**4. The past, present, and future**

The discovery of DNA as hereditary material and the helical structure base paring of DNA have opened the new avenue toward the current understanding and use of nucleic acids for the development of oligonucleotide-based therapies [6, 34]. The antisense oligonucleotides bind to RNA through Watson-Crick base pairing theory and modulate the function of the targeted RNA. This leads to the key discovery and direct importance of development of oligonucleotide-based drugs and medicine [6, 34]. The initial discovery of antisense technology is to enhance or improve the protein and small molecule-based technology by targeting RNA instead of protein. As a therapeutic strategy, it effects the RNA processing and modulates protein expression by binding to RNAs encoding difficult-to-target proteins. However, translating this technology into the clinic had some disadvantage such as inadequate target

**7**

*Antisense Therapy: An Overview*

*DOI: http://dx.doi.org/10.5772/intechopen.86867*

arrangement, insufficient biological activity, and off-target toxic effects over two decades, but this strategy significantly develops the numbers and types of targets that can be approached for therapy. Further, this technology has a great advantage to develop drugs for the treatment of both monogenic and polygenic diseases as well as influence the human genetics and genomics [12, 34]. The novel chemical modifications of antisense oligonucleotides have been engaged to address these limitations over the years. Both antisense alterations and their mechanism of their action have not only improved the clinical trial design but also provide the breakthrough toward the translation of antisense oligonucleotide-based strategies into therapies [6, 34]. Additionally, multiple clinical trials have clearly confirmed the clinical uses of antisense pharmacology in humans and demonstrated their safety by using various mechanisms. The current antisense drugs, which are under clinical development phase, target different tissues both systemically and locally. The advanced oligonucleotide chemistry further enhances the antisense drug properties by increasing potency, safety, and broader tissue distribution [6]. The advancement of oligonucleotide designs had improved the antisense mechanism of binding to RNA to inhibit their function with and without out RNA degradation. Recently, several researchers demonstrated that antisense oligonucleotides can also be utilized to overexpress or suppress the protein production [34, 35]. Many nonpathogenic disease conditions lack an effective treatment; the rapid development of new improved nextgeneration antisense oligonucleotide-based drugs bear the potential of intense clinical application and therapeutic impact on the treatment soon, demonstrating that this new class of molecular medicines/drugs has several potential and advantageous applications in the clinic. There was always a lag period from the early discoveries till the therapy enters into the clinical trials and further in the market [34]. RNA-based precision medicine and gene therapy still need the improvement because of their off-target effects. The future applications of antisense technology will solely depend on the performance of novel molecules in the clinical trials. Till date tremendous progress has been made toward the antisense technology, despite the fact this has yet to deliver its full potential [36]. There are still more unanswered questions which need further improvement of the technology. The establishment of first-generation phosphorothioate oligodeoxynucleotides is a great asset and valuable pharmacological tool, which has shown the promise of new therapies to the patient. Further development of new improved second- and third-generation antisense oligonucleotides with novel formulation will result in better therapies for patients. Even though the tremendous progress has been made in antisense technology, there are still more questions that remain unanswered for the technology and opportunities to further improve upon the platform [36]. This technology has provided solutions and confidence to diseases which were earlier considered untreatable. The high-cost factor has prevented this treatment mode inaccessible for the general masses. To fulfill the clinical need, future innovations to this technology might help in finding better and affordable cure to many more diseases which is available to all. Furthermore, the development of CRISPR, an RNA-guided gene-editing technology, and the delivery of mRNA transcribed in vitro are a major development of the RNA therapeutics. The clinical applications and validations of RNA-based antisense drugs for modulation of gene/protein expression and genome editing are currently being investigated both in the laboratory and in the clinic [1]. The CRISPR-Cas genome editing has transformed the field and impacted the biomedical science field which has stimulated the development of RNA-based antisense delivery approaches to facilitate clinical translation of CRISPR-Cas technology [1]. For cancer therapy, the first US-based human trial using CRISPR-Cas9 ex vivo is isolated from the T cells of cancer patients by knocking out the genes encoding PD1 and T-cell receptor alpha/beta [1]. Unquestionably, the field of antisense RNA

#### *Antisense Therapy: An Overview DOI: http://dx.doi.org/10.5772/intechopen.86867*

*Antisense Therapy*

is in clinical trial phase [30, 31].

**4. The past, present, and future**

**3.5 Antisense therapy for infectious diseases**

for targeting other exons, which will broaden the treatment of the patient population. **Myotonic dystrophy type 1 (DM1)** is a multisystemic disease caused by a triplet repeat expansion (CTG) in the 3′ untranslated region of myotonic dystrophy protein kinase (DMPK) gene [26]. IONIS-DMPK-2.5Rx is a chimeric antisense oligonucleotide and is currently under a randomized controlled study trail in DM1 patients. **Transthyretin amyloidosis** is a form of systemic amyloidosis caused by misfolded transthyretin protein (TTR), in multiple tissues, including peripheral nerves, the gastrointestinal tract, and the heart [27]. Three different antisense drugs, IONIS-TTRRx, RNase H–dependent, and patisiran, are currently in development for the treatment of TTR amyloidosis, as well as for both familial amyloid polyneuropathy and cardiomyopathy [28]. **Spinal muscular atrophy (SMA)**, a progressive motor neuron disease, usually occurs in infancy or childhood caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene [29]. Nusinersen drug is a fully modified oligonucleotide designed to bind to a specific sequence in intron 7 of the SMN1 and 2 pre-mRNAs, enhancing exon 7 inclusion and increasing the production of SMN protein [30, 31] which is under review for market authorization. **Huntington's disease (HD)** is an autosomal dominant neurodegenerative disorder resulting from an expanded CAG repeat in the huntingtin (HTT) gene, which causes a toxic gain of function due to an expanded polyglutamine tract in the resulting protein. Antisense oligonucleotide designed to lower total HTT has been shown to provide a prolonged improvement in HD, and the drug

Various antisense mechanisms can be utilized to inhibit viral replication—for example, by binding to viral mRNA to block the translation of the protein or to degrade the viral RNA through an RNase mechanism or by blocking host microR-NAs that support viral replication [6]. Several antisense therapies are currently undergoing clinical trials for various infectious diseases. MicroRNA-122 (miR-122) is highly abundant in the liver and is essential to the stability and propagation of hepatitis C virus (HCV) [32]. This mRNA binds to a highly conserved 5′ untranslated region of the HCV genome, protecting it from degradation and host innate immune responses [32]. Additionally, miR-122 is also believed to play a major role in inflammatory activity in the liver [33], and RG-101, a GalNAc-conjugated oligonucleotide drug, is designed to inhibit miR-122 and HCV replication. The results from this clinical trial were very encouraging and support continued study

The discovery of DNA as hereditary material and the helical structure base paring of DNA have opened the new avenue toward the current understanding and use of nucleic acids for the development of oligonucleotide-based therapies [6, 34]. The antisense oligonucleotides bind to RNA through Watson-Crick base pairing theory and modulate the function of the targeted RNA. This leads to the key discovery and direct importance of development of oligonucleotide-based drugs and medicine [6, 34]. The initial discovery of antisense technology is to enhance or improve the protein and small molecule-based technology by targeting RNA instead of protein. As a therapeutic strategy, it effects the RNA processing and modulates protein expression by binding to RNAs encoding difficult-to-target proteins. However, translating this technology into the clinic had some disadvantage such as inadequate target

**6**

of the drug.

arrangement, insufficient biological activity, and off-target toxic effects over two decades, but this strategy significantly develops the numbers and types of targets that can be approached for therapy. Further, this technology has a great advantage to develop drugs for the treatment of both monogenic and polygenic diseases as well as influence the human genetics and genomics [12, 34]. The novel chemical modifications of antisense oligonucleotides have been engaged to address these limitations over the years. Both antisense alterations and their mechanism of their action have not only improved the clinical trial design but also provide the breakthrough toward the translation of antisense oligonucleotide-based strategies into therapies [6, 34]. Additionally, multiple clinical trials have clearly confirmed the clinical uses of antisense pharmacology in humans and demonstrated their safety by using various mechanisms. The current antisense drugs, which are under clinical development phase, target different tissues both systemically and locally. The advanced oligonucleotide chemistry further enhances the antisense drug properties by increasing potency, safety, and broader tissue distribution [6]. The advancement of oligonucleotide designs had improved the antisense mechanism of binding to RNA to inhibit their function with and without out RNA degradation. Recently, several researchers demonstrated that antisense oligonucleotides can also be utilized to overexpress or suppress the protein production [34, 35]. Many nonpathogenic disease conditions lack an effective treatment; the rapid development of new improved nextgeneration antisense oligonucleotide-based drugs bear the potential of intense clinical application and therapeutic impact on the treatment soon, demonstrating that this new class of molecular medicines/drugs has several potential and advantageous applications in the clinic. There was always a lag period from the early discoveries till the therapy enters into the clinical trials and further in the market [34]. RNA-based precision medicine and gene therapy still need the improvement because of their off-target effects. The future applications of antisense technology will solely depend on the performance of novel molecules in the clinical trials. Till date tremendous progress has been made toward the antisense technology, despite the fact this has yet to deliver its full potential [36]. There are still more unanswered questions which need further improvement of the technology. The establishment of first-generation phosphorothioate oligodeoxynucleotides is a great asset and valuable pharmacological tool, which has shown the promise of new therapies to the patient. Further development of new improved second- and third-generation antisense oligonucleotides with novel formulation will result in better therapies for patients. Even though the tremendous progress has been made in antisense technology, there are still more questions that remain unanswered for the technology and opportunities to further improve upon the platform [36]. This technology has provided solutions and confidence to diseases which were earlier considered untreatable. The high-cost factor has prevented this treatment mode inaccessible for the general masses. To fulfill the clinical need, future innovations to this technology might help in finding better and affordable cure to many more diseases which is available to all. Furthermore, the development of CRISPR, an RNA-guided gene-editing technology, and the delivery of mRNA transcribed in vitro are a major development of the RNA therapeutics. The clinical applications and validations of RNA-based antisense drugs for modulation of gene/protein expression and genome editing are currently being investigated both in the laboratory and in the clinic [1]. The CRISPR-Cas genome editing has transformed the field and impacted the biomedical science field which has stimulated the development of RNA-based antisense delivery approaches to facilitate clinical translation of CRISPR-Cas technology [1]. For cancer therapy, the first US-based human trial using CRISPR-Cas9 ex vivo is isolated from the T cells of cancer patients by knocking out the genes encoding PD1 and T-cell receptor alpha/beta [1]. Unquestionably, the field of antisense RNA

#### *Antisense Therapy*

therapeutics is presently undertaking foremost development, and the potential for using RNA antisense drugs for personalized medicine and immunotherapy as well as to address genetic, infectious, and chronic diseases will ensure the continued development of antisense RNA therapeutics for years to come [1].
