Delivery Systems of siRNA Drugs and Their Effects on the Pharmacokinetic Profile

With the increasing approval of oligonucleotide drugs, they attract increasing attention in the pharmaceutical industry. Although several oligonucleotide drugs have been successfully approved, their target tissue delivery remains challenging. Due to their physicochemical characteristics, unmodified free oligonucleotide drugs may be rapidly cleared by the body after dosing and pose a risk of off-target and toxic side effects. Therefore, nucleic acid-based drugs usually require chemical modifications and appropriate delivery systems to exert therapeutic effects. Although chemical modification plays an essential role in the delivery of oligonucleotide drugs, it is challenging for small interfering RNAs (siRNAs) drugs to reach the target sites by chemical modifications alone, and it is similar for the delivery of antisense oligonucleotides (ASOs). Therefore, an effective delivery system is vital to successful oligonucleotide drug development. At present, the leading companies in siRNA drug development all possess licensed delivery technology platforms. In the future, a breakthrough in delivery systems will undoubtedly become key factors for companies to dominate the market, facilitating the development of various nucleic acid drugs. This article focuses on nucleic acid drug delivery technology with the beginning of the delivery system of siRNA drugs.

Delivery Systems for Approved siRNA Drugs

In vivo delivery of siRNA drugs could be divided into three main phases, and the body sets different biological barriers for siRNA drugs in each stage:

• Reaching target tissues: After dosing, the siRNA first needs to evade degradation by nucleases in plasma and tissues and capture by the immune system, successfully entering the target tissue.

• Entering cells: Due to their high molecular weights and negative charges, siRNA drugs do not readily cross cell membranes through passive diffusion and thus require cellular internalization through endocytosis to enter the cells.

• Lysosomal escape: siRNA needs to escape from endosomes before they fuse with lysosomes, entering the cytoplasm where they bind to target mRNA to induce gene silencing.¹

The main task of a delivery system is to protect the siRNA against all biological barriers to reach the cytoplasm and bind to target mRNA, thus exerting drug efficacy.

(1) Lipid nanoparticle (LNP) delivery system

Due to the charge characteristics of siRNAs, cationic liposomes appear to be the best candidate delivery systems. However, the toxicity of traditional cationic liposomes impedes significantly the development of siRNA drugs. Alnylam has achieved a breakthrough in the successful delivery of oligonucleotides using Arbutus’ ionizable cationic lipid materials. 2018 saw the approval of Onpattro, the first siRNA drug using lipid nanoparticles (LNP) as a delivery system, which is used for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis (hATTR).

The major success factor of lipid nanoparticle delivery systems is using ionizable cationic lipids. These lipids exhibit different charging characteristics under different PH conditions, being positively charged in acidic pH environments and nearly neutral charged at physiological pH. The ionizable properties of lipids provide intelligent protection for siRNAs across biological barriers.² (Figure 1)

Firstly, siRNA drugs must be produced under low pH conditions. During production, the ionizable lipids are positively charged to achieve stable and optimal siRNA drug encapsulation rates. Polyethylene glycol (PEG) lipids are added to the liposomes to obtain liposomes of 100 nm or less sizes. Liposome size (20–100 nm) can be regulated by adjusting the proportion of PEG lipids to other lipids.

After administration, the lipid nanoparticle (LNP) delivery system presents an essentially neutral charge surface in plasma, which helps the siRNA drug to evade capture by the immune system and degradation by nucleases in conjunction with the PEG lipids.

As PEG lipids dissociate from the liposome surface, the siRNA-carrying liposomes enter cells via endocytosis. Due to the low pH in endosomes, the ionizable lipids protonated and their positive charge destabilizes the endosome phospholipid bilayer structure, facilitating siRNA escape from endosomes. This releases siRNA into the cytoplasm where it binds to target mRNA to silence target gene expression.

Figure 1. Lipid nanoparticle (LNP) mediated delivery of siRNAs in vivo

From among more than 300 candidate ionizable lipid materials, Onpattro selected the ionizable lipid material Dlin-MC3-DMA with the most desirable pKa characteristics. In clinical trials, Dlin-MC3-DMA-based liposomes showed superior therapeutic efficacy compared to first-generation ionizable lipids, with over 80% reduction in thyroxine transporter protein in serum.

(2)  N-Acetylgalactosamine (GalNAc) delivery system

A delivery system based on GalNAc-siRNA conjugation has received attention from nucleic acid drug development organizations in recent years for its efficient liver targeting and good safety profile. N-Acetylgalactosamine (GalNAc) is a ligand for the asialoglycoprotein receptor (ASGPR), which is predominantly expressed on hepatocyte cell membranes (~500,000 ASGPR per cell). GalNAc-siRNA conjugates transport siRNA from the cell surface into the cell through specific binding of ASGPR followed by endocytosis. Consequently, the GalNAc-siRNA conjugates dissociate from ASGPR. ASGPR recycles back to the cell surface, while GalNAc-siRNA conjugates further dissociate, releasing free siRNAs that exert pharmacological effects by silencing genes in the cytoplasm (Figure 2).^{3, 4} The 3ʹ end of the siRNA sense strand chemically bonds to GalNAc with a trivalent structure, and GalNAc-siRNA conjugate stability is increased by enhanced stabilization chemistry (ESC) technology to achieve increased liver exposure and prolonged gene silencing time.^4,5 Currently, several GalNac-siRNA drugs have received FDA and EMA approval(Table 1).

Figure 2. In vivo GalNAc-siRNA conjugate delivery process.

Table 1. EMA/FDA Approved GalNac-siRNA conjugate drugs.

Influence of Delivery Systems on siRNA Drug PK Profile

The delivery system assists siRNA drugs from the administration site to the target cell, and the siRNA exists mostly as a complex with the delivery system during the delivery process. Therefore, the delivery system influences siRNAs in vivo processes and distribution. Some researchers determined the tissue distributions of ³H-labeled free siRNAs and lipid nanoparticle-encapsulated siRNAs using quantitative whole-body autoradiography (QWBA) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) techniques, respectively. The QWBA study revealed that the animal kidneys and salivary glands exhibit the highest radioactivity after a single intravenous dosing of free siRNAs. However, there were the highest radioactivity levels in the tissue distribution of livers and spleens after the dosing with lipid nanoparticle-encapsulated siRNA, with radioactivity lasting >168 h. MALDI-MSI imaging results showed that the distribution of cationic lipid material was consistent with that of radiolabeled siRNAs, suggesting that siRNA may circulate together with lipid material and distributes to tissues in the form of nanoparticle complexes.⁶ By altering free siRNA tissue distribution characteristics, the delivery system achieves targeting and effectively prolongs the in vivo retention time of siRNA drugs.

Lipid nanoparticle (LNP) delivery systems primarily exhibit passive hepatic targeting. However, GalNac-siRNA conjugate delivery systems achieve active hepatic targeting by binding ligand GalNac to the asialoglycoprotein receptor. Adjusting the structure, proportions, or particle size of components or modifying oligonucleotide structures may result in significant changes in tissue distribution and gene-silencing efficacy of siRNA drugs. In Onpattro, the optimal pKa value for ionizable lipids is around 6.4, and a deviation in pKa of only 0.5 may lead to a 100-fold reduction in its efficacy. To enable the rapid distribution of Onpattro to target tissues in the liver, scientists have developed C14 alkyl chain PEG lipids, which are short-chain PEG lipids that can dissociate more rapidly from the liposome surface in vivo than conventional PEG lipids. The C14 alkyl chain PEG lipids allow liposomes that are not encapsulated by PEG to rapidly adsorb endogenous apolipoprotein E (ApoE), triggering ApoE receptors on the surface of hepatocytes to endocytose these liposomes. In GalNac-siRNA delivery systems, trivalent and tetravalent ligands exhibit a higher affinity for ASGPR than monovalent or divalent ligands. Therefore, trivalent GalNAc was selected to conjugate with siRNA for this delivery system. Exposure of GalNac-siRNA conjugates in the liver was significantly increased after structural modification of siRNA by Advanced ESC technology.⁷ Refinement of the structure and proportion of each component is key to optimizing the targeted distribution and efficacy of oligonucleotide drugs.

Prospects for the Development of Oligonucleotide Drug Delivery Systems

The indications for siRNA drugs that have been approved or entered clinical stages mainly concentrate on the liver or liver-related disease areas, which is closely associated with the successful development of liver-targeted delivery systems. Expanding indications to target other tissues and organs has become the focus of commercial oligonucleotide drug R&D at this stage.

Studies indicate that siRNAs can distribute and release in tissues including the heart, lungs, fat, and muscle by conjugating with various lipids (saturated fatty acids, unsaturated fatty acids, cholesterol, vitamins, etc.). Arrowhead’s TRiM™ (Targeted RNAi Molecule) platform ⁸ may facilitate this expansion, targeting multiple tissues, including the liver, lung, and tumors. This platform utilizes ligand-mediated delivery for tissue-specific targeting. The TRiM™ platform includes a highly potent RNA trigger that can optimize high affinity targeting ligands for each drug candidate, enhancing drug structure and pharmacokinetics. It also contains specific nucleic acid sequences that assist in achieving targeted drug delivery and maintaining optimal pharmacological activity and safety. Another startup, utilizing Cyclic Cell-Penetrating Peptides as core technology, should also be paid attention to. The Endosomal Escape Vehicle (EEV™) platform of Entrada Therapeutics ⁹ aims to develop intracellular therapeutics. The EEV™ technology platform conjugates and delivers various drugs, including oligonucleotides and antibodies, to the cytoplasm of target cells through chemical modifications. Preclinical studies have shown that more than 90% of EEV conjugates can be absorbed by the body, and those conjugates that enter a target cell can escape from early endosomes efficiently and rapidly. Scientists have observed that approximately 50% of EEV conjugates reach intracellular targets, which shows a significant improvement compared to the less than 2% escape rate of many current biologics. siRNAs targeting tumors and various diseased tissues via peptide nanoparticles, viral vectors, or binding antibody fragments are also current R&D hotspots.

In the development of delivery systems, quantifying the effects of changes in the delivery system on tissue distribution using PK studies combined with pharmacodynamic validation is key to unlocking the “smart” oligonucleotide delivery. This involves targeting different diseased tissues and treating multiple indications by adjusting the structure or proportion of delivery system components.^10,11

In recent years, siRNA drug delivery systems have also attracted the attention of companies involved in the development of other nucleic acid drugs. By conjugating with delivery systems, the efficacy of ASOs can be significantly enhanced, and several GalNac-ASO conjugate drugs are already in clinical trials. A lipid-based nanoparticle delivery system has also been successfully adopted by Pfizer and Moderna for the development of an mRNA vaccine against coronavirus disease. ¹²

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Authors: Xiang Li, Jing Jin

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