*Review* **Delivery of Oligonucleotides: Efficiency with Lipid Conjugation and Clinical Outcome**

**Phuc Tran <sup>1</sup> , Tsigereda Weldemichael <sup>1</sup> , Zhichao Liu <sup>2</sup> and Hong-yu Li 1,\***


**Abstract:** Oligonucleotides have shifted drug discovery into a new paradigm due to their ability to silence the genes and inhibit protein translation. Importantly, they can drug the un-druggable targets from the conventional small-molecule perspective. Unfortunately, poor cellular permeability and susceptibility to nuclease degradation remain as major hurdles for the development of oligonucleotide therapeutic agents. Studies of safe and effective delivery technique with lipid bioconjugates gains attention to resolve these issues. Our review article summarizes the physicochemical effect of wellstudied hydrophobic moieties to enhance the cellular entry of oligonucleotides. The structural impacts of fatty acids, cholesterol, tocopherol, and squalene on cellular internalization and membrane penetration in vitro and in vivo were discussed first. The crucial assays for delivery evaluation within this section were analyzed sequentially. Next, we provided a few successful examples of lipid-conjugated oligonucleotides advanced into clinical studies for treating patients with different medical backgrounds. Finally, we pinpointed current limitations and outlooks in this research field along with opportunities to explore new modifications and efficacy studies.

**Keywords:** oligonucleotide; lipid conjugates; LNP; delivery; cholesterol; fatty acid; tocopherol; squalene

## **1. Introduction**

#### *1.1. Background of Oligonucleotide*

Oligonucleotide (ON) is a short strand of nucleic acid polymers mostly comprising of thirteen to twenty-five nucleotides, which can hybridize to targeted DNA or RNA. They are categorized into classes including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNAs), and aptamer. Watson–Crick base pairing is quintessential for ON mechanisms to act on targeted mRNA, which leads to the following consequences: (1) RNase H activity degradation, (2) inhibiting the formation of matured mRNA, and (3) conjuring steric blockage from ribosome interaction [1]. Therefore, ONs are preferable therapeutic strategies to prevent and treat various disorders via selective inhibition of deleterious gene expression. It is indeed shifting the era of drug discoveries into an exciting new field—oligonucleotide therapy. Comparatively, the ease of manufacturing, base-pairing specificity/sensitivity potential, and its long duration of action give higher preference than the conventional therapy. Longer duration of action which varies from weeks to months of post-administration outweighs the technical hitches of being only in an injectable formulation. Given the knowledge of genes and their role accessibility, incurable genetic disorders are made possible through this novel approach. Its application is not merely limited to drug discovery but also pertinent for investigations of the mechanism and stereochemistry of biochemical reactions, mapping of nucleic acid-protein interactions, and diagnostic applications [2]. ON therapy is well aligned to play a noteworthy role in speeding up drug discovery against traditionally undruggable targets. Figure 1 displays

**Citation:** Tran, P.; Weldemichael, T.; Liu, Z.; Li, H.-y. Delivery of Oligonucleotides: Efficiency with Lipid Conjugation and Clinical Outcome. *Pharmaceutics* **2022**, *14*, 342. https://doi.org/10.3390/ pharmaceutics14020342

Academic Editor: David Brayden

Received: 29 December 2021 Accepted: 27 January 2022 Published: 1 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

some pros and cons of oligonucleotide-based drugs versus conventional small molecules. Hence, ON has gained its deserved attention in research in a wide range of disease indications from oncology to anti-viral therapy. However, the biggest concern for ON is cellular membrane penetration. This hurdle is the result of ON's highly hydrophilic nucleoside combing of anionic phosphate backbone. Thus, passive transport is not an effective option, and conquering this issue is not a straightforward task.


**Figure 1.** Pros and cons comparison of oligonucleotide versus small molecule drugs. Some highlighted advantages of oligonucleotide would be inhibitory specificity, expedient manufacture, and low in drug–drug interaction. Disadvantages of cost, stability, and formulation remained.

The assistance with external delivery systems such as liposomes, nanoparticles, or micelles were proposed and experimented thoroughly; however, toxicity was frequently reported due to the immunogenicity caused by polycationic material [3,4]. Alternatively, chemical conjugation to neutral lipid/hydrophobic moiety can overcome this backlash. Hypothetically, these naturally occurring biomolecules that are familiarized with the human system can reduce the risk of toxicity while enhancing cellular penetration and systemic stability. Different forms of lipid structures were incorporated and evaluated in vivo for improvement in pharmacokinetic and pharmacodynamic properties. In this review article, we will summarize the studies of characterizing hydrophobic moiety in the relationship of improving delivery efficacy. Additional discussion about potential therapeutic application and future outcomes of lipid-conjugated oligonucleotides will be highlighted.

#### *1.2. Oligonucleotide-Based Drug Mechanism of Action*

A comprehensive review composed by Crooke et al. has highlighted the fundamental aspects of ON mechanism of action. We would like to briefly summarize his work by discussing in two distinctive groups. Occupancy-only mechanism and occupancy-mediated degradation as illustrated in Figure 2.

**Figure 2.** An illustration of main oligonucleotide mechanism of action as divided into two main groups: (1) Occupancyonly and (2) occupancy-mediated degradation. In occupancy only, oligonucleotide would act as steric blocker or inhibitor **Figure 2.** An illustration of main oligonucleotide mechanism of action as divided into two main groups: (1) Occupancy-only and (2) occupancy-mediated degradation. In occupancy only, oligonucleotide would act as steric blocker or inhibitor preventing any upcoming interaction with precedent targets. Meanwhile, occupancy-mediate degradation activates cleavage the targeted RNA via RNase or AGO-2.

discussing in two distinctive groups. Occupancy-only mechanism and occupancy-

In the occupancy-only approach or direct inhibition mechanism, ON will bind specifically to mRNA molecules via Watson**–**Crick base-pairing, which induces steric block from any followed-up interaction with proteins, nucleic acids, or transcription factors. In consequence, it can conjure either downregulation (translational arrest or cap inhibition) or upregulation (splicing modulation or RNA activation) processes. The most utilized, well studied, and therapeutically beneficial approach would be splicing inhibition. Once targeted mRNA hybridized with ON, a complex of small nuclear ribonucleoprotein (snRNP) would be sterically blocked from intron binding, thus halting the maturation of mRNA [2]. Splicing inhibition was successfully applied for Duchenne muscular dystrophy treatment, for which eteplirsen was approved by the FDA. Another plausible mechanism would be the disruption of RNA structural integrity following by ON hybridization. As result, abnormality in three-dimensional conformation interrupted its stability and halted sequential protein expression. Vickers et al. on disruption of HIV's TAR element would be a prime example. This research group implemented an ON to destabilize TAR's (trans-activating response sequence) stem loops, which followed by

mediated degradation as illustrated in Figure 2.

tarnishing HIV replication efficacy [5].

In the occupancy-only approach or direct inhibition mechanism, ON will bind specifically to mRNA molecules via Watson–Crick base-pairing, which induces steric block from any followed-up interaction with proteins, nucleic acids, or transcription factors. In consequence, it can conjure either downregulation (translational arrest or cap inhibition) or upregulation (splicing modulation or RNA activation) processes. The most utilized, well studied, and therapeutically beneficial approach would be splicing inhibition. Once targeted mRNA hybridized with ON, a complex of small nuclear ribonucleoprotein (snRNP) would be sterically blocked from intron binding, thus halting the maturation of mRNA [2]. Splicing inhibition was successfully applied for Duchenne muscular dystrophy treatment, for which eteplirsen was approved by the FDA. Another plausible mechanism would be the disruption of RNA structural integrity following by ON hybridization. As result, abnormality in three-dimensional conformation interrupted its stability and halted sequential protein expression. Vickers et al. on disruption of HIV's TAR element would be a prime example. This research group implemented an ON to destabilize TAR's (trans-activating response sequence) stem loops, which followed by tarnishing HIV replication efficacy [5].

On the other hand, occupancy-mediated degradation was often emphasized with two major mechanisms: RNase or AGO-2 mediated degradation. Both mechanisms can result in deteriorating targeted mRNA but with slight differences in recruitment algorithms. For RNase, it is universally well documented as an enzyme responsible for degrading a single RNA strand, or RNA:DNA hybrid [6]. It has two isoforms (H1 and H2) which are identified in mammalian cells with expression in the cytoplasm and especially in the nucleus. RNase H1 participates more enthusiastically in cleavage than H2, though H2 is more abundant [7]. Recruitment of RNase is accompanied by a gapmer or even a short tetramer ON. Additionally, binding with an RNA metabolic protein such as P32 can enhance cleavage specificity which provided a good glimpse for optimizing chimera to accelerate RNase H1 activity. AGO-2 protein is one of the four argonaute family members, which facilitates endonuclease cleavage at the targeted RNAs and contains three domains. Mid and PIWI domains confer catalytic actions and perform simultaneously with Paz domain, which is responsible for small RNA binding. Being an indispensable component of the RISC, it operates with highly complementary at the translational or posttranscriptional level. Thus, it exerts RNA-based silencing mechanisms by altering protein synthesis and affecting RNA stability. Precise complementarity between guide RNA and the target is essential for the efficient cleavage of targets. Studies show that mismatches at the 50 regions are less tolerated than the 30 region of guide RNA or cleavage site [8,9].

## *1.3. Biological Barriers That Challenged Druggability/Pharmacokinetic Profile of Oligonucleotide In Vivo*

Dated back in 1998, oligonucleotide was an ultimate breakthrough in the discovery of a new drug modality. Significantly, Fomivirsen [10], a cytomegalovirus (CMV) retinitis ONbased treatment for AIDS immunocompromised patients, was recognized and approved by the FDA. Thus, marked the beginning of its massive emergence in the drug industry. Accounting in 2021, additional ON-based therapies were introduced into the market for non-cancerous indications as shown in Table 1; while there are still numerous entries examined in clinical trials for oncogenic targets [11]. Before approaching this height, the first unmodified ON was deemed expendable due to its high clearance rate after in vivo delivery. Agrawal et al. conducted the first report on the ON pharmacokinetic profile that showed unfavorable properties of unmodified ON after intravenous injection to a monkey with a dose of 30 mg/kg [12]. Quantification of polyacrylamide gel (PAGE) determined short systemic retention of approximately 15 min with a half-life of only 5 min. Structurally, the unmodified ON was identical with the endogenous mRNA in nature. Its highly polyanionic and hydrophilic characters still hampered the ability to penetrate the phospholipid membrane with the addition of high renal clearance.

From numerous pharmacokinetic data and mechanistic studies, scientists such as Juliano et al. [13] outlined four possible biological barriers that instigate unfavorable conditions for the efficacy of ON therapies as illustrated in Figure 3. We begin the discussion with the first barrier known as nuclease, especially 30 -exonuclease. It is an enzyme that is widely expressed in plasma and induces hydrolyzing reaction by cleaving phosphodiester bond at either at 30 or 50 ends. It can directly target ON indiscriminately and catalyze degradation reaction, which leads to complete loss of the therapeutic effect of ON before reaching the targeted site. The second barrier would be the reticuloendothelial system (RES) or mononuclear phagocyte system (MPS). It can easily be defined as a homogenous collection of phagocytotic cells that act as cellular securities to process and clear any form of alienated particles such as toxins, bacteria, or any xenobiotic. Therapeutic ONs are no exception as macrophages engulf ON, which endangers its survivability. Once these phagocytotic cells fused with the lysosome, therapeutic ON are considered dead. Consequently, long-term degradation of ON can lead to detrimental effects such as renal tube degradation, splenomegaly, and elevation of liver transaminase [14]. The third barrier is the thickness of endothelial tissue. The lining of endothelium must be sturdy to enclose safe blood flow; however, in the case of therapeutic ON (which is only administered via injection), the wall

of the endothelium can prevent leakage of ON macromolecules. Thus, most therapeutic ON still lingers within circulation while an infinitesimal amount can escape from vascular lumen to interstitial fluid. The final barrier would be the cellular uptake mechanism in which scientists continued to manipulate for ON delivery. They studied the key concept of internalization mechanisms such as clathrin-based coated, caveolar, clathrin-independent carriers (CLIC), or micropinocytosis. As crucial as understanding the uptake mechanism, we beg a question: "How come the cellular uptake can be posed a challenge?" The answer lies in the late endosomal stage after ON is encapsulated in the cytoplasm. Late endosomes are usually fused with the lysosome to break down debris or recycle necessary material; hence, therapeutic ONs are victims of degradation and required to escape for maintaining a longer lifespan [15].


**Table 1.** List of FDA approved oligonucleotide drugs.

As these hurdles tamper ON effectiveness, alternative solution such as direct nucleic acid modification was applied to overcome exonuclease cleavage. However, it was not adequate since these nucleic acid derivatives were continuously recognized by the immune systems to digest and excrete as foreign invaders. Nanoformulation and direct conjugation (with GalNac, lipid, or antibodies) were strongly recommended to mask ON and avoid from RES associating clearance. Additionally, both techniques could improve membrane penetration, which assist ON to permeate through endothelial lining. Enhancement of ON to escape late endosome-lysosomal degradation remained controversial and not understood clearly. Co-administration with chemical enhancers to disrupt encapsulating vesicle was suggested; however, it concurred with high risk of toxicity. Our Table 1 of FDA approved ON drugs also updated with the modificative modalities to achieve maximal clinical outcome and to bypass the mentioned challenges.

**Figure 3.** Four biological barriers preventing activity of therapeutic oligonucleotide. (**A**) Endolysosomal entrapment. ON required escape from late endosome before subjected to lysosomal degradation. (**B**) Exonuclease cleavage. Initiate hydrolysis of phosphodiester backbone, which deteriorate ON. (**C**) Reticuloendothelial system. Digestion of ON by macrophage can terminate its activity. (**D**) Endothelium lining. Blockage of transverse ON from vascular lumen to interstitial fluid.

#### *1.4. Early Attempt of Oligonucleotide Chemical Modification*

Direct chemical modification was conceptualized to battle the discussed biological barrier to safely deliver therapeutic ON to its site of action. The earliest attempt was sulfurization of phosphodiester backbone into phosphorothioate (PS), which altered its physiochemical properties. With the low electronegative element of sulfur, phosphorothioate would be less susceptible to be nucleophilic attacked by nuclease. Improvement from the first-generation phosphorothioate was documented with pharmacokinetic data showing extension of half-life up to 1 or 2 h. Moreover, the clearance rate was significantly decreased with less than 5% ON detected in urine or feces after murine dosing for 12 h [16,17]. This high systemic retention was accompanied by a high affinity to plasma protein with 95% bound. Even with phenomenal achievement, there is some evidence of relevant phosphorothioate potential flaws: (1) degradation still can occur via other mechanisms such as transesterification or pyrophosphatase [18] and (2) an excessive amount of phosphorothioate on ON can negatively impact the binding affinity of targeted RNA. This first-generation modification is still frequently applied in modern ON synthesis, but it is incorporated with the second-generation modification at 20 ribose.

For RNA, the 20 hydroxyl group is a critical component for RNase to recognize and catalyze hydrolysis. Thus, protection of this group is necessary, which can perform via methoxylation. Moreover, 20 ribose modification was reported to diminish unwanted immune stimulation [19]. Researchers explored this protection technique by starting with naturally occurring 20 -O-methyl (20 -OMe) RNA, which exudes the improvement of nuclease resistance and binding affinity. A bulkier group such as 20 -O-methoxylethyl (20 -MOE) emerged as the most prominent candidate, which can be confirmed via thermal shift assay revealing stronger binding affinity as ∆Tm increased from 0.9 ◦C to 1.7 ◦C per modification counts. From these encouraging findings, the third generation was developed by introducing a constraint that hindered the nucleotide's conformational flexibility. The first attempt was bridging 20 -oxygen to 40 -carbon ribose to produce locked nucleic acid (LNA), which showed intense elevation of binding affinity (increasing of ∆Tm from 4 ◦C to 8 ◦C per modification units binding to RNA) [20]. Its derivatives with an additional methyl group, constrained ethyl (cET), were believed to conduct tighter binding. However, 20 ribose modification caused therapeutic ON the incompetency to recruit and facilitate RNase cleavage mechanism due to inability to identify and covalently bind to 20 -hydroxyl group [21,22]. A clever solution to this drawback is implementing these second-generation to flank at both sides of the gapmer, an ON consisting of a central DNA region recruiting RNase H.

Moreover, ribose moiety can be completely substituted with less rotatable structures such as tricycle DNA (tcDNA) or cyclohexene nucleic acid (CeNA). A fully modified tcDNA, which is equipped with three extra carbons between C(50 ) and C(30 ), lifted the thermal stability up by 1.2 ◦C and 2.4 ◦C per modification while interacting with DNA and RNA, respectively [23]. Similarly, the replacement of a furanose ring with cyclohexene also restricts some flexibility while exhibiting superior serum stability from degradation and enhancing RNase recruitment capability [24]. Nonconventional nucleic acid modification is illustrated via phosphorodiamidate morpholino oligomer (PMO). The ribose moiety retains the traditional oxidative oxygen while being re-closed with an additional ammonia unit. The phosphodiester backbone is replaced with phosphorodiamidate linkage. This modification demonstrates exceptional degradative resistance either from protease, esterase, or 13 different hydrolases in serum and plasma. With the uncharged character, PMO prevents unwanted hybridization with surrounding protein, which exacerbated ON effectiveness [25]. Figure 4 illustrates the representative variation of ON modification segregated by their generation.

**Figure 4.** Three generations of common nucleic acid modifications. First generation replaced phosphodiester (PO) backbone to phosphorothioate (PS) to enhance degradative resistance. Second generation includes modification of 20 ribose into 20 -O-methyl (20 -OMe) and 20 -O-Methoxyethyl (20 -MOE), which are popular in gapmer synthesis. Third generation restricts conformational flexibility by introducing a methyl bridge between 20 -O and 40 of ribose. Locked nucleic acid (LNA) and constraint ethyl (cET). Ribose moiety would be completely replaced with tricyclic DNA (tcDNA) or cyclohexene (CeDNA). Alternative modification was phosphorodiamidate morpholino oligomer (PMO). This neutral nucleic acid was described with an additional amine accompanied with phosphorodiamidate backbone.

Despite these exciting discoveries, systemic toxicity inherited by nucleic acid modification plaque ON therapies as reported in vivo and significantly, clinical trials. For instance, P.S modification was known for enhancing protein plasma binding, however, excessive repetition of P.S in a single ON unit impacted the affinity to mRNA and promiscuously developed off-target toxicity after long-termed exposure [11,21,26]. The second-generation such as 20 -MOE, are encountered in vivo toxicity in mice reported by Zanardi et al. However, there were no significant increases in toxicity for longer treatment duration. cET was the candidate believed to reduce much toxicity compared to other 20 ribose modifications [27]. Finally, the third generation cannot escape this trauma such as report in LNA with associating liver toxicity. Therefore, the modification must be considered with moderation to avoid unwanted adverse effects and needed additional sources of delivery.
