UNIVERSITAT POLITÈCNICA DE VALÈNCIA

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UNIVERSITAT POLITÈCNICA DE VALÈNCIA

ETSIAMN

MODULAR DESIGN OF BACTERIAL CANCER

THERAPIES USING SYNTHETIC BIOLOGY

Author: Ivan Alarcon Ruiz

Directors: Eloisa Jantus Lewintre, PhD & Alejandro Vignoni, PhD

BACHELOR’S DEGREE IN BIOTECHNOLOGY

2019 – 2020

License Creative Commons

Valencia, July 2020

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TÍTULO

Modular design of bacterial cancer therapies using synthetic biology

ABSTRACT

Synthetic biology pursues the application of engineering principles to design and re-design living organisms, with the aim of controlling and modulating their behavior. Synthetic biology allows us to design and construct genetic circuits in a rational way, converting microorganisms into effective tools with many applications in a wide variety of fields. Oncology, within biomedicine, is one of them.

Moreover, cancer is a challenge in the current society, with more than 18 million of new cases and almost 10 million of death only in 2018. However, cancer treatment and therapies, the focus of oncology, have presently serious side effects, derived from the systemic action originated from the involved drugs. For that reason, the development of new systems providing localized action of antitumoral drugs, minimizing the damage that they cause to the healthy tissues, is essential.

The main objective of this work is the design of a synthetic biology based bacterial cancer therapy, with focus on the requirements and fundamental characteristics that bacteria must accomplish to become a therapy.

After an exhaustive analysis of the scientific literature, six modules or requirements have been identified that should be present in these bacteria: (1) Tumor-targeting Module; (2) Payload Module; (3) Release Module; (4) Memory Module; (5) Genetic circuit stability Module; and (6) Biocontainment Module. For each module, several systems have been proposed.

In the second part of this work, a specific modular design of engineered bacteria has been proposed as a therapy against Non-Small-Cell Lung Carcinoma. A part of this design has been experimentally studied: the lysis circuit controlled by Quorum Sensing (Oscillator). We improved the experimental conditions achieving an oscillatory behavior of the bacterial population by using a low enough initial concentration of bacteria. Moreover, it has been proven that the population’s decline is caused by the expression of the lysis protein. Finally, the robustness of the lysis circuit has been proved in several culture media, including bacteria and tumorspheres media.

Although the applications of synthetic biology in medicine are almost new, the rapid development of new systems and the advance in their efficacy and security would lead to a fast growth of this discipline in the next years, not only in cancer, but applied to other many malignancies, such as infections, inflammatory, immune or metabolic disease.

Key words: Synthetic Biology; Cancer; Bacteria; Cancer therapy; NSCLC; Lysis circuit; Oscillator. Author: Iván Alarcon Ruiz

Location & date: Valencia, July 2020 Director: Eloisa Jantus Lewintre, PhD Codirector: Alejandro Vignoni, PhD

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TÍTULO

Diseño modular para terapias antitumorales basadas en bacterias usando biología sintética.

RESUMEN

La biología sintética busca aplicar los principios de la ingeniería a los organismos vivos, con el objetivo de controlar y modular su comportamiento. Esta disciplina ha permitido el diseño y construcción de circuitos genéticos de forma racional, convirtiendo a los microorganismos en potenciales herramientas con aplicaciones en muchos campos, entre ellos la biomedicina. Por otro lado, el cáncer es un reto de la sociedad actual, con más de 18 millones de nuevos casos y casi 10 millones de muertes solo en 2018. Sin embargo, muchos de los tratamientos antitumorales actuales llevan asociados importantes efectos secundarios, derivados de la acción sistémica que ejercen muchos de estos fármacos. Por ello, es necesario el desarrollo de sistemas que permitan la acción localizada de los agentes anticancerígenos sobre el tumor, evitando o disminuyendo el daño que ejercen sobre el resto de tejidos sanos.

El principal objetivo de este Trabajo Final de Grado es el diseño de una bacteria sintética enfocada al tratamiento de cáncer de pulmón no microcítico, poniendo especial atención en los requerimientos y características fundamentales que cualquier bacteria modificada debe cumplir para poder convertirse en una terapia antitumoral.

Tras un exhaustivo análisis de la bibliografía científica, se han identificado seis módulos o requisitos que deben cumplir estas bacterias: (1) Módulo para la focalización tumoral; (2) Módulo para la actividad antitumoral; (3) Módulo para la liberación de moléculas (4) Módulo de memoria; (5) Módulo para la estabilidad del circuito genético; y (6) Módulo para la biocontención. Dentro de cada módulo se han propuesto diferentes sistemas.

En la segunda parte de este trabajo y basándonos en el enfoque modular expuesto previamente, se ha propuesto un diseño de bacteria sintética enfocada al tratamiento del cáncer de pulmón no microcítico. Una parte de este diseño ha sido estudiada experimentalmente: el circuito de lisis controlado por Quorum Sensing (Oscilador). Se han mejorado las condiciones experimentales relativas al cultivo de las bacterias, lográndose un comportamiento oscilatorio de la población bacteriana cuando la concentración inicial de bacterias es lo suficientemente baja. También se ha demostrado que la disminución de la población es causada por la expresión de la proteína de lisis. Además, se ha demostrado la robustez de este comportamiento en diferentes medios de cultivo, tanto de bacterias como de tumoresferas.

Aunque la aplicación de la biología sintética a la medicina es muy reciente, la rápida evolución de nuevos sistemas y los avances en eficacia y seguridad aseguran un rápido crecimiento de esta disciplina en los próximos años, no solo en cáncer, sino también en una gran variedad de enfermedades, como infecciones o enfermedades inflamatorias, inmunitarias o metabólicas.

Palabras clave: Biología Sintética; Cáncer; Bacteria; Terapia antitumoral; NSCLC; Circuito de

lisis; Oscilador.

Autor: Iván Alarcon Ruiz

Localidad y fecha: Valencia, Julio de 2020

Tutor académico: Prof. Dña. Eloisa Jantus Lewintre Cotutor: Prof. D. Alejandro Vignoni

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AGRADECIMIENTOS

Es difícil condensar tantos años y tantas personas en una sola página, especialmente cuando hay tanta gente a la que estoy agradecido y a la que debo muchísimo. Quiero y debo empezar por mis amigos, los del pueblo y los de la carrera, esos que ahora, cuatro años después de conocernos, terminan junto a mí el Grado en Biotecnología convertidos en prometedores científicos que en adelante deben luchar por hacerse un pequeño hueco en esta difícil profesión. Gracias por amenizar cada tarde, cada clase, cada examen o trabajo y por ser una fuente inagotable de risas y de inspiración. Una de las peores cosas de finalizar esta etapa es, sin duda, tener que aceptar que ya no nos podremos ver todos los días, pero estoy seguro de que siempre encontraremos tiempo para una quedada, una cervecita (para mí una Coca-Cola, por favor) o una charla por videollamada en la que hablemos de todo y de nada. Ojalá la vida os de todo lo bueno que os merecéis, que no es poco.

A mi familia, siempre atenta, paciente y entregada. En especial a mis padres Fina y Juan, por enseñarme a luchar por lo que quiero y a no rendirme ante la adversidad, por ayudarme a ver ese pequeño atisbo de luz que siempre aparecía en los momentos más oscuros y por poner todo su empeño en entender este proyecto sin ser científicos. A mis hermanas Lidia y Almudena, por ser mis compañeras de vida y por alegrarme cada instante. Os voy a echar muchos de menos el año que viene. Estos últimos años los estudios y el laboratorio me han absorbido demasiado, os pido disculpas por no haberos prestado toda la atención que merecéis, ya sabéis que mi amor por vosotros es incondicional. A Salvi y Emilio, por su confianza ciega en mi desde antes de que aprendiese incluso a caminar, por ser mis segundos abuelos y por todo su apoyo durante estos largos años. También a mis abuelos Carmen y Pedro, pero sobre todo a Maruja y Felipe. Estaba decidido a ser arquitecto, pero vuestra historia, vuestras ganas de vivir y vuestra lucha sin final feliz me hicieron replanteármelo todo y dirigir mis pasos hacia la investigación biomédica. Ojalá hoy fuese arquitecto y no biotecnólogo, porque eso significaría que seguiríais aquí conmigo. No puedo olvidarme de los miembros del sb2_{cLab, dirigido por el Prof. Jesus Picó y a quien} siempre estaré inmensamente agradecido por haberme abierto las puertas de su laboratorio y haberme permitido iniciarme en este extraño mundo de la biología sintética. Permitidme una mención especial a dos personas que se han convertido en parte de mi vida: Ale y Yadi, mis mentores. Gracias por vuestro esfuerzo incansable, vuestra pasión por la investigación y el trabajo bien hecho y, sobre todo, por dejarme traspasar la frontera de lo profesional y ser siempre una mano amiga a la que acudir cuando me surge el más mínimo problema. Iniciarse en el mundo de la investigación es difícil, no me cabe duda, pero vosotros me lo habéis puesto muy fácil, y eso es algo que nunca olvidaré. Confiasteis en mi desde el primer momento y os habéis dejado la piel en enseñarme cada paso en esta compleja profesión, empezando hace unos años con aquel “te voy a enseñar a abrir los tubos con una mano”, y vaya si me lo enseñaste. Estoy seguro de que nuestros caminos volverán a cruzarse en un futuro, solo espero que sea más pronto que tarde. Entretanto, me conformo con encontrarme por el camino a gente la mitad de buena que vosotros, porque iguales seguro que no los encuentro.

Finalmente, a mis tutores, los dos pilares de este proyecto: la Dra. Eloisa Jantus Lewintre y el Dr. Alejandro Vignoni. Siempre me imaginé a los responsables de laboratorio como personas super estrictas, cuyas órdenes tienes que obedecer sin la más mínima réplica, pero vosotros me habéis sacado de mi error. Desde el primer momento habéis escuchado mis propuestas y me habéis guiado y aconsejado con una profesionalidad y un trato increíbles. Gracias por ser unos grandísimos guías, siempre os tendré de referentes.

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LIST OF FIGURES

Figure 1. SynBio cycle ... 1

Figure 2. Modular design of bacterial cancer therapies ... 6

Figure 3. Tumor-targeting systems I ... 7

Figure 4. Tumor-targeting systems based on controlled expression of target genes ... 11

Figure 5. Payload systems ... 12

Figure 6. Release systems ... 16

Figure 7. Payload Delivery Device ... 18

Figure 8. Memory systems I ... 19

Figure 9. Memory systems II ... 20

Figure 10. Systems for the maintenance of the genetic circuit I ... 22

Figure 11. Systems for the maintenance of the genetic circuit II ... 23

Figure 12. Genetic circuits used as a biocontainment system ... 25

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LIST OF TABLES

Table 1. Experimental settings of the plate reader ... 4

Table 2. Inducible promoters and their inductors ... 10

Table 3. Enzyme/prodrug systems ... 14

Table 4. Bacterial cancer therapies I ... 31

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ABBREVIATIONS

6MeP 6-methylpurine

6MePdR 6-methylpurine 2-deoxyriboside 9ACG 9-aminocamptothecin glucuronide

9AC Topoisomerase I inhibitor 9-aminocamptothecin AHL N-acyl homoserine lactone

Amp Ampicillin

BCG Bacillus Calmétte-GuerinSALMONEL Cb Carbenicillin

CB1954 5-(aziridine-1-yl)-2,4-dinitrobenzamide CDA Ciclic di-AMP

CDase Escherichia coli cytosine deaminase ClyA Cytolysin A

Cm Chloramphenicol CPG2 Carboxipeptidase G2 CPP Cell-Penetrating Peptides

BDEPT Bacterial Directed-Enzyme Prodrug Therapy FasL Fas Ligand

FlaB Flagellin B

FNR Fumarate and nitrate reduction regulator HIP-1 Hypoxia-Inducible Promoter

IFN Interferon IS Immune System

MEFL Molecules of Equivalent Fluorescein MTD Mitochondrial targeting domain (of Noxa) PNP Purine Nucleoside Phosphorylase

QS Quorum Sensing RBS Ribosome Binding Site

SAH Staphylococcus aureus α-hemolysin sFlt-1 Soluble fms-like tyrosine kinase receptor ssDNA single-stranded DNA

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STING Stimulator of interferon genes T3SS Type III Secretion System tkRNAi trans-kingdom RNA interference TLR5 Toll-Like Receptor 5

TNF Tumor Necrosis Factor TNF-α Tumor Necrosis Factor-α

TRAIL TNF-Related Apoptosis-Inducing Ligand UBPs Unnatural Base Pairs

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Introduction

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1. INTRODUCTION

Synthetic biology or SynBio seeks to programme living cells with artificial genetic circuits rationally designed to perform user-defined functions (Wu et al., 2019). One of the key features of synthetic biology is the application of engineering principles to biological systems through a four-steps cycle: (1) Design and modelling of the genetic circuit in silico; (2) Construction and implementation of the genetic circuit; (3) test of the system with in vivo experiments; and (4) learning process and enrichment of the system (Boada, 2018) (Figure 1). The foundations of SynBio is considered to be the design, construction and implementation of the first toggle switch (Gardner et al., 2000). Since then, more complex systems have been developed (Cameron et al., 2014) with a broad range of application, such as industrial processes (Gao et al., 2019), agriculture (Wurtzel et al., 2019) or medicine (Leventhal et al., 2020).

Cancer is a current challenge in our society, with 18,1 million new cases and more than 9,5 million of deaths from cancer in 2018. Cancer is an important cause of morbidity and mortality worldwide (Bray et al., 2018), the first cause of death in high-income countries, whereas cardiovascular disease is the second (Dagenais et al., 2020). Moreover, lung cancer is the most common type of cancer, followed by breast, prostate and colon cancer (Bray et al., 2018). Bacteria has been frequently regarded as the causative agents of infectious disease, although most bacteria are non-pathogenic and could have positive effects in our health. In fact, bacteria have been tested as anticancer therapies (Piñero-Lambea et al., 2015b). The use of bacteria as a cancer treatment is not new.German physician W. Busch seems to be the promoter of that, when in 1868 purposefully infected with Streptococcus pyogenes (that produces erysipelas) a woman with an inoperable sarcoma. The tumor shrunk, but the patient died from the infection 9 days after. In 1883, Fehleisen reported the use of Streptococcus in a breast cancer patient, who experimented a complete tumor regression and six months later remained free of the malignancy (Coley, 1891). After that, hundreds of patients with sarcoma were treated with Coley’s toxin (Coley et al., 1898), whose developer is broadly recognised as the founder of cancer immunotherapy. However, the use of bacteria originated infections, erypsipelas and other malignancies and many patients died as a result of these side effects. For this reason, the idea of using bacteria against cancer disappeared with the advance of chemotherapy, radiotherapy and other safer techniques with less side effects (Pawelek et al., 2003). Now, with the development of genetic engineering, synthetic biology and a deeper knowledge about both microbiology and oncology, it is possible to improve bacterial therapies, making them safer and more effective.

Figure 1. SynBio cycle. There are four main steps in synthetic biology, starting with the design of the genetic circuit in

silico, followed by the build and implementation of the genetic circuit and its test in vivo. Once the experimental results have been obtained, they are studied to learn and improve the design.

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Introduction

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Furthermore, bacterial cancer therapies overcome some drawbacks of the current cancer therapies: (1) the high cost of these therapies, whereas bacteria can be produced cost-effectively providing an alternative treatment for clinical use (Jiang et al., 2010); (2) the side effects derived from drug systemic action, while the goal of targeted drug delivery by bacteria is to maximize drug accumulation within the tumor, minimizing off-target effects (Ozdemir et al., 2018) and allowing the use of antitumor molecules with severe side effects when administered systemically (Rensing-Ehl et al., 1995; Zhu et al., 2011); and (3) development of resistances to the current therapies. In fact, many of the bacterial cancer therapies under study have shown to develop immunological memory and, consequently, resistance to secondary tumor challenge and longer cancer-free time interval (Leventhal et al., 2020).

The rational design of microorganism for biomedical applications requires the availability of genetic modules with predictable phenotype that could be integrated in complex cellular assemblies with an orthogonal behaviour and that should be inherited through cell generations (Piñero-Lambea, 2014).

The present work gains insight into the interface between SynBio and oncology, exploring the current situation of bacterial cancer therapies from a new perspective, and combining the current bacterial therapies under development with the study of novel systems that could be useful for the progress and improvement of more complex systems. So far, nobody has examined the bibliography exhaustively to determine the basic requirements needed by engineered bacteria to fight cancer.

Here, the modular design of bacteria is proposed, where the modules represent the requirements that all bacteria must achieve to become a successful bacterial cancer therapy and to be able to leave the laboratory and reach clinical use. For each module, several systems have been proposed. Moreover, some limitations in current bacterial cancer therapies have been detected, such as the shortage of bacteria with a biocontainment module, and several systems have also been proposed to overcome them. This work aims to ease the development of new engineered bacteria against cancer through different combinations of one or more systems for each module.

Additionally, and as a demonstration, we apply this modular approach to design a specifically engineered bacterium with a focus on the treatment of Non-Small-Cell Lung Carcinoma (NSCLC). So, the best system for this particular purpose is discussed at the end of each module.

Finally, as a complementary part of this work, we have started the construction and implementation of this proposed bacterium, focusing on the lysis circuit or oscillator as a release module. For that, the lysis circuit implemented by Requena (2019) has been used and the experimental conditions of the experiments have been optimized to achieve the desired performance of the system. These engineered bacteria have been tested in several culture media, both bacterial media and cancer cell media. Furthermore, ten different variations of this system are under development and we propose to test them in a tumorspheres culture. The selection of tumorspheres instead of other classical cultures is its similarity with tumors. Patient-derived tumorspheres retain several characteristics of the original tumor and show stemness features (self-renewal and unlimited exponential growth potential). The Cancer stem-like cells can survive after conventional cancer treatments and, consequently, regenerate tumors, so that they are a promising target. Tumorspheres also exhibit resistance against chemotherapeutic agents, a key point of this work because of the ability of engineered bacteria to overcome them. Finally, tumorspheres show invasion and differentiation capacities in vitro and overexpress genes related to these features (Herreros-Pomares et al., 2019). Therefore, these characteristics offer a better model of real tumors and improve the extrapolation of the results, and these are the reasons why we suggest the use of 3D cultures instead of 2D cultures.

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Objectives

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2. OBJECTIVES

The main objective of this work is the design of a synthetic biology based bacterial cancer therapy, with focus on the requirements and fundamental characteristics that bacteria must accomplish to become a therapy. To achieve this goal, the following specific objectives have been established:

1. Revise the state-of-the-art of the interface between synthetic biology and oncology to collect the main bacterial cancer therapies developed so far.

2. Understand the purpose of each system used in each engineered bacterium against cancer.

3. Analyse the challenges and limitations of the current bacterial cancer therapies and search of useful systems developed by SynBio to overcome them.

4. Integrate the identified systems and classify them into modules in order to simplify the decision-making process in the design of bacterial cancer therapies.

5. Apply this modular approach to develop a bacterial cancer therapy focused on Non-Small-Cell Lung Cancer.

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Materials and methods

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3. MATERIALS AND METHODS

3.1. BIBLIOGRAPHIC SEARCH

In order to conduct this Project, an in-depth analysis of the published literature has been accomplished. The main sources of scientific articles have been the databases PubMed and Google Scholar.

However, not all the papers related with the subject of this review have been included, but a series of criteria have been established to ensure the quality of the sources used. In general, only scientific papers published in the last 10 years in indexed scientific journals with an impact factor higher than 4 have been included. Nevertheless, some references have been selected without meeting these requirements because their quality have been considered subjectively high enough to form part of this review.

For the bibliographic search, in addition to the criteria previously exposed, certain key words have been used, which have varied throughout the process depending on the objective of the search. Among the key words used, there are: bacteria, cancer treatment, tumor, synthetic biology, tumor targeting bacteria, engineered bacteria, programable bacteria, synthetic bacteria, biocontainment, lysis circuit, plasmid stability, artificial genetic bases, auxotrophy or synthetic genetic circuit.

3.2. EXPERIMENTAL PROCEDURE

Engineered bacterium Escherichia coli DH5α carrying the plasmid pARKA1-O1 was used in this work, provided by Synthetic Biology and Biosystems Control Lab (from Technical University of Valencia) and developed by Requena (2019). Culture media employed were sLB (purchased to Condalab), M9CA (purchased to VWR), and SOC (purchased to Condalab), RPMI and DMEM (both purchased to Gibco). RPMI and DMEM are the culture media used in tumorspheres cultures. They were supplemented with BSA 0,4%, bFGF, EGF and ITS. Moreover, Kanamycin (Km) were added to all the culture media. All experimental measurements were taken with the plate reader Biotek CytationTM_3.

All the experiments were done following the same protocol, which consist of these main steps: (1) Inoculation of 3 mL of sLB with bacteria from glycerol stock stored at -80ºC; (2) overnight incubation in the orbital shaker at 37 ºC and 200 rpm; (3) dilution from sLB to new sLB at 0,1 OD; (4) incubation for 4 hours in the orbital shaker at 37 ºC and 200 rpm; (5) cooling of the bacterial culture for 30 minutes; (5) dilution from sLB to the specific culture medium of the experiment; and serial dilutions to fill the 96-well plate; and (6) incubation and measurements in the plate reader with the specifications indicated in Table 1.

Table 1. Experimental settings of the plate reader

Total time of the experiments 18 hours Time between measurements 5 minutes

Shaking Double orbital (Continuously) Absorbance wavelength 1 600 nm

Absorbance wavelength 2 450 nm Excitation wavelength 485 nm Emission wavelength 528 nm

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Materials and methods

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Finally, data were transformed from blanked Abs600 to particles and from blanked GFP (arbitrary units) to MEFL (Molecules of equivalent of fluorescein), in order to standardize these results and easier their comparison with results obtained by other laboratories. To do that we applied the equations 1 and 2, previously developed by our research group (Boada et al.¸ 2019).

𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 = 108,062_{· (𝐴𝐴𝐴𝐴𝑝𝑝}

600)1,185 (Equation 1)

𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 = 1012,28_{· (0,00229 ∗ 𝑁𝑁}

𝐺𝐺𝐺𝐺𝐺𝐺)1,038 (Equation 2)

where Abs600 is the measurement of absorbance at 600nm and NGFP is the measurement of the fluorescence (λex = 485nm; λem = 528nm).

Log(MEFL/Particle) have also been calculated. MEFL/Particle is relative to the amount of fluorescence protein in each individual cell. Log is applied for graphical considerations.

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Modules for engineered bacterial cancer therapies

Tumor-targeting Module

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4. MODULES FOR ENGINEERED BACTERIAL CANCER THERAPIES

In this work, we have identified six modules or requisites that any bacteria must integrate to be suitable for clinical applications in cancer treatment (Figure 2). The objective of these modules is the development of an engineered bacteria which would be able to fight cancer effectively without any risk for the patient and the environment. To achieve it, each module play an important and specific role: (1) a Tumor-targeting module, which directs the bacterial action only to the tumor, avoiding off-target effects; (2) a Payload module, being responsible for the antitumor activity; (3) a Release module, that delivers the payload to tumor tissue; (4) a Memory module, which is not a requirement but could be useful in some application which requires to maintain the target gene expression once specific stimuli have disappeared; (5) a Genetic circuit stability module, to avoid the loss of the genetic circuit or its defunctionalisation; and (6) a Biocontainment module to ensure the therapy safety for both the environment and the patient.

Figure 2. Modular design of bacterial cancer therapies. Each engineered bacterial cancer therapy needs to accomplish

some requirements or modules. For each module, several systems are available. It is compulsory to choose one system in each module (green point). Memory module is optional.

4.1. TUMOR-TARGETING MODULE

One of the most important advantages of bacterial therapies is their ability to avoid off-target effects and focus only on tumor cells and metastases. Many bacterial strains have shown a wild tropism for the tumor microenvironment and, consequently, they can bypass problems associated with poor selectivity and limited tumor penetrability of conventional cancer chemotherapies (Piñero-Lambea et al., 2015b). Moreover, several strategies have been developed to achieve tumor-targeting by bacteria and two or more systems can be combined to ensure and enhance tumor-targeting effect. These strategies can be classified into two groups depending on their underlying mechanisms. On the one hand, the systems that focus on the specific proliferation of bacteria within tumor tissue, such as wild tumor tropism, adhesion molecules and synthetic adhesins, quorum sensing systems, or engineered chemoreceptors. On the other hand, when the systems have a controlled expression of a target gene only in the tumor tissue, so that if bacteria reach a healthy tissue this gene would not be expressed. Among these systems one can find inducible promoters, toehold switches and miRNA and p53 sensing.

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4.1.1. WILD TUMOR TROPISM

Hypoxia decreases the efficiency of some currently used anticancer strategies, such as surgery, chemotherapy and radiotherapy. However, many bacteria can take advantage of this physiology (Nuyts et al., 2002). Over the years many species of (facultative and strict) anaerobic bacteria were reported to proliferate preferentially within solid tumors (Figure 3a). Several reasons may explain this wild tropism of bacteria to tumors. Firstly, the tumor microenvironment is immune-deficient and lacks macrophage and neutrophil clearance mechanisms, helping bacteria to escape of the immune system attack. Secondly, solid tumors are hypoxic environments that could enhance bacterial replication. Finally, the chaotic and leaky vasculature in tumors and the presence of chemotactic compounds increase bacterial entry and retention within tumoral mass (Pawelek et al., 2003; Piñero-Lambea et al., 2015b).

Despite the immune-deficient tumor microenvironment, it has been observed that bacteria accumulate almost exclusively in large necrotic areas and spare a rim of viable tumor cells, and that the necrotic and viable areas are separated by a barrier of host neutrophils that have migrated inside the tumour but are unable to reach the necrotic core (Westphal et al., 2008). This may explain why the therapeutic effect of engineered bacteria can be enhanced by radiotherapy, which would destroy the tumors cells in the normoxic area, whereas bacteria would destroy those in the hypoxic area (Jiang et al.,2010). In addition, depletion of host neutrophils has led to an increase in the spread and in the total number of bacteria inside the tumor and, consequently, to an improved bacteria-mediated tumor therapy. However, the depletion of neutrophils must be done very carefully, otherwise it could cause a systemic infection. So that, systemic neutrophil depletion seems unrealistic in humans, but a local depletion could be achieved by engineering bacteria that secretes depleting antibodies only in tumor tissue (Westphal et al., 2008).

Figure 3. Tumor-targeting systems I. (a) Wild tumor tropism. Many bacterial strains growth preferentially within

tumor tissue and not in healthy tissue. (b) Engineered chemoreceptors integrated by a ligand-binding

domain (orange) and a DNA binding domain (red). (c) Adhesions molecules. (d) Quorum Sensing. Bacteria

produce LuxR (orange molecules) and AHL (pink and purple circles). When the population level is higher than a certain threshold, the internal concentration of AHL (purple circles) is high enough to allow the formation of a transcription factor (LuxR·AHL) that drives the expression of a target gene (red molecule).

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Some of the bacteria identified with wild tumor tropism are Salmonella typhimurium (Yoon et al., 2017; Ganai et al., 2009; Loeffler et al., 2007), Salmonella enterica (Royo et al., 2007), Bifidobacterium infantis (Zhu et al., 2011), Bifidobacterium longum (Hu et al., 2009), Escherichia coli (Jiang et al., 2010; Leventhal et al., 2020), Clostridium acetobutylicum (Theys et al., 2001) and Clostridium oncolyticum (Lambin et al., 1998).

4.1.2. ENGINEERED CHEMORECEPTORS

Bacteria use chemotaxis signaling pathways to sense environmental changes. To do that, they have chemoreceptors, which are highly specific signaling proteins that allow them to respond to environmental chemoeffectors. Despite the wide range of chemoeffectors available in nature, medical application often requires novel sensors with enhanced or new specificity to desirable molecules. In fact, engineered chemoreceptors development is possible nowadays because of the extensive knowledge about receptors sequences, functions and mechanisms (Bi and Lai, 2015).

There are many methods to develop new chemoreceptors. The most widely used are the rational design strategy by site-directed mutagenesis, the directed evolution strategy using PCR prone or gene fragment recombination, and hybrid chemoreceptors created by gene fusion (Bi and Lai, 2015).

Other systems are the modular transcriptions factors that repress the expression of a gene when a certain ligand is absent. This engineered transcription factors are composed by two domains: a ligand binding domain and a DNA binding domain. Changing the first one, you can produce novel repressors; and changing the second one you can modify the promoter that binds to the transcription factor. The transcription factor is a repressor and its binding to the promoter inhibits the gene expression. In addition, the binding of the ligand to the repressor inhibits this inhibition. Consequently, the target gene is expressed if the ligand is present within the cell. Moreover, it is possible to combine many transcription factors with the same DNA binding domain and different ligand binding domain into orthogonal AND gates, in which a certain gene is expressed only when all the ligands are inside the cell. If any ligand is missing, the gene expression is repressed (Shis et al., 2014).

On the other hand, to increase the repertoire of ligands that can be detected, it is possible to develop a synthetic receptor through the fusion of a single domain antibody (ligand binding domain) and a DNA binding domain. This receptor forms a dimer after the binding of the ligand to the single domain antibody (CHH). The dimerization of the receptor causes his activation, that controls gene expression. So that, gene network can be connected to ligand detection (Figure 3b). The advantage of this method is that there are large libraries of nanobodies that can be used in this approach to relate a specific ligand with the control of a synthetic gene network. Moreover, this receptor system can be applied to engineer cytosolic and transmembrane receptors, allowing the detection of intracellular and extracellular ligands (Chang et al., 2018).

4.1.3. ADHESION MOLECULES AND SYNTHETIC ADHESINS

Other tumor-targeting strategy is the expression of adhesion molecules in the bacteria surface to bind other target-molecules in cancer cells. An ideal application of this system would be to target molecules that are overexpressed in tumor cells, but not in normal cells (Figure 3c). Recently, an E. coli Nissle 1917 has been engineered to express Histone-like protein A (HlpA), which binds to Heparan Sulphate proteoglycan, overexpressed on colorectal cancer cells (Ho et al., 2018).

Moreover, β1-integrin is overexpressed in many types of solid cancers because it takes part in tumor progression, participating in neoangiogenesis, migration and invasion into the

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surrounding stroma, extravasation through neoangiogenic vessels and homing in new tissues to form metastasis (Blandin et al, 2015). Invasin is a protein encoded for the inv gene of Yersinia psudotuberculosis. It binds to these β1-integrins, leading to the phagocytosis of the bacterial cells. In fact, this is a release module very used in bacterial cancer therapies (Ahmed and Lage, 2018)

Beyond natural molecules, synthetic adhesins can be designed to drive an effective and specific adhesion of bacterium to a target cell. The construction of synthetic adhesins is achieved by the fusion between the outer membrane anchoring β-domain of Intimin A and a nanobody (VHH) recognizing a specific antigen in the surface of the target cell. The advantage of these engineered synthetic adhesins is the wide variety of nanobodies available, allowing the targeting of many molecules on the cancer cell surface (Piñero-Lambea, 2014). An engineered E. coli expressing a synthetic adhesin has been tested in mice to measure its efficiency in tumor colonization. The results show that lower doses of engineered bacterium are required to colonize more than 90% of the target tumors, only 2% of the dose required for wild type E. coli (Piñero-Lambea et al., 2015a). Finally, the activation of the promoter sequence of the yeeE gene follows the bacterial adhesion to their target, allowing the development of genetic circuits that could respond to specific adhesion (Piñero-Lambea et al., 2014).

4.1.4. QUORUM-SENSING SYSTEMS

Quorum sensing (QS) is a property discovered in Vibrio fischeri that acts as a density-dependent switch where gene expression depends on bacterial population density. As bacterial accumulation is usually higher in tumor tissue than in healthy tissue, a system that only activates gene expression when a high population density is reached would help to restrict the gene expression to the tumor tissue, avoiding off-target effects (Swofford et al., 2015).

The QS system taken from V. fisheri is composed by three genes: (1) luxI, an enzyme that produce the autoinducer or communication molecule N-Acyl Homoserine Lactone (AHL); (2) luxR, a protein that binds AHL to form a transcription factor; and (3) target gene. LuxR is constitutively expressed, whereas LuxI and target gene expression is driven by the Plux promoter, activated by

LuxR·AHL. Autoinducer molecules are able to passively diffuse across the membrane following a concentration gradient. When bacterial population density is low, the concentration of AHL in the surroundings of the cells and inside the cells is also very low and there is not target gene expression. In contrast, if bacteria reach the tumor site and start to proliferate, the concentration of AHL in the environment increases making the AHL within the cell also increase until the activation of Plux promoter is achieved, leading to the expression of the target gene and

establishing a positive feedback to produce more LuxI and, consequently, more AHL (Boada et al., 2017) (Figure 3d). The QS system has been used to express a reporter protein only in tumors (Swofford et al., 2015) and control the lysis of the bacteria (Chowdhury et al., 2019).

4.1.5. CONTROLLED EXPRESSION

In the next systems, off-targets effects are avoided because the expression of the target gene is only allowed in the tumor tissue, and not in the healthy one. There are different mechanisms, but all of them are based in the presence of tumor-specific molecules or induction.

4.1.5.1. INDUCIBLE PROMOTERS

Systems with a constitutive gene expression difficult the control over the amount of therapeutic compound produced because it depends on several aspects, such as the population density. One of the easier ways to control it is the use of promoters and Ribosome Binding Sites (RBS) with a known strength (Ozdemir et al., 2018). In fact, there are a wide range of genetic components available, as well as characterization methods that allow the detailed description of a specific

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genetic part (Boada et al., 2019). The development of standard and well-characterized genetics elements allows the design, construction, and implementation of more complex systems (Lou et al., 2012). However, with a constitutive gene expression, off-target effects may increase. A further control can be achieved with inducible promoters which drives the expression of a target gene only under specific conditions. The main benefit of inducible promoters is their ability of the promoter to act as a switch, allowing for temporal and spatial control of gene expression. Several inducible promoters have been developed and applied to bacterial therapies (Table 2).

Table 2. Inducible promoters and their inductors

Promoter Inductor Reference

recA Radiotherapy (Ganai et al., 2009; Nuyts et al., 2001b) PompC Red light (Tabor et al., 2009)

PompC L-arabinose (Jeong et al., 2014 ; Zheng et al., 2017)

PrhaBAD L-rhamnose (Loessner et al., 2009)

Ptet Anhydrotetracycline (Loessner et al., 2009)

PfnrS Hypoxia (Leventhal et al., 2020)

HIP-1 Hypoxia (Mengesha et al., 2006) PIL-8 Inflammation (Castagliuolo et al., 2015)

PnorV Nitric Oxide (inflammation) (Archer et al., 2012)

PphsA Thiosulphate (Daeffler et al., 2017)

PttrB Tetrathionate (Daeffler et al., 2017)

Psal Salicylate (Royo et al., 2007)

Moreover, radio-inducibility of recA promoter can be increased or reduced by adding or eliminating Cheo Box in the promoter region; and a Cheo Box could be added to constitutive promoters to become them into radio-inducible promoters (Nuyts et al., 2001a).

Temperature can also be used as an input signal, using temperature-dependent transcriptional repressors (TlpA, TcI, TetR mutants and LacI mutants) and heat shock promoters (GrpE, HtpG, Lon, RpoH, Clp and DnaK). This system has been proposed to selective activation within a mammalian host using focused ultrasounds, sensing and responding to fever and self-destroying at ambient temperatures to avoid biocontainment concerns (Piraner et al., 2017).

However, the usage of a single promoter may offer limited control of specificity and efficacy. To improve them, more complex systems have been designed, such as the dual-promoter approach (Figure 4a), in which there are two different inducible promoters and each one drives the expression of a part of a transcription factor. When the two inductors are in the environment, the two parts are expressed and an active transcription factor is produced, generating the expression of a target gene. (Nissim and Bar-Ziv, 2010)

4.1.5.2. TOEHOLD SWITCH

Despite many independent genetic switches have been engineered, the design of circuits interconnecting switches is more complicated because each switch is sensitive to its own predetermined trigger compound (Ausländer and Fussenegger, 2014). Toehold switches are a

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de-novo-designed prokaryotic riboregulators that regulate the expression of a target gene based on the presence/absence of a specific RNA (trigger RNA) in the host cell. In this system, the mRNA that contains the target gene (switch RNA) forms a loop when a specific trigger RNA is absent. This loop blocks the RBS and, consequently, inhibits gene expression. In contrast, if the trigger RNA is in the cell, it binds to the Toehold sequence and prevents the loop formation. So that, the RBS is accessible and the translation is allowed (Green et al., 2014; Green et al.,2017) (Figure 4b). Furthermore, the response to the toehold switch can be modulated by introducing a supplementary inhibitory hairpin, varying the length of the Toehold sequence and controlling the expression of triggers RNAs with inducible promoters (Kim et al., 2019).

Figure 4. Tumor-targeting systems based on controlled expression of target genes. (a) Complex inducible promoters.

Each inductor (blue and pink) activates an inducible promoter. If both inductors are present in the environment, an active transcription factor is produced, and a target gene is expressed. (b) Toehold switch.

The expression of the target gene is repressed because its mRNA forms a loop that blocks the Ribosome Binding Site. If a trigger RNA (light blue) is present in the tumor cell, its binds to the complementary sequence of the switch RNA (dark blue) and unlock the RBS, allowing gene expression. (c) miRNA sensing. The target

gene is expressed only when a certain combination of miRNAs is given in the tumor cell. miR-141 must be absent because it inhibits the mRNA of the target gene. The purple molecule also inhibits the expression of the target gene, so it must be repressed and, to achieve that, miR-17 and miR-30 must be present.

4.1.5.3. miRNA AND p53 SENSING

Using an appropriate release system that allow the transference of a genetic circuit to a mammalian cell from a bacterium, we can engineer a genetic circuit in which a target gene is expressed only when a specific combination of miRNA is given in the mammalian cell (Wu et al., 2019) (Figure 4c). The miRNAs profile of a cancer cell is different of that of a normal cell, so that this system can be adapted to allow the expression of the target gene only when a tumor cell is detected. This approach has been tested in HeLa cancer cells, inducing apoptosis without affecting non-Hella cell types (Xie et al, 2011).

Moreover, synthetic circuits can be engineered to sense levels of p53, whose function is the DNA repair and it is a very important protein to avoid tumor development. This circuit drives the expression of a target gene (such as a therapeutic protein) in those cells with low levels of p53 (Mircetic et al., 2017).

4.1.6. SELECTION OF TUMOR-TARGETING SYSTEM

NSCLC is a solid tumor with necrotic areas where bacteria can proliferate. Consequently, we decide to use the wild tumor tropism as a tumor targeting system in our engineered bacteria. Among the bacteria with this characteristic, in this work we select Escherichia coli DH5α, which have shown selective growth in tumor tissue (Yu et al., 2004) and is broadly known. To further increase the tumor-targeting, we also utilise the Quorum Sensing system, that drives the expression of a lysis protein when the bacterial population is high.

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4.2. PAYLOAD MODULE

One of the most important modules in bacterial cancer therapies is the payload module, the mechanism or molecule that bacteria uses to attack and kill tumor cells. There is a broad range of possibilities and, in many cases, it depends on the specific tumor type which is being treated. Other times, however, bacteria could encode a payload which is cytotoxic for different tumors. Many bacteria have shown a wild stimulation of the immune system and, in addition, they can be engineered with immunomodulatory proteins to enhance this effect or with other proteins that have a cytotoxic effect on its own. Other option is the expression of an enzyme that catalyse the transformation of a non-toxic prodrug to a cytotoxic molecule, which is called enzyme/prodrug therapy. Finally, genetic material can be transferred from bacteria to tumor cell, such as miRNAs to silence oncogenes or a DNA engineered for its expression in tumor cells.

4.2.1. WILD STIMULATION OF THE IMMUNE SYSTEM

Many bacteria are detected as a threat by the host organism, leading to the host immune system stimulation. In fact, this is the mechanism used in Bacillus Calmette-Guérin (BCG) immunotherapy, which is broadly applied in the clinic to treat bladder cancer and as a vaccine for tuberculosis. This bacteria induce the up-regulation of PD-L1 on both macrophages and dendritic cells through the secretion of IL-6 and IL-10 and the consequent activation of STAT-3, leading to superior CD4+_{T-cell responses to recall antigen (Copland et al., 2019).}

E. coli also exhibits a moderate antitumor activity because of the immune system stimulation with an increment in IL-6 and TNFα levels inside the tumor (Leventhal et al., 2020). Furthermore, the infiltration of S. typhimurium in colon tumour have demonstrated to stimulate the immune system because of the activation of macrophages with M1 phenotypes (Zheng et al., 2017) (Figure 5a).

Figure 5. Payload systems. (a) Wild stimulation of the immune system. (b) Immunomodulatory proteins that stimulate

the immune system. (c) Production of cytotoxic proteins. (d) Enzyme/prodrug system. Bacteria produce an

enzyme that transform a prodrug in an antitumor compound. (e) tkRNAi. Transference of RNAi from bacteria

to tumor cell to downregulate the expression of target oncogenes. (f) Transference of a DNA engineered for

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Despite of many engineered bacteria has been design with other type of active compound as a main resource against cancer, almost all of them have shown to stimulate the immune response, that contributes to tumor regression (Loeffler et al., 2007; Loeffler et al., 2008; Zhu et al., 2011; Leventhal et al., 2020).

4.2.2. IMMUNOMODULATORY PROTEINS

As previously discussed, some bacteria have the ability to stimulate the immune system and, consequently, originate tumor regression. In addition, this ability can be strengthened with an engineered bacterium producing an immunomodulatory protein, whose function is the attraction, activation and stimulation of immune cells in the tumor microenvironment (Figure 5b). One example is the LIGHT-expressing Salmonella, with whom successful results have been obtained (Loeffler et al., 2007). LIGHT (also known as TNFSF14 or HVEM-L) is a cytokine known to promote tumor rejection and generate lasting antitumor immunity by normalizing tumor vasculature, driving tertiary lymphoid structures neogenesis and improving effector tumor infiltrating lymphocytes infiltration (Skeate et al., 2020).

Moreover, an engineered S. typhimurium expressing the proapoptotic cytokine Fas ligand (FasL) has shown tumor regression in murine models of breast carcinoma, colon carcinoma, melanoma and lung metastases (Loeffler et al., 2008).

IFN-γ has also been engineered in S. typhimurium and used against melanoma. INF-γ inhibits tumour cell growth because it allows the recruitment and activation on NK cells and macrophages. It also enhances antigen processing and presentation. IFN-γ increases tumour recognition and its destruction by T cells. It can also induce an anti-angiogenic effect through the induced expression of chemokines, such as protein-10 and monokines, and through the development of INF-γ-dependent CD4+_{T-cells. Finally, IFN-γ activate macrophages, which} produce nitrite and Reactive Oxygen Species, that have an antitumorigenic effect (Yoon et al., 2017).

Other immunomodulatory proteins that has been used in bacterial cancer therapies are STING agonists. Activation of STING results in activation of Antigen-Presenting Cells (APCs) and production of IFNα and IFNβ (type I IFNs), that are required for the generation of antitumor CD8+ T cells. It has been demonstrated that administration of STING agonists stimulates reversion of immune-suppression and, consequently, the tumor regression (Jing et al, 2019). Recently, this strategy has also been used in an engineered E. coli expressing dacA gene, that encode a CDA-producing enzyme. CDA or cyclic di-AMP is an agonist of STING (Leventhal et al., 2020).

4.2.3. CYTOTOXIC PROTEINS

Bacteria can also be engineered with genes encoding cytotoxic proteins that would kill the tumor cells when released (Figure 5c). To be effective, a payload protein must be secreted, diffuse through tissue, and efficiently kill cancer cells (Jean et al., 2014). However, the barrier between the immunomodulatory and the cytotoxic proteins is not clear in some cases, because some molecules have both therapeutics effects at the same time.

In this regard, proteins from TNF family have been widely used. TNFα can induce both apoptotic and necrotic forms of cell death through the selective action on the neovasculature of tumors, stimulation of T cell-mediated immunity and direct cytotoxicity to tumor cells (Nuyts et al., 2001b), although it can also induce metastasis in some types of tumors (Ho et al., 2012). Another member of the TNF family is TRAIL, that causes apoptosis through the activation of caspase-3. TRAIL has been expressed in a non-pathogenic S. typhimurium under the control of RecA promoter against mammary tumors (Ganai et al., 2009).

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ClyA is a pore-forming toxin of E.coli, that is able to disrupt the membrane of target cells (Benke et al., 2015). Another pore-forming toxin is the α-hemolysin from Staphylococcus aureus (SAH), which has been expressed in E. coli and tested successfully in mice bearing mammary carcinomas (Jean et al., 2014).

On the other hand, Noxa is a transcriptional target of p53 that induces apoptosis through the activation of mitochondrial damage and the intrinsic apoptosis pathway. Its mitochondrial targeting domain (MTD) has been shown to cause massive necrosis and it has been fused with a CPP and introduced in S. typhimurium along with a lysis system (Jeong et al., 2014).

Vibrio vulnificus flagellin B (FlaB) has also been used successfully against TLR5-negative colon cancer in murine models (Zheng et al., 2017). Finally, soluble fms-like tyrosine kinase receptor (sFlt-1) is the extracellular part of VEGFR-1 and it has an antitumor effect because it competes with VEGFR-1 for binding VEGF and inhibits its function. B. infantis expressing sFlt-1 have reach promising results against Lewis Lung Cancer (LLC) in mice (Zhu et al., 2011).

4.2.4. ENZYME/PRODRUG SYSTEM

Bacterial directed enzyme/prodrug therapy (BDEPT) is a system in which bacteria encode a prodrug converting enzyme. This enzyme catalyses the transformation of a prodrug in the antitumor compound (Figure 5d). The advantage of this system is that the anticancer agents usually are toxic at therapeutic doses and using BDEPT it is possible to administer a prodrug with less toxicity, that afterwards would be transformed in the anticancer agent only in tumor tissue, where bacteria is located because of the tumor-targeting module. In this regard, it is possible to achieve higher doses of the anticancer agent in tumor tissue, without side effects in healthy tissues (Lehouritis et al., 2013; Friedlos et al., 2008). Several options are available (Table 3).

Table 3. Enzyme/prodrug systems

Enzyme Prodrug _compoundAntitumor References

Thymidine

kinase Ganciclovir Unknown (Tang et al., 2009) CDase 5-Fluorocytosine 5-Fluorouracil (Nuyts et al., 2002; Theys et _{al., 2001; Royo et al., 2007)} Myrosinase Glucosinolate Sulphoraphane (Ho et al., 2018)

CPG2 Derivatives from _{Glutamic acid} DNA interstrand _crosslinking (Friedlos et al., 2008) Nitroreductase CB1954 DNA interstrand _crosslinking (Nuyts et al., 2002; _{Swe et al., 2012)} Nitrate / Nitrite

reductases NO3- (and C3N4) Nitric Oxide (Zheng et al., 2018). β-Glucuronidase 9ACG 9AC (Cheng et al., 2008; _{Cheng et al, 2013).}

PNP 6MePdR 6MeP (Chen et al., 2012).

4.2.5. TRANSKINGDOM RNA INTERFERENCE (tkRNAi)

Transkingdom RNA interference (tkRNAi) are systems that can modifygene expression within the host genome. They have been used to silence oncogenes that are responsible for a tumorigenic process (Xiang et al., 2006). This approach needs a Release Module that allows the

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transference of tkRNAi from the bacteria to the cancer cell (Figure 5e).

RNAi is a small molecule of RNA which selectively downregulates the expression of genes with complementary sequence, through degradation of the target mRNA and prevention of protein expression (Nguyen et al., 2009). The main difficulty of this system is to transfer the RNAi effector to the tumor cell. This problem has been solved with E. coli encoding the gene inv (discussed in Release Module) and its efficacy delivering shRNAi has been proven successfully in oral squamous carcinoma cell in vitro (Ahmed and Lage, 2018) and in vivo against human colon cancer xenografts in mice (Xiang et al., 2006).

4.2.6. DNA

Finally, another option is the transference of a DNA to the cancer cell, using the Type III Secretion System (T3SS) discussed in Release Module. This DNA should be designed to allow its expression in mammalian cells, and it can encode any of the previous active payloads strategies, such as proapoptotic molecules (Figure 5f). The key point is to deliver a killer DNA to cancer cells or an antigen so that cancer cells could be recognised by immune system (Castagliuolo et al., 2005).

4.2.7. SELECTION OF THE PAYLOAD

For our engineered bacteria against NSCLC we proposed the enzyme/prodrug system, using the enzyme thymidine kinase from Herpes simplex virus and the prodrug ganciclovir. The effectivity of this payload against NSCLC have already been demonstrated (Määttä et al., 2004). Moreover, E. coli also shows wild stimulation of the immune system (Leventhal et al., 2020), enhancing the therapeutic effect of our bacteria.

4.3. RELEASE MODULE

The payload module is very important for the therapy, but it is equally important to engineer a system to the release of this payload, as in most therapies the molecules need to be released outside the bacterium to exert their anticancer effect. However, the release system chosen for a specific programable bacterium is not arbitrary, it depends on the type and mechanism of payload. Some type of release system could be ideal for a specific bacterial cancer therapy but may not work in another one. Over the years, some release systems have been described or developed. In this regard, several secretion systems are available, highlighting the T3SS which allows the transference of molecules directly from bacteria to host cell. Cytoplasmatic content can also be released through bacterial lysis, controlled by external induction or by quorum sensing systems (oscillator). Finally, bacteria can invade tumor cells to deliver its content.

4.3.1. SECRETION SYSTEMS

Gram-positive bacteria allow protein secretion directly to the extracellular medium because they have not outer membrane. They do that using the General Secretory (Sec) pathway. However, Gram-negative bacteria need more complex systems because of the outer membrane (de Keyzer et al., 2003).

The protein transport across cellular membranes in bacteria is a challenging biochemical feat. So far, seven secretion systems have been described in bacteria (Type I to Type VI and type VIII, T1SS to T6SS and T8SS). Five of them (T1SS, T2SS, T3SS, T5SS and T8SS) have shown its ability to secrete recombinant proteins (Burdette et al., 2018). Strategies based on the secretion systems can be divided into four categories: (1) modification of the secretion tags; (2) engineering the secretion system machinery; (3) transference of a secretion system to a different bacteria; and (4) the manipulation of the genetic regulation of this system (Burdette et al., 2018).

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These systems can be classified in two groups: One-step systems, that deliver the protein directly to the extracellular space; or Two-steps systems, that use an intermediate step in which the protein is exported to the periplasm firstly (Figure 6a). T1SS and T3SS are One-step systems, whereas T2SS, T5SS and T8SS are Two-step systems. The first step for all the Two-step systems are the Sec or Tat (Twin arginine traslocation) pathways (Sibbald et al., 2006). Almost all the secretion system requires a signal sequence or a peptide secretion tag for protein release. In fact, the efficacy of the secretion systems have been already checked in murine tumor models, using YebF secretion tag fused to myrosinase (Ho et al., 2018), pelB leader sequence fused to FlaB (Zheng et al., 2017) or residues 1 to 160 of SipB fused to IFN-γ (Yoon et al., 2017).

Figure 6. Release systems. (a) Secretion systems. (b) Type III Secretion System, that allow the transference of

molecules directly between bacteria and tumor cells. (c) Externally controlled lysis. A lysis gene is expressed

in response to an external inductor. (d) Bacterial invasion of a tumor cell through the expression of inv and

hly genes. Inv (red molecule) binds to β1-integrin (blue receptor) overexpressed in many tumor types.

4.3.2. TYPE III SECRETION SYSTEM (T3SS)

Among all the Secretion Systems mentioned above, one of the most used in engineered biotherapeutics is the third type. T3SS system has been described in Gram-negative bacteria and, contrary to the other six types, it forms a channel that connect the bacterial cytoplasm directly with the cytoplasm of the host cell, allowing trans-kingdom interactions, such as the transference of proteins or a genetic circuit designed for its expression in the cancerous cell (Deng et al., 2017) (Figure 6b). Proteins that will be secreted should incorporate a secretion tag, although there are several tags that can be used. This system has been applied in engineered S. typhimurium expressing immunomodulatory molecules to treat breast cancer, colon cancer, melanoma and lung metastases (Loeffler et al., 2007; Loeffler et al., 2008). To further control of this system, genes responsible for the T3SS formation could be placed under the control of an inducible promoter (Reeves et al., 2015).

4.3.3. EXTERNALLY-CONTROLLED LYSIS

The bacterial lysis is a very direct way to release all the bacterial content in the tumor microenvironment. It can be achieved with bacteria encoding a lysis gene under the control of an inducible promoter (Figure 6c). For example, the ‘Lys’ fragment of the phage iEPS5 have been placed under the control of the PBAD promoter, which is externally induced by L-arabinose. In this system, the bacterial lysis can be induced through the external addition of L-arabinose. This induction has been proved in vitro and in vivo using mice (Jeong et al., 2014).

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4.3.4. LYSIS CIRCUIT (OSCILLATOR)

The lysis circuit or oscillator is also a lysis system, but more complex than the previous one, because it is activated by Quorum Sensing and does not need external induction (Din et al., 2016).

Lysis circuit principle is based on the ability of the bacteria to lyse themselves when the population grows and reaches a certain density threshold. This synthetic genetic circuit is based on Quorum Sensing system and it is formed by 4 genes: (1) luxI, an enzyme that produces AHL; (2) luxR, a transcription factor activated by his binding to AHL; (3) ϕX174E, a bacteriophage lysis gene; and (4) the therapeutic protein or GFP as a reporter (Figure 13a).

The expression of the lysis protein is driven by a promoter activated by LuxR·AHL (Plux). So that, when bacteria reach the tumor, the population level is low (low AHL concentration too) and no self-lysis occurs. As bacteria proliferate, AHL accumulates until a specific threshold density is reached and the circuit lysis is activated because AHL binds to LuxR, and the active transcription factor LuxR·AHL triggers the expression of the lysis protein, causing the lysis of the bacteria and the release of all his content, the therapeutic protein included (Din et al., 2016). One of the aspects that makes this system so interesting is that the lysis circuit gets activated when the population density is high and becomes inactivated when it decreases, allowing the population growth again. With this approximation, the population density is kept at constant levels, while the release cycles of antitumoral compounds occur through the bacterial lysis, obtaining a treatment that can be maintained over time. For this reason, this system is also called Oscillator (Danino et al., 2010). Recently a programable E. coli has been engineered with this lysis circuit to release an anti-CD47 nanobody, leading to tumor progression in murine cancer models of lymphoma, mammary carcinoma and melanoma (Chowdhury et al., 2019).

Another type of Oscillator has been generated without bacterial lysis, using luxI and aiiA genes. LuxI produces AHL and aiiA encode an enzyme that hydrolyses AHL (Prindle et al., 2012a; Prindle et al. 2012b).

4.3.5. CELL INVASION

Some payloads need to be delivered inside the tumor cells. To ensure that the bacteria are able to penetrate the tumor cell, the inv locus of Yersinia pseudotuberculosis can be expressed in the synthetic bacteria. This gene encodes invasin, a bacterial surface protein that binds to β1-integrin in the target cell surface (Huh et al., 2013; Ahmed and Lage, 2018). Afterwards, bacteria are introduced within the host cell with endocytic vesicles. Moreover, bacteria should also encode the hly gene from Listeria monocytogenes, which produces listeriolysin O, a perforin cytolysin able to perforate phagosomal membranes after bacteria uptake, allowing the release of its content, such as a cytotoxic protein or a DNA designed for its expression in the host cell (Castagliuolo et al., 2005; Xiang et al., 2006) (Figure 6d).

This release system has been used in an engineered E. coli to deliver a tkRNAi against oncogenic HPV16-E7 protein to oral squamous carcinoma cells (Ahmed and Lage, 2018). In fact, a plasmid (TRIP) has been developed to ease the transference of a shRNA to tumor cells. TRIP contains three key elements: the shRNA expression cassette, inv gene and hly gene (Nguyen et al., 2009). β1-integrins are over expressed in several solid tumors, focusing bacterial action on tumor cells and not in healthy tissues (Blandin et al., 2015). To further control of the cell invasion, the inv gene have been expressed under the control of an inducible promoter by hypoxia or L-arabinose and the Quorum Sensing system (Anderson et al., 2006).

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On the other hand, some bacteria with immunomodulatory payloads have been demonstrated to have an antitumoral effect in phagocytosis-dependent manner, although the mechanism is not clear yet (Leventhal et al., 2020).

Other option is the use of cell-penetrating peptides (CPPs) fused to the target protein. CPPs are short, membrane-permeable, cationic peptides capable of targeting intracellular proteins and carrying cargo proteins into tumor cells. The best studied CPPs are those derived from the TAT protein of HIV-1, as well as DS4.3 derived from the potassium channel Kv2.1. The last one has been used successfully to deliver the prodeath domain MTD of Noxa to tumor cells in a murine mammary carcinoma model, using an engineered S. typhimurium (Jeong et al., 2014).

4.3.6. PAYLOAD DELIVERY DEVICE (PDD)

The Payload Delivery Device (PDD) is an adaptation of the Cell invasion previously discussed. It has been developed to improve the payload delivery inside the tumor cell, because it senses when the bacterium have been internalized and, then, it lyses bacterium and vacuole, leading to a better release of the bacterial content in the tumor cell cytoplasm (Huh et al., 2013). The PDD has 5 modules: (1) PLD or Payload Device; (2) ID or Invasion Device, formed by the inv gene constitutively expressed; (3) VSD or Vacuole Sensing Device, that sense the vacuole microenvironment and detects when the bacteria have been internalized, activating the SLD; (4) SLD or Self-Lysis Device, that cause bacterial lysis in response to VSD activation; and (5) VLD or Vacuole Lysis Device. In this system, bacteria produce the payload and invade the tumor cell. After that, the vacuole microenvironment is detected and the bacteria lyses itself and the vacuole, delivering the payload to the host cell. (Huh et al., 2013) (Figure 7).

Figure 7. Payload Delivery Device. Bacteria produce the payload (red circle). At the same time, it express invasin in its

surface (ID: Invasion Device), that binds to the β1-integrin in the surface of the tumor cell. Then, bacteria are phagocyted. Bacteria detect the low concentration of magnesium inside the vacuole, which activates the PMg promoter (VSD: Vacuole Sensing Device), driving the expression of the SLD (Self-Lysis Device) and VLD

(Vacuole Lysis Device). SLD is formed by 4 genes, which cause the bacterial lysis: lysozyme (S), holing (H), antiholin (H) and bacteriocine release protein (BRP). VLD is formed by two genes, which cause vacuole lysis: perfringolysin (pfo) and phospholipase C (plc). Once the bacteria and the vacuole have been lysed, the payload is released inside the tumor cell.

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