Preface

*"It's time for scientists to shout about RNA therapies" Lorna Harries (Nature 574, S15. 2019)*

*RNA Therapeutics - History, Design, Manufacturing, and Applications* addresses recent advances in RNA-based drug discovery and commercialization. The design and development of RNA drugs have resulted in several distinct classes of treatment known as nucleic acid therapies. Chapter 1: "Introductory Chapter: RNA Drugs Development and Commercialization" describes and discusses these therapies, which include an impressive list of mRNA, SARNA, miRNA, siRNA, RNA analogs, ribozymes, antisense oligonucleotides, and CRISPR-based drugs.

The COVID-19 pandemic spurred a burst of development of mRNA vaccines, which culminated in rapid mRNA vaccine testing and approval for use in humans. Chapter 2 "Prophylactic Ribonucleic Acid Vaccines to Combat RNA Viral Infections in Humans" discusses these vaccines, while Chapter 3 "Ribozymes as Therapeutic Agents against Infectious Diseases" discusses therapeutic ribozymes.

Applications of RNA therapeutics range from infectious disease prophylaxis to treating cancer and chronic conditions, improving organ transplant outcomes, and correcting inherited mutations. Chapter 4: "A New Era of RNA Personalized Vaccines for Cancer and Cancer-Causing Infectious Diseases" discusses RNA therapeutics for various types of malignancies originatingfrom chronic viral infections or somatic mutations. Chapter 5: "Perspective Chapter: RNA Therapeutics for Cancers" reviews distinct classes of RNA therapeutics as well as examines the challenges in RNA drug engineering, delivery, and improvement of pharmacological effectiveness.

The optimization of RNA-based therapeutics enables countless opportunities in our pursuit of achieving the goals of individualized medicine. This is particularly applicable to rare genetic disorders for which RNA drugs may provide a cure. As such, Chapter 6: "RNA Interference Applications for Machado-Joseph Disease" discusses applications of RNA interferences for a rare neurodegenerative disease caused by abnormal expansion of trinucleotide repeats in non-coding regions of RNAs.

RNA therapeutics have significantly impacted medicine, economy, and overall public health and thus hold great promise to modernize health care.

#### **Irina Vlasova-St. Louis, MD, Ph.D.**

Section 1

Introduction

Individualized Genomics and Health Program, Johns Hopkins University, Baltimore, MD, USA

Genomics Specialist, Molecular Branch of Newborn Screening Program, Texas Department of State Health Services, Austin, Texas, USA

Section 1 Introduction

### **Chapter 1**

## Introductory Chapter: RNA Drugs Development and Commercialization

*Irina Vlasova-St. Louis*

#### **1. Introduction**

RNA therapeutics are chemically synthesized biomolecules with broad clinical applications, ranging from correcting inherited mutations to treating cancer, chronic conditions, improving organ transplant outcomes, and infectious disease prophylaxes (**Figure 1**).

#### **2. Applications of RNA-based therapeutics in medicine**

The development of RNA therapeutics has been an intense journey, with numerous stories of success and failure. The potential, and suitability, of recently discovered RNAs stemmed from several Nobel Prize-winning discoveries. For example, the Nobel prize for messenger RNA discovery was awarded to F. Jacob, J. Monod, and A. Lwoff in 1965 [1]. Almost 30 years later, P. Sharp and R. Roberts were presented with the Nobel Prize for the discovery of alternative mRNA splicing. The idea for mRNA

**Figure 1.**

*Applications of RNA-based therapeutics in medicine that are discussed in this book.*

technologies as biopharmaceuticals for infectious and oncological diseases materialized in the early twenty-first century. Two companies, BioNTech and Moderna, which were founded two years apart (in 2008 and 2010, respectively), began working on the commercialization of mRNA-based vaccines against flu and subsequently Ebola disease [2]. The COVID-19 pandemic has speeded up mRNA technologies and culminated in rapid mRNA vaccine testing and approval for use in humans. RNA-based therapeutic vaccines (e.g., those developed to fight against SARS-CoV-2 infection) have been proven to be safe and effective. Several of the vaccines were approved by the FDA and the European Commission (EC).

An interesting formulation of double-stranded RNAs is one which activates TLR-3 receptors. This drug is sold under the generic name Rintatolimod in South America and Canada. The drug is indicated for treatment of patients with chronic fatigue syndrome, a poorly understood complication of many viral infections [3]. RNA drugs have great therapeutic potential to modulate inflammatory responses and combat oxidative stress to prevent tissue and organ damage during severe infections; however, the investigations of RNA drug utility are still at the pre-clinical stage [4, 5]. Greater attention has been devoted to antiviral RNA therapeutics, several of which have progressed to clinical phases 2 and 3, including Favipiravir (against Ebola Disease) and siRNA drugs for the treatment of chronic hepatitis B and HPV virus infections [6–8]. The anti-SARS-CoV-2 RNA analogs Ledipasvir and Remdesivir have recently been granted FDA approval to treat COVID-19 infection [9–11].

RNA therapies are evolving as individualized treatment solutions for cancer. In 2006, Nobel Prize was shared by Professors A. Fire and C. Mello for their discovery of gene silencing by double-stranded RNA interference (RNAi) [1]. siRNAs (as well as miRNAs) have been tested to inhibit overexpressed genes in various malignant tumors, including multiple myeloma, pancreatic, and hepatocellular carcinomas [12]. Unfortunately, the side effects that were observed in the studies' participants along with poor efficacy resulted in the termination of many studies.

Antisense Oligonucleotides (ASO) became the number one choice for therapeutic design in the early twenty-first century to treat cancers that resulted from oncogene duplication or overexpression (e.g., C-MYB, BCL, IGF1R) [13]. Several ASO therapeutics have been incorporated into the conventional treatment of oncological diseases, including chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), and glioblastoma [14]. More recently, RNA aptamers and raptamers have been tested as multifunctional RNA drugs in the field of oncology [15]. For example, bi-specific aptamers were designed to activate receptors on tumor-infiltrating T cells against cancer-associated receptors. The aptamers linked to a siRNA against the gene of interest can downregulate the target gene directly in tumor cells or modulate tumor cell immunogenicity, thus enhancing anti-tumor immune response. Aptamers conjugated to chemotherapeutic molecules can be delivered in a cell-specific manner (e.g., if designed to bind tumor oncomarkers) [15]. Such properties significantly expand the portfolio of malignant diseases, including cancers with immunosuppressive properties.

Human trials of non-formulated mRNA- and mRNA-based dendritic-cell cancer vaccines have been taking place since the mid-2000s. Several dozens of ongoing clinical trials are well described in [16]. The majority of them is designed as study arms in combination with standard immune checkpoint therapies or individualized biologics to treat devastating cancers such as glioma, melanoma, prostate cancer, and nonsmall-cell lung, pancreatic, and colorectal neoplasms. The future goal is to achieve targeted delivery, attune kinetics of mRNA expression, overcome cancer mutation rate, and reduce unintended host-immune reactions [17].

#### *Introductory Chapter: RNA Drugs Development and Commercialization DOI: http://dx.doi.org/10.5772/intechopen.109951*

siRNA drugs has become invaluable in the field of transplantology, where life-saving hematopoietic stem cell transplantation is accompanied by numerous pre- and posttransplant complications [18, 19]. One of the complications is hepatic veno-occlusive disease/sinusoidal obstructive syndrome (VOD/SOS), which has been successfully treated by the drug Defibrotide, which was formulated as a mixture of single- and double-stranded oligonucleotides [20]. Patients who undergo transplantation procedures are often at high risk for GVHD and acute kidney injury, which now can be mitigated by siRNA against p53 mRNA (Teprasiran, Quark-Pharmaceuticals) [21].

Alnylam, a U.S.-based company, is pioneering siRNA treatments against rare hereditary diseases. Several siRNA drugs have already been approved by the FDA and granted orphan drug designation [22]:


Another group of rare diseases, hemophilia A and hemophilia B, are being evaluated for management with monthly subcutaneous administration of siRNA-based therapy fitusiran (Sanofi) [29]. Currently, novel siRNA drugs are entering clinical trials almost daily; information about them can be found at clinicaltrials.gov and ema.europa.eu/en/medicines. Many pre-clinical studies are in progress at academic institutions and biopharmaceutical companies [30].

S. Altman and T. Cech were awarded a Nobel prize for the discovery of catalytic RNAs, now named Ribozymes [1]. This diverse group of single-stranded RNAs acts as enzymes when folded into secondary and tertiary structures [31]. Several clinical trials investigated the utility of Ribozymes in the treatment of HIV-infected individuals [32, 33]. Therapeutic Ribozymes were designed and tested against angiogenic factor VEGF1, which is often overexpressed in cancer; however, due to higher interest in the commercialization of RNAi-based therapies, Ribozyme trials eventually stopped.

E. Charpentier and J. Doudna received the Nobel Price for the discovery of CRISPR-Cas in the middle of the COVID-19 pandemic [34]. CRISPR technology, which was initially designed to disrupt the gene of interest for experimentation, now is thought to be applied to treat inherited diseases. CRISPR-Cas is becoming a great alternative to siRNA therapeutic applications [35].

There are estimated 5000–8000 rare monogenic diseases that can be cured by gene therapies, including CRISPR-Cas [36]. Commercialization of CRISPR technology leads to several clinical trials that utilize CRISPR-Cas9 modalities to correct mutations that cause sickle cell anemia, β-thalassemia, cystic fibrosis, Duchenne muscular dystrophy, Huntington's chorea, and hereditary retinal degenerative diseases [24, 37]. The versatility of CRISPR-Cas therapeutic applications is wide and has the potential to provide twenty-first-century cures to newborns. Additionally, it may even provide cures, preconceptionally, to families affected by the genetic disease.

#### **3. Conclusions**

This book presents distinct classes of RNA therapeutics, ranging from singlestranded antisense oligonucleotides (ASOs) and subclasses of RNA interferences (miRNAs and other RNAi) to *in vitro* transcribed mRNAs and RNA vaccines. Also presented are some of the challenges in RNA drug engineering, delivery, and specificity. Additionally, the improvement of pharmacological effectiveness is discussed.

RNA therapeutics have already had a significant impact on medicine, the economy, and overall public health. They are becoming prescription drugs, and this holds great promise for modernizing healthcare [38]. National Genome Research Institute has recently launched a genotype-first approach to trace genomic variants back to human disorders. Accumulated data on human genome sequencing may inevitably lead us to a preventive medicine mindset. Monumental breakthroughs in molecular biology, computational chemistry, bioinformatics, and individualized genomics, which undoubtedly propelled RNA therapeutics through the commercialization stage, are also examined in this book.

#### **Acknowledgements**

I am thankful for the grant support received from the Association of Public Health Laboratories (APHL) during the editing of this book.

#### **Conflict of interest**

None declared.

#### **Acronyms and abbreviations**


*Introductory Chapter: RNA Drugs Development and Commercialization DOI: http://dx.doi.org/10.5772/intechopen.109951*

#### **Author details**

Irina Vlasova-St. Louis1,2

1 Individualized Genomics and Health Program, Johns Hopkins University, Baltimore, MD, USA

2 Molecular Branch of Newborn Screening Program, Texas Department of State Health Services, Austin, Texas, USA

\*Address all correspondence to: stlouis.irina@gmail.com; istloui1@alumni.jh.edu

© 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.

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Section 2

## RNA Vaccines and Drugs against Infectious Diseases

#### **Chapter 2**

## Prophylactic Ribonucleic Acid Vaccines to Combat RNA Viral Infections in Humans

*Irina Vlasova-St. Louis and Jude Abadie*

#### **Abstract**

Vaccines have evolved as widely applicable and available prophylaxes against infectious diseases. Advances in ribonucleic acid technologies revolutionized the biopharmaceutical field of vaccine manufacturing. Numerous novel mRNA-based vaccines that have been approved by the United States and European regulatory agencies are proven to be safe and effective in preventing disease. This chapter presents the history of RNA vaccine development in the context of preventing diseases caused by RNA viruses such as SARS-CoV-2, HIV, influenza, Chikungunya, Zika, RSV, PIV, HMPV viruses, Rabies, and Ebola. Advantages, disadvantages, and challenges in mRNA vaccine engineering, delivery, and safety are discussed. The formulation, safety, long-term effectiveness, and requirements for booster immunizations are presented using data from clinical trials. The results of these clinical trials highlight important milestones, setbacks, and ultimate advancements in vaccine development. mRNA vaccines have significantly impacted public health in a relatively short time, and they demonstrate great potential in serving as clinical public health prophylaxis against current and future pandemics. Future development is likely to include polyvalent, mosaic, and strain/lineage-specific individualized vaccines.

**Keywords:** prophylactic vaccines, mRNA vaccines, SARNA vaccines, RNA viruses, SARS-CoV-2, influenza, HIV, RSV, parainfluenza type 3 virus, human Metapneumovirus, chikungunya, Zika virus, COVID-19, Ebola, epidemic, pandemic, clinical trials, genomics surveillance, public health, emergency preparedness

#### **1. Introduction**

The history of vaccines development continually demonstrates their evolution as prophylaxes agents against the spread of disease. For example, as demonstrated with annual flu immunizations, vaccinations have been key in establishing herd immunity and preventing outbreaks of infectious diseases. The pandemic resulting from SARS-CoV-2 infections necessitated vaccine development in a fashion that was accelerated compared to standard regulatory approval processes. Biopharmaceutical companies initiated vaccine research and development as soon as SARS-CoV-2 sequencing data become available. This led to rapid and seamless transitions to clinical trials using conventional vaccine candidates, as well as mRNA vaccines.

Next-generation RNA sequencing continues to evolve as the primary method public health laboratories use to conduct genomic epidemiology surveillance. This is particularly important for novel zoonotic infections that can cross inter-species barriers with the potential to cause epidemics and, perhaps, pandemics. RNA viruses demonstrate continual genetic recombination, in conjunction with the rapid accumulation of mutations, due to the inefficient proofreading ability of viral replicases. Therefore, real-time viral genotyping is of critical importance to public health during outbreaks resulting from virulent RNA viruses. Genotyping data became the fundamental basis for vaccine design. Furthermore, it provided insight into vaccine breakthroughs and allowed vaccine optimization through transgene sequence modifications.

The four types of conventional vaccines include live-attenuated vaccines, wholepathogen inactivated vaccines, toxoid vaccines, and recombinant protein vaccines [1]. Inactivated vaccines and live-attenuated vaccines contain the whole pathogen. Live-attenuated vaccines (for example, against yellow fever, chickenpox, rotavirus, smallpox, or combined vaccine against measles, mumps, rubella (MMR)), are produced through various attenuation procedures [2]. These vaccines are quite immunogenic, and they can induce long-lasting humoral (systemic or mucosal) and cellular immune responses. However, whole virion vaccines are costly because viruses must be grown in cell cultures during commercial production [3]. There is a risk of reversion of live attenuated vaccines to a wild form, and this is why they are contraindicated for immunocompromised individuals. Poliovirus, hepatitis A, influenza, and rabies are the most successful inactivated vaccines. They can be conveniently freeze-dried for transport; however, large doses of virion administration are required, which can cause unintended adverse events due to host immune reactions. Additionally, the inactivation process may alter immunogenic epitopes confirmation, which makes vaccines less effective [4]. Toxoid vaccines immunize against toxins, which are produced by some bacterial pathogens (e.g., tetanus).

Recombinant DNA technologies produced recombinant protein vaccines. These vaccines were considered safer, with fewer adverse events in clinical trials. However, the identification of the best immunogenic antigen and the complexity of manufacturing design lengthened pre-clinical studies from several years to decades [4]. Protein vaccines often require adjuvants or conjugates to improve immunogenicity, stabilizers to maintain antigen conformation, and other nanomaterials – to improve internalization by antigen-presenting cells (APCs) *in vivo* [4]. The examples of most recent protein vaccines are hepatitis B and human papillomavirus (HPV). Traditionally, the development and production of these conventional vaccine types have been laborious and costly; furthermore, many of them lacked the efficacy to attain post-market approval.

Advances in nucleic acid technologies revolutionized the biopharmaceutical field of vaccine manufacturing. The ability of two novel types of vaccines, mRNA and DNA-based, to produce protein inside the immunized organisms, opened a new era in vaccinology [5]. However, unlike protein vaccines that are formulated without cargo, the DNA and mRNA vaccines required vehicles so that they could be delivered into cells [6]. Upon immunization, DNA vaccines use either plasmid or viral vectors to deliver the transgene into cells. Various lipid nanoparticle cargos have recently been developed for mRNA vaccines to increase the efficiency of cytoplasmic delivery. The poor stability of mRNA molecules (*ex vivo* and in *vivo*) requires additional considerations for formulation and storage (**Table 1**) [7]. Several biochemical solutions for RNA chemistries and lipid nanoparticle design have been proposed and thoroughly reviewed [8–11]. The major challenge identified for mRNA-based vaccines is achieving


#### **Table 1.**

*Advantages and disadvantages of DNA and RNA-based vaccines.*

effective *in vivo* translation and identifying the correct/optimal dose of immunogen [12]. Therefore, despite the cost-effectiveness of *in vitro* synthesized mRNA vaccines and the potential for attaining large-scale manufacturing, the formulation of mRNA vaccines for delivery was an obstacle for several decades that has recently been overcome [13]. The history of successes and failures in vaccine development against infections caused by RNA viruses is elucidated throughout the literature review for infections caused by the Ebola virus, SARS-CoV-2, rabies, Zika, HIV, influenza, and the respiratory syncytial virus (RSV).

#### **2. SARS-CoV-2 RNA vaccines**

Coronaviruses are enveloped and contain between 25 and 32 Kb of non-segmented positive-sense RNA. Before the emergence of SARS-CoV-2, coronaviruses caused sporadic epidemics around the world [14, 15]. As described in [16], early during the COVID-19 outbreak, next-generation sequencing (NGS) of SARS-CoV-2 RNA provided valuable data about viral genome, its molecular origin, and a deeper understanding of pathogenicity.

As the COVID-19 pandemic spread, the world anxiously anticipated vaccine countermeasures [17]. At that time, mRNA vaccine development was the scientific leader in our fight to end the pandemic. It is nothing short of spectacular heroism and scientific acuity that novel, effective mRNA vaccines were developed in less than 1 year and awarded emergency use authorization (EUA) in the United States. EUA authority allows the Food and Drug Administration (FDA) to approve medical products in order to diagnose, treat, or prevent life-threatening diseases during times or circumstances when no viable alternatives exist during public health emergencies. The Secretary of the US State and Human Services declared the COVID-19 public health emergency on January 31, 2020.

The first batch of Moderna's mRNA-1273 was released for Phase 1 study in the United States in February 2020. This vaccine targeted the receptor binding domain of the Spike protein subunit and was encapsulated in lipid nanoparticles. The cytosolic delivery and temporary presence of mRNA in the cytoplasm improved the safety profile of these nucleic acid vaccines. To assess safety, Pfizer and BioNTech launched phases 1 and 2 clinical trials with the mRNA vaccine during the subsequent months. The primary goal for the phase 2 trial was to achieve *in vivo* protein translation and induction of humoral immune responses upon intramuscular injection. When phases 1 and 2 were successfully completed, the FDA approved phase 3 in conjunction with EUA-authorized vaccine use [18, 19]. While perhaps not expected, it was quickly realized that mRNA vaccines were neither 100% effective nor 100% safe. Subsequent infections, caused by SARS-CoV-2 lineage Omicron, were accompanied by numerous vaccine breakthroughs. Fortunately, novel variants have been associated with milder diseases demonstrated by lower rates of morbidity and mortality. Investigational findings after showed that the anti-SARS-CoV-2 neutralizing antibody titers declined about six months after initial vaccination, which supported recommendations for a booster vaccination. Booster vaccines, like the initial vaccinations, were neither fully effective nor safe. Adverse reactions reported among vaccinations include myocarditis, thyroiditis, systemic vasculitis, and vaccine-associated pulmonary immunopathology [20–22].

Another new type of vaccine known as self-amplifying RNAs (SARNA) has recently completed pre-clinical studies [23]. SARNAs are synthetic RNAs capable of *in vivo* self-amplification for 40 to 60 rounds, a feature supported by their delivery with an alphavirus replicase gene that encodes an RNA-dependent RNA polymerase (RdRP) [24]. SARNA and RdRP can be synthesized as two different amplicons or formulated as one *cis*-amplicon sequence in the lipid nanoparticle cargo. The ability to undergo several rounds of replication *in vivo* makes the SARNA vaccine more costeffective than mRNA. However, SARNAs constructs are larger than those of mRNAs, and that feature may adversely alter the effectiveness of delivery. This concern is currently being addressed in phase 1, open-labeled trial NCT05155982. The study design includes 8 arms in which participants are administered 25 to 50 micrograms of SARNA-based COVAC-1 vaccine or placebo [25]. Two other SARNA vaccine candidates entered phase 2 clinical trials in the United Kingdom (randomized-controlled ISRCTN17072692, and NCT04758962) to assess the safety and measure the titers of vaccine-induced serum (IgG type) binding antibody responses to the SARS-CoV-2 S glycoprotein [26, 27].

Interestingly, both types of vaccines (mRNA and SARNA) elicited not only antigen-specific antibody responses but also antigen-specific T-cell responses, while SARNA elicited a stronger response at lower doses in mice [28]. A novel self-amplifying messenger ribonucleic acid (SAmRNA) trial by Gritstone Bio, Inc. is recruiting HIV-infected individuals to assess vaccine safety. Vaccines use a codon-optimized cassette covering multiple epitopes from the SARS-CoV-2 spike and non-spike proteins and additional T cell epitopes (NCT05435027) [29].

#### *Prophylactic Ribonucleic Acid Vaccines to Combat RNA Viral Infections in Humans DOI: http://dx.doi.org/10.5772/intechopen.108163*

In lieu of fast changes in SARS-CoV-2 lineages, a variety of RNA vaccine reformulations may be needed to maintain emergency preparedness for future responses. A new development has recently been announced: the FDA granted emergency use authorizations (EUAs) to new formulations of both Pfizer-BioNTech and the Moderna COVID-19 vaccines. Authorized bivalent formulas, so-called "updated boosters" now contain two mRNA components of SARS-CoV-2 virus: the first is the originally approved (against lineage A of SARS-CoV-2); the second is common between the BA.4 and BA.5 lineages of the Omicron variant of SARS-CoV-2 [30]. Ongoing genomic surveillance of SARS-CoV-2 variants of concern allows real-time detection of immune escape mutations and prediction of vaccine breakthroughs [16].

#### **3. Vaccines against human immunodeficiency virus infection**

Human Immunodeficiency Virus (HIV) continues to present a serious global health threat since it made its appearance as a human-to-human transmitted pathogen, causing acquired immunodeficiency syndrome (AIDS) [31]. HIV1 and HIV2 are single-stranded positive-sense RNA retroviruses that are subdivided into several distinct classes [16]. HIV vaccine designs appeared to be the most challenging among other RNA viruses, due to frequent mutations, integration into the human genome, and a long latency phase [16].

There were more than two dozen HIV clinical trials conducted since early the 1990s that tested plasmid DNA-based protein vaccines as prophylactic or therapeutic types. These clinical trials were successful in phases 1 and 2; however, they were stopped in mid-phase 3 for futility by Data Safety Monitoring Board (DSMB) [32]. DSMB data analysis study did not find a statistically significant decrease in the number of HIV infections in the vaccine compared to placebo groups [33]. It was determined that the elicitation of humoral immune response was not robust enough to prevent infection or stop the progression of AIDS [34].

Numerous recombinant DNA-based vaccines were tested against several immunogenic epitopes (e.g., HIV protease, gag, env, gp120/140, or reverse transcriptase proteins) [35–37]. Various routes of administration (mucosal, intradermal, intravenous, and intramuscular) were also tested [33], and intramuscular injections were found to be the most efficacious. Recombinant DNA-based HIV vaccines generated only modest HIV-specific T cell and humoral responses, and that was insufficient for protection [38, 39]. Research studies on therapeutic vaccines continue to be performed. The randomized, double-blind, placebo-controlled dose escalation trimer-4571 vaccine (against HIV envelope protein) in combination with alum adjuvant has been the most widely reviewed study [40].

Ongoing challenges in HIV vaccine development include frequent HIV virus mutations that can lead to a glycan shield that covers HIV immunogens prompting Scripps Research Institute and Moderna's team to design a trimeric mRNA vaccine against HIV/AIDS (NCT05217641). The study focuses on recruiting participants who will be immunized with various doses of a modified trimeric vaccine composed of mRNA against glycan shields, CD4KO, and gp151 [41, 42]. Another phase 1 trial (NCT05001373) evaluates the safety and immunogenicity of two mRNA vaccine types after intraperitoneal administration. That trial aims to detect antigen-specific epitopes on CD4+ T cells and B cells in peripheral blood and in the germinal centers of secondary lymphoid organs [43]. Both mRNA trials are designed to induce Broadly Neutralizing Antibodies (BNAbs) in HIV-uninfected adult participants [44].

Different studies' interpretations differ in opinion with respect to the benefits and ability of HIV vaccines in activating endogenous single/double-stranded RNA sensing molecular machinery [45, 46]. It has been shown that in patients with advanced HIV infection, the immune system functions in absence of a sufficient amount of cytokine interferon-gamma (IFNG), and the innate immune branch often exhibits exaggerated responses to antigenic stimulation [47, 48]. Clinically, such responses are seen in the form of immune reconstitution inflammatory syndrome (IRIS) toward persistent antigens from previously treated opportunistic infections [49, 50]. Because they are not specific, vaccines can cause exaggerated systemic innate immune responses that lead to adverse events in immunocompromised individuals through activation and overexpression of TLR 3,7,8 OAS1–11, MDA1–5, IRFs, IFI, type 1 interferon genes, and the components of the inflammasome [49–52]. Adequate levels of interferon-gamma are necessary to establish appropriate virus-specific cytotoxic lymphocyte responses after therapeutic vaccination [53, 54]. IFNG is primarily produced by mature CD4+ T helper cells, which demonstrates low counts in immunocompromised individuals [55]. Therefore, the response to vaccines that are supplemented with adjuvants can be unpredictable [55]. The NCT04177355 trial evaluates the safety and immunogenicity of the HIV1BG505SOSIP.664gp140 vaccine in healthy HIV-uninfected adults [56]. This vaccine is formulated in combination with TLR-7/8 agonists and alum adjuvant (inflammasome activator). Yet, the safety of the vaccine/agonist/adjuvant combinations is needed to be assessed in HIV-infected populations to demonstrate clinical utility.

The major disadvantage of *in vitro*-transcribed mRNA vaccines is the unstable nature of mRNA molecules which often leads to their degradation by intracellular enzymes ribonucleases (i.e., RNases) [57]. mRNA synthesized by *in vitro* preparations can generate a small percentage of double-stranded RNAs that trigger activation of pathogen-associated molecular pathways through induction of interferon response genes [11]. The end result is enhanced mRNA degradation and a decrease in antigen production [58]. This is the main reason why formulations that used naked mRNA were unsuccessful [55]. Additionally, the poor thermal stability of mRNA vaccines requires product refrigeration. Those logistical constraints can present with problems during the distribution of the product in resource-limited settings (**Table 1**).

Perhaps the vaccine formulation for prophylactic and therapeutic vaccination should be different as the goal of the latter is to prevent the infection via various routes, and the former is to control localized viral replication. Researchers remain hopeful that novel self-amplifying vaccine formulations will lead to effective mosaic anti-HIV vaccines that completely interrupt HIV transmission and prevent subsequent infection [11].

#### **4. Influenza vaccines**

Influenza viruses are negative-sense, enveloped, segmented single-stranded RNA viruses that are encapsidated by nucleoproteins [59]. Several approved influenza vaccines were developed through recombinant DNA technology. These vaccines are reformulated annually based on predicted hemagglutinin (HA) and neuraminidase (NA) gene mutations (drifts and shifts). Constructs are delivered with baculovirus vector into host cells and recombinant HA protein is manufactured as a vaccine [60]. Influenza type A HA is subdivided into heterosubtypic groups 1–18, and influenza B - into 9. Several vaccines from four main biopharmaceutical companies are cleared by FDA: Fluad Quadrivalent and Flucelvax Quadrivalent are inactivated vaccines (Seqirus), Fluarix Quadrivalent is also inactivated vaccine (GlaxoSmithKline

*Prophylactic Ribonucleic Acid Vaccines to Combat RNA Viral Infections in Humans DOI: http://dx.doi.org/10.5772/intechopen.108163*

Biologicals), and Flublok Quadrivalent is a recombinant influenza vaccine (Protein Sciences Corporation).

Many more vaccines are in clinical trials measuring primary outcomes as the humoral immune protection against surface viral proteins of seasonal avian influenza strains/subtypes/groups [61].

Pre-clinical trials in 2009 tested DNA plasmid carriers that contained genes that express viral antigens [62]. The poor success of those DNA-based vaccines may have been due to inefficient delivery of nucleic acids to cell nuclei and subsequent failure of DNA amplification in those target cells. Replication-competent and non-replicating adenoviral vectors offered improved delivery platform for influenza vaccines and achievement of systemic and mucosal immunity [63, 64]. As for mRNA and selfreplicating RNA vaccines, they are delivered into the cytoplasm of cells but do not require nuclear translocation [65]. When formulated into lipid nanoparticles, RNA vaccines are efficiently delivered into the cytoplasm.

The first human clinical influenza mRNA-based trial employed a non-chemically modified mRNA construct, where the intent was to induce antibody titers against multivalent targets of four different influenza strains [66]. ModernaTX, Inc. is in recruitment of participants to evaluate modified mRNA-1647 to assess sero-responses in comparison to adjuvanted inactivated, quadrivalent seasonal influenza vaccine [67]. Subsequent vaccine goals include the development of multiplexed vaccine candidates into one dose of SARS-CoV-2, respiratory syncytial virus, or other formulations. Pfizer led a clinical research study of six SARNAs preparations of hemagglutinin antigens that were designed against four influenza strains. The proportion of participants achieving hemagglutination inhibition titers for each strain had been measured in the context of secondary outcomes [68]. It remains to be established if RNA vaccines will provide long-term protection with an established frequency of booster administration.

The global initiative on sharing all influenza data (GISAID) established the first repository of shared influenza sequences in 2006. GISAID has been instrumental for WHO and National Influenza Centers in providing bi-annual recommendations on strain selection for influenza vaccines [69]. Moreover, GISAID provides bioinformatics workspaces like FluSurver to allow scientists to perform assessments of the positions of novel mutations, changes in antigenic properties or glycosylation, and even predict viral susceptibility to drugs [70]. The geographical assessment of currently circulating strains can be visualized, as well as the phylogeny of current clades and the molecular clock of viral evolution (**Figure 1a, b**) [70]. For present strains of epidemiological importance, the frequency projections of currently circulating A/H3N2 clades are calculated from a fitness model based on the current frequency and estimated fitness [71]. The strain fitness is estimated by a combination of antigenic novelty and mutational load. Antigenic novelty is based on inferred measurements of antigenic advance from hemagglutination inhibition assay [71]. Mutational load is calculated by the number of amino acid mutations each strain carries at putative non-epitope sites relative to its most recent ancestor from the previous season (see **Figure 1** and pull down menus under Black "X" in: https://www.gisaid.org/epiflu-applications/ influenza-genomic-epidemiology/).

a.Real-time tracking of influenza A/H1N1 evolution.

**Top left**: Rectangular phylogenetic tree of influenza A/H1N1 shows color-coded clades (by genotype). The black line represents linear regression of divergence. Black **X** represents an interactive drop-down menu with information about the date, specific nucleotides changes, amino acid changes, calculated divergence score, and potential vaccine selection. **Top right**: geographical distribution of A/H1N1 clades.

**Bottom**: Molecular clock representation of clades divergence since 2013, with an estimated rate of 3.7x10−3 substitutions per site per year.

b.Real-time tracking of influenza A/H3N2 evolution.

**Top left**: Rectangular phylogenetic tree of influenza A/H3N2 shows color-coded clades (by genotype). The black line represents linear regression of divergence. Black **X** represents interactive drop-down menu with information about the date, specific nucleotides changes, amino acid changes, calculated divergence score, and potential vaccine selection. **Top right**: geographical distribution of A/H3N2 clades.

**Bottom**: Molecular clock representation of clades divergence since 2013, with an estimated rate of 4.06x10−3 substitutions per site per year.

As more and more public health laboratories upload the sequencing results to GISAID, the global real-time tracking of influenza became possible. As a result, RNA vaccines can be re-designed just in a few days, and produced in just a few weeks.

*Prophylactic Ribonucleic Acid Vaccines to Combat RNA Viral Infections in Humans DOI: http://dx.doi.org/10.5772/intechopen.108163*

**Figure 1.** *Visualization of influenza phylogeny, geographical distribution, and divergence of clades.*

#### **5. Respiratory syncytial virus vaccines**

The human respiratory syncytial virus (RSV) is a negative-strand RNA, enveloped, non-segmented virus of the order Mononegavirales, genus Pneumovirus and family Paramyxoviridae [72]. The human respiratory syncytial virus represents a significant public health burden in two main populations that includes young children and older adults. Previously, only passive immuno-prophylaxis with neutralizing antibodies was considered minimally protective against severe disease. The RSV-live attenuated vaccines did not prevent subsequent RSV disease [73]. Moreover, whole-virus inactivated vaccines were associated with enhanced

respiratory disease in the lungs, presenting with monocytic eosinophilic pulmonary inflammation on histologic evaluation [74].

Despite the diversity of antigens, human RSV infection produces some serotypes that can be divided into two antigenic subgroups, with the RSV A being more diverse than B subgroup [75]. Elucidation of the atomic structure in conjunction with the identification of the fusion (F) glycoprotein was of critical importance for vaccine development and clinical trials. Unfortunately, these protein vaccines did not meet clinical expectations in robustness for preventing subsequent disease progression [76]. The development of a new generation of RSV-F protein, stabilized in the perfusion conformation, allowed GlaxoSmithKline and Medimmune to launch four phase-3 clinical trials testing pregnant mothers and infants [77]. Within 6 months after immunization, these vaccines were found to be protective against severe lower respiratory tract infections in infants and mothers [77].

An RSV-targeting recombinant virus-like particle vaccine trial (NCT04519073) conducted in Belgium demonstrated promising preliminary results of increased titers of micro-neutralizing antibodies against RSV A and B [78]. Additionally, a Phase 3 randomized, observer-blinded study evaluated the safety, tolerability, and immunogenicity of the mRNA-1345 (RSVictory) vaccine targeting RSV (NCT05127434) [79]. The vaccine was successfully tested in adults ≥50 years of age when administered alone or when co-administered with inactivated quadrivalent influenza vaccine (Afluria®) [80]). Outcome data will evaluate the percent of participants with sero-responses, who are defined by a ≥ 4-fold increase in RSV-A neutralizing antibody titer between one and six months after vaccination. This study has been conducted by ModernaTM, and the main outcome goal is to achieve long-term immunity to both infections. These vaccines are yet to show reliable prophylactic and therapeutic efficacy.

#### **6. Combination vaccines against parainfluenza type 3 virus (PIV3) and human Metapneumovirus (HMPV)**

Like RSV, PIV is also a negative-strand RNA virus from the Paramyxoviridae family and Paramyxovirinae subfamily. Bi-directional high-throughput RNA sequencing technology elucidated several types of parainfluenzas (1–5), with PIV3 as most predominant [81]. Another, more recently identified member of the order Mononegavirales, family Paramyxoviridae, subfamily Pneumovirinae is a human metapneumovirus (HMPV) [82]. HMPV became a part of infectious disease genomic surveillance after development of whole-genome tiled amplicon sequencing technology. This methodology allowed the identification of two major phylogenetic subtypes of HMPV, each containing two sublineages (A1, A2, B1, B2) [83, 84]. The use of this new knowledge in vaccine manufacturing led to multi-viral vaccine research and development.

Human subfamilies (Paramyxovirinae and Pneumovirinae) are known to cause hospital-acquired infections, infections in young and elderly adults, and pneumonia in immunocompromised individuals [85]. Antiviral medication or vaccinations against these globally spread viral infections, including multiple re-infections that occur throughout life, did not exist until recently. Two clinical trials conducted by Moderna TX are recruiting participants to assess the safety, reactogenicity, and Immunogenicity of the mRNA-1653 vaccine. This is a combined design against PIV and HMPV, which will be tested in healthy adults (NCT03392389) and children 12

to 59 months of age (NCT04144348) [86, 87]. If these trials are successful, other Paramyxoviridae infections can be targeted with the same polyvalent vaccine design.

#### **7. Chikungunya and dengue viruses' vaccine trials**

Chikungunya, Dengue, and Zika viruses are transmitted by mosquitos of the *Aedes* genus. These infections had little attention in Western World prior to travel-related epidemics spreading from tropical countries of equatorial Africa, South America, India, or the Polynesian region.

The mosquito-borne Chikungunya fever is caused by RNA arbovirus that belongs to the alphavirus genus of the family Togaviridae. Patients usually present with relatively mild disease; however, debilitating chronic arthritis has been reported in some patients who recover from the infection [88].

Phase 1 and 2 clinical trials of Chikungunya virus-like recombinant protein vaccines (aluminum hydroxide-adjuvanted) have been completed [89–91]. One study, conducted by Emergent BioSolutions (PXVX0317) reported promising results related to satisfactory safety outcomes and sufficient neutralizing antibody titer responses (NCT0348369; NCT03992872) [92]. Phase 3 was initiated in August 2022, and the focus was to test PXVX031 in adults ≥65 years of age [93].

DNA-based vaccines have been designed and tested (based on mumps and rubella viral vectors), but those vaccines failed to demonstrate long-term immunogenicity [94]. Two years before the COVID-19 pandemic, Moderna launched the first Phase 1 trial of the mRNA-1388 vaccine and subsequently the second trial of mRNA-1944 [95, 96]. Although these trials were interrupted by the COVID-19 pandemic, preliminary results showed favorable tolerability of mRNAs in healthy volunteers. mRNA-1388 is a prophylactic vaccine that consists of a single mRNA encoding the full native structural polyprotein (C-E3-E2-6k-E1 peptides). This polyprotein is naturally processed into C and E structural viral proteins that assemble into viral-like particles before being released from cells [97]. mRNA-1944 encodes the heavy and light chains of the Chikungunya antibody formulated in proprietary lipid nanoparticles and can be used as biotherapeutics [97]. More information about these vaccines and trial designs can be found in the archives of the United States Security and Exchange Commission reports [97].

Sequencing of the full 10 kB Chikungunya virus genome is important for epidemiological investigation and genomic surveillance; however, few Public Health Laboratories are pursuing these investigations [98]. Understanding genetic diversity and rates of *de novo* mutations will allow estimates of higher and lower fitness of circulating variants (those that have sufficient fitness to cause epidemics and those that can be naturally purified during transmission bottlenecks) [98, 99]. Similar analogies can be made with the 10.7-kB ribonucleic acid virus Dengue. The incidence of Dengue disease is increasing globally and is attributed to the exportation of the disease from tropical countries via tourism and inefficient mosquito controls. Significant concerns about the spread of this emerging disease, as well as potential solutions are elucidated in comprehensive reviews on dengue vaccine development [100–103]. The development of effective vaccines and mandatory vaccination of international travelers has already proven to be the most effective way in preventing the transmission of vectorborne diseases like yellow fever [104]. Thus, vaccination certificates may be required in the future for travelers as a condition of entry to specific countries, and this would facilitate safer international travel.

#### **8. Zika virus vaccines**

Zika is an eleven-kilobases-long single-stranded positive-sense RNA virus. Zika's genome encodes for one open-reading frame, which is translated into 20 functional proteins. There are seven nonstructural and 13 structural proteins, including premembrane, envelope, and capsid. Like most flaviviruses, Zika is transmitted by mosquitos. Intercontinental travel has facilitated Zika virus spread out of Africa, as well as it is being spread from human to human via sexual contact. Pregnancy, in conjunction with gestational Zika infection, is strongly associated with microcephaly and other congenital abnormalities in newborns. Preventing congenital Zika infections has been the subject of vaccine research in animal models [105].

Pre-clinical Zika studies with the modified-nucleoside mRNA vaccines have been designed to target the envelope and pre-membrane proteins [106]. Recently initiated Moderna's phase 1 and 2 human clinical trials have shown a near 90% seroconversion rate in adult participants after booster vaccination [107]. Phase 2 of this study is expected to be completed in 2024, with the primary outcome measure focusing on systemic reactogenicity while reducing adverse events, and achieving measurable serum-neutralizing antibodies against Zika virus [108].

Various formulations of SARNA vaccine studies in animal models have been compared with the efficacy of DNA and mRNA vaccines [109]. SARNAs have shown to be more effective in smaller doses compared to the other vaccines. One reason is attributed to the double-stranded SARNA being able to induce innate immune interferon type 1/2 responses, which serve as an endogenous adjuvant. This has been proposed to eliminate the administration of a second dose that is required for mRNA vaccines. In comparison, the second and third exposures to DNA vaccines elicit host immune response against the vectors that contain the vaccines' DNA (**Table 1**). Conversely, this is not known side-effect for mRNA or SARNAs because the majority of those vaccines are encapsulated into non-immunogenic neutral or charged lipid nanoparticles [110]. Seventy other DNA, RNA, and conventional Zika-vaccine studies are currently registered with clinicaltrials.org in the assessment of safety and preliminary efficacy (phases 1 and 2). Future studies are required to demonstrate which vaccine could be more robust, providing longer-lasting immunity against Zika infection.

#### **9. Rabies virus vaccines**

The rabies virus is an RNA virus transmitted through mammalian vectors. The genome of the rabies virus encodes 5 proteins (N, P, M, G, and L), and the sequencing of the single-stranded RNA genome classified the viral structure within the Lyssavirus family. Due to the neurotropic properties of the virus and a lack of effective treatment, rabies exposure, if not immediately addressed, is lethal in humans and other mammals within three weeks from infection [111]. Furthermore, vaccine portfolios have not significantly advanced, and that may be in part due to the endemic and sporadic nature of the disease. While DNA vaccines against rabies have been developed, they have proven to be poorly immunogenic in humans [112]. Thus, conventional types of inactivated rabies virus vaccines (RabAvert, Rabipur, Imovax, etc.) are most common for vaccination of individuals in specific professions who are at high risk of rabies exposure [111].

mRNA rabies vaccines CureVac and CV7201 entered phase 1 clinical trial in order to assess their safety and reactogenicity [113, 114]. These mRNA vaccines also encode rabies virus glycoprotein G and have shown promise to be safe and effective

*Prophylactic Ribonucleic Acid Vaccines to Combat RNA Viral Infections in Humans DOI: http://dx.doi.org/10.5772/intechopen.108163*

as a pre- and early post-exposure prophylactic vaccine for humans (NCT03713086; NCT02241135). Several novel self-amplifying RNA (SARNA) have been tested in combination with diverse nanoparticle formulations. Preclinical studies of proprietary cationic nanoemulsion-formulated glycoprotein G-encoding self-amplifying RNA (RG-SAM [CNE]) showed that the vaccine was well tolerated following multiple intramuscular injections in animals [112]. The rabies SARNA is a virus glycoprotein G RNA that showed promising results in phase 1 clinical studies through protecting neutralizing antibody responses (IgM and IgG) against viral glycoprotein. SARNA vaccines are well tolerated and cause mild side effects comparable to those in conventional vaccines trial (GlaxoSmithKline, NCT04062669) [115]. Establishing clinical efficacy is the next step for this type of SARNA vaccines, as they hold great promise to become valuable pre-exposure prophylactics. SARNA technology offers distinct advantages because they are highly amenable to mass- *in vitro*- transcription in GMP-level facilities.

#### **10. Ebola virus disease vaccines**

Ebola is a single-stranded, negative-sense RNA virus that causes the Ebola virus disease. Ebola is subdivided into five immunologically different subspecies based on surface envelope glycoprotein spikes and the virion proteins of nucleocapsid [116]. UCSC Genome Browser and GISAID contain the most comprehensive genomic information on Ebola subspecies sequence variations and phylogeography [117, 118].

More than four dozen vaccine trials were initiated after the Ebola outbreak of 2014 [119]. At least half of them were DNA-based transgenes, delivered with non-replicative viral vectors like Venezuelan equine encephalitis virus, human replication-defective adenovirus, recombinant chimpanzee adenovirus type 3, modified vaccinia strain Ankara, or Kunjin replicon virus-like particle vaccine. The other vaccine trials utilized replicative vectors, including human parainfluenza virus type 3-based vaccine, recombinant vesicular stomatitis virus-based vaccine (rVSV-EBOV), recombinant rabies virus, or recombinant cytomegalovirus. All of these vaccines were designed against envelope spike glycoproteins [120].

The first Ebola vaccine (rVSV-ZEBOV, Merk) was approved in the United States in 2019 and had been used in the 2018 Ebola epidemic in the Democratic Republic of the Congo as part of clinical trials. Subsequently, it had been used under criteria for compassionate use that included children and pregnant women [121]. rVSV-ZEBOV showed the ability to generate protective immunity in a form of anti-glycoprotein immunoglobulin G antibody titers that lasted at least 2 years of observation [122]. Several other DNA-based vaccines are being tested by Inovio Pharmaceuticals via routes of prime intramuscular injection with subsequent boost electroporation [123]. Electroporation is less invasive; however, it requires a specialized medical device to deliver brief electrical pulses during intradermal gene transfer [124]. Challenges remain with DNA vaccine platforms. These challenges include immune response to viral vectors after booster vaccination, safety concerns about replication-capable viral cargo (e.g., human genome integration), and slow optimization of antigen sequences to make multivalent vaccines against all sub-strains of the Ebola virus (**Table 1**).

mRNA vaccines can respond to these challenges quicker because the manufacturing process and formulations allow multi-sequence delivery and, therefore, avoids safety issues associated with booster immunization. The lipid nanoparticle (LNP) encapsulation technology and the design of glycoprotein mRNA with posttranscriptional modifications have the potential to exhibit durable immune responses in pre-clinical and phase 1 studies [125]. Due to lower doses requirement, and lower cost, SARNA vaccines may have a higher potential to rapidly respond to future Ebola outbreaks. Like DNA vaccines, SARNAs are stable and can be delivered intradermally via electroporation. Non-human primate experiments showed that SARNA induces sufficient protective responses against Ebola after a single primed immunization [126]. Future expectations are that SARNA vaccines will be successfully delivered with electroporation (intradermal) and will not require boost immunization.

#### **11. Future directions**

Epidemics caused by genetically recombined or mutated RNA viruses will continue to evolve and pose health threats locally and globally. Because of this, RNA vaccinology will continue to strive to develop new manufacturing processes to improve RNA transcript stability by incorporating modified synthetic nucleotides during *in vitro* transcription, optimizing delivery formulations, and adjusting the adjuvants' potency. Additionally, next-generation viral genotyping conducted by CDC and Public Health Laboratories will provide real-time pathogen surveillance data for rapid modifications and manufacturing of RNA vaccines. Mosaic vaccines against multiple viral strains or multi-pathogen vaccines are a goal that needs to be achieved to prevent pandemics, epidemics, and endemic infections.

#### **Acknowledgements**

IVS is sponsored by APHL/CDC COVID-19 Laboratory Associate Fellowship grant, and Ronald H. Laessig Memorial Newborn Screening Fellowship fund.

### **Conflict of interest**

None declared.

#### **Abbreviations**


*Prophylactic Ribonucleic Acid Vaccines to Combat RNA Viral Infections in Humans DOI: http://dx.doi.org/10.5772/intechopen.108163*

#### **Author details**

Irina Vlasova-St. Louis1,2\* and Jude Abadie3,4

1 Individualized Genomics and Health Program, Johns Hopkins University, Baltimore, MD, USA

2 Molecular Branch of Newborn Screening Program, Texas Department of State Health Services, Austin, Texas, USA

3 El Paso Public Health Laboratory, USA

4 Texas Tech University Health Sciences Center El Paso, USA

\*Address all correspondence to: stlouis.irina@gmail.com

© 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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#### **Chapter 3**

## Ribozymes as Therapeutic Agents against Infectious Diseases

*Bao Chi Wong, Umama Shahid and Hock Siew Tan*

#### **Abstract**

Ribozymes, also known as RNA enzymes, are catalytic RNA molecules capable of cleaving specific RNA sequences, leading to decreased expression of targeted genes. Recent studies suggest their role in cancer therapeutics, genetic diseases and retroviral infections. This book chapter will focus on ribozymes acting as therapeutic agents against infectious diseases caused by viral and bacterial pathogens. Firstly, we will introduce a brief history of ribozymes and a general overview of ribozymes and their characteristics. Next, different types of ribozymes will be explored regarding their targets and mechanisms of action. After that, ribozymes specific to viral and bacterial infections will be explored. We will briefly discuss the current status of ribozymes as therapeutic agents. Finally, the roadblock and challenges ribozymes face before being developed into therapeutic agents—such as their delivery and efficacy issues—will be discussed.

**Keywords:** ribozymes, therapeutic agent, antiviral, antibacterial, infectious diseases

#### **1. Introduction**

Proteins have always been the undefeated champions in most stories that any molecular biologist has to tell. A classic textbook elaborates extensively on these molecules, their structures, localisations and functions, followed by an essential section on enzymes. The Central Dogma of Molecular Biology states that deoxyribonucleic acid (DNA) precedes protein. DNA encodes important information, is converted into ribonucleic acid (RNA) and finally translated into the master molecule, protein [1]. So, in principle, proteins cannot exist without nucleic acids. However, the precursor here, i.e. DNA, is not even capable of replicating, much less forming a protein by itself, because it is found in a double-stranded form and hence is functionally inert. Therefore, DNA requires something capable of catalysing these reactions. Biologists have tried to explore the players involved in this phenomenon for years until a relatively recent discovery of catalytic RNAs by Thomas Cech and Sidney Altman proposed a possible explanation [2].

In 1978, Thomas Cech (University of Colorado) and his team decided to study RNA splicing, a considerably new field at the time. To explore RNA processing, they started working with a ciliated protozoan, *Tetrahymena thermophila*. Ribosomal RNA was chosen owing to its abundant amount in the selected system [3]. The 26S rRNA gene in *Tetrahymena* includes an intron of about 400 nucleotides, which must

be removed before the gene product can localise and function as required. However, they observed a 9S RNA fragment (approximately 400 nucleotides) in all their phenol-based nuclear extractions, leading them to assume the possibility of protein contamination in the nuclear extracts, which was responsible for the splicing of this intron. Extreme efforts were made to remove/denature the protein that was thought to be either contaminant or strongly attached to the RNA molecules. The samples were subjected to high salt concentrations and exposed to high temperatures (both being something that a protein does not like), but splicing was observed, nonetheless. This result hinted that no protein could be present/responsible for the processing. However, this was not enough evidence. Kruger et al. described the cloning of the *Tetrahymena* rRNA gene in the *Escherichia coli* plasmid, followed by its *in vitro* transcription in their 1982 article [4]. The rRNA thus transcribed was also capable of excising the intron itself, proving that it was, in fact, a self-splicing molecule and did not require any protein for the processing. In the same year, Cech and his team released an article explaining the actual working of rRNA self-splicing where they showed that a GTP was required as a co-factor [5]. A detailed mechanism of selfsplicing will be explained later in this chapter.

The discovery of self-splicing RNA molecules raised consciousness in the molecular biology world. Where one set of researchers dismissed it by calling the finding 'not a big deal', others started investigating the possibility of more reactions that were catalysed by RNA. Sidney Altman, Norman Pace and their respective teams studied ribonuclease P, an enzyme responsible for tRNA processing. Ribonuclease P is an interesting molecule since 80% of its content is RNA, and only 10% is protein. Initially, the RNA part of ribonuclease P was considered leftover contamination from protein purification with no significance. However, both teams demonstrated that reactions could occur without the protein section of ribonuclease P, proving that the RNA component catalysed the cleavage [6]. In 1989, Cech and Altman shared a Nobel prize in chemistry for demonstrating the catalytic activity of RNA. Many terms were coined for these special RNA molecules, now named Ribozymes (Ribonucleic acids that act as enzymes). Though not as common in vertebrates, RNA catalysis is now known to be widely spread amongst bacteria, viruses, some lower eukaryotes and even plants. One is also found in humans [7]. The naturally occurring ribozymes are reported to aid in reactions such as Ribosyl 2'-O mediated cleavage [8], RNA cleavage and ligation [9], DNA cleavage and ligation [10], etc. In addition, researchers worldwide are generating artificial ribozymes through combinatorial screening of random RNA sequences, which has increased the catalytic repertoire to an even larger range, including phosphorylation [11], acyl transfer reaction [12] and an amazing RNA polymerase ribozyme capable of polymerising complex RNA structures such as aptamers, ribozymes and even tRNA, amongst others [13].

#### **2. General characteristics of ribozymes**

Catalytic RNAs, like proteins, form a 3-D structure to be functionally sound for catalysis. Metal ions such as K+ or Mg2+ are required for the proper folding of ribozymes to recompense for the high negative charge of the oligonucleotides [14]. Ribozymes typically contribute to self-targeted reactions (such as self-cleavage, selfsplicing, ligation and template-directed polymerisation) except for one, i.e. RNase P (involved in the processing of tRNA) [15]. RNA has a limited range of chemical functionalities with just four similar nucleotides as building blocks. Despite this,

*Ribozymes as Therapeutic Agents against Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.107141*

RNA can catalyse phosphoryl transfer reactions by about a million-fold, if not more [16]. Generally, naturally occurring ribozymes catalyse these reactions by attacking sugar 2′ or 3′-hydroxyl on a phosphodiester linkage. This nucleophilic attack involves activation of the nucleophile, stabilisation of an electronegative transition state and stabilisation of the leaving group.

Ribozymes can be categorised into two categories based on their size and whether a ribozyme uses its sugar -OH group to target the 3′ phosphodiester bond or requires an exogenous nucleophile [15]. The first group is the small ribozymes (approximately 35–155 nucleotides) that utilise 2′-hydroxyl of an adjacent nucleotide for the nucleophilic attack. The second group is the large ribozymes (approximately 200–3000 nucleotides) that attack using exogenous groups such as water, hydroxyl group from a mononucleotide or even a distantly located nucleotide from the same stretch [17]. Ribozymes perform phosphoryl transfer reactions using two main mechanisms, which are acid-base catalysis (seen in hammerhead, hairpin and *glmS* ribozymes) and metal-ion-assisted catalysis (seen in RNase P, group I, group II introns, HDV ribozymes) [17].

#### **2.1 Small self-cleaving ribozymes**

In general, small self-cleaving ribozymes act on the same strand, i.e. act in *cis* and hence, have a single catalytic turnover. These classes work on general acid-base catalysis. They use adjacent nucleobases or external co-factors as the general base or acid. The base takes a proton from the 2′-hydroxyl group, thereby increasing oxygen's nucleophilicity, which can then attack the nearby phosphorous. As a result, a transition state is formed. On the other hand, 5′-oxygen gets protonated by a general acid leading to the release of leaving group and thus the formation of 2′,3′-cyclic phosphate and a free 5′-hydroxyl group (**Figure 1**) [18]. Below are the different classes of small self-cleaving ribozymes.

With a size of about 40–50 nucleotides, the **hammerhead ribozyme** is, by far, the most extensively studied. Originally found in plant viroids and satellites, they are a widely spread class of self-cleaving RNAs known to catalyse the conversion

#### **Figure 1.**

*Mechanism of catalysis in ribozymes: Ribozymes perform reversible nucleophilic reactions. (A) General Acid-Base catalysis. The general base (blue) deprotonates the 2*′*-hydroxyl in the cleavage reaction (or the 5*′*-hydroxyl in the reversed ligation reaction). The general acid (red) donates a proton to the 5*′*-oxyanion leaving group for cleavage (or the 2*′*oxyanion for ligation). A trigonal bipyramidal phosphorane is formed in the transition state (shown in the centre). B) RNA metalloenzymes***.** *Large ribozymes, including RNase P and self-splicing introns, catalyse the phosphodiester bond breakdown via metal-ion catalysis. The figure is a representative group I intron where three metal ions bind to the transition state to bring about catalysis.*

of their trimeric and dimeric forms into monomeric RNAs [19]. They are made up of three helical regions (Stem I, II, III), which are variable and a universally conserved junction sequence made up of three single strands (**Figure 2**) [20]. Hammerhead ribozymes cleave after an NUH [21] or NHH [22] triplet, where N can be any nucleotide, and H is any nucleotide except guanosine. They utilise N1 of G12 from stem II in their catalysis as a nucleophile. It forms a hydrogen bond with 2′ hydroxyl of C17 [23]. Some studies report that a divalent metal cation helps activate G12. Stabilisation of G8 occurs due to its base pairing with G3 [24]. Earlier, the 2′-hydroxyl group of G8 was thought to be the acid in this acid-base catalysis. However, a recent study reports that Mn2+-bound water is the general acid during cleavage [25].

**Hairpin ribozymes**, like hammerhead ribozymes, are also found in plants' satellite viruses such as the tobacco ringspot (best studied), chicory yellow mottle and Arabis mosaic virus also catalyse the self-cleavage of multimeric RNA [26]. They comprise four stems that, when aligned, resemble a hairpin (**Figure 2**). A10 and G11 and A24 and C25 assemble as a ribose zipper and form a catalytic centre. The general base, in this case, is G8 (stem B), and A38 a (stem A) acts as a general acid, respectively. Rigorous *in vitro* selection of active mutants has shown that hairpin ribozymes prefer G at the +1 position of their cleavage site. N\*GUY emerged as the best agreedupon cleavage site, where N is any nucleotide, G is guanine, U is uracil and Y is any pyrimidine [27]. Later studies showed that substrates with G\*GUN, G\*GGR (R is any purine) and U\*GUA could also be cleaved but with a considerably lower catalytic activity [28]. A crowded environment near the hairpin ribozymes increases their activity by stabilising the active conformation [29].

**Hepatitis delta virus-like ribozymes** are self-cleaving ribozymes present in the genomic strand and the complementary/anti-genomic strand found in Hepatitis delta virus (HDV) (a single-stranded RNA virus that infects mammalian hepatocytes) [30]. These ribozymes also catalyse a transesterification reaction through a nucleophilic attack by a 2′ hydroxyl on the adjacent phosphate and result in the formation of a 2′–3′ cyclic phosphates and the release of 5′ hydroxyls. Their structure consists of five paired regions of helices, which, when coaxially aligned, are stacked over each other (P1 over P1.1 and P4; P2 over P3). Single-stranded joining strands link these helices. Crystallography reveals that they assume an extremely stable structure resembling a double pseudoknot. HDV-like ribozymes cleave at the first guanosine residue at the base of the P1 helix [31].

The **glucosamine-6-phosphate synthase (***glmS***) ribozyme** is found in several Gram-positive bacteria in the 5' UTR region of the *glmS* gene [32]. It regulates the expression of glutamine-fructose-6-phosphate transaminase and is the only known ribozyme which requires glucosamine-6-phosphate (GlcN6P) as a co-factor [33]. The *glmS* ribozyme comprises three parallel helices stacking each other (P1 on P3.1, P4 on P4.1 and P2.1). It also forms a core resembling a double pseudoknot. P3 and P4 are not essential for catalysis. However, they provide structural stability and enhance the activity of ribozymes. The P2.2 forms the binding site for GlcN6P, and the correct folding of P2.2 brings the ribozyme into active conformation [34]. A co-factor is required for the protonation of the 5′ oxygen leaving group, activation of the 2′-oxygen nucleophile and charge stabilisation [35].

Largest nucleolytic RNA with a length of ~150 nucleotides, the **Varkud Satellite (VS) ribozymes** are found in certain strains of Neurospora and help in the replication of single-stranded RNA [15]. VS ribozymes comprise seven helices (1–7), forming a three-way junction (2-3-6, 3-4- and 1-7-2). The inner loops of stem 1 act as their

*Ribozymes as Therapeutic Agents against Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.107141*

#### **Figure 2.**

*Consensus secondary structures ribozymes. A, U, G and C represent adenine, uracil, guanine and cytosine. N represents any nucleotide. R stands for any purine and Y for any pyrimidine. The black arrows show the cleavage site, orange-coded nucleotides represent conserved bases near the cleavage sites, and the solid line shows a variable stretch of nucleotides.*

cleavage site, while stems 6 and 1 harbour the catalytic centre [36]. A kissing loop forms between GUC in stem 1 and GAC in stem 5 to form an active site, bringing the cleavage site to the catalytic centre. The residues A756 and G638 act as the general acid and base, respectively. Additionally, Mg2+ is reported to interact with scissile phosphate and activate G638 [37].

**Hatchet ribozymes** are one of the bioinformatically revealed ribozymes, and very little is known about them. They comprise four stems (P1–P4). P1 and P2 are linked with highly conserved residues, whereas internal loops (L2 and L3) connect the other three stems [38]. X-ray crystallography reveals that they appear as pseudo symmetrical RNA and form long-range interactions of conserved residues near scissile phosphate. The cleavage site is located at the 5′ end of the P1 stem. N7 of G31 acts as the general base to deprotonate the 2′-OH of C (−1) for nucleophilic attack. In addition, Mg2+ is required for proper folding and catalysis [39].

**Twister ribozymes** are widely spread among many species of bacteria and eukaryotes [40]. They are made up of five stems (P1–P5) and internal loops held together by two pseudoknots. Twister ribozymes cleave folding-dependently, where central pseudoknot opens and closes at variable Mg2+ concentration [41]. These cations help position the phosphate oxygen at the U-A cleavage site and stabilise the transition stage to form an intermediate. Guanine is a conserved residue at the cleavage site. It acts as a general base in the general acid-base catalysis, whereas an adenine residue plays as the general acid. The active site comprises at least 10 conserved nucleotides, harbouring scissile phosphate between A and U joining P1 [42].

**Twister sister ribozymes** are highly similar to twister ribozymes in sequence and secondary structure. The only difference is that they do not have a double pseudoknot interaction. Long-range interactions that bring conserved nucleotides closer to the core are mediated by Mg2+ cations [43]. C62 and A63 flank the cleavage site on the internal loop between P1 and P2. Hydrogen bonds (N1H of G5, inner-sphere water of Mg2+ and phosphate oxygen) keep the scissile phosphate in its place [44]. The substrate specificity of these ribozymes has not been studied in detail yet.

**Pistol ribozymes** were discovered bioinformatically through comparative genomic analysis to search hammerhead and twister ribozymes-related sequences. Pistol ribozymes consist of three helical stems (P1–P3) connected by three loops (loops 1–3) and one pseudoknot. P1 and pseudoknot form a stacked structure [45]. N1 of G40 acts as a general base, and A32 acts as a general acid. Crystallography shows that Mg2+ cations have a significant role in catalysis. All information on this ribozyme is limited; some studies suggest that residues at positions 32 and 40 might affect the substrate specificity [46].

**Hovlinc ribozymes** are a recently discovered class of ribozymes that came up in a genome-wide search of human catalytic RNAs [47]. Although very little is known about hovlinc ribozymes, structure analysis shows that their catalytic core comprises two stem loops and two pseudoknots. They are pH-dependent and require divalent cations where their activity was shown to be highest in the presence of Mn2+ (Mn2+ > Mg2+ > Ca2+) [48]. Further studies will be required to properly reveal its characteristic folding, cleavage site, catalytic centre and functioning.

#### **2.2 Large ribozymes**

These are often called 'true catalysts' because they can act on a substrate in a *trans* manner and thus have a catalytic turnover. In contrast to the small self-cleaving ribozymes, large catalytic RNAs act as metalloenzymes [7]. Metal ions are usually found in the active sites of these ribozymes and form inner-sphere complexes with oxygen atoms of the RNA (**Figure 1**).

Introns are intervening noncoding regions between a gene's exon (coding regions). When a gene is transcribed, the pre-RNA thus formed undergoes removal, i.e. splicing all the introns to obtain mature RNA [49]. Naturally found in bacteria and bacteriophages, nuclear rRNA genes and chloroplast DNA, **Group I introns** are selfsplicing in nature and can excise themselves without a protein enzyme [50]. These can migrate and insert themselves at different positions of the host genome, thus acting as mobile genetic elements [51]. Although widespread, the group I ribozymes have

very less sequence similarity. However, they all can fold into a conserved secondary structure with 10 paired segments (P1–P10). The catalytic core comprises P3, P4, P6 and P7 [9]. The intron is spliced from the pre-RNA by a two-step transesterification reaction. First, 3′ hydroxyl makes a nucleophilic attack on a guanosine co-factor at the 5′ splice site. The exon-intron phosphodiester bond is cleaved, and guanosine forms a 3′,5′-phosphodiester bond at the 5′ ends of the intron. Finally, a nucleophilic attack of now free 3′ hydroxyl of the 5′ at the 3′ splice site to form the ligated exons results in the release of an intron with the nonencoded guanosine. The intron is circularised by making a nucleophilic attack with its highly conserved 3′ terminal guanosine at a phosphodiester bond of C-15 or C-19. Each step is fully reversible and follows the SN2 reaction mechanism [52].

The self-splicing **group II introns** are widespread amongst the mRNA, tRNA and rRNA genes of plant and fungal mitochondria and chloroplasts (including algae and protists) [51]. The secondary structure of Group II introns was initially revealed via computational modelling and phylogenetic comparisons. They are composed of six helices (I–VI) radiating from a centrally located wheel [53]. Out of the six helical domains present, only I and V are crucial for their activity. Domain V, the most variable region, harbours the active site. A conserved Adenosine residue is present in the domain, which initiates the splicing reaction. 2′-Hydroxyl of adenosine performs the nucleophilic attack and forms a structure known as a lariat, which contains 3′–5′ and 2′–5′ phosphodiester bonds at the adenosine branch site. Following this, free 3′-hydroxyl of 5′ exon makes a nucleophilic attack at the 3′ splice site resulting in ligated exons and spliced out intron (as a lariat). Though not common, a nucleophilic attack may sometimes be initiated by water, resulting in a linear intron [54].

**RNase P** is a widespread enzyme processing tRNA precursors [55]. It is known to exist in a ribonucleoprotein complex consisting of about 350–400 nucleotide long RNA stretch and about 14kDa of small protein subunit [56]. Though protein moiety is important for catalysis *in vivo*, the RNA component is enough *in vitro*. The reaction occurs at a high salt concentration, and protein was assumed to promote RNA enzyme and substrate interaction. However, studies have shown that the protein component of RNase P plays a significant role in site specificity and turnover [57]. No high sequence conservation is observed in RNase P across different organisms. However, they all can fold into a similar secondary structure [56]. RNase P from *E. coli* M1 RNA consists of 18 paired helices, but RNase P from *Bacillus subtilis* lacks P6, P13, P14, P16 and P17 but contains a few extra helices (P5.1, P10.1, P15.1 and P19). Despite these differences, comparative analyses of RNase P secondary structure have deduced a catalytic core composed of P1–P5, P7–12 and P15. This ribozyme uses water to make a nucleophilic attack [58].

In eukaryotic cells, intron removal occurs through a ribonucleoprotein complex called **spliceosomes**. These complexes are not preformed; five RNAs and about 100 proteins assemble directly into a spliceosome on their substrate [59]. Splicing events can be divided into four main reaction steps: assembly, activation, catalysis and disassembly [60, 61]. The catalytic centre of spliceosomes highly resembles Group II introns, and even the splicing mechanism is quite like the latter [62].

The **ribosome** is a protein translating machinery formed by 30S and 50S subunits in bacteria and 40S and 60S subunits in eukaryotes, respectively [63]. The larger subunit contains the peptidyl transferase centre (PTC), which forms peptides by joining amino acids. X-ray crystallography and electron microscopy have elucidated two main reactions involved in protein synthesis: aminolysis to form peptide bonds and

peptidyl hydrolysis to release protein after synthesis. The catalysis does not occur via nucleobase-mediated catalysis, rather is mediated by 2′-hydroxyl of tRNA [64]. Both these reactions occur in the PTC, known to be made completely of RNA [65].

#### **3. Ribozymes as antiviral and antibacterial infection alternatives**

The potential of ribozymes as therapeutic agents has been explored from other perspectives, including cancer and inherited diseases. Ribozymes downregulate the expression of the target gene(s) through the cleavage of mRNA transcripts. If the expression of a gene could lead to pathogenesis, then the downregulation of that gene expression via ribozymes can be performed as a therapeutic option. Previous studies have selected a few important genes responsible for viral replication as targets. By decreasing the viral replication, the application of ribozymes will inevitably treat the viral infection.

Multiple viruses have been used as targets in antiviral ribozyme research, including the human immunodeficiency virus (HIV), herpes simplex virus (HSV) and human cytomegalovirus. Different types of ribozymes were used, demonstrating their potential to be used as therapeutic agents in both *in vitro* and *in vivo* conditions (**Table 1**). There are different strategies for studying the efficiency of antiviral ribozymes. If a target gene is shortlisted and the cleavage site is determined, the ribozyme can be designed rationally. If the cleavage site is undetermined, the potential target cleavage site can be screened to discover any region exposed to the ribozyme for easy binding. Another method is to use a library of ribozymes to find any ribozymes with



#### **Table 1.**

*Examples of antiviral ribozymes.*

high binding or cleavage activity towards the target virus. Finally, two main delivery methods exist for introducing ribozymes into the system. While some studies propose the potential of ribozymes as therapeutic agents for viral infections, there is still a distinct lack of ribozymes that successfully passed their pre-clinical or clinical trials.

To our best knowledge, there are currently no studies on using ribozymes to cleave specific target genes in bacteria to treat bacterial infections. Instead, Felletti et al. [85] successfully cleaved the bacterial 3′-untranslated region (UTR) using twister ribozymes, affecting the expression of the gene downstream. By designing the ribozymes specific to the 3′-UTR of essential bacterial genes, these ribozymes have potential as antibacterial agents.

#### **4. Current status of ribozymes**

As of 2022, only four clinical trials are registered on ClinicalTrials.gov for using ribozymes as therapeutic agents (**Table 2**). Among these four, three clinical trials are targeted towards human immunodeficiency virus (HIV), while the other ribozyme is targeted towards kidney cancer.

Two clinical trials were conducted for OZ1, a ribozyme designed to target the overlapping region between two essential genes. The multifunctional viral protein R (vpr) is involved in host infection, immune system evasion and infection persistence [86]. The tat protein is also involved in viral replication, enhancing the efficiency of viral expression [87]. The ribozyme OZ1 is a hammerhead ribozyme encoded within a Moloney murine leukaemia gammaretroviral vector LNL6 [74]. By cleaving the overlapping region in the *vpr* and *tat* gene, the ribozyme could inhibit the replication of HIV-1. A phase I clinical trial was conducted by delivering the OZ1 ribozyme through a retroviral vector to the mature CD34+ hematopoietic cells [74]. It was determined that the gene expression of ribozyme was detected within the patients, demonstrating that the ribozyme OZ1 can be maintained. Another Phase I study was done using a similar delivery vector to CD4+ T lymphocytes, demonstrating similar results whereby the cells can express the ribozyme long term [88]. A Phase II clinical trial (NCT00074997) was conducted with OZ1 ribozymes targeting the CD34+ hematopoietic cells. They did not achieve their primary efficacy endpoint as the mean


#### **Table 2.**

*Clinical trials of ribozymes registered on ClinicalTrials.gov. All trials were completed in phase 2 trials.*

*Ribozymes as Therapeutic Agents against Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.107141*

plasma HIV-1 viral load difference was lower but not significantly different from the placebo. However, no serious adverse events were linked to OZ1 gene transfer, indicating that using the retroviral vector to perform this gene therapy is safe, albeit with low efficacy. A second Phase II clinical trial (NCT01177059) was performed with the same group of patients from the previous trials to investigate the long-term effect of the ribozymes. There was no serious adverse effect on the participants due to the treatment. The OZ1 and the retroviral vector LNL6 marking analysis showed that they were only detected in a few participants. Unfortunately, there are no further studies on this ribozyme, perhaps due to its low efficiency in the human system.

Another Phase II clinical trial (NCT00002221) also investigated the usage of ribozymes against HIV. In this trial, a retrovirus containing two ribozyme sequences named L-TR/Tat-neo that target the tat and rev region of the virus RNA was used. Like the tat protein, the rev protein is also essential for viral replication [89]. The ribozymes were delivered to the participants of the clinical trials through *ex vivo* retroviral modified CD34+ stem cells. However, no results have been provided for this clinical trial.

Finally, RPI.4610 (ANGIOZYME), a ribozyme that targets vascular endothelial growth factor receptor 1 (VEGF1) was used to treat patients with metastatic kidney cancer. VEGF is an angiogenesis-promoting molecule, and when its preRNA is cleaved, it can inhibit angiogenesis and tumour growth [90–92]. Clinical trials with ANGIOZYME have demonstrated that it is well tolerated. However, due to its lack of efficacy, this drug could not proceed with further development [93].

#### **5. The roadblock to commercialisation**

While ribozymes have the potential to be one of the alternatives to treat infectious diseases, it cannot be denied that there are still multiple roadblocks before they can be developed as marketable drugs. Like other nucleic-acid therapeutics, ribozymes' challenges include selecting the appropriate ribozyme type and target mRNA sequence, delivery to the target site, efficiency *in vivo* and potential side effects as therapeutic drugs.

#### **5.1 Selection of target and ribozymes**

There is a wide variety of genes to choose from within the target pathogen, be it virus or bacteria, which can be used as a ribozyme target. The selection of these targets would thus depend on the aim of the ribozyme. An antiviral ribozyme may target the mRNA of genes important for viral replication, while an antibacterial ribozyme to decrease antibiotic resistance may target antimicrobial resistance genes (AMR) instead. More importantly, the cleavage site within the mRNA transcript must be carefully determined for the best cleavage efficiency. Designing sequence-specific ribozymes can be done through rational design or by *in vitro* selection.

To design a ribozyme that targets a specific gene, it needs a target-specific sequence that leads the ribozyme to the target mRNA transcript and cleaves it. Different ribozymes have different target cleavage sites due to their structural variety. For instance, hammerhead ribozymes have an NUH or NHH sequence specificity. In comparison, hairpin ribozymes catalyse site-specific reversible cleavage on the 5' side of a GUC triplet [94]. Another criterion to consider is the accessibility of the cleavage site to the ribozymes. RNAs can fold to specific three-dimensional structures; multiple methods exist to study these structures [95]. One of them is the usage of dimethyl sulfate (DMS), a chemical that can covalently modify both purines and pyrimidines

that are accessible [96, 97]. Through DMS probing and footprinting, it is possible to detect the RNA secondary and even tertiary structure, determine the potential region most accessible to DMS modification and presumably ribozyme binding.

On the other hand, Zhang et al. used a random pool of ribozymes to find accessible target sites [81]. As we progress into the post-genomic era, some may look towards in-silico analysis and bioinformatics to determine the best cleavage site, shortlisting a few for wet lab validation. RiboSoft [98] and RiboSubstrates [99] are some web applications that allow a comprehensive ribozyme design. Unfortunately, these two websites are not maintained. RNAiFold is another web server used to design a hammerhead ribozyme through computational design with experimental validation, showing that this method can be used for synthetic ribozymes [100].

Other than rational design, another method to obtain specific and efficient ribozymes is through an *in vitro* selection process using a ribozyme library [101]. Multiple studies have used this process to identify ribozymes with high cleavage efficiency. A putative self-cleaving hairpin ribozyme library was used whereby ribozymes that successfully bind and cleave a target sequence were identified [102]. Not only does this method allows the identification of effective target sites within the target mRNA, but it can also identify the most efficient ribozyme for a particular target site. The in vitro selection was used by Maghami et al. [103] to identify efficient trans-acting adenylyl transferase ribozymes that can label specific RNA sites. The ribozymes developed can be modified to target other RNA sequences by changing the sequence-specific region of the ribozymes. This method can be modified to different types of ribozymes and towards different targets.

Finally, it is worth noting that while the discovery of ribozymes is not recent, there is still undiscovered land in this field. Firstly, ribozyme variants may provide higher efficiency in their catalytic activity, which can be discovered through *in vitro* selection from a random pool of ribozymes. Deep sequencing of a ribozyme library [104] or a high-throughput analysis [105] can help elucidate novel ribozymes and their properties. Secondly, new types of ribozymes are continually being discovered and studied. A new RNA polymerase ribozyme discovered can also act as a reverse transcriptase enzyme [106]. In contrast, a type of novel ribozyme called hatchet ribozyme was reported in 2019 [38], while a pseudoknot-type hammerhead ribozyme was studied in 2020 [19]. These discoveries demonstrate that new ribozymes with improved potential still continuously emerge in recent times.

#### **5.2 Stability and delivery of ribozymes**

Like most nucleic acids, Ribozymes are vulnerable to nuclease attacks by the host cells. An unmodified ribozyme would be rapidly degraded and would not be effective when exposed to nuclease-rich fluids and tissues. Additionally, some ribozymes require co-enzymes or a certain concentration of metal ions for sufficient stability and efficiency. For example, the *glmS* ribozyme-riboswitch requires the presence of the intracellular small molecule co-enzyme GlcN6P for effective catalysis [107]. On the other hand, divalent metal ions, such as magnesium ions, are generally required by ribozymes to form a tertiary structure or catalytic activity [108]. Certain modifications or delivery vectors are needed to ensure their efficiency *in vitro* and *in vivo*.

Ribozymes can be modified to improve their stability and resistance towards nucleases. Some modifications include using locked nucleic acids (LNAs) [109], cholesterol [83], nanoparticles [110], or low-molecular-weight polyethyleneimine [111]. Modifications to the ribozyme tertiary structure or interactions can improve their stability. For instance, a tertiary interaction between a GAAA tetraloop and a

tetraloop receptor within a hammerhead ribozyme showed higher activity even under low magnesium conditions [75]. Another method of modification is to simply conduct an *in vitro* selection to determine which variants of ribozymes can remain effective. An RNase P ribozyme from *in vitro* selection showed a higher cleavage efficiency than the wild-type ribozyme. This variant was used towards the thymine kinase [66] and major transcription activator ICP4 [67] or the herpes simplex virus, as well as the assembly (AS) of murine cytomegalovirus [73]. A coenzyme-independent variant of *glmS* ribozyme was also successfully isolated through *in vitro* selection [112]. This variant contains the wild-type structure that can catalyse the cleaving reactions effectively with the presence of divalent cations alone. These studies demonstrate that it is feasible to develop variants of known ribozymes and modify their requirements for co-enzymes or increase their efficiency.

There are two ways to deliver the ribozymes into the cells: exogenous delivery (as preformed ribozymes) or endogenous delivery (as ribozyme genes). The preformed ribozymes can be delivered through electroporation or lipofection for exogenous delivery. A ribozyme stabilised by GAAA tetraloop and its receptor motif was transfected into human HeLa cells using Lipofectamine 2000 and showed effective target gene silencing [75]. A chimeric DNA-RNA hammerhead ribozyme was transfected using a polyethylenimine reagent into the cells [84]. Due to the vulnerability of ribozymes within the biological system, exogenous delivery relies on modifications that improve the stability of ribozymes. Other studies utilise endogenous delivery. In endogenous delivery, the ribozymes are introduced through ribozyme genes carried within plasmids or expression vectors. These plasmids can then be introduced through transfection to the cells, allowing the cells to express the ribozyme within the cytoplasm. The ribozymes can then catalyse the intended cleavage reaction within the cells [80, 81]. Besides plasmids, the ribozyme genes can be inserted in retroviralderived or adeno-associated viral-derived vectors (refer to **Table 1**: Delivery). While unsuccessful, the clinical trials of multiple ribozymes using Moloney murine leukaemia virus retroviral vector LNL6 demonstrated its feasibility as delivery agents of ribozymes [113]. Endogenous delivery also benefits from modifications aiming to improve ribozyme stability. Peng et al. used a novel scaffold RNA to stabilise the ribozyme structure, improving its catalytic activities [114]. However, modifications performed on the ribozymes require further investigation. Czapik et al. showed that modifications such as adding a hairpin motif to the hammerhead ribozyme decreased their catalytic activity compared with the unmodified ribozymes [79].

The delivery methods of ribozymes are not limited to these traditional methods. Rouge *et al.* successfully transfected ribozymes into cancer cells without auxiliary transfection agents using the spherical nucleic acid (SNA) architecture to stabilise the ribozymes [110]. The ribozyme, targeted towards a gene involved in chemotherapeutic resistance of solid tumours, increased the sensitisation of the cancer cells towards therapy-mediated apoptosis. On the other hand, an attenuated strain of Salmonella that contains the expression vector encoding the ribozymes was used to deliver these ribozymes to mice [71]. The success of Salmonella-mediated oral delivery of the ribozymes introduced an alternative delivery method other than those mentioned before.

#### **5.3 The efficiency of ribozymes under in vivo conditions**

It is easily shown that they can cleave their target mRNA transcripts *in vitro* through the direct cleavage of RNAs or *in vitro* studies. However, it is not as simple to translate these data from *in vitro* conditions to *in vivo*. Multiple studies have used animal models to prove the potential therapeutic use of ribozymes, and they have successfully demonstrated that in models such as rats and rabbits. Nevertheless, there are still some challenges before the ribozymes can be used in the human body.

Ribozymes, like all enzymes, also require co-factors for their optimal function. One crucial co-factor is the divalent ions, such as magnesium ions. Mainly, these ions are required for the ribozymes to achieve the correct folding of the active site and their tertiary structures [108]. However, the requirements differ between ribozymes. For instance, magnesium is essential for the catalysis activity of hammerhead ribozymes, but hairpin ribozymes do not require magnesium [77, 115].

Further research into the effects of ion concentration on the catalytic core or structure of the ribozymes allowed specific modifications to be made. A section of the ribozyme responsible for substrate-binding and tertiary stabilisation functions can be separated into discrete structural segments to ensure that trans-cleaving hammerhead ribozymes can be used in intracellular applications [116]. This separation provided the resulting ribozymes with an efficient catalytic activity at lower magnesium ion concentration. Additionally, with careful selection, ribozymes may be evolved to require a lower concentration of metal ions for their efficient activity *in vitro* and *in vivo* [117].

Finally, the efficiency of the ribozymes to cleave their targets within the *in vivo* system is also a key to the success of ribozymes as antiviral or antibacterial therapeutic agents. As mentioned previously, the ribozyme ANGIOZYME, while showing promising results in pre-clinical trial studies, did not manage to proceed further than Phase 2 clinical trials due to their lack of efficiency in the patients [93]. Other studies have also highlighted the difficulty in translating the efficiency of ribozymes from *in vitro* to *in vivo*. Due to their rapid degradation during *in vivo* conditions decreasing their concentration within the system, it was proposed that ribozymes are more suitable for acute diseases and not chronic diseases [84]. There were also significant differences in the ribozyme efficiency in recognising and cleaving the target sequences when comparing *in vitro* and *in vivo* cells [77]. Due to these challenges, ribozymes' development as therapeutic agents, in general, has slowed down in the past years. More research must be conducted to improve the feasibility of ribozymes in the *in vivo* system by focusing on their stability and efficiency to bring ribozymes back to the table.

#### **6. Conclusion**

Ribozymes are catalytic RNAs that can catalyse reactions similarly to protein enzymes. There is a wide variety of ribozymes classes with different characteristics and structures, and even now, novel ribozymes are being discovered through research. Ribozymes have the potential to be used as therapeutic agents for infectious diseases. While there is a lack of actual ribozymes for antibacterial purposes, multiple ribozymes are tested to successfully target viruses such as human immunodeficiency virus (HIV), human cytomegalovirus and herpes simplex virus. Unfortunately, their uses have not been translated into real-world applications, mostly due to their vulnerability to nucleases in the biological system and the difficulty in translating their efficiency from the *in vitro* system to the *in vivo* system. However, progress has been made in improving their stability and delivery, and it is hoped that with more research, ribozymes can be the next therapeutic agent used for infectious diseases.

*Ribozymes as Therapeutic Agents against Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.107141*

#### **Acknowledgements**

School of Science, Monash University Malaysia.

#### **Conflict of interest**

The authors do not have any conflict of interest.

### **Author details**

Bao Chi Wong, Umama Shahid and Hock Siew Tan\* Monash University Malaysia, Selangor, Malaysia

\*Address all correspondence to: tan.hocksiew@monash.edu

© 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 3
