized sample. For sample designation see Table III.1.1. As seen from Figure III.1.1 B, the average size of the ovoid PA4 neat MPs (Figure III.1.1 B(a-c)) is much smaller than in the PA6 neat carrier, being in the range of 6-10 µm, displaying pore sizes with visible diameters of 150-200 nm. In the ASO entrapped samples (Figure III.1.1 B(d-f)), the size of the PA4-Ent-ON sample is maintained around 10 µm; however, the shape of the particle’s changes from ovoid to platelets with sharper edges, mostly due to different nucleation mechanisms during MPs crystallization. The shape and size of the PA4-Imm-ON sample (Figure III.1.1 B(g-i)) is remarkably similar to the neat PA4 used for its preparation, with analogous surface topography and average diameters of the pores on the surface ca. 200 nm. The variations in the shape, size, and surface topography of the PA6and PA4-based samples can be explained with the different polymerization conditions of the polyamide carriers required by the different monomer activity and crystallization behavior during the polymerization. FTIR Spectroscopy An FT-IR spectral comparison between the PA6-based and PA4-based samples of this study is presented in Figure III.1.2a,b, respectively. In all samples, the bands at 3300 cm-1 were assigned to the valence stretching vibrations of hydrogen atoms in secondary NH groups of the polyamide carriers. Also,
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 96 Figure III.1.6 Relative cell viability of (A) PA4-based polyplexes and (B) PA6-based polyplexes microparticles determined by the absorbance values (Abs (490 nm) cm-2)) of the formazan product obtained from 3T3 cells in contact with 6 and 10 mg mL-1 microparticles. The control refers to cells without any treatment. Error bars represent standard deviation. c. Controlled release of antiEFG1 2’OMe ASO from polyplexes MPs Figure III.1.7 presents the cumulative release concentration of antiEFG1 2’OMe ASO from PA4 and PA6 MPs over 48 h. For all polyplexes MPs, the release of antiEFG1 2’OMe ASO increases over the time; however, the release rate was polyplex type dependent. Figure III.1.7 Cumulative controlled release profiles of antiEFG1 2’OMe ASO immobilized (Imm) and entrapped (Ent) into (A) PA4-based polyplexes and (B) PA6-based polyplexes over time. In the case of PA4-Imm-ON, the release rate was similar to PA4-Ent-ON until the first 24 h (Figure III.1.7 A). However, after 24 h, it was observed a significant increase on antiEFG1 2’OMe release from PA4-Imm-ON. These results seem to be consistent with the ASO molecule localization on the surface of MP after the immobilization process, suggesting that they can be more easily freed. The profile of antiEFG1 2’OMe ASO released from PA6 MPs was significantly different from that observed from PA4 MPs.
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 97 It was observed that antiEFG1 2’OMe ASO is more easily released from PA6-Ent-ON than from PA6-ImmON over the time, despite reaching similar values after 48 h of incubation (Figure III.1.7 B). In other instance, the release rate of PA6-Ent-ON (Figure III.1.7 B) was higher than that observed in the case of PA4-Ent-ON (Figure III.1.7 A). This is an expectable result, once as SEM images revealed that the size pore of PA6 is higher than that observed on PA4 (Figure III.1.1). The release curves of PA4Imm-ON (Figure III.1.7 A) and PA6-Imm-ON (Figure III.1.7 B) were remarkably similar. d. Effect of antiEFG1 2’OMe ASO released from polyplexes MPs on C. albicans The antiEFG1 2’OMe ASO was designed to bind to the EFG1 mRNA in order to degrade it, blocking its translation into proteins and, consequently, reducing the transition of C. albicans from yeast to filamentous forms. As previously shown, the antiEFG1 2’OMe ASO significantly reduced EFG1 gene expression and Efg1p translation and consequently reduced C. albicans cell filamentation. In the present work, was evaluated the possibility to use PA4 and PA6 carriers for antiEFG1 2’OMe ASO cargo and delivery. For that, the efficacy of antiEFG1 2’OMe released from polyplexes MPs (PA4-Imm-ON, PA4-EntON, PA6-Imm-ON, PA6-Ent-ON) on C. albicans cells filamentation was determined, as well as their effect on C. albicans filament length (Figure III.1.8). Figure III.1.8 A presents the results as the number of nonfilamentous cells per µM of ASO released. Importantly, it was observed an effect on C. albicans cells filamentation for all polyplexes MPs, however dependent on the polyplex type and time. In accordance with Figure III.1.7, all polyplexes MPs presenting different release rates and consequently ASOs molecules could not be immediately available to interact with C. albicans cells. To note that at 24 h, the highest performance was observed in the case of PA6-Imm-ON and the lower in the case of PA6-Ent-ON (Pvalue<0.05). The lower performance of PA6-Ent-ON in terms of C. albicans filamentation reduction could be related with the temperature (80-85 °C) used to polymerize the polymer. Concerning, the number of non-filamentous cells counting after incubation with PA4-Imm-ON and PA4-Ent-ON, it was observed a similar pattern (P-value>0.05) (Figure III.1.8A). After 48 h of incubation, it was observed in general a decrease on the effect of the ASO release from all polyplexes MPs, with exception of PA4-Ent-ON and PA6-Ent-ON, which maintains its low performance (P-value>0.05). The loss of effect observed over the time may reflect excess ASOs molecules free that are competed to cross the C. albicans cells wall. The highest effect at 48 h was observed in the case PA4-Ent-ON (Figure III.1.8 A) which is the polyplex MPs with lower values of ASO released (Figure III.1.7 A).
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 98 In Figure III.1.8 B, is presented the epifluorescence images for all MPs after 24 h and 48 h. The examination of epifluorescence microscopy images confirms the results of reduction on filamentation obtained and it is also possible to observe a consistent relevant decrease in terms of the filaments’ lengths. As can be seen, after 24 h, PA6-Ent-ON presented the major reduction of the filament length of around 60 % (135 to 57 µm) and subsequently the highest release rate of ASO (Figure III.1.7 B). In contrast, the more pronounced reduction of hyphae length after 48 h was in PA4-Imm-ON, with around 74 % (270 to 69 µm), presenting the highest release rate of ASO (Figure III.1.7 A). Figure III.1.8 Effect of antiEFG1 2’OMe ASO released from immobilized (Imm) and entrapped (Ent) polyplexes microparticles on C. albicans cells filamentation. (A) Number of non-filamentous C. albicans cells normalized by the quantity of ASO released at each time; (B) Epifluorescence microscopy images of C. albicans stained with Calcofluor after 24 h and 48 h in contact with the polyplex microparticles and the average of the hyphae size for each condition. The assays were performed for C. albicans SC5314. Error bars represent standard deviation. *Significant differences between samples of the same polymer at each time (P-value<0.05). +Significant differences between both times analysed for the samples of the same polyplex microparticle (Pvalue<0.05). Arrows highlight yeast cells.
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 99 AntiEFG1 2’OMe is an ASO able to recognize and block the EFG1 gene and to control C. albicans filamentation. The delivery of these molecules remains a challenge and there is no optimal delivery strategy. This data confirms that PA4 and PA6 polyplexes MPs are feasible carriers for antiEFG1 2’OMe ASO molecules either using the entrapped and immobilized strategies, once all the ASO released maintains its activity against C. albicans cells. References 1 Juliano R L. (2016) The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 44, 6518–6548. 2 Yin H, Kanasty R L, Eltoukhy A A et al. (2014) Non-viral vectors for gene-based therapy. Nat Rev Genet. 15(8), 541–555. 3 Crespo-Barreda A, Encabo-Berzosa M M and González-Pastor R. (2016) Chapter 11 - Viral and nonviral vectors for in vivo and ex vivo gene therapies. In Translating Regenerative Medicine to the Clinic ; Laurence, J., Ed.; Academic Press: Boston, MA, USA, pp. 155–177 4 Hattori Y. (2017) Progress in the development of lipoplex and polyplex modified with anionic polymer for efficient gene delivery. J. Genet. Med. Gene Ther. 1, 003–018. 5 Dencheva N, Denchev Z, Lanceros-Méndez S and Sanz T E. (2015) One-Step in situ synthesis of polyamide microcapsules with inorganic payload and their transformation into responsive thermoplastic composite materials. Macromol. Mater. Eng. 301, 119–124 6 Dencheva N, Braz J, Nunes T G, et al. (2018) One-pot low temperature synthesis and characterization of hybrid poly(2-pyrrolidone) microparticles suitable for protein immobilization. Polymer 145, 402–415 7 Dencheva N, Oliveira F D, Braz J F and Denchev Z Z. (2020) Bovine serum albumin-imprinted magnetic poly(2-pyrrolidone) microparticles for protein recognition. Eur. Polym. J. 122, 109375 8 Dencheva N, Braz J and Scheibel D. (2020) Polymer-assisted biocatalysis: polyamide 4 microparticles as promising carriers of enzymatic function. Catalysts 10, 767 9 González J B, González N, Colldelram C, et al. (2015) NCD-SWEET beamline upgrade. In: Proc. 10th Mech. Eng. Des. Synchrotron Radiat. Equip. Instrum. pp. 374–376. 10 Ashiotis G, Deschildre A, Nawaz Z et al. (2015) The fast azimuthal integration Python library: pyFAI. J. Appl. Crystallogr ., 48: 510–519. 11 Williams D W, Wilson M J, Lewis M A and Potts A J. (1995) Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA. J. Chin. Microbiol. 33, 2476-2479. 12 DeVos S L and Miller T M. (2013) Antisense oligonucleotides: treating neurodegeneration at the level of RNA. Neurotherapeutics 10(3), 486-97. 13 Wang H, Jiang Y, Peng H et al. (2015) Recent progress in microRNA delivery for cancer therapy by nonviral synthetic vectors. Advanced Drug Delivery Reviews 81, 142–160. 14 Roberts T C, Ezzat K, Andaloussi S E, Weinberg M S. (2016) Synthetic SiRNA delivery: Progress and prospects. Methods in Molecular Biology 1364, 291–310 15 Kim N, Kim J H, Nam S W, et al. (2015) Preparation of nylon 4 microspheres via heterogeneous polymerization of 2-pyrrolidone in a paraffin oil continuous phase. J. Ind. Eng. Chem. 28: 236-240. 16 Tachibana K, Hashimoto K, Tansho N and Okawa H. (2011) Chemical modification of chain end in nylon 4 and improvement of its thermal stability. J. Polym. Sci. Part A: Polym. Chem . 49, 2495-2503. 17 Dencheva N, Nunes T, Oliveira M J and Denchev Z. (2005) Microfibrillar composites based on polyamide/polyethylene blends. 1. Structure investigations in oriented and isotropic PA6. Polymer 46, 887901. 18 Fredericks J, Doyne T H and Spague R S. (1966) Crystallographic studies of nylon 4. II. On the β and δ polymorphs of Nylon 4. J. Polym. Sci. Polym. Phys. 4, 913-922. 19 Bellinger M A, Waddon A J, Atkins E D T and MacKnight W J. (1994) Structure and morphology of nylon 4
Chapter III.1 Polyamide microsized particulate polyplex carriers for 2’-OMethylRNA EFG1 ASO 100 chain-folded lamellar crystals. Macromolecules 27(8), 2130-2135. 20 International Organization for Standardization ISO 10993-5:2009. (2009) Biological evaluation of medical devices. Part 5: tests for in vitro cytotoxicity, third edition, (2009-06-01).
101 Chapter III.2 Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’- O MethylRNA antisense oligonucleotide Main goal To develop lipid-based formulations for antiEFG1 2’- O MethylRNA antisense oligonucleotide cargo and delivery, prepared with cationic and neutral lipids, and to evaluate its efficacy to control Candida albicans filamentation in vitro and in vivo . Conclusions The results obtained showed that all lipid-based formulations are feasible nanocarriers for antiEFG1 2’OMe ASO cargo, specially the DOTAP/DOPC 80/20 ρ=3 formulation that considerable contributed to the increase of the G. mellonella survival infected with C. albicans .
Chapter III.2 Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’-OMethylRNA antisense oligonucleotide 102 III.2.1 Introduction As mentioned in Chapter III.1, the cargo and delivery of ASOs to its site of action remains a key challenge. For the last years, various non-viral vectors have been engineered for improved gene and drug delivery strategies [1–3]. Some of the most promising strategies for delivery of nucleic acids encompass complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge [4– 8]. Due to the attractive interactions between cationic liposomes and nucleic acids, new nanostructured complexes tend to be formed, and are referred to as lipoplexes [9–12]. Besides facilitating the formation of nanostructured particles with high encapsulation efficiency, the cationic charge of liposomes also favour attractive interactions with cell and endosomal membranes, improving cellular uptake and endosomal release [13–15]. Typically, cationic liposomes used in nucleic acid delivery are composed by at least two lipid components: a cationic lipid (e.g. 1,2-dioleoyl-3-trimethylammoniumpropane - DOTAP) and a neutral or zwitterionic lipid (e.g. dioleoylphosphocholine - DOPC). The role of the neutral lipid is to help in the adjustment of the amount of positive charge per liposome area (i.e. membrane charge density - σM), and can also have fusogenic properties that help the lipid-nucleic acid complex to fuse with endosomal membranes, as in the case of dioleoylphosphatidylethanolamine (DOPE) and monoolein (MONO) [2,16,17]. Both the ability to modulate membrane charge density (σM) and fusogenicity properties are known to influence the transfection efficiency [13,14]. So, the main purpose of this section of the work was to develop lipid-based formulations for antiEFG1 2’- O Methyl ASO cargo and delivery, using the DOTAP as cationic lipid and the DOPC, DOPE or MONO as neutral lipids. The antiEFG1 2’OMe ASO lipid-based formulations efficacy was evaluate in terms of its capability for controlling C. albicans filamentation in vitro and in vivo (using the Galleria mellonella model). III.2.2 Materials and Methods a. Materials The antiEFG1 2’OMe ASO with the sequence 5’-mG mG mC mA TACCGTTA mU mU mG mU-3’ (m2’OMe), was designed and synthesized as described in the previous Chapter II.1. For that, a stock of ASO at 4 µM was prepared in sterile ultrapure water and stored at -20 °C for later use. The lipids 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dioleoylphosphocholine (DOPC), and dioleoylphosphatidylethanolamine, (DOPE), in chloroform, were purchased from the Avanti Polar
Chapter III.2 Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’-OMethylRNA antisense oligonucleotide 103 Lipids (USA). Monoolein (MONO) was purchased from Nu-Chek Prep (Elysian, MN, USA). All lipids were used as received. b. Liposomes and lipoplexes preparation Liposomes were prepared according to the thin film hydration method followed by sonication. For this end, DOTAP (cationic lipid) and the helper lipids DOPC, DOPE and MONO were mixed in chloroform to the different molar ratios as described in Table III.2.1. The resulting mixture was dried using a constant nitrogen gas stream, and then placed in vacuum overnight. The lipid film was resuspended in ultrapure nuclease free Milli-Q water to a total lipid concentration of 2 mM. The suspensions were vortexed and sonicated using a tip sonicator for 1 min, with 10 % amplitude and 50 % duty cycle using a Branson Digital Sonifier 250 Model. Table III.2.1 Different liposomes prepared and its molar ratios, cationic-to-anionic charge ratio (ρchg) and type of lipoplexes structure Cationic Lipid Helper Lipid Cationic/helper lipid molar % Typical lipoplex structure DOTAP DOPC 80/20 Lamellar [18] 30/70 Lamellar [18] DOPE 80/20 Lamellar [19] 30/70 Inverted hexagonal phase [19] MONO 80/20 Lamellar [20] 30/70 Inverted hexagonal phase [20] For lipoplex formation, equal volumes of liposomes and ASO solutions previously diluted to the right concentrations were mixed for a final antiEFG1 2’OMe ASO concentration of 40 nM. The resulting mixture was promptly vortexed for 30 sec and left at least 30 min under stirring conditions. The formed complexes were stored at 4 °C. Lipoplexes were prepared with a cationic-to-anionic charge ratio (ρchg) of 3 and 10. The ρchg is calculated as the total number of positive charges (from the number of DOTAP molecules) divided by the total number of negative charges (from the number and valence of ASO molecules).
Chapter III.2 Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’-OMethylRNA antisense oligonucleotide 104 c. In vitro effect of the lipoplexes against C. albicans cells Microorganisms and growth conditions The Candida strain used in this study was C. albicans SC5314, which is a reference strain from Candida collection of the Biofilm group of the Centre of Biological Engineering. The identification of all strains was confirmed using a chromogenic medium, specifically CHROMagarTM Candida , through the distinction of colonies’ colours and by PCR-based sequencing with specific primers (ITS1 and ITS4) [21]. For all experiments, the yeast strain was subcultured on sabouraud dextrose agar (SDA; Merck, Germany) and incubated for 24 h at 37 ᵒC. Cells were then inoculated in sabouraud dextrose broth (SDB; Merck, Germany) and incubated overnight at 37 °C and 120 rpm. After incubation, cells’ suspensions were centrifuged for 10 min at 3000 g and 4 ᵒC, and washed twice with phosphate-buffered saline (PBS; pH 7, 0.1 M). Pellets were suspended in 5 mL of Roswell Park Memorial Institute (RPMI; Sigma, St Louis, USA), and the cellular density was adjusted for each experiment using a Neubauer chamber (Paul Marienfild, Lauda-Königshofen, Germany) to 1x106 cells mL-1. Effect on C. albicans filamentation The lipoplexes performance was evaluated in terms of the ability of the antiEFG1 2’OMe ASO released from each formulation to control C. albicans filamentation. For that, on 24-well polystyrene microtiter plates (Orange Scientific, Brainel’Alleud, Belgium) 500 µL of each lipoplex was added to 500 µL of a suspension of C. albicans at 1x106 cells mL-1. The solutions were incubated at 37 °C and 120 rpm during 72 h. A control was prepared with C. albicans cells treated with 40 nM of ASO-free. Moreover, the positive control was prepared only with C. albicans cells and the negative controls with the empty lipoplexes together with C. albicans cells in RPMI. The results were presented as percentage (%) of filamentation reduction, as previously described in Chapter II.1. In addition to the filamentation inhibition studies, the length of the filaments was quantified through fluorescence microscopy analysis as previously described in Chapter II.1.
Chapter III.2 Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’-OMethylRNA antisense oligonucleotide 105 d. In vivo effect of the lipoplexes using the G. mellonella model Galleria mellonella larvae Galleria mellonella larvae were reared on a pollen grain and bee wax diet at 25 °C in the darkness and used in a final stage with a weight of approximately 250 mg. The larvae were injected into hemolymph via the hindmost left proleg, previously sanitized with 70 %(v/v) ethanol, using a micro syringe adapted in a micrometer to control the volume of injection [22,23]. Toxicity evaluation The lipoplexes for in vivo assays were selected based on the in vitro results. Namely, the DOTAP/DOPC 80/20 ρ=10; DOTAP/DOPE 80/20 ρ=10; DOTAP/MONO 80/20 ρ=10 for presenting a better performance and the DOTAP/DOPC 80/20 ρ=3 for presenting the worst performance in vitro . To test in vivo the lipoplexes toxicity, 10 G. mellonella larvae were injected with 5 µL of each formulation. As control a set of larvae were injected with the same volume of free-ASO and only with PBS. Larvae were placed in petri dishes and stored in the dark at 37 °C. Larvae survival was recorded over 72 h and survival curves were constructed. Galleria mellonella survival To study the in vivo effect of lipoplexes, G. mellonella larvae were previously infected with a lethal dose of C. albicans cells (7x107 cells mL-1 in PBS) and randomly allocated to different experimental groups (a set of 10 larvae). A set of larvae was treated with a single-dose of lipoplex (0 h post infection) and another with a double dose of each lipoplex (0 h and 12 h post infections). As control larvae were treated with free ASO or only with PBS. Larvae were placed in petri dishes and stored in the dark at 37 °C over 3 days, and consequently, the survival curves were constructed. Caterpillars were considered dead when they displayed no movement in response to touch [23]. e. Statistical analysis Data are expressed as the mean ± standard deviation of at least three independent experiments. Results were compared using two-way ANOVA and Tukey’s multiple comparisons tests. 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® (GraphPad Software, San Diego, CA, USA).
Chapter III.2 Cationic lipid-based formulations for encapsulation and delivery of antiEFG1 2’-OMethylRNA antisense oligonucleotide 112 the single-dose studies). To note, the double-dose administration of DOTAP/DOPC 80/20 ρ=3 enhances the G. mellonella survival on around 1.5 times more comparing to the single-dose administration, increasing the larvae survival into 40 % after 48 h and into 25 % after 72 h of treatment (P-value<0.005) (Figure III.2.5). It is important to point out that the double-dose administration also potentiates the effect of ASO-free on around 1 time more (18 % after 24 h and 13 % after 72 h of infection (P-value<0.05)). Figure III.2.5 Double-dose antiEFG1 2’OMe ASO lipid-based formulations effect on Galleria mellonella survival infected with Candida albicans. Survival curves of infected larvae treated with a doubledose (0 h and 12 h post infection) of DOTAP/DOPC 80/20 ρ=3 lipoplex. As control larvae infected were injected only with antiEFG1 2’OMe ASO-free and only with PBS. Effectively, the survival rate of G. mellonella when treated with the antiEFG1 2’OMe ASO lipidbased formulations was significantly higher than when treated with ASO-free. This evidence confirms the importance of the encapsulation process for protecting ASOs against the degradation by serum proteases and nucleases. Among non-viral vectors, cationic liposomes have been more commonly used [1,2] and this data confirms also its viability to antiEFG1 2’OMe ASO cargo and delivery into C. albicans cells. In accordance with the in vitro results, seems to be irrelevant the proportions between cationic and neutral lipids, and between cationic charges of the liposomes and ASOs negative charges on the performance of antiEFG1 2’OMe ASO lipid-based formulations. However, by considering the in vivo results, the excess of cationic charges as is the case of DOTAP/DOPC 80/20 ρ=10 seems to have a negative impact with a lower rate of larvae survival. This study demonstrated the successful delivery of antiEFG1 2’OMe ASO encapsulated in lipidbased formulations, providing valuable information for further assays. The DOTAP/DOPC 80/20 ρ=3 formulation has the potential to improve nanodrug administration for C. albicans species.
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115 Chapter IV General conclusions and work perspectives
Chapter IV General conclusions and work perspectives 116 IV.1 General conclusions The incidence of Candida infections has increased remarkably in the last years, being attributed to the rise of the elderly population, to the number of immunocompromised patients, and to the widespread use of indwelling medical devices. Candida albicans remains as the most prevalent of all Candida species and its pathogenicity is promoted by several virulence factors, being the ability to switch from yeast to filamentous forms one of the most alarming. This phenomenon is regulated by a complex regulatory network of genes and EFG1 is one of the most important virulence determinants. It is well known that the high levels of morbidity and mortality related to C. albicans is mainly due to the rise in antifungal resistance and the limited number of efficient antifungal drugs. In this sense, it is urgent to develop new strategies to prevent and control C. albicans infections. The key hypothesis that supports this research is: “If a particular gene, as is the case of EFG1 gene, is known as a determinant of C. albicans filamentation, it could be the target by antisense oligonucleotides, which will bind to the respective mRNA causing its inactivation and translation into protein and thus it will be possible to control C. albicans virulence”. Antisense therapy (AST) is being applied in a large number of human genetic diseases, however poorly explored in case of microbial infections, particularly candidiasis. Therefore, the main goal of this research was to promote the development of novel therapeutic approach based on AST to track C. albicans filamentation employing the established and the emerging generations of ASOs. To reach on such aim, two complementary objectives were addressed: I) Exploitation and application of ASOs to control the EFG1 gene and consequently C. albicans filamentation; II) Creation of strategies for C. albicans ASOs cargo and delivery. The unmodified ASOs have a limited clinical use since they are rapidly degraded by intracellular endonucleases and exonucleases, being destroyed before binding their respective target. To overcome these issues, ASOs have been chemically modified and up to now there are three different generations of chemical modifications. The first aim of this researcher was achieved through the development of ASOs based on the second and the third generation of ASOs targeting the EFG1 gene (Chapter II). Regarding the second generation of ASOs, it was projected the antiEFG1 2’- O MethylRNA ASO (Chapter II.1), applying the 2’- O Methyl chemical modification. This modification is a sugar modification that is described as improving the nuclease resistance and increasing the hybridization affinity for target mRNA. The results revealed that the antiEFG1 2’OMe ASO is able to significantly reduce the levels of EFG1 gene expression and of Efg1p protein translation (both approximately 60 %), and effectively prevent filamentation of C.
Chapter IV General conclusions and work perspectives 117 albicans cells (by 80%). Moreover, it was also verified that antiEFG1 2’OMe ASO keeps the efficacy in different simulated human body fluids. To test whether antiEFG1 2’OMe ASO could have a therapeutic potential in vivo , its efficacy was assessed using a G. mellonella caterpillar model of infection (Chapter II.2). It was clear that treatment with a single-dose (0 h post infection) of antiEFG1 2’OMe is able to enhance the larvae survival over 24 h (by 20-30 %). It was also evident, that a double-dose (0 h and 12 h post infection) of the antiEFG1 2’OMe ASO is needed to prolong its effectiveness until 72 h of infection (by 30 %). The third generation of ASOs was developed to further enhance biostability and pharmacokinetics of the molecules in addition to enhance nuclease resistance and the target affinity. In this sense, a set of LNA-ASO were projected (Chapter II.3), in the so-called gapmer constitution, to control also the EFG1 gene expression and to reduce in vitro C. albicans filamentation. It is important to address that in vitro all LNA-ASOs projected were able to reduce C. albicans filamentation (by 50 %) and the levels of EFG1 gene expression (by 40 – 80 %) as in the case of antiEFG1 2’OMe. Although, the in vivo studies using the G. mellonella model revealed important differences among the LNA-ASOs performances. The inclusion of PS-linkage and palmitoyl-2’-amino-LNA chemical modification into the same gapmer demonstrated to be the most promising combination. In fact, an increasing on G. mellonella survival of around 40 % (at 72 h) was observed with a single-dose administration in comparison to the treatment with the antiEFG1 2’OMe (no effect was observed at 72 h with a single-dose administration). Given these findings, with this first part of the research, it is possible to conclude that the second and third generation of ASOs are viable approaches to target specific genes and control C. albicans virulence. The second goal of this research was to create strategies for C. albicans ASOs cargo and delivery (Chapter III). It is known that the delivery of ASOs to its site of action remains a key challenge. The development of non-viral vectors has been widely studied to ensure that these molecules become effectively protected from the environmental body conditions and to deliver them to their site of action. Polymers and liposomes become the most non-viral vectors researched in this sense, and specifically, the cationic polymers and liposomes due to their favourable interactions with the negatively charged RNA or DNA and cell membranes. Aiming for the future to coat medical devices with the antiEFG1 2’OMe ASO, novel anionic and cationic polyplexes microparticles based on poly(γ-butyrolactam) (PA4) or poly(εcaprolactam) (PA6) respectively, were developed using the entrapped and immobilized approaches (Chapter III.1). To note, that PA4 and PA6 polyplexes microparticles proved to be feasible carriers for antiEFG1 2’OMe ASO either using the entrapped and immobilized strategies, once all the ASO released maintains its activity against C. albicans cells. In another instance, aiming hereafter to develop a liposomal
Chapter IV General conclusions and work perspectives 118 nanocarrier to oral or cutaneous administration, lipid-based formulations to antiEFG1 2’OMe ASO cargo and delivery, prepared with DOTAP (cationic lipid) and DOPC, DOPE or MONO (neutral lipids) were developed (Chapter III.2). Importantly, all the lipid-based formulations revealed to be also feasible nanocarriers for antiEFG1 2’OMe ASO cargo, specially the DOTAP/DOPC 80/20 ρ=3 formulation that considerably contributed to increase G. mellonella survival when infected with C. albicans (in to 40 % after 48 h and 25 % after 72 h with a double-dose administration). The real importance of the development of nanocarriers for C. albicans ASOs cargo and the lipoplexes and polyplexes feasibility become clear with this part of the work. In summary, the research developed under this work provides potentially valuable information for future research into the management of Candida infections, regarding the development of a credible and alternative method to control C. albicans infections, based on AST methodology. Undoubtedly, this work validates the in vitro and in vivo therapeutic potential of ASO for controlling C. albicans infections. IV.2 Work perspectives The work described in this thesis provided a useful approach to control Candida infections. However only a part of the work was performed, and some important facts have been left behind, leading to interesting new questions for further research. Some of these suggestions should be considered for further investigations: a) To extend the studies performed with PA4 and PA6 microparticles to their application on medical surfaces (e.g. latex, silicone, acrylic) to control C. albicans proliferation. b) To further investigate other strategies to improve the lipoplexes formulations. For example, the incorporation of PEGylated lipids into the lipoplexes studied to overcome specific cellular barriers and immune defense mechanisms innate. In addition to the ratios between the cationic and neutral lipids, it is intended to vary the degree of PEG coverage to optimize ASOs stability and efficiency, and to evaluate its impact on C. albicans filamentation in vitro and in vivo . c) To extend the in vivo studies performed on the G. mellonella larvae to other in vivo models (e.g. mouse-models). The mouse-model will enable a realistic approach of the ASOs effect, and it will be interesting to evaluate the pharmacokinetics, pharmacodynamics and toxicology events behind molecules administration. d) As in the case of antiEFG1 2’OMe, further studies to improve the stability with the antiEFG1 LNA-gapmer5 should be performed. It would be interesting to study the encapsulation
Chapter IV General conclusions and work perspectives 119 of antiEFG1 LNA-gapmer5 on polyplexes and lipoplexes formulations as a strategy to improve its delivery and its effect on C. albicans filamentation. Moreover, to further investigate if the administration of a double-dose of this LNA-gapmer will enhance the control of C. albicans filamentation on G. mellonella larvae and to extend these studies to other in vivo models.
Annex I 120 Annex I Figure AI.1 AntiEFG1 2’OMe Candida albicans strains sensitivity determined by fluorescence in situ hybridization (FISH). The images were obtained by epifluorescence microscopy with the same exposure time for cells incubated in absence and presence of ASO. The values of exposure time used varied between 32.05 ms and 340.7 ms. Negative controls were prepared only with 20 µL of hybridization solution without probe.
Annex I 121 Annex AI.2 Evaluation of C. albicans filamentation and EFG1 gene expression in the untreated cells. (A) Percentage of filamentous forms (%) of Candida albicans SC5314 at different time points (4, 6 and 8 h) in RPMI (Without ASO); (B) Percentage of EFG1 gene expression obtained from Candida albicans SC5314 at different time points (4, 6 and 8 h). Error bars represent standard deviation. *Significantly differences between 4 h and the other times tested (P-value<0.05). Annex AI.3 Scrambled ASO effect on Candida albicans filamentation. Percentage of inhibition of filamentous forms (%) at different time points (4, 6 and 8 h) with 40 nM of scrambled ASO in RPMI.