Anti-Cancer RNA Therapeutics

#### **Chapter 4**

## A New Era of RNA Personalized Vaccines for Cancer and Cancer-Causing Infectious Diseases

*Ana Ayala Pazzi, Puneet Vij, Nura Salhadar, Elias George and Manish K. Tripathi*

#### **Abstract**

RNA vaccines for cancer and cancer-causing infectious agents are recognized as new therapeutics and are perceived as potential alternatives to conventional vaccines. Cancer is a leading cause of death worldwide, and infections (certain viruses, bacteria, and parasites) are linked to about 15–20% of cancers. Since the last decade, developments in genomics methodologies have provided a valuable tool to analyze the specific mutations, fusions, and translocations of the driver genes in specific cancer tissues. The landscape of the mutations identified by genome sequencing and data analysis can be a vital route to personalized medicine. This chapter will discuss the present state of mRNA vaccine development and ongoing clinical trials in oncology.

**Keywords:** mRNA, therapeutics, cancer, clinical trials, vaccine

#### **1. Introduction**

Conventional vaccine approaches were adopted for infectious diseases, but the RNA (mRNA) vaccine developed for COVID-19 changed the vaccine development landscape, providing global recognition and a new alternative. Moreover, RNA vaccines consist of rapid development, scalability, and cell-free manufacturing [1]. RNA vaccines are the clinical reality and are being studied to treat cancer, diseases like HIV, influenza, and genetic disorders [2]. mRNA cancer vaccines have received lots of attention, and efforts have resulted in some rapid developments, especially in the last 5 years [3, 4].

Cancer is not an infectious disease; vaccines for cancer aim to clear active disease instead of preventing disease, the only exception being the recently approved vaccine that prevents cancers caused by the human papillomavirus (HPV) [5]. Cancer is a particularly unpredictable disease that occurs due to random genetic events, and mutations are the driving force [6, 7]. Even though most potentially detrimental mutations are eliminated or neutral in nature, one mutation may cause a single somatic cell to develop an advantage over the rest, generating a pattern of amplified proliferation and progression that, over time, gives rise to a cancerous tumor [8]. Genome profiling provides insight into the diversity and heterogeneity within each type of cancer, which is a significant challenge in finding the right therapy for each patient [9, 10].

#### **1.1 What is mRNA?**

Messenger RNA is a versatile, single-stranded molecule that mediates protein translation, posttranscriptionally regulates genes, and has other regulatory properties inside the cell [11, 12]. A mature mRNA will have a protein-encoding region, or open reading frame (ORF), between a start and a stop codon enclosed in a single strand with a 7-methyl-guanosine and untranslated region at the 5′ end and a poly-A tail with its respective untranslated region at the 3′ end. Both the 5′ cap and the poly-A tail are essential for mRNA maturation and stability, therefore heavily regulating the efficiency of protein translation and mRNA degradation [13, 14]. Generally, once the mRNA enters the cell, it has a short time to produce the protein it is encoding for before it starts to degrade [15]. This is a challenge when studying mRNA as a therapeutic delivery, especially in hereditary diseases [16, 17].

#### **1.2 RNA therapeutics**

mRNA presents a viable option for patient therapeutics comparable to existing cancer therapies [13, 18]. Since the inception of RNA-based cancer vaccination, many preclinical and clinical studies have explored the idea of mRNA-based anticancer vaccines using autologous RNA-transfected dendritic cells or direct injection into the organism. For instance, mRNA acts outside the cell nucleus, eliminating the need to bypass this membrane while still being a messenger for genetic information. In the cytoplasm, the exogenously delivered mRNA starts protein translation, whereas DNA must reach the nucleus first and then be transcribed into mRNA to produce an effect in the cell [15 19, 20]. Additionally, mRNA does not incorporate into the genome; instead, it produces proteins for a short period, significantly minimizing the risk of mutations in the patient and long-term side effects [21]. Moreover, mRNA drugs can be manufactured relatively inexpensively to express any protein for virtually any disease. Multiple research studies conducted during the past few decades have demonstrated the curative properties of this technology and its ability to target various health conditions [22–25]. This is particularlytrue in the case of synthetic mRNA-based vaccines that were developed rapidly

#### **Figure 1.**

*Key discoveries and advances in mRNA-based therapeutics. Created with BioRender.com.*

#### *A New Era of RNA Personalized Vaccines for Cancer and Cancer-Causing Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.110905*

during the COVID-19 pandemic, and many years of research in RNA biology paved the way for this unparalleled achievement. The first mRNA vaccine approved for emergency use for infectious disease (COVID-19) by the FDA was created by BioNTech and Pfizer [26]. The candidates for the vaccine (BNT162b1 and BNT162B2) were initially identified in Germany and were further studied in the United States [27]. These targets were chosen as they encoded the spike protein of the SARS-CoV-2 virus. The delivery method for this vaccine consisted of lipid nanoparticles [28]. The Moderna vaccine also targeted a similar gene product and was delivered intramuscularly to the patient. **Figure 1** shows the history of RNA and the recent development of mRNA-based COVID-19 vaccines.

#### **2. Challenges and advantages of mRNA vaccines**

The delivery of mRNA into a cell is particularly challenging due to the size of 300 to 5000 bp, in contrast to microRNA and silencing RNA, which only go up to 5–15 bp in size. Additionally, instability due to charges in the molecule is another factor that impairs its functionality as a therapy, as it cannot penetrate the cell membrane. However, some cells can uptake naked mRNA, a relatively inefficient process, because most cells have a low rate of mRNA uptake [29, 30]. In contrast, the immature dendritic cell is an exception, which can take up mRNA through the macro pinocytosis pathway and accumulate mRNA efficiently [15].

One advantage of mRNA vaccines is a simplified development process, which only requires a few laboratory techniques and resources. In contrast, the production of biologics such as plasmid DNA vaccines can be time-consuming and expensive compared to mRNA vaccines, thereby augmenting the interest in mRNA therapeutics. However, in the initial stages of the study surrounding mRNA vaccines, researchers struggled to stabilize the product and increase its safety profile [31, 32]. Some solutions to these issues included chemical modification of mRNA sequences (e.g., via nucleoside manipulations) and packaging into nanocarriers [33, 34]. RNA-active vaccines (protamine-formulated mRNA vaccines) encoding six prostate cancer-specific antigens (CV9104) and five non-small cell lung cancer (NSCLC) tumor-associated antigens (CV9201) have been investigated clinically for safety, overall survival, and progression-free survival [35].

The challenges that must be overcome in the production of mRNA vaccines include the negative charge of the RNA (which must cross the hydrophobic cell membrane) and the strong immune reaction of exogenous RNA, which can cause cell toxicity [29, 36]. Recent research has overcome these obstacles by personalization of vaccines for their ability to target specific diseases [16, 37]. Moreover, once synthetic mRNA is translated into protein in the cytoplasm, it is subsequently degraded within a few minutes or hours, thereby preventing any harmful effects.

Various forms of mRNA therapy include replacement therapy (to synthesize a defective protein), vaccination, and cell therapy (which entails ex vivo transfection) [16]. Another challenge is that antigen presentation is often short-lived, as mRNA can be degraded by exogenous RNases [21]. However, this can be addressed using self-amplifying RNA sequences utilized by alphaviruses, which prolong antigen expression [38].

#### **3. Immunology of vaccination**

The human immune system is comprised of innate and adaptive immune cells that play unique roles in eliminating a pathogen. The innate immune system serves as a

first-line response to a pathogen and acts via lysis or phagocytosis [39, 40]. Since it is possible for pathogens to evade this first-line defense, the adaptive immune system can prompt the activation of humoral and cell-mediated immunity (see **Table 1**) [33, 41]. Humoral immunity consists of B-cells that produce antibodies, which can eliminate a pathogen via various mechanisms. Antibodies may envelop the pathogen with their Fc (constant fragment) portions which are subsequently recognized by phagocytic cells [42]. Other mechanisms include the creation of immune complexes which trigger the complement cascade, expressing receptors on phagocytic cells and directly attaching antibodies to viruses via receptor binding sites [33]. Cell-mediated


#### **Table 1.**

*Immune response, products, and associated infectious diseases [33].*

#### **Figure 2.**

*Administration of vaccine leading to immunity production steps. Macrophages and dendritic cells are phagocytic antigen-presenting cells (APCs). Upon vaccine administration, these APCs take up the contents of the vaccine. After activation of APCs by specific antigens, the migration occurs toward lymph nodes (LNs) as shown. Within the LNs, the antigen is presented to lymphocytes for further activation. Antigen-specific B- and T-cells then multiply clonally to create their progenitors by recognizing the same antigen. Long-term protection is also achieved by the production of memory B- and T-cells against pathogen infection. Created with BioRender.com.*

#### *A New Era of RNA Personalized Vaccines for Cancer and Cancer-Causing Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.110905*

immunity clears infected cells via cytotoxic T-cells and T-helper cells. The B- and T-cells of the adaptive immune system are more specific to the pathogen, and vaccines seek to build up this response to evade the severe consequences of infection. Upon infection, the innate immune system prompts B-cells and T-cells (specific to the virus) increase in number, thereby strengthening their degree of protection [33, 43]. The vaccine entry requires uptake *via* antigen-presenting cells, which deliver the vaccine to secondary lymphoid organs where T- and B-cells are produced (see **Figure 2**).

Once the infection has cleared, some of the B- and T-cells will undergo apoptosis, but some may persist and will be able to respond if re-infection of the same pathogen

#### **Figure 3.**

*Adaptive immune responses after two different scenarios: (A) infection: This part of the figure represents the response after primary and secondary infection. The primary infection causes disease manifestation, as there is a lag in developing T- and B-cells. The secondary infection causes the memory T-cells to respond quickly and helps develop antibodies to fight the infection or pathogen. (B) Administration of vaccination follows a similar pattern without the manifestation of the disease. Created with BioRender.com.*

occurs (see **Figure 3**). Thus, the aim of achieving a faster immunological response to a pathogen is achieved through this mechanism [44].

For effective antibody production, the coordinated actions of CD4-positive follicular helper T-cells and B-cells depend on the successful presentation of a protein antigen, which is recognized by its specific B-cell clone in secondary lymphoid organs such as the lymph node and provides the first signal for B-cell activation [45]. This specific B-cell clone processes an extracellular protein antigen by uptake into endosomes and lysosomes for proteolytic digestion into peptides of varying length for incorporation into highly diverse HLA Class II molecules, which are imported from the endoplasmic reticulum [46] and can bind antigenic peptides of 10 to 30 residues in length. The mature HLA Class II molecule bearing its antigenic peptide is then expressed on the surface of the B-cell for presentation to CD4-positive follicular helper T-cells at the periphery of the follicles of secondary lymphoid organs. The interaction between the antigen-presenting B-cell and the follicular T-cell depends on specific recognition of the mature HLA Class II molecule containing its peptide antigen by its T-cell receptor. It provides a second signal for the activation of the B lymphocyte resulting in its proliferation and differentiation into antibody-secreting plasma cells and memory B-cells [47], with the latter capable of rapid response to a second exposure to its specific antigen resulting in antibodies of higher affinity.

Cell-mediated immunity targets cells functioning as reservoirs of infection or displaying foreign peptides. The mechanism of antigen presentation is analogous to the Class II pathway described above but differs in several ways. First, the protein antigen is present in the cytoplasm, which is processed by ubiquitin-mediated proteasomal digestion resulting in small peptide fragments about nine residues in length that are then imported into the endoplasmic reticulum. Here, they may bind to HLA Class I molecules if the fragments contain sufficient antigenicity. The mature HLA Class I molecules with their bound antigenic peptides are then displayed on the antigen-presenting cell surface for recognition by an activated CD8-positive cytotoxic T cell specific for this complex [48, 49]. Delivery of the cytotoxic payload of this effector T-cell results in the activation of the apoptotic pathway of the target cell and its elimination.

A second exposure to an antigen, such as a booster, is often required for a more robust and effective immune response. Thus, a successful vaccine design strategy requires this immunologic knowledge and characteristics of its protein target, where computational methods to determine peptide antigenicity among the highly polymorphic HLA molecules are helpful [50, 51].

#### **4. Clinical development of mRNA vaccines for the prevention of cancer-causing infectious diseases and as cancer therapeutics**

#### **4.1 mRNA vaccines for the prevention of cancer-causing infectious diseases**

Microbial infection accounts for around 15% of all human cancers, totaling approximately two million yearly cases [52]. Bacterium Helicobacter pylori, human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), and Epstein–Barr virus (EBV) are primarily responsible for 97% of these cancers [53]. Besides cancer-causing infectious diseases, mRNA vaccines are also being studied as a preventive treatment against influenza A, zika, cytomegalovirus, respiratory syncytial, and rabies [16].

#### *A New Era of RNA Personalized Vaccines for Cancer and Cancer-Causing Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.110905*

Currently, mRNA vaccines have been designed for two of seven viruses that can cause cancer (oncoviruses). One of the examples is the liposome-encapsulated mRNA vaccine for human papillomavirus type 16 (HPV-16). It encodes for the oncoproteins E6 and E7, which have the potential for immunomodulation and antineoplastic activities [54]. Upon intravenous administration, the liposomes protect the RNA degradation within the bloodstream leading to uptake by APCs [55]. Translocation to the cytoplasm leads to the translation of E6 and E7 oncoproteins. After the processing of the proteins, the peptide complexes are presented to the immune system and hence induce antigen-specific T-cell responses (CD8+ and CD4+) against HPV16 E6 and E7 [56]. The associated clinical trial is mentioned in **Table 2**. Another example is mRNA-1189 Epstein–Barr virus (EBV) vaccine. This encodes EBV's envelope glycoproteins (gH, gL, gp42, and gp220), which mediate viral entry into B-cells and epithelial surface cells, the primary targets of EBV infection [57, 58]. The viral proteins in mRNA-1189 are expressed in their native membrane-bound form for recognition by the human immune system.



#### **Table 2.**

*Clinical trials of mRNA encoding TAAs and TSAs (clinical trials.gov).*

Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of three human malignancies: Kaposi's sarcoma, primary effusion lymphoma, and the plasma cell variant of multicentric Castleman disease. Currently, there are no well-developed KSHV vaccine candidates. One of the clinical trials completed in 2019 looked at the impact of Valganciclovir on severe immune reconstitution syndrome (S-IRIS)-Kaposi Sarcoma (KS) mortality: an open-label, parallel, randomized controlled trial, in which 40 patients were randomized and 37 completed the study. It was concluded that Valganciclovir significantly reduced the episodes of S-IRIS-KS. Although attributable KS mortality was lower in the experimental group, the difference was insignificant. Mortality was significantly lower in EG patients with pulmonary KS [59].

#### **4.2 Development of mRNA vaccines as cancer therapeutics**

Several widely used conventional cancer therapies, such as chemotherapy and hormone therapy, have proven effective in treating cancer [60]. Chemotherapy involves a series of drugs that impair DNA synthesis, thus fatally interrupting the physiological processes of cancerous and healthy cells [61, 62]. However, the success rates for this treatment are most effective only in highly proliferative and low heterogeneity cancers. Alternatively, hormonal or endocrine therapy targets growth signaling pathways by interfering with hormone receptors in cancer cells [63]. Thus, it is suitable for low-proliferating cancers such as breast and prostate [64].

Among immunotherapeutic treatments, mRNA vaccines stand out due to their superior specificity and potential for adaptability according to the genetic profile of each patient's cancer. To produce an efficient, individualized cancer vaccine, specific genetic mutations in the cancerous cells are identified to produce neoantigens that

#### *A New Era of RNA Personalized Vaccines for Cancer and Cancer-Causing Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.110905*

could bind to T-cells and elicit an immune response in the patient more specifically than traditional systemic and local methods [37]. However, this treatment has faced challenges, such as a need to enhance the identification of potential genetic markers that could provide the specificity needed for cancer vaccines [23, 65].

RNA vaccines targeting various cancers are in the development and undergoing clinical trials. Examples of RNA cancer vaccines include CV9202 (CureVac), which targets multiple antigens found in non-small cell lung cancer [13]. Moderna is also developing an mRNA vaccine that targets the K-RAS proto-oncogene that plays a role in the pathogenesis of non-small cell lung cancer, colorectal cancer, and pancreatic adenocarcinoma [66]. The mRNA-4157 against melanoma, created by Moderna, and the BNT122 vaccine against prostate cancer, created by BioNTech, targets various solid tumors and are individualized vaccines [35, 67]. These specific vaccines are designed to elicit the immune response toward tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs) in malignant tumor cells. These vaccines used nextgeneration sequencing technology to identify and isolate antigen epitopes unique to each patient, creating a more refined vaccine. Various clinical trials exist for different cancer vaccines (see **Table 2**) [2]. TAAs are present in both normal tissues and tumors, as these are non-mutated self-antigens. For a few tumors, TAAs are desirable vaccine targets. However, immune tolerance responses, such as central and peripheral, may be triggered by vaccines that can express TAAs and can reduce clinical vaccination efficacy [68]. Therefore, these kinds of vaccines are still in a phase where they are used in combination with immune checkpoint inhibitors [69]. With many ongoing clinical trials in different phases and preexisting clinical information or data, personalized vaccines can potentially be effective in cancer treatment. BioNTech vaccine BNT122 RO7198457) and Moderna vaccine mRNA-4157 are two personalized mRNA-based cancer vaccines in phase II clinical trials.

There is a significant increase in ongoing or completed studies/clinical trials in mRNA vaccines. In addition, various other clinical trials evaluate the tolerability, safety, immunogenicity, and/or efficacy of mRNA-personalized vaccines in participants with tumors. In this way, we are stepping into a new era of therapeutic mRNA-based cancer vaccines or prevention and treatment of currently incurable malignant diseases.

#### **5. Summary**

This chapter describes the technology, the basics of the immune response, and examples of developing mRNA vaccines for cancer and cancer-causing infectious agents. They can be used for preventive and therapeutic purposes. This information is of value to interdisciplinary researchers, engineers, and healthcare professionals as it may impact the prospects of medical care. Built on the highly fueled interest and potential, we have complete confidence to predict an accelerated pace in mRNA therapy studies and development in the next decade, possibly providing many solutions for the prevention and treatment of currently incurable diseases.

#### **Funding acknowledgment**

This study is supported by NIH/NIGMS R16GM146696 and UTRGV SOM Startup funds to MKT and partially supported by AARG-NTF Alzheimer's Association and KSA International Collaboration grant from Saudi Arabia to MKT.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Ana Ayala Pazzi1,2†, Puneet Vij3,4†, Nura Salhadar5 , Elias George2,6 and Manish K. Tripathi2,7\*

1 Department of Biology, College of Sciences, The University of Texas Rio Grande Valley, McAllen, TX, USA

2 Department of Immunology and Microbiology, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX, USA

3 Department of Pharmaceutical Sciences, St. John's University, Queens, NY, USA

4 Michigan Public Health Institute, Okemos, MI, USA

5 School of Medicine, The University of Texas Rio Grande Valley, Edinburgh, TX, USA

6 Department of Medical Education, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX, USA

7 South Texas Center of Excellence in Cancer Research, School of Medicine, The University of Texas Rio Grande Valley, McAllen, TX, USA

\*Address all correspondence to: manish.tripathi@utrgv.edu

† Equal contribution.

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*A New Era of RNA Personalized Vaccines for Cancer and Cancer-Causing Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.110905*

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*A New Era of RNA Personalized Vaccines for Cancer and Cancer-Causing Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.110905*

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#### **Chapter 5**

### Perspective Chapter: RNA Therapeutics for Cancers

*Michiko Kurikawa, Marimu Sakumoto and Akihide Yoshimi*

#### **Abstract**

RNA therapeutics represent a promising class of drugs and some of the successful therapeutics have been recently transformed into clinics for several disorders. A growing body of evidence has underlined the involvement of aberrant expression of cancerassociate genes or RNA splicing in the pathogenesis of a variety of cancers. In addition, there have been >200 clinical trials of oligonucleotide therapeutics targeting a variety of molecules in cancers. Although there are no approved RNA therapeutics against cancers so far, some promising outcomes have been obtained in phase 1/2 clinical trials. We will review the recent advances in the study of cancer pathogenesis associated with RNA therapeutics and the development of RNA therapeutics for cancers.

**Keywords:** nucleic acid therapeutics, antisense oligonucleotide, cancer, aptamer, clinical trial

#### **1. Introduction**

Nucleic acid plays a central role in biology and it is an attractive tool for therapeutic applications due to multiple reasons. One of the major obstacles is the low *in vivo* stability of nucleic acid therapeutics due to nuclease sensitivity. Numerous synthetic oligonucleotides have been developed to overcome this obstacle using chemical modifications, phosphate backbone, and many other technologies. Some of these technologies have been shown to potently protect the oligonucleotides from degradation and enable efficient cellular uptake, which could be translated into the clinic. In fact, some RNA therapeutics have shown dramatic effects on neurodegenerative disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. Although there has been no approved RNA therapeutics in oncology so far, researchers have obtained a number of promising results from preclinical and clinical studies. In this chapter, we will concisely summarize the general characteristics of RNA therapeutics and review recent advances in the development of RNA therapeutics in the oncology field.

#### **2. RNA therapeutics**

RNA therapeutics represents a therapy with the use of RNA-based molecules to modulate molecular and biological processes to cure a specific disease or improve

symptoms. There are multiple classes of RNA therapeutics and each of them has its own strengths which would be difficult to achieve by using other drug modalities.

#### **2.1 Classification of RNA therapeutics**

Oligonucleotide therapeutics that have been investigated in clinical trials include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs) and aptamers (**Figure 1**).

#### *2.1.1 Antisense oligonucleotide (ASO)*

ASOs are small (~18–30 nucleotides), synthetic, single-stranded nucleic acid polymers that are complementary to the specific RNA through Watson-Crick base-pairing [1]. They are highly sensitive to degradation by nucleases in their naked form. In addition, their phosphodiester backbone makes it difficult to go through the plasma membrane. To resolve these issues, numerous efforts have been made to improve these situations by chemically modifying ASOs. As a result, there are currently three generations of modified ASOs. Chemical modifications and pharmacological profiles were reviewed in detail elsewhere such as in [2]. The main mechanisms of approved ASOs are classified into the following two categories [3]:

i.ASOs in the first category induce the cleavage of a target mRNA by binding to the target sequence. When this category of ASOs binds to the target mRNA, RNase H endonuclease recognizes the RNA**-**DNA heteroduplex, degrades the mRNA and downregulates gene expression.

#### **Figure 1.**

*A variety of RNA therapeutics and their mechanisms. Endocytosis is the main pathway for oligonucleotides to enter cells. Antisense oligonucleotides (ASOs) block the translation of target messenger RNA (mRNA) in RNase H-dependent and -independent manners. ASOs are also able to modulate RNA splicing. Mature mRNA is targeted by small interfering RNAs (siRNAs). The roles of microRNAs (miRNAs) are mainly classified into two types: miRNA mimetics that restore the levels of miRNAs and antagomiRs that suppress expression levels of target miRNA. Aptamers functions to block receptors, protein-protein interactions, etc. like antibodies, but they are smaller in size and easier to pass through the cell membrane compared to antibodies.*

ii.ASOs in the second category regulate splicing of pre-mRNAs generally by blocking the binding of splicing factors to cis-element such as splice sites, exonic splicing enhancer (ESE) and intronic splicing silencer (ISS). This category of ASOs is the most widely used strategy.

#### *2.1.2 Small interfering RNA (siRNA)*

siRNAs are non-coding RNAs that degrade the mRNA of the targeted gene. Exogenous double-stranded precursor siRNAs are taken up into the cell and processed by Dicer into 20–25 bp long, which are passed to Argonaut (Ago) protein and the sense strand is released [4]. The remaining antisense strand and Ago then form an RNA-induced silencing complex (RISC). Finally, the RISC seeks out and binds to the target mRNA and degrades it [5].

#### *2.1.3 microRNA (miRNA)*

In addition to siRNA, miRNA is another RNA therapy based on RNA interference. miRNA is a small non-coding RNA that degrades mRNA in the same way as siRNA. However, its mechanism is slightly different from that of siRNA. Transcripts expressed from miRNA genes are single-stranded RNAs, taking a hairpin structure. In the nucleus, miRNA transcripts undergo primary processing by Dorsha [6], which has an RNase III domain, and after that, Exportin5 transports them to the cytoplasm. In the cytoplasm, miRNAs receive secondary processing by Dicer and are cleaved as double-stranded miRNAs [7]. As with siRNAs, the single-stranded miRNAs then bind to Ago protein and form RISC. In addition, GW182 protein is required for target RNA degradation [8]. Via GW182, some RNA degrading enzymes assemble on the RNA and RISC destabilizes RNA instability.

#### *2.1.4 Aptamers*

The other category is RNA therapy targeting proteins. Aptamers are short singlestranded nucleic acids that bind to proteins. Its properties are achieved by its tertiary structures. Aptamer can have a wide range of functions including agonists [9, 10], antagonists [11, 12], bispecific aptamers [13, 14] and carriers for other drugs [15, 16]. Although its function is similar to antibodies, RNA aptamers are smaller in size and easier to pass through the cell membrane.

#### **2.2 Advantages of RNA therapeutics**

RNA therapy has several valuable strengths, which make the development of RNA technologies a worthwhile investment. These advantages could be summarized below:

#### *2.2.1 Targeting the untargetable, treating the untreatable*

One of the greatest advantages of RNA therapeutics is nicely condensed in the phrase above. RNA drugs can target "undruggable" molecules that are difficult or impossible to target with small molecule-based drugs or other modalities. Only about one-third of proteins can be targeted by common drugs such as small molecules and antibodies [17]. In addition, many proteins share similar structures, which makes it difficult to target specific proteins. On the other hand, as RNA drugs can indirectly

act on proteins before the translation, they function independently of protein structure. Furthermore, small molecules and monoclonal antibodies exert their effects by binding to the active site pocket of receptors or enzymes. For this reason, it is impossible for conventional drugs to target non-coding RNAs that are not translated. RNA drugs can target non-coding RNAs and are expected to greatly expand the range of therapeutic targets in the future [3]. We will review some examples of previously "undruggable" targets for which clinical trials are currently ongoing.

#### *2.2.2 Quick production*

As we all enjoyed the significant benefits from mRNA vaccines for coronavirus disease-2019 (COVID-19) in recent years, the next important advantage of RNA therapeutics is that RNA drugs can be designed and synthesized rapidly for clinical tests. Given that the development of small molecule or antibody-based drugs takes several years, this characteristic of RNA therapy is the biggest reason that we were able to control the COVID-19 pandemic by significantly reducing the rate of infection and the severity of the disease. By simply changing the sequence of RNA drugs according to the target genes/diseases, researchers can quickly create a novel RNA therapeutic for further testing within a short period of time. This leads to another advantage below.

#### *2.2.3 Patient-customized therapy*

Pharmaceutical companies generally hold the back investment for rare diseases as the market is small and the cost-benefit ratio is normally not attractive. However, RNA therapy might be a game changer in this scenario. A landmark trial of patient-customized ASO therapy for neuronal ceroid lipofuscinosis 7 (CLN7), a fatal neurodegenerative disorder (a form of Batten's disease) was reported in 2019 [18]. In this case, a mutation located in intron 6 of MFSD8 creates a novel acceptor, leading to a cryptic exon with a premature stop codon. The authors developed a tailored ASO to rescue the mis-splicing event and delivered it to the patient within 1 year after first contact with the patient. This led to a reduction in seizures without any serious adverse events. The fact that rare diseases affect approximately 30 million persons in the United States alone [19] highlights the importance of such rapid development of patient-customized treatments.

#### **3. Current advances in the development of RNA therapy for cancers**

Targeted therapies have greatly improved cancer management by specifically targeting the genetic alterations and consequent molecular disturbances that play an essential role in cancer initiation and maintenance. One of the major therapeutic successes would be inhibitors that specifically target constitutively active tyrosine kinases, such as imatinib and its second- and third-generation inhibitors specifically targeting BCR-ABL against Philadelphia chromosome-positive chronic myeloid leukemia (CML) [20] and acute lymphoblastic leukemia (ALL) [21]. Before the development of imatinib, treatment with interferon alfa plus cytarabine was standard care for patients with CML. In the landmark clinical trial of imatinib, newly diagnosed chronic-phase CML patients were treated with either imatinib or interferon alfa plus cytarabine. After a median follow-up of 19 months, the major cytogenetic response was 87.1% in the imatinib group versus 34.7% in the combination therapy group (P < 0.001) [20]. Based on the clearly superior therapeutic outcome, imatinib

*Perspective Chapter: RNA Therapeutics for Cancers DOI: http://dx.doi.org/10.5772/intechopen.107136*

became the first-line therapy in newly diagnosed chronic-phase CML. Other successful targeted therapies include vemurafenib for the constitutively active form of the BRAF kinase (BRAFV600E) in BRAF-V600E mutated metastatic melanoma [22] and the blocking antibodies such as anti-EGFR antibody for metastatic colon cancer [23] and anti-HER2 antibody for breast cancer with HER-2 amplification) [24].

On the other hand, targeted therapies remained to be developed for many other cancer-associated genes, especially for other 'undruggable' targets such as RAS and MYC. Although there has been no approved RNA therapy for cancers so far, extensive efforts have been focused on targeting such 'undruggable' targets by using a variety of RNA therapeutics, which will be introduced in this section.

#### **3.1 ASO therapy**

Targeted therapies through ASO have been most actively studied among RNA therapeutic and approximately half of ongoing clinical trials on RNA therapeutics are classified as this modality. Recently developed ASO therapies against cancers are summarized in **Table 1**.


*Abbreviations: AR, androgen receptor; AML, acute myeloid leukemia; Ph-ALL, Philadelphia-chromosome positive acute lymphoblastic leukemia; DLBCL, diffuse large B-cell lymphoma; MDS, myelodysplastic syndromes.\* The table does not include all the recent clinical trials on ASO therapies in oncology. This equally applies to* **Tables 2***–***4***.*

#### **Table 1.**

*Recent ASO therapy in clinical trials.\**

Some of the landmark trials in this field were performed or are currently performed as follows:

#### *3.1.1 ASO therapy against MYB*

Historically, the first clinical trial of ASO in oncology was a phase II study back in 1993, which evaluated G4460, an ASO targeting MYB in CML (NCT00002592). *MYB* is a proto-oncogene that encode a transcription factor. As evidenced by the discovery of translocations and duplications of *MYB* in a subset of T-cell acute lymphoblastic leukemia (T-ALL) [25, 26], MYB activation was shown to contribute to the leukemogenesis via differentiation block [25]. In addition, early studies using an antisense oligodeoxynucleotide and dominant-negative form of MYB have demonstrated that MYB activation is important for the proliferative capacity of myeloid malignancies such as AML and CML. Another study indicated that an oligomer complementary to the sequence of *MYB*-encoded mRNA resulted in significant growth inhibition in several leukemic cell lines [27, 28]. Based on these observations, G4460 was designed to bind the MYB mRNA and trigger RNase H-dependent degradation [29]. In a pilot study, CD34+ marrow autografts were purged with G4460 in allograft-ineligible CML patients. Although the clinical efficacy of G4460 could not be assessed in this pilot study, MYB mRNA levels were significantly reduced in approximately 50% of patients, suggesting the feasibility of transplanting G4460-treated autografts [29]. As described above, the standard treatment strategy for CML has been dramatically changed since imatinib and other tyrosine kinase inhibitors were developed. Nonetheless, MYB is an attractive target, considering that overexpression of MYB is associated with cellular proliferation and differentiation in multiple cancers including several types of leukemias and breast cancers [30].

#### *3.1.2 ASO therapy targeting BCL2*

BCL2 family of proteins have long been identified for their roles in apoptosis. BCL2 was initially discovered in the context of B-cell lymphoma in the 1980s, followed by the identification of a variety of homologous proteins [31–33]. The role of the BCL2 family is typically understood as the anti-apoptotic and pro-apoptotic members. By regulating outer mitochondrial membrane (OMM) integrity and function, BCL2 facilitates oncogenesis through cell death resistance [34]. In cancer, increased expression of BCL2 protein is frequently found [35] and is commonly associated with reduced susceptibility to chemotherapy and increased radioresistance [36]. These observations provided a rationale to target BCL2 in a variety of cancers.

Genasense (oblimersen, G3139) would be a representative ASO targeting BCL2, which targets codon 1–6 of BCL2 mRNA and triggers RNase H-dependent degradation [37]. More than 40 clinical trials have been performed on this ASO in a variety of types of cancers and Genasense obtained orphan drug designation for CLL in 2001. However, overall and progression-free survival was not affected and the primary endpoint was not reached by the treatment of Genasense in the following eight phase III studies. For example, combined fludarabine + cyclophosphamide + Genasense therapy resulted in a better response (complete + partial response) rate over fludarabine + cyclophosphamide therapy in CLL, which fulfilled only the second endpoint of the NCT00024440 trial [38]. Following these unsatisfactory results, Genasense was not approved and the production of Genasense was ceased.

#### *Perspective Chapter: RNA Therapeutics for Cancers DOI: http://dx.doi.org/10.5772/intechopen.107136*

Several other ASOs such as SPC2996 and PNT2258 have been developed to target BCL2. SPC2996 is a gapmer that targets the first six codons of the BCL2 mRNA. Although the phase 1/2 trial for evaluating SPC2996 was performed in CLL, approximately 40% of patients experienced painful inflammatory reactions [39]. PNT2258 is a liposome-encapsulated ASO that targets the BCL2 promoter to suppress its transcription. Although the safety of PNT2258 was confirmed in the phase 1 study, the following phase 2 trial targeting patients with diffuse large B-cell lymphoma (DLBCL) resulted in an unsatisfactory outcome with a very low response rate of 8.1%.

Following these failures of ASOs targeting BCL2, the development of ASOs against BCL2 slowed down. In 2016, the selective BCL2 inhibitor ABT-199 (venetoclax), a BH3 mimetic was approved as the first small molecule drug targeting a protein-protein interaction for chronic lymphocytic leukemia (CLL) [40]. Venetoclax has been also approved for the treatment of AML in combination with other chemotherapeutic agents such as DNA demethylating agents and low-dose cytarabine [41].

#### *3.1.3 ASO therapy targeting IFG-1R*

Results from a unique clinical study were reported in 2021 [42]. In this phase IB clinical trial, the safety and efficacy of IMV-001, an antisense oligodeoxynucleotide against IGF type 1 receptor (IGF-1R) mRNA were evaluated in adults with newly diagnosed glioblastoma. Glioblastoma is one of the most aggressive forms of brain cancer which represents approximately 15% of all brain tumors [43]. Despite intensive treatment, glioblastoma almost always recurs, leading to a dismal prognosis with a median survival of 10–13 months [44]. On the other hand, IFG-1R is highly expressed in a variety of malignancies, which regulates transformation and antiapoptotic effects and are essential for the survival and progression of malignant cells [45–48]. However, previous efforts to target IGF-1R alone were not successful [48]. Interestingly, IMV-001 had an off-target effect to activate Toll-like receptor 9 (TLR9) in antigen-presenting cells [49, 50], which stimulates the immune response. Therefore, the research group from Thomas Jefferson University designed a phase IA trial of IGV-001 to use an autologous cell combination product therapy [51]. More specifically, 12 patients underwent MRI-based image-guided tumor resection (which resulted in partial resections in all the cases). After diagnostic confirmation, an abdominal acceptor site between the rectus sheath and rectus abdominis muscle was created. On the other hand, the resected tumor cells were treated with IMV-001 *ex vivo* and encapsulated in several chambers. Immediately after irradiation to the tumor cells, chambers were implanted in the acceptor site and removed after 24 h (**Figure 2**).

While 3 of 12 patients were re-treated after the approval from FDA was obtained, 8 patients received no other treatment except surgical resection and/or best support care (and the other one exceptional case received temozolomide). As a result, there were no unexpected treatment-related complications except deep vein thrombosis, which was successfully managed by enoxaparin prophylaxis. Post-treatment observation identified two and four patients with complete and partial responses, respectively, which were atypical for the nature of aggressive glioblastoma. Among the patients with these responses with disease recurrence, three patients had unusual regression spontaneously or after surgical resection. Interestingly, perivascular lymphocytic infiltration was observed in some patients who did not have such infiltration at diagnosis, strongly suggesting a contribution of the immune response. Based on these results, IGV-001 was granted Orphan Drug designation by FDA in 2017. A total 33 newly diagnosed patients with glioblastoma were enrolled in the subsequent Phase

#### **Figure 2.**

*Study design for the IGV-001 treatment. After MRI-based image-guided tumor resection and diagnostic confirmation, an abdominal acceptor site between the rectus sheath and rectus abdominis muscle was created. The resected tumor cells were treated with IMV-001 ex vivo and encapsulated in several chambers. Immediately after irradiation to the tumor cells, chambers were implanted in the acceptor site and removed.*

IB study (ClinicalTrials.gov: NCT02507583). In this study, patients received IGV-001 and standard care which consists of maximal safe resection, adjuvant radiotherapy and temozolomide and maintenance therapy with temozolomide. Median progression-free survival (PFS) in the intent-to-treat population was 9.8 months, which was significantly better than that of patients who received standard care in published studies (6.5 months; P = 0.0003). Because the promoter methylation status of the *MGMT* gene was previously shown to positively predict the therapeutic efficacy of temozolomide [52, 53] and overall survival (OS) [54], the authors quantified the methylation levels of *MGMT* and revealed that the *MGMT* methylation status is a potent biomarker for PFS and OS. Furthermore, they assessed serum cytokines and identified that some of the pro-inflammatory cytokines such as IFNγ and IL-2 were elevated after IGV-001 treatment (before initiation of standard care). Although these responses were not associated with therapeutic outcomes, these results suggested that IGV-001 treatment induces a local environment at implantation which promotes a proinflammatory innate immune response [42].

#### **3.2 siRNA therapy**

Although most clinical trials on siRNA drugs in oncology are currently phase 1, there are some promising results from these trials. In addition, some phase 2 trials have been recently initiated (**Table 2**).

#### *3.2.1 siRNA therapy targeting MYC*

*MYC* is one of the most famous and most commonly activated oncogenes and has thus far been considered one of the major "undruggable" targets in cancers. As described above, a therapeutic approach using RNA interference (siRNA) is a

*Perspective Chapter: RNA Therapeutics for Cancers DOI: http://dx.doi.org/10.5772/intechopen.107136*


#### **Table 2.**

*Recent siRNA therapy in clinical trials.*

promising strategy because a number of studies have shown that silencing MYC induces growth inhibition in MYC-activated tumors in multiple cellular and animal models. An anti-MYC siRNA formulated in lipid nanoparticles called DCR-MYC has shown anti-tumor potential *in vivo* across several tumor models [55]. In phase 1 doseescalation study, 19 patients with a variety of cancers were treated with DCR-MYC. DCR-MYC was well tolerated and demonstrated promising clinical efficacy across various dose levels, including a complete response in one patient and tumor regression in several other patients, validating the hypothesis that siRNA targeting *MYC* is a potential therapeutic strategy to make the "undruggable" target druggable.

Recently, another strategy to pharmacologically target MYC was reported [56]. In this study, the authors performed a pan-cancer transcriptome and splicing analysis of RNA sequence data generated from cancer patients with or without hotspot mutations in *SF3B1*, which is the most frequently mutated splicing factor across cancer [57, 58]. In this study, detailed molecular and biological experiments using isogenic murine models and cancer patient samples revealed that a mis-splicing event in *PPP2R5A* induces MYC activation via post-translational modifications. More specifically, mutant SF3B1 induced 3′ alternative splice site in *PPP2R5A*, which led to a reduced protein expression of PPP2R5A, a regulatory B subunit of PP2A phosphatase complex. PP2A complex containing PPP2R5A was shown to regulate phosphorylation of MYC protein which was critical for the regulation of protein stability. Therefore, loss of PPP2R5A function stabilized MYC protein. Importantly, FDA-approved activator FTY-720 suppressed mutant SF3B1 leukemogenesis *in vivo*, providing a preclinical insight into the use of PP2A activators in SF3B1 mutant cancers [56]. Furthermore, the mis-splicing event in *PPP2R5A* can be potentially targeted by a specific ASO, which will also create a therapeutic opportunity for pharmacological intervention toward activated MYC.

#### *3.2.2 siRNA therapy targeting mutant KRAS*

Another "undruggable" target commonly detected across cancers, especially in pancreatic cancers is a hotspot mutation in *KRAS*. Based on the results that siRNA-mediated KRAS silencing resulted in growth inhibition of pancreatic cancer cells *in vitro* and *in vivo*, Silenseed Ltd. has developed a siRNA drug named siG12D-LODER, which is a siRNA targeting KRAS G12D and other additional G12X mutations such as G12C and G12V with a miniature biodegradable polymeric matrix. LODER™ allows slow and prolonged local release of the encapsulated drug. siG12D-LODER was designed to keep releasing the drug for 4 months, which can be inserted into the pancreatic tumor via a standard endoscope ultrasound-guided biopsy procedure.

In the phase 1/2a dose escalation and expansion study, patients with pancreatic cancer received a one-time dose of siG12D-LODER via endoscopic intervention with chemotherapy including gemcitabine or FOLFIRINOX. The combination of chemotherapy and siG12D-LODER was safe and well-tolerated, with five of 15 treated patients experiencing serious adverse events including grade 3–4 neutropenia and cholangitis. Regarding efficacy, the median OS was 15.1 months. Tumor progression was not observed in any patients at 8 weeks after the treatment. In addition, in 10 patients whose tumor marker CA19-9 levels were elevated at enrollment, more than 20% decrease in CA19-9 levels were observed in seven patients [59]. Following these promising results, a phase 2 clinical trial is recruiting patients with both borderline resectable and locally advanced pancreatic cancer [60].

#### **3.3 miRNA therapy**

Compared to the ASO and siRNA modalities, the number of clinical trials for evaluating miRNA therapeutics is limited as below (**Table 3**). However, research on miRNA or miRNA therapeutics are being greatly increased in number, according to a survey by Bonneau et al. [61].

Here are some examples of miRNA therapeutics developed or being developed. Therapeutic strategies using miRNA are mainly classified into the following two groups: (1) AntagomiRs to repress overexpressed miRNAs (Example: MRG-106), and (2) miRNA mimetics to restore downregulated miRNAs (Example: MRX34).


#### **Table 3.**

*Recent miRNA therapy in clinical trials.*

#### *3.3.1 miRNA therapy against miR-155*

miR-155 is overexpressed in various malignancies, especially in cutaneous T-cell lymphoma (CTCL) including Mycosis fungoides (MF) [62–64], and is associated with enhanced cell proliferation and survival [65–67] and genomic instability [68, 69]. In addition, in a number of studies, genetically engineered mice with overexpression of miR-155 murine homolog in lymphoid cells had an increased susceptibility to develop lymphomas and leukemias [64, 70–72]. Molecularly, miR-155 directly targets SHIP1 [73], SOCS1 [74] and some other cancer-associated genes. Overexpression of miR-155 is also related to activation of the PI3K-AKT [75], NF-κB [76] and JAK/STAT [77] pathways. Collectively, these observations provided a rationale to target miR-155 in cancer therapy.

Evidenced by these scientific results, miRagen therapeutics has developed cobomarsen (MRG-106), an oligonucleotide inhibitor of miR-155 which is optimized for efficient uptake in CD4<sup>+</sup> T-cell and MF cells with lipid nanoparticles. Cobomarsen was shown to de-repress direct miR-155 target genes as well as de-activate multiple survival pathways in MF cell lines *in vitro* [78]. The phase 1 trial of cobomarsen recruited 15 patients with biopsy-proven stage I-III MF [79]. Intratumoral or subcutaneous administration of cobomarsen resulted in almost no clinically significant adverse events. On the other hand, histological examination of pre- and posttreatment tissue revealed a reduction in cell density and depth in most patients. In addition, a gene expression analysis on these specimens demonstrated significant inactivation of PI3K-AKT, NF-κB and JAK/STAT pathways. This led to the Orphan Drug Designation of cobomarsen for MF type CTCL in 2017 and the initiation of phase 2 trials.

#### *3.3.2 miR-34a based therapeutic*

Accumulating evidence has demonstrated the presence of a normally small fraction of cancer cells, cancer stem cells (CSCs) which share stem-like properties with normal stem cells such as self-renewal and differentiation capacities. miR-34 is a tumor suppressive miRNA whose expression is frequently downregulated in many cancers [80] and CSCs.

miR-34 family is one of the three major tumor suppressive miRNA families consisting of miR-34a, miR-34b and miR-34c. Among them, miR-34a is known to repress the expression of >200 target genes and loss of miR-34a biologically regulates tumor growth by inhibiting multiple processes such as cell cycle, epithelial-to-mesenchymal transition, metastasis, immune response and stemness [81–83].

In addition, the loss of miR-34a is associated with CSC regulation in multiple cancer types. For example, MET, NOTCH1 and NOTCH2 were identified as direct targets of miR-34a in glioma stem cells [84] and restoration of miR-34a expression induced differentiation of glioma stem cells with increased expression of astrocyte and oligodendrocyte markers [85]. Another example comes from colorectal cancer where miR-34a functions as a cell-fate determinant of CSCs in this malignancy. Bu et al. identified that high miR-34a expression decreased both symmetric and asymmetric division (resulting in decreased CSCs and increased more differentiated daughter cells), while low miR-34a expression enhanced symmetric CSC-CSC division and suppressed asymmetric division [86].

The first-in-human phase 1 study was initiated to evaluate the maximum tolerated dose, safety, pharmacokinetics and clinical activity of MRX34, a liposomal miR-34a

mimic in 47 patients with advanced tumors [87]. Although MRX34 demonstrated some clinical response, including one patient with hepatocellular carcinoma exhibiting a prolonged partial response for 48 weeks and four patients with stable disease for more than 16 weeks, the trial was halted by FDA in 2016 due to severe immune reactions and deaths in four patients in the expansion cohort.

#### **3.4 Aptamer therapy**

Although there are only a limited number of clinical trials for Aptamer therapy as is miRNA therapeutics (**Table 4**), there are some promising results, especially from the studies on the aptamer targeting CXCL12.

#### *3.4.1 Aptamer therapy targeting CXCL12*

CLL is the most common adult form of leukemia in Western countries which is characterized by the expansion of mature monoclonal B-cells. It has been known that the tissue microenvironment confers survival advantage and drug resistance to the CLL cells via CXC chemokine ligand CXCL12 and other factors such as BAFF, APRIL and CD40 ligand [88–90]. Therefore, drug development has been focused on strategies that interrupt the crosstalk between CCL cells and the stroma such as bone marrow (BM) stroma cells (BMSCs). Importantly, the migration of CLL cells in the tissues is controlled by tissue gradients of chemokines. In the BM, CLL cells are attracted by the CXCL12, which is continuously secreted from BMSCs. The close proximity between CLL cells and BMSCs protects CLL cells from spontaneous- and drug-induced


*Abbreviations: AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; MM, multiple myeloma; HSCT, hematopoietic stem cell transplantation.*

#### **Table 4.**

*Recent aptamer therapy in clinical trials.*

*Perspective Chapter: RNA Therapeutics for Cancers DOI: http://dx.doi.org/10.5772/intechopen.107136*

#### **Figure 3.**

*Schema representing the roles of the CXCL12-CXCR4 axis in CLL. Constitutively secreted CXCL12 from the bone marrow stromal cells attract CLL cells via the chemokine receptor CXCR4, which creates leukemic niche in the bone marrow. Molecularly, CXCR4 activates the downstream multiple cancer-associated pathways such as PII3K/ AKT/mTOR, RAS/RAF/MEK/ERK and NF-κB pathways.*

apoptosis [90–93]. Besides these protective effects, CXCL12 enhances the expansion of BMSC-dependent pre-B cell clones [94] as well as activates multiple pro-survival pathways associated with ERK1/2, STAT3 and AKT (**Figure 3**) [90, 95, 96].

Because CLL cells are attracted via CXCR4, the chemokine receptor of CXCL12, the first small molecule targeting the CXCL12-CXCR4 axis was developed. A multicenter phase 1 study of plerixafor in combination with the anti-C20 antibody rituximab was performed in 24 patients with relapsed/refractory CLL. In this study, a median 3.3-fold increase of CLL cells in the peripheral blood was observed after the first administration of plerixafor, strongly supporting the mobilizing capacity of the drug on CLL cells or the CXCL12-CXCR4 axis and suggesting that plerixafor would contribute to the sensitization of CLL cells [97].

Another therapeutic approach to target the CXCL12-CXCR4 axis is the blockade of CXCL12. However, CXCL12 is highly evolutionary conserved, which hinders the development of antibody-based drug development for CXCL12. NOX-A12 (Spiegelmer), an RNA oligonucleotide successfully bypassed this issue by using a mirror image configuration of naturally occurring RNA. More specifically, the Spiegelmer technology enables an RNA oligonucleotide to bind target molecules with high affinity and specificity [98, 99]. The major merits of using a mirror-image configuration would be summarized as follows: (i) Spiegelmer is resistant to degradation by nucleases, (ii) Spiegelmer does not hybridize with native nucleic acids, (iii) Spiegelmer is immunologically "cold". NOX-A12 is a Spiegelmer that was designed to bind and antagonize CLCX12.

After a phase 1 trial in healthy volunteers was completed, two clinical trials were initiated. In a phase 1/2 trial (NCT01486797) [100], 28 patients with relapsed/refractory CLL were treated with NOX-A12 (olaptesed pegol) in combination with bendamustine and rituximab (BR). NOX-A12 was well-tolerated and there was no additional toxicity when patients were treated in combination with

chemoimmunotherapy. In addition, an overall response rate of 8%, including a complete response of 11% was obtained, with a median PFS of 15.4 months and a 3-year OS of >80%. These results compare favorably with those reported by BR alone and other recent BR combination trials [100–102], warranting further clinical development.

Similarly, NOX-A12 was evaluated in 28 patients with relapsed/refractory multiple myeloma (MM) in phase 2 clinical trial (NCT01521533) [103]. This was based on the scientific observations that CXCL12 plays an essential role in supporting myeloma cells in the bone marrow microenvironment and in mobilizing myeloma cells to the peripheral [104, 105]. Patients with MM were treated with NOX-A12 alone for 2 weeks in the pilot phase, followed by the combination treatment (NOX-A12 + bortezomib and dexamethasone) for up to 8 cycles. There were no unexpected adverse events. The overall response rate was 68%, including a complete response of 7% and a very good partial response of 18%. The median PFS and OS were 7.2 months and 28.3 months, respectively. Given that overall response rates in the previous MM studies of bortezomib and bortezomib-based combination treatment for relapsed/refractory MM patients were mostly within the range of 43–63% [106–111], the outcome of this phase 2 study is favorable. In addition, the overall response rates of CXCR4 inhibitor ulociplumab or plerixafor with bortezomib + dexamethasone were 40% and 51%, respectively [112, 113], suggesting that NOX-A12 is a promising approach to target the CXCL12-CXCR4 axis in MM. The results of these clinical trials emphasize the importance of further evaluation of NOX-A12 in MM.

#### **4. Conclusion**

Numerous efforts to develop RNA therapeutics against cancers have been made as we partly introduced in this chapter. Although there is currently no approval of RNA therapeutics in oncology, some of the phase 2 studies yielded promising results, which greatly encourages investigators in the field. On the other hand, oligonucleotide drug delivery has now almost matured to the position of clinical utility (there are excellent reviews on this topic such as [39, 114]). Therefore, it is possible that the outcome of a previously failed oligonucleotide therapeutic could be improved with the use of nextgeneration oligonucleotide or with a novel drug delivery system. These developments would provide expectation that RNA therapy for many cancers will be soon available through the use of precision genetic medicine.

#### **Acknowledgements**

This study is partly supported by the following grants awarded to A.Y.: Science and Technology Platform Program for Advanced Biological Medicine (grant number JP22am0401007) and the Japan-Canada Joint call for Strategic International Collaborative Research Program (SICORP; grant number JP22jm0210085) from the Japan Agency for Medical Research and Development (AMED), Grant-in-Aid for Scientific Research (A) (grant number 21H04828) and Home-Returning Researcher Development Research (grant number 19 K24691) from the Japan Society for the Promotion of Science (JSPS), Fusion Oriented Research for disruptive Science and Technology from Japan Science and Technology Agency (JST) (grant number 22-221036124), National Cancer Center Research and Development Funds

(grant number 2020-A-2), ASH Global Research Award from American Society of Hematology (ASH), CDP Special Fellow Achievement Award from Leukemia and Lymphoma Society (LLS), and grants from Shimadzu Science Foundation, The Yasuda Medical Foundation, the Chemo-Sero-Therapeutic Research Institute, The Sumitomo Foundation, the Uehara Memorial Foundation, Princess Takamatsu Cancer Research Fund, Takeda Science Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research and Astellas Foundation for Research on Metabolic Disorders.

#### **Author contributions**

A.Y. designed the manuscript. M.K., M.S. and A.Y. wrote the manuscript. M.S. and A.Y. prepared all the figures and tables.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Michiko Kurikawa, Marimu Sakumoto and Akihide Yoshimi\* Division of Cancer RNA Research, National Cancer Center Research Institute, Tokyo, Japan

\*Address all correspondence to: ayoshimi@ncc.go.jp

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 4
