Cancer

1 INTRODUCTION

1.1 Cancer

Cancer is one of the most tenacious global diseases, representing an immense burden to our society. One in five men and one in six women worldwide will get cancer during their lifetime, and one in eight men and one in eleven women will die from it. In 2018 alone, 9.6 million deaths were recorded. The number of cancer cases will constantly rise with the growing and aging population. Therefore, cancer represents a big threat to our health care.⁽¹⁾ The most commonly used cancer treatments are chemotherapy and radiotherapy the main purpose of which is to induce the apoptosis of tumor cells. Unfortunately, this kind of therapy has a high level of toxicity due to a limited specificity and not all types of cancers being curable with such an approach. Moreover, tumor resistance to chemotherapeutic drugs has been observed. Some drug-resistant tumors had overexpressed drug-efflux pumps that drive anticancer agents out of the tumor cells.⁽²⁾ That is why new ways of treating this persistent disease need to be found. In the last decades, two new promising ways of treatment have come to the fore; cancer immunotherapy (tumor vaccines, CAR-T cells therapy and cytokine gene therapy) and therapy based on nucleic acids. They present a lot of potential for curing previously incurable cancers or improving their therapeutic efficiency and minimizing treatment side effects. In gene therapy, therapeutic agents encompass; antisense oligonucleotides (ASOs), microRNA (miRNA), interference RNA (siRNA), and short hairpin RNA (shRNA) to regulate gene expression, mRNA, synthesis of an exogenous protein, or therapeutics based on gene and genome editing.^(3,4) They are silencing oncogenes such as cMYC or KRAS, and genes involved in drug-resistance (multi-drug resistance 1/MDR1) or restoring the expression of tumor suppressor genes.⁽⁴⁾ About 22 nucleotide-long noncoding RNA molecules, so-called miRNA can be found in some cancer tissues – negatively regulating gene expression and modulating a range of biological functions, from cell survival, proliferation, apoptosis, tumor growth, metastasis – alongside oncogenes and suppressor genes.⁽⁵⁾ miRNAs which promote tumor progression (oncomirs) can be overexpressed or those that reduce the inhibitory control over oncogenes, differentiation, and apoptosis can be downregulated. The miR‑34 family of miRNAs is a crucial regulator of cell growth in various types of cancers, including liver and lung cancers, and inhibits cell growth by targeting a group of oncogenes that are involved in cell cycle control, proliferation, apoptosis, and metastasis. The miRNA let‑7 is involved in the inhibition of RAS, which is a critical oncogene for lung cancer development. miR‑21 suppresses several

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key tumor suppressor genes, such as PTEN, tropomyosin 1 (TPM1), and programmed cell death protein 4 (PDCD4) while miR‑221 was found to be upregulated in many cases of human hepatocellular carcinoma and overexpression stimulates the growth of tumor.⁽⁶⁾ 1.2 Nucleic acid therapeutics

1.2.1 Gene therapy

Gene therapy is quite a novel field with substantial therapeutic potential and much room for improvement with which a variety of inherited and acquired diseases can be treated. The main aim is to modify, delete or replace abnormal gene(s) in target cells. After many setbacks, gene therapy is rapidly improving and many drugs based on gene therapy have been approved in Europe and the USA, mainly for diseases like hemophilia, lymphoid cancers, and neurodegenerative disorders.⁽⁷⁾ In 2003, Gendicine, developed by Shenzhen SiBiono GeneTech Co. Ltd, was approved by the China Food and Drug Administration (CFDA), becoming the first-ever approved gene-therapy drug in the world. It was based on an adenovirus vector for treating head and neck cancer. Gendicine-transduced cells express wild-type p53 protein, which mediates DNA repair cell-cycle arrest or induces apoptosis according to the cellular stress. From 2012, three gene therapeutics based on the adeno-associated virus were approved. Glybera in 2012, for treating the hereditary genetic disease lipoprotein lipase deficiency (LPLD), LUXTURNA in 2017, for treating inherited retinal disease (IRD), and ZOLGENSMA in 2019, for treating spinal muscular atrophy (SMA).⁽⁸⁾ Additional two therapeutics based on CAR-T technology were registered, Kymriah™ and Yescarta™. In the majority of clinical trials, more than 65 % have addressed cancer and 11 % inherited monogenic diseases to name a few.⁽⁹⁾ Gene editing is one of the new prospects that enables insertion, deletion, modification, or can replace a specific sequence in the genome. The CRISPR/Cas9 system exhibited stable and efficient genome editing and can be delivered as a plasmid or linear DNA encoding Cas9 and sgRNA using viral or non-viral vectors. Due to rapid progress in recent years, it is highly likely thatit will be possible to delete or replace genes that cause cancer or other genetic diseases soon.⁽¹⁰⁾ Insertion of a suicide gene, such as a toxin gene or a gene encoding an enzyme that can convert a non-toxic prodrug into a cytonon-toxic drug, into tumor cells, is also another possibility that can be used to fight cancer, but for this kind of therapy, highly specific delivery vehicles are necessary.⁽⁴⁾ Another promising kind of gene-based cancer therapy is through the theranostic sodium iodide symporter (NIS), which can deliver genes as a plasmid, enabling effective

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anticancer radioiodide therapy for hepatocellular cancer, and can also provie noninvasive imaging of the functional NIS gene expression.⁽¹¹⁾

1.2.2 Modulation of gene expression

Gene expression can be modulated by antisense oligonucleotides (ASOs), microRNA (miRNA), or interference RNA (siRNA). With siRNA, which is of exogenous origin, and miRNA, complementary mRNAs can be targeted, and gene expression can be modulated.

They are easy to synthesize and allow a large-scale synthesis and uniform production.⁽¹²⁾ Recently, a new drug based on siRNA technology has been granted marketing approval under a commercial name Leqvio® (Inclisiran). It is the first and only approved small-interfering RNA (siRNA) treatment in Europe for lowering LDL-C.^(13,14) Based on antisense oligonucleotides two drugs have been granted marketing approval. Alicaforsen for treating inflammatory bowel disease which is caused due to the upregulation of intracellular adhesion molecule-1 (ICAM-1), which is a cell surface receptor involved in the process of inflammation that can be downregulated with antisense oligonucleotide Alicaforsen.⁽¹⁵⁾ Mipomersen for treating familial hypercholesterolemia due to downregulation of apolipoprotein B leading to a decrease of very low-density lipoprotein and LDL levels.⁽¹⁶⁾ miRNA, which is about 22 nucleotides long, is of endogenous origin and its purpose is fine-tuning gene expression in cells. Long primary miRNA transcripts (pri-miRNAs) are generally transcribed in the cell’s nucleus by RNA polymerase II and processed by the RNase III enzyme Drosha into ~70 nucleotides stem-loop structures known as pre-miRNAs.

Then, exportin 5, dsRNA-binding protein, transport the pre-miRNA from the nucleus to the cytoplasm, where Dicer and its dsRNA-binding protein partners, TAR RNA-binding protein and protein activator of protein kinase R, process the pre-miRNA, longer siRNAs (27 nucleotides) or shRNAs (29 nucleotides, delivered with plasmid or viral vector and transported into cytoplasm.) and facilitate loading of the siRNA or about 22 nucleotides long mature miRNA duplex into AGO2 and RISC. If the loaded RNA duplex in RISC has perfect sequence complementarity, AGO2 cleaves the passenger strand. Active RISC contains the guide strand, which is complementary to the target sequence. If the loaded RNA duplex has imperfect complementary sequence, a bypass mechanism is used, in which helicase unwind the passenger strand from the guide strand and generate a mature miRNA strand and consequently active RISC. The mature miRNA strand targets 3′ untranslated regions (3′ UTRs) of mRNAs with which they share partial sequence complementarity. When the

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miRNA/siRNA strand recognizes target sites, it directs mRNA cleavage through catalytic domain AGO2 and silences gene expression.⁽¹²⁾ On the other hand, with the ASOs technlogy that targets miRNAs directly (anti-miRs), miRNA function can be inhibited. ASOs specifically bind with high complementarity to miRISCs and thereby block binding to endogenous mRNA targets. There are three well known strategies; miRNA sponges (Multiple miRNA-binding sites that serve as decoys for the targeted miRNA), chemically modified miRNA-targeting antisense oligonucleotides (anti-miRs), small-molecule inhibitors that can interfere with the transcription of primary miRNAs, inhibit pri-miRNA Dicer processing and loading into AGO2 and interactions between RISC and target mRNA.

However, there are still many obstacles that have to be surmounted in regard to hybridization-associated (anti-miRs are not able to differentiate between miRNAs within the same family or the siRNA guide strand might function as a miRNA if it comprises a seed-sequence that matches to the mRNA 3′ UTR regions), hybridization-independent off-target effects (immunostimulatory off-target effects), and delivery-related issues.⁽⁶⁾

1.3 Ways of molecular therapeutic delivery 1.3.1 Naked nucleic acids

As mentioned in the previous chapter, nucleic-acid therapeutics represent a huge potential for the fight against a wide variety of diseases, from cancer to viral infections, insulin resistance, multiple myeloma, hemophilia, Parkinson’s disease, lipoprotein lipase deficiency to name a few.^(6,7) However, there are always benefits and drawbacks. To begin with, nucleic acids have huge molecular weight and are much larger in comparison to conventional drugs.

In addition, they are hydrophilic and very negatively charged. Hence, their passive diffusion across the negative-charged lipid membranes into cells is practically impossible.

Furthermore, they are unstable in blood and serum, highly susceptible to degradation by exo- and endonucleases and prone to innate immune response via toll-like receptors/TLRs in the endosome, especially degradation by phagocytes. Therefore, their half-life is very short in vivo.⁽¹⁷⁾ To improve their stability, some chemical modifications at 2ʹ sugar position have been made (2ʹ-O‑methyl (2ʹ-OMe), 2ʹ-O‑methyoxyethyl (2ʹ-MOE), 2ʹ-fluoro (2ʹ-F) and locked nucleic acid), phosphate groups have been replaced with phosphorothioate and asymmetrical Dicer–substrate siRNAs have been introduced. These modifications increase stability, improve binding to targets and transport, may impede degradation from minutes to hours and can reduce immunogenicity.⁽⁶⁾ Some naked siRNA can be administrated by direct

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injection if local tumors, retina, skin or the central nervous system are being targeted or it can be administered topically to the lung epithelium.⁽¹⁸⁾ Other physical methods beside injecting naked nucleic acid therapeutics are particle bombardment, hydrostatic pressure, electroporation and magnetofection.⁽¹⁹⁾ However, injecting nucleic acid therapeutics into targeted tissues is not possible. Therefore, systemic delivery following intravenous injection is the only remaining option. To protect unstable nucleic acids and prolong their stability in blood circulation, chemical modifications can be made. Nonetheless, there is still practically no cellular uptake, no specificity to the targeted tissue, high susceptibility to glomerular filtration (5 min after administration, siRNA was observed in mouse urine.⁽¹⁸⁾) and high doses of the therapeutic agent are needed.⁽¹⁷⁾

1.3.2 Simple delivery systems

Chemical conjugation with cholesterol to the 3′ hydroxyl group of the chemically modified siRNA can facilitate incorporation into high-density lipoproteins (HDLs) or low-density lipoprotein (LDL) and lead to receptor-mediated endocytosis cellular uptake. This approach was very successful for the delivery into the livers or jejunum of mice. Another option for liver targeting is a conjugation with α‑tocopherol and a peptide that is able to penetrate the skin. It was reported that conjugated siRNAs were successfully delivered to the keratinocytes.⁽⁶⁾ For more specific targeting, chemically modified siRNA can be conjugated with antibody fragments or aptamers.⁽¹²⁾ Antibodies possess a high affinity and binding specificity, which makes them attractive vehicles for specific delivery. A commonly used approach is linking an RNA-binding protein to Fab or scFv fragments. However, this kind of delivery still has many drawbacks, such as low half-life, susceptibility to glomerular filtration and the need for high doses of the therapeutic.⁽¹⁷⁾ The observed problems have led to the search for other kinds of delivery.

1.3.3 Viral vectors

Another way of delivery is using a viral vector, by far the most often used vehicle for gene therapy. It has been used in over two thirds of gene therapy clinical trials.⁽⁹⁾ In the past and ongoing clinical trials, several different viral vectors have been utilized, such as the adenovirus (Ad), adeno-associated virus (AAV), herpes simplex virus (HSV), lentivirus (LV), and murine g-retrovirus. So far, three AAV therapies that treat spinal muscular atrophy (Zolgensma), a form of congenital blindness (Luxturna) and lipoprotein lipase deficiency (Glybera) which was later withdrawn after commercial flop, have been granted marketing

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approval.^(20,21) AAV therapies are favorable for in vivo gene transfer because of their safety profile in comparison to other vectors, broad in vivo biodistribution, ability to transduce a battery of tissues, lower capsid immunogenicity compared to adenoviruses and availability of different tropism of viral capsids. In addition, purification methods resulted in high yields and good transduction capacity. The main limitation is their packaging capacity. Overall, recombinant viral vectors have no replicating or pathogenic characteristics, and are still able to cross the cell and nuclear membranes to efficiently deliver the therapeutic gene and ensure long-lasting therapeutic expression. They are considered as the most efficient delivery vehicles because of a high degree of transfection. Modification of the viral structure brings greater specificity to the targeted tissue or cell and to gene expression.⁽²²⁾ On the other hand, they are susceptible to the immune system, which causes an innate immune response through pattern recognition receptors and an adaptive immune response. Viral vectors that contain viral proteins, to which humans were exposed in the past may be neutralized, by neutralizing antibodies because of pre-existing immunity.⁽¹⁰⁾ Additional drawbacks, such as limited genetic-load, cancer mediated by insertion of payload near cell-growth-controlling genes, and limited mass-production of viral vectors have led to development of other, non-viral delivery systems.⁽⁴⁾

1.3.4 Non-viral delivery systems

Non-viral delivery systems encompass a broad range, from organic to inorganic nanoparticles. Some inorganic nanoparticles that need to be mentioned are carbon nanotubes, which are biocompatible and formed by covalent carbon linkages through different C-C hybridizations, silica-based delivery systems, especially mesoporous which exhibit attractive characteristics of high cargo loading, gold nanoparticles that show low toxic and immunogenic nature, high permeability and retention effect that facilitates penetration and accumulation of drugs at the tumor sites, calcium phosphate nanoparticles that enable incorporation of nucleic acids therapeutics on their surface, and metal−organic frameworks.^(23–27)

Lipid nanoparticles, the leading non-viral delivery systems for nucleic acid therapeutics, represent a large group of organic nanoparticles. The most useful are a large unilamellar sub-class of liposomes in a size range of 100 nm. They possess a highly efficient reproducible encapsulation process, scalable, robust manufacturing, and stability of the product for at least one year at 4 °C. Because of their neutral surface, they can avoid rapid accumulation with

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serum proteins and macrophages. A step forward was made with introduction of cationic lipids, which can be easily assembled with negatively charged RNA and DNA molecules.

However, they were unstable, too large, too positively charged and expressed toxic side effect. These drawbacks were reduced by ionizable cationic lipids such as 1,2-dioleoyl-3-dimethylammonium propane (DODAP) and the gold standard for delivery into the liver dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA DLin-MC3-DMA is three times more potent than DODAP, despite only a slight difference in chemical structure. They form small nanoparticles in the size range of 100 nm or less, with low surface charge (ideal pKa between 6.2–6.8) at a physiological pH value that protect them from clearance by the immune system, minimalize nonspecific interaction of LNPs, and prevent aggregation with proteins.^(28,29) Their uptake is also facilitated through ApoE which associates preferably with neutral particles and mediates uptake via receptors containing ApoE binding ligands in the liver and brain tissue. They enter the cell through endocytosis where ionizable nanoparticles get protonated because of a higher endosomal pH (pH 5.5) and consequently combine with the phospholipids that are negatively charged, forming a cone shape that disrupts the endosomal bilayer and allows the DNA molecule to escape into the cytosol.⁽²⁹⁾ Unsaturated acyl chains are also supposed to have beneficial effects regarding the endosomal-escape theory. Circulation in the blood can be prolonged with PEG-lipids. However, potency of gene silencing is reduced but with introduction of dissociating PEG lipids, silencing can be improved. The most promising is their potential for delivery of mRNA to the liver and utilizing them as a factory to produce proteins, neutralizing antibodies, and as vaccines. They also show low toxicity. Yet, their uptake mediated by endocytosis is less than 5 %, as up to 60 % is recycled in the extracellular medium and the rest is degraded in lysosomes.⁽²⁸⁾ The second largest group are particles based on polymers such as polymeric nanoparticles, polymer micelles, polymersomes, polyelectrolyte polyplexes, polymer-lipid hybrid systems, and polymer-drug/protein conjugates. They mainly consist of self-assembled multivalent cationic polymers and polyanionic nucleic acids, which are formed by entropy-driven ionic interactions. Due to versatile polymer chemistry, a variety of polymers can be integrated into new carriers. Some polymeric systems are already approved for clinical use, still, macromolecular drug formulations have to correspond to the pharmaceutical standards and many challenges need to be surmounted regarding precision chemistry, purification, and high-end analytics. Furthermore, tumor heterogeneity represents another challenge.^(19,30)

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There are also some more exotic materials that can be used as non-viral vectors, such as black phosphorus-based, reactive oxygen and nitrogen species (RONS) scavenger-based systems, perfluorocarbon nanoparticles, sulfide-based systems, and oxide-based systems.

Properties of these systems are mostly related to the characteristics of the material.⁽³¹⁾ 1.4 Polyplexes as delivery systems

1.4.1 Delivery pathways

Polyplexes can be administrated only intravenously, their size ranging from 6 nm up to 400 nm which mostly depends on the number and type of nucleic acids. Polyplexes containing one molecule of pDNA can have a size of 25 nm, while those containing siRNA, only 6 nm.⁽¹⁹⁾ Nanoparticles, whose hydrodynamic parameter is smaller than 5-10 nm, are rapidly cleared by the kidneys, whereas nanoparticles above 200 nm are recognized by reticuloendothelial system and consequently degraded by macrophages. Particles of size from 10 to 400 nm, can passively accumulate in tumors by enhanced permeability and retention effect (EPR), which is a consequence of the tumor’s leaky blood vessels that enable penetration into the tumorous tissue. Nevertheless, different types of cancers are not equally permeable and that is why polyplexes of different sizes should be applied when targeting specific tumors.^(19,32) Due to their positive charge, their stability in blood circulation can be limited. Although a highly positive charge of nanoparticles can prevent aggregation due to electrostatic repulsion, it may cause non-specific interactions with negatively charged groups of plasma proteins, vessel endothelium and blood cells that can lead to partial or complete dissociation. They can form large self-aggregates or aggregates with blood components (mostly accumulated in pulmonary capillaries), such as erythrocytes or other blood cells, which can lead to serious conditions on larger scale. Moreover, they can also be recognized by innate immune system. This can be avoided by appropriate shielding with hydrophilic polymers as PEG, hydroxyethyl starch or pHPMA. They enable prolonged blood circulation, reduced unspecific cell uptake, cytotoxicity and aggregation, and increase accumulation of polyplexes in tumorous tissue. However, shielding polymers may affect the efficiency of endosomal escape, which can be solved with introduction of a pH-labile bond that can be cleaved in slightly acidic environment such as endocytic vesicles and tumors, thus restoring the endosomolytic capability.⁽³³⁾ The main way polyplexes enter the cytosol is by engulfment into the endosomal compartment and subsequent endosomal escape. There are three proposed mechanisms of endosomal escape for polyplexes: endosomal escape using osmotic

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pressure, escape induced by nanoparticle swelling and membrane destabilization.⁽³⁴⁾

pressure, escape induced by nanoparticle swelling and membrane destabilization.⁽³⁴⁾