Nucleic Acid Therapy - BiopharmaDirect

RNA Therapy

From the central point of view, DNA is transcribed into RNA, and RNA is translated into protein. Theoretically, treatment can be targeted at DNA, RNA, and protein levels, respectively. Taken into consideration the current treatment technologies, possible treatment options might be as follows.

Figure 1 DNA → RNA → Proteins (image from M. Dwars, Quora)

(1) Small molecules and monoclonal antibodies target the protein level for treatment, which has the highest maturity and belongs to short-acting treatment.
(2) ADC, bispecific antibody (BsAb), Protac are improved versions of treatments, but are less mature.
(3) Gene therapy (excluding gene editing), RNAi, and mRNA therapy lasts longer, and can continue to express regulatory genes, but cannot permanently modify and change genes. Compared with gene editing and cell therapy, they have the advantages of being more durable and safer, and thus are advocated by many pharmaceutical companies like Alnylam Pharmaceuticals.
(4) Cell therapy and gene editing are permanent changes to modify DNA, which are newly emerging technologies with the lowest maturity. Although the treatment is for permanent change, it is currently facing safety issues such as cytokine release syndrome (CRS).

The different of small molecules, antibody drugs, and RNA drugs are compared in the following three aspects:

Small molecular targets are mainly intracellular proteins and cell surface proteins. The former includes KRAS, TKI, etc., while the latter includes GPCRs, EGFR, etc.

Antibody drug targets are secreted proteins such as TNFalpha, AB, IL and cell surface proteins such as PD1, HER2.

RNAi drug targets are targeted mRNA, which indirectly regulates the expression of various proteins, including secretory, intracellular and cell surface proteins and other broad-spectrum targets, including dystrophin, Bcl-2, TTR, PCSK9, ApoC3, EGFR, GPCR, etc.

Small molecule targeting requires a drug "pocket" or “site”, which can achieve direct or allosteric regulation. Antibody drugs directly target surface antigens while RNAi only requires mRNA sequence.

Small molecule drugs are designed through high-throughput screening and computationally assisted optimization methods such as SAR analysis. The route of administration includes oral and injection, and the half-life is short, measured in hours. Generally, the specificity is low, the binding is weak, and the off-target probability is greater than that of antibody drugs and RNAi. Metabolic processes and metabolites often have liver and kidney toxicity, and usually there is no immunogenicity problem.

Antibody drug design uses phage display and high-throughput live testing to find candidate clones with good affinity. The mode of administration is subcutaneous or intravenous injection. The half-life is medium, measured in weeks. Generally, the specificity and binding ability are strong, the off-target rate is low, the metabolites are amino acids, and the safety is good. There might be immunogenic problems.

RNAi drugs are programmed to design candidate sequences according to the given target sequence. The mode of administration is mainly subcutaneous or intravenous or intrathecal injection. Oral administration is still under development. The half-life is longer, measured in months. Generally, it has strong specificity, strong binding ability and low off-target rate. With the improvement of technology, immunogenicity and cytotoxicity caused by chemical modification have been reduced.

The development of RNA therapy has met with twists and turns. In the nearly 18 years from 1998 to 2016, some large pharmaceutical companies abandoned RNA therapy mainly for safety problems. For example, in 2014, Arrowhead's Phase 2 HBV RNAi clinical trial was suspended due to safety issues. In 2016, Revusiran, a collaboration of Sanofi and Alnylam, failed in clinical phase 3. But recently Blackstone have invested $2 billion in Alnylam this year, which manifested the strong potentials of RNA therapy.

From the technical difference, it can be divided into the following categories.

The mechanism is through complementary binding with the targeted mRNA to induce its degradation, thereby silencing gene expression. There are many ways, common ones are ASO, siRNA, miRNA, shRNA, etc. Representative drugs include Kynamro from Ionis, Patisiran from Alnylam, and cobomarsen from miRagen

The mechanism is to target specific gene promoter regions through saRNA, thereby up-regulating gene expression.

The mechanism is to use the three-dimensional mechanism formed by the folding of single-stranded oligonucleotides to achieve specific binding to the target protein, and to play the role of inhibition or activation.

The mechanism is to express the target protein (therapeutic or absent) by introducing mRNA, including non-replicating mRNA and self-amplifying mRNA. mRNA is currently used in antiviral vaccines, tumor vaccines, etc. After the emergence of coronavirus this year, the application of mRNA in viral vaccines has been accelerated. Currently, no mRNA therapeutic drugs and vaccines have been approved for marketing, and breakthroughs in technology and applications are needed. Representative drugs are Translate Bio's MRT5201 and Moderna's mRNA-1273.

The mechanism is based on RNA as the target, thereby up-regulating or down-regulating the target translation efficiency.

The mechanism is based on RMPs, nRNA regulatory proteins, as the target to change the body's transcription level, thereby realizing the regulation of disease-related protein expression.

Among the above six platforms, RNAi is the most mature technology platform that currently has new drugs approved. RNAi includes ASO, siRNA, miRNA, etc. Among them, ASO is developed earliest, and siRNA is currently most popular.

The differences of three RNAi technology subtypes, ASO, siRNA, and miRNA, are compared as follows.

ASO is a single-stranded DNA that directly complements targets such as mRNA, miRNA, or premRNA, and regulates expression by inducing degradation, hindering translation, and shearing and capturing.

siRNA is a double-stranded RNA, which is combined with AGO2 to form a siRNA-induced silencing complex siRISC. The antisense strand is completely complementary to the targeted mRNA and induces degradation. siRNA can be amplified by RdRP.

miRNA is a single-stranded RNA with an endogenous sequence. By forming miRISC, the antisense strand is partially complementary to the targeted mRNA and hinders translation.

The technical advantages of ASO are as follows:
-Clear targeting and strong specificity.
-Relatively enriched in liver, kidney and other tissues.
-Various functions such as to up-regulate or down-regulate gene expression and to regulate subtype expression.
-Stable pharmacokinetics, with a certain long-term effect, up to 1-3 months.

The limitations of ASO are as follows:
-Sometimes the affinity with the target sequence is insufficient.
-It may be off-target and toxic.
-Targeted delivery is difficult.

The technical advantages of siRNA are as follows:
-There is a cascade effect. Gene silencing efficiency is high, and the efficiency is usually higher than ASO (3-5 times).
-Clear targeting and strong specificity.
-Good pharmacokinetics: long-term effect is better than ASO.

The limitations of siRNA are as follows:
-Usually can only down-regulate genes.
-Potential off-target effects caused by double strands.
-Targeted delivery is difficult.

The technical advantage of miRNA are as follows:
-It can target multiple mRNA targets at the same time, and has the potential of synergy and network regulation.

The technical limitation of miRNA are as follows:
-It is still at the early stage of development and there might be a non-specific risk.
-The down-regulation effectiveness is limited.

Similar to any other new drug development, efficacy and safety are eternal issues that RNAi development faces. Currently, the industry are mainly focused on delivery methods and sequence optimization.

In terms of delivery methods, different companies have different technology platforms.

The principle is based on the high expression of ASGRP on the surface of some liver cells and high affinity with GalNAc, so it can be widely used in liver transport diseases. It is the current method with high maturity and rich clinical pipeline, such as Alnylam's GalNAc platform, Dicerna's GalXC platform, and Ionis' LICA platform.

The principle is to encapsulate drug molecules through liposomes to reduce renal clearance and at the same time have a certain specificity for specific organs. The first RNA drug on the market used LNP technology, but the overall technology maturity is not high, and the tissue targeting is weak.

Endogenous nanovesicles are easier to penetrate biological barriers (such as the blood-brain barrier) and achieve tissue targeting, and have the potential for oral administration.

From the point of view of nucleic acid modification, a single nucleic acid and a full-length sequence can both be modified. The modification of a single nucleic acid has been widely used in ASO and siRNA. At an advanced level, both Alnylam and Arrowhead have developed related technologies.

EvalutePharma and BCG predict that the RNA nucleic acid therapy market will reach US$8.6 billion in 2024, with a CAGR of approximately 35% in 2018-2024. The expansion of the RNA therapy market comes from the approval of the research and development of various innovative drugs and the expansion of new targets and indications. Current indications include genetic diseases, heart diseases, infectious diseases, central nervous system CNS, ophthalmology, etc.

Till now ten RNA therapy drugs have been approved.

MACUGEN^® (pegaptanib sodium injection) was first approved by the FDA in 2004. The target is VEGF. The indication is wet age-related macular degeneration wAMD. The medication is intravenous Intravitreal Injection. Macugen is a PEG-modified 28nt long oligonucleotide covalent conjugate. It is an aptamer with a molecular weight of about 50kD. The structure is as follows and the manufacturer was Bausch + Lomb/Valeant Pharmaceuticals at the time of approval.

Figure 2 Strcture of pegaptanib sodium injection. (image from Drug Infomation)

KYNAMRO (mipomersen sodium), first approved by the FDA in 2013, is administered by subcutaneous injection. The target is apolipoprotein B-100, which is a nucleotide inhibitor, and the indication is homozygous familial hypercholesterolemia. ApoB is the main apolipoprotein of low-density lipoprotein LDL. Mipomersen inhibits the expression of ApoB by binding to the mRNA sequence of ApoB. Kynamro is 20nt long and is methylated at the 5-end of bases C and U. The underlined bases are 2'-O-(2-methoxyethyl), and the rest are 2'-deoxynucleosides. The structure of Kynamro is as follows, and its molecular weight is 7594.9 g/mol. Kynamro's manufacturer is Genzyme, a subsidiary of Sanofi.

Figure 3 Strcture of mipomersen sodium. (image from drugs.com)

EXONDYS 51 (eteplirsen), first approved by the FDA in 2016, is indicated for Duchenne muscular dystrophy (DMD). It is derived from exon 51 skipping of the DMD gene, and the medication is intravenous. Exondys 51 is an ASO antisense oligonucleotide, a type of phosphorodiamidate morpholino oligomer (PMO). The technical principle is to replace the five-carbon sugar ring in natural DNA and RNA with a six-ring morpholine ring. Natural DNA and RNA are negatively charged, but PMO is neutral and uncharged and cannot be recognized by enzymes, so it is more stable. Eteplirsen is 30nt long and has a molecular weight of 10305.7 daltons. The molecular structure is as follows and the manufacturer is Sarepta Therapeutics.

Figure 4 Strcture of eteplirsen. (image from Drug Infomation)

SPINRAZA (nusinersen), first approved by the FDA in 2016, is indicated for spinal muscular atrophy (SMA). The administration method is intrathecal intrathecal injection, and the target is survival motor neuron-2 (SMN2). Spinraza is the antisense nucleotide ASO of the exon 7 sequence of SMN2 gene. The 2'hydroxyl group of ribofuranosyl is replaced by 2'-O-2-methoxyethyl groups and the phosphate chain is replaced by phosphorothioate. Spinraza has a molecular weight of 7501.0 daltons and its molecular structure is as follows. The manufacturer is Biogen.

Figure 5 Strcture of nusinersen. (image from FDA)

ONPATTRO (patisiran), first approved by the FDA in 2018, is indicated for hereditary transthyretin-mediated amyloidosis and is for intravenous use. Onpattro is a double-stranded siRNA (small interfering ribonucleic acid), and the target is transthyretin (TTR). Onpattro binds to the 3’ untranslated region (3’UTR) of the TTR gene mRNA and acts to cause mRNA degradation. The molecular weight of Onpattro is 14304 Da, and the structure is as follows. The manufacturer is Alnylam Pharmaceuticals.

Figure 6 Strcture of patisiran. (image from FDA)

TEGSEDI (inotersen), first approved by the FDA in 2018, is indicated for polyneuropathy of hereditary transthyretin-mediated amyloidosis, which is exactly the same as the above-mentioned Onpattro indication. The mode of administration is subcutaneous injection, the target is TTR, the drug form is antisense nucleotide ASO, and the molecular weight is 7600.73 Da. The molecular structure of the drug is as follows. The manufacturer is Ionis Pharmaceuticals.

Figure 7 Strcture of inotersen. (image from healthgrades.com)

Waylivra (volanesorsen) was not yet approved by FDA, but has been approved by in 2018. The indication is Familial Chylomicronemia Syndrome (FCS), the target is apolipoprotein C-III (ApoC- III), and the drug form is ASO. The manufacturers are Akcea Therapeutics and Ionis.

Figure 8 Strcture of volanesorsen. (image from ema.europa.eu)

GIVLAARI (givosiran), first approved by the FDA in 2019, is indicated for acute hepatic porphyria (AHP), injected subcutaneously in the form of siRNA, and the target is aminolevulinate synthase-1 (ALAS-1). The drug is delivered to the liver by covalent coupling of N-acetylgalactosamine (GalNAc). The molecular weight of the drug is 17,245.56 Da, and the molecular structure is as follows. The manufacturer is Alnylam Pharmaceuticals.

Figure 9 Strcture of givosiran. (image from newdrugapprovals.org)

VYONDYS 53 (golodirsen), first approved by the FDA in 2019, is indicated for Duchenne muscular dystrophy (DMD). It is derived from exon 53 skipping of the DMD gene, and used by intravenous injection in the form of ASO. The molecular weight is 8647.28 daltons and the structure is as follows. The manufacturer is Sarepta Therapeutics.

Figure 10 Strcture of golodirsen. (image from newdrugapprovals.org)

VILTEPSO (viltolarsen), first approved by the FDA in 2020, is indicated for Duchenne muscular dystrophy (DMD). It is derived from exon 53 skipping of the DMD gene, and is used by intravenous injection in the form of ASO withPMO technology. The molecular weight of the drug is 6924.82 daltons. Although the target and indications are the same as that of VYONDYS 53, the molecular weight is different. The structure is as follows. The manufacturer is NS Pharma.

Figure 11 Strcture of viltolarsen. (image from newdrugapprovals.org)

The innovative companies focusing on RNA therapy that have been listed include: Alnylam, Arrowhead, Ionis, Sarepta and Dicerna. Visit their websites to learn more about their latest progress on RNA therapy.