RNAI therapy

RNA therapy is a term used to describe the use of RNA-based molecules to modulate biological processes with the aim of treating specific diseases. In general, the RNA sequence is the key to influencing the expression or activity of its target molecules. Once the nucleic acid chemistry and delivery method are established, the production of RNA-based drugs for a new target can be accomplished in a relatively short time using these established methods.

Historical development:

However, there was a second, equally important but less recognized discovery in the past that was crucial for RNA research: the realization that two RNAs can form base pairs. Although this information is taken for granted today, early RNA researchers did not believe that RNA could form a double helix structure. However, in 1956, Rich and Davies published the first nucleic acid hybridization reactions, which showed that RNA can form a similar configuration to DNA due to the same base complementarity. This discovery formed the basis for the subsequent discovery of microRNAs (miRNAs) in 1993 and RNA interference in 1998, in which the formation of RNA duplexes is a key step in RNA interference.

The first application of RNA base pairing for therapeutic purposes was described by Stephenson and Zamecnik in 1978. They developed an antisense oligonucleotide that targeted the 35S RNA sequence of Rous sarcoma virus (RSV) to inhibit viral replication.

About 20 years later, the first drug containing an antisense oligonucleotide was approved by the US Food and Drug Administration (FDA) for the treatment of cytomegalovirus retinitis (Roehr B 1998).

In contrast to the long development history of antisense oligonucleotide-based drugs, comparatively little time elapsed between the discovery of small interfering RNAs (siRNAs) and their use as drugs.

The phenomenon of RNA interference (RNAi) was first described in 1998. It was shown that the treatment of Caenorhabditis elegans embryos with a mixture of sense and antisense RNAs led to a strong and specific inhibition of endogenous target mRNAs. Because RNA interference (RNAi) is simple and effective, it was rapidly adopted and widely applied by the scientific community e.g. (McCaffrey AP et al. 2002).

In 2010, the first clinical trials based on RNAi technologies were used to treat a patient with advanced melanoma (Davis ME et al. 2010). The successful cleavage of the target mRNA by the administration of siRNA was described. Subsequently, several siRNA-based drugs were developed. The first siRNA drug for patients with hereditary transthyretin-mediated amyloidosis was approved in 2018.

In 2013, mRNA vaccines against infectious diseases (rabies) were used for the first time. Treatment with this mRNA vaccine led to functional antibodies against these viral antigens (Alberer M et al. 2017). In 2020, the first mRNA-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was approved in most countries around the world.

Note: One of the biggest advantages of RNA-based drugs is their ability to target almost any genetic component within the cell. RNA-based drugs, including antisense RNA and siRNA, work by sequence-specific binding to their targets. This suggests that these drugs may be particularly effective in targeting non-coding RNAs (Frankish A et al.2019; Esteller M 2011; Song J et al. 2012). In this respect, it can be assumed that the importance of RNA drugs will continue to increase.

Antisense oligonucleotides

Antisense oligonucleotides modulate the expression of target RNAs through sequence-specific binding. Although the structure of these antisense oligonucleotides is primarily determined by their specific sequence, their chemical structure can be modified to achieve new effects. These modifications can increase the specificity and stability of antisense oligonucleotides (Roberts TC et al. 2020). Fomivirsen was the first antisense oligonucleotide drug approved by the FDA in 1998 and was used to treat cytomegalovirus retinitis, particularly in HIV-positive patients. Fomivirsen binds to the cytomegalovirus (CMV) mRNA that codes for the immediate-early-2 protein, which is essential for CMV replication (Roehr B 1998). However, the FDA withdrew the drug from the market in 2001, as antiretroviral therapies for HIV patients significantly reduced the demand for fomiviruses.

Small interfering RNAs

siRNAs use the endogenous RNAi signaling pathway to modulate the expression of their target RNAs. In native RNAi, endogenous small RNAs form a complex with the Argonaute protein (Ago) and thus produce an RNA-induced silencing complex (RISC). This then suppresses the expression of their target mRNAs through sequence-specific binding (Ha M et al. 2014). siRNA-based drugs use the same mechanism to achieve a similar effect. siRNA duplexes join the native RNAi signaling pathway after their integration into the RISC. Only the guide strand is bound by the Ago protein, while the passenger strand is discarded. The guide strand then directs the RISC activity specifically against its target.

Aptamers

Aptamers are nucleic acid constructs that bind specific proteins in order to modulate their function. To date, only one RNA-based aptamer drug (aptamers are named by adding the suffix "-apt-") has been approved by the US Food and Drug Administration (FDA). Pegaptanib is a 28 nucleotide long construct with two polyethylene glycol (PEG) residues at its end (Gragoudas ES et al 2004). This aptamer binds to isoform 165 of vascular endothelial growth factor (VEGF), blocking its interaction with its receptor protein and thereby inhibiting cell proliferation, the main downstream effect of VEGF signaling. Due to this effect, pegaptanib was developed as a therapeutic agent for wet (neovascular) age-related macular degeneration (AMD) (Gragoudas ES et al 2004). Although pegaptanib is currently rarely used due to competition from various antibody-based drugs with similar efficacy, it is a good example of how RNA-based aptamers can be used as therapeutics. Several RNA-based aptamers are in development and further aptamer-based therapeutics are expected to be available in the future.

Already approved RNAi therapeutics:

• Patisiran (Onpattro): This was the first approved RNAi therapeutic. It was approved in the EU in 2018 for the treatment of hereditary transthyretin-mediated amyloidosis (hATTR). This disease leads to an accumulation of faulty proteins in various organs, resulting in severe symptoms. Common side effects include infusion reactions (such as redness, itching, shortness of breath), headache, peripheral edema, nausea and infusion site reactions. Severe allergic reactions are also possible, but less common.
• Givosiran (Givlaari): Approved in the EU in 2020, givosiran is used to treat acute hepatic porphyria, a group of rare genetic metabolic disorders. In the treatment of acute hepatic porphyria, givosiran can cause side effects such as nausea, darkening of the urine, skin rash and elevated liver enzymes. In rare cases, severe allergic reactions may also occur.
• Lumasiran (Oxlumo): This medicine was approved by the EU Commission in 2020 for the treatment of primary hyperoxaluria type 1, a rare genetic disorder that leads to excessive production of oxalate. Side effects: injection reactions, abdominal pain, upper respiratory tract infections and fever.
• Inclisiran (Leqvio): Approved in 2021, inclisiran is used to treat hypercholesterolemia or mixed dyslipidemia. Side effects: Injection reactions, joint pain, urinary tract infections and respiratory tract infections.

RNA therapeutics work by influencing the expression and activity of specific target molecules, enabling the treatment of diseases that do not respond to conventional drugs. Such therapies can be adapted to a wide range of different RNA and protein forms and could pave the way for personalized medicine and therapies for rare diseases. However, RNA-based drugs are larger molecules than other therapeutics, which makes them difficult to target in the body. Kim therefore emphasizes that ensuring effective RNA drug delivery should be a key concern for future research (Kim YK 2022).

RNA interference (RNAi) is a natural biological process that is used in the development of RNAi therapeutics to specifically modify gene expression in cells. This process begins with the introduction of small interfering RNAs (siRNAs) or microRNAs (miRNAs) into the cell. These RNA molecules are specially designed to attach to the messenger RNA (mRNA) of a specific target gene. Once the siRNA or miRNA is introduced into the cell, it is incorporated into the RNA-induced silencing complex (RISC). Within this complex, the remaining RNA strand binds to its complementary sequence in the target mRNA, resulting in either degradation of the mRNA or inhibition of its translation into a protein.

This inhibition or degradation prevents the production of the corresponding protein, resulting in "silencing" of the target gene. Through this targeted gene regulation, RNAi can be used to suppress the expression of disease-causing genes, with the specificity of the process helping to minimize side effects. RNAi therapeutics therefore offer a precise and effective way to treat genetic and other diseases at the molecular level.

Genetic disorders: RNAi therapeutics can treat genetic disorders caused by overactive or faulty genes. For example, RNAi can be used to reduce the production of an abnormal protein that plays a role in certain genetic diseases.

Cancer: In some types of cancer, oncogenes or their products that promote tumor growth can be switched off. RNAi can be used specifically to suppress the expression of these genes in cancer cells, which can lead to slower tumor progression or even cell death.

Infectious diseases: RNAi therapies can also be developed to suppress genes that are important for the replication of viruses within infected cells. This could be particularly important in the treatment of viral infections such as HIV, hepatitis B and C or influenza.

Eye diseases: RNAi can be used in the treatment of certain eye diseases, such as age-related macular degeneration (AMD), by switching off the genes that contribute to the harmful growth of blood vessels in the eye.

Neurodegenerative diseases: In diseases such as Alzheimer's or Huntington's disease, RNAi could be used to reduce the production of toxic proteins associated with disease progression.

Cardiovascular diseases: RNAi could help regulate genes involved in cardiovascular diseases, such as those that help regulate cholesterol levels.

Metabolic disorders: There are potential applications for RNAi in the treatment of metabolic disorders, for example by regulating genes involved in glucose and lipid metabolism.

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