Progress on siRNA-based gene therapy targeting secondary injury after intracerebral hemorrhage

Abstract

Intracerebral hemorrhage (ICH) is a life-threatening condition with a high mortality rate. For survivors, quality of life is determined by primary and secondary phases of injury. The prospects for injury repair and recovery after ICH are highly dependent on the extent of secondary injury. Currently, no effective treatments are available to prevent secondary injury or its long-term effects. One promising strategy that has recently garnered attention is gene therapy, in particular, small interfering RNAs (siRNA), which silence specific genes responsible for destructive effects after hemorrhage. Gene therapy as a potential treatment for ICH is being actively researched in animal studies. However, there are many barriers to systemic delivery of siRNA-based therapy, as the use of naked siRNA has limitations. Recently, the Food and Drug Administration approved two siRNA-based therapies, and several are undergoing Phase 3 clinical trials. In this review, we describe the advancements in siRNA-based gene therapy for ICH and also summarize its advantages and disadvantages.

1. Introduction

Hemorrhagic stroke is a devastating event that can cause severe brain injury, potentially leading to lifelong disabilities or death. Hemorrhagic stroke has two subtypes, subarachnoid hemorrhage and intracerebral hemorrhage (ICH) (1). ICH is the second most frequent cause of stroke after ischemic stroke(2). It accounts for 10–15% of all stroke types but has the highest mortality (2, 3). Nearly 50% of patients die within the first month. Only 20% of patients who survive ICH achieve functional independence within 6 months of symptom onset(2, 4). Each year, ICH affects over 1 million people worldwide, and the incidence is projected to increase with the increase in life expectancy(3). Knowledge and understanding of the pathophysiologic mechanisms that underlie the injury cascade after ICH has improved in recent years; however, effective therapy for ICH is still lacking, with only supportive treatment options available (5, 6).

2. Molecular Mechanisms of ICH

ICH occurs when a blood vessel of the brain ruptures. The blood released into the surrounding area sets off a cascade of widespread injury. The initial damage that follows ICH is the consequence of mechanical compression from the spreading hematoma. Within the first few hours, the hematoma increases intracranial pressure and reduces cerebral blood flow, causing mechanical disruption of neurons and glial cells and damaging the blood-brain barrier (BBB). These events are preceded by mitochondrial dysfunction, membrane depolarization, brain edema, and neuronal death and are collectively called the “primary injury”(1, 7–11). Secondary injury develops over a period of time (from hours to days) after initial symptoms, and is characterized by an inflammatory response against the release of blood components (hemoglobin and iron) and coagulation factors (thrombin). This inflammation further exaggerates BBB damage. Secondary injury is considered to be the second most important contributor to delayed effects of brain injury after ICH (1). Thrombin release activates microglia and induces apoptosis in neurons and astrocytes. Expression levels of toll-like receptors (TLRs) TLR-4 and TLR-2 in microglia increase by 6 h after ICH onset and remain elevated for 3 days(12). TLRs recruit cytoplasmic adaptor proteins, such as TIRAP, TRIFs, and MyD88. These adaptor proteins then prompt microglia to release interleukin (IL) 1β, tumor necrosis factor α (TNF-α), and IL-6 via the NFкB pathway(13–15). Evidence for this signaling pattern is also supported by a clinical study showing that upregulation of TLR4 and TLR2 is associated with a larger hematoma volume and poor neurologic outcomes in ICH patients(16). Activated microglia also produce chemokines that attract leukocytes (peripheral inflammatory cells) into the brain, thus causing an accumulation of inflammatory mediators(17–21). Other factors responsible for the inflammatory cascade in the brain, as observed in animal and human studies, include the activation of leukocyte adhesion molecules on the luminal surface of endothelial cells and the upregulation of matrix metalloproteinases (MMPs). Notably, astrocytes also contribute to the progressing inflammation by expressing MMPs. Reactive astrocytes and microglial cells interact with one another and subsequently activate MMPs(18). Various clinical studies have shown that MMP-9 levels increase in patients with acute spontaneous ICH. MMP-9 levels increase within 12 to 24 h of ICH and are associated with lesion volume expansion, followed by worsening neurologic function, greater inflammation, and increased perihematomal edema(18, 22). Iron released from dying cells is another culprit that induces large amounts of lipid reactive oxygen species (ROS), which accumulate in neurons and cause ferroptosis(23). Furthermore, ICH induces various cell death mechanisms in the perihematomal region, such as apoptosis, necrosis, necroptosis, autophagy, and pyroptosis(24). Our in vivo and in vitro studies of secondary injury have shown that cofilin, a cytoskeletal protein, participates in the inflammatory cascade and mediates neuroinflammation after ICH(25, 26). Collectively, these events dictate the extent and severity of the secondary injury and ultimately the patient’s outcome(1, 27–33). Therefore each step of the pathway can become a target for therapeutic intervention with biologics.

As most small-molecule neuroprotective agents that have shown promise in animal studies have failed in clinical trials, the thrust of research has shifted toward developing gene therapies. Initially, gene therapies met with severe criticism for their lack of translational value, but their recent success in clinical trials has led to more favorable views. Many gene therapies are now being used for a variety of diseases, and the most promising, small interfering RNA (siRNA), is gaining clinical prominence. Consequently, interest has increased in translating siRNA into clinical settings as a novel next-generation medicine. To this end, the U.S. Food and Drug Administration (FDA) has approved two RNA interference (RNAi)-based therapies: 1) patisiran for the treatment of hereditary transthyretin amyloidosis with polyneuropathy and 2) givosiran for the treatment of acute hepatic porphyria (34). The preclinical success of siRNA-based studies could completely alter the clinical management of complex diseases like ICH. Therefore, in the next section, we discuss the progress of studies aimed at identifying and developing siRNA-based therapies for ICH-induced secondary brain injury.

3. siRNA-based therapies targeting ICH-induced secondary brain injury

The discovery of RNA interference in the early 1990s triggered a sequence of studies that helped to advance the field of siRNA gene therapy. However, studies on the use of siRNA therapeutics for ICH have not been widespread. To date, only a handful of in vivo studies have been conducted to investigate siRNA as a potential therapeutic strategy for secondary brain injury after ICH. As discussed above, the ICH-induced secondary injury cascade involves a multitude of deleterious mechanisms, such as inflammation, immune system activation, oxidative stress, excitotoxicity, microglial activation, and neuronal apoptosis(17, 35). Therefore, treatment strategies that target these processes might reduce ICH-related secondary injury and improve neurologic deficits. In this section, we describe in vivo research in which naked siRNA was administered intracerebroventricularly as a therapeutic agent in different models of hemorrhagic brain injury (see Table 1 and Figure 1).

Table 1.

In vivo studies that used siRNA-based therapies to target ICH-induced secondary brain injury

Study	siRNA target	In vivo model	Target	Delivery time point	Target pathway
Ma et al. [41]	Inflammation	ICH (autologous blood)	NLRP3	24 h before ICH	Decreased NLRP3 inflammasome
Feng et al. [42]	Inflammation	ICH (collagenase model)	P2X7R	24 h before ICH	Prevented NLRP3 inflammasome activation
Wang et al. [43]	Inflammation	ICH (autologous blood)	PTPN22	24 h before ICH	Alleviated Inflammation via PTPN22/NLRP3 pathway
Ma et al. [44]	Inflammation	ICH (collagenase model)	VAP-1	48 h before SAH	Decreased neuroinflammation by inhibiting VAP-1
Zhang et al. [45]	Inflammation	ICH (collagenase model)	Snhg3	24 h after ICH	Decreased inflammation by targeting TWEAK/Fn14/STAT3 pathway
Li et al. [46]	Inflammation	ICH (autologous blood)	Foxo-1	24 h before ICH	Decreased Inflammation by blocking the Foxo1/TLR4/NF-κB signaling pathway
Yang et al. [47]	Inflammation	ICH (autologous blood)	PDGF receptor-β	24 h before ICH	Decreased inflammation by preventing plasmin-induced brain injury after ICH
Wu et al. [48]	Microglial phenotype polarization	ICH (autologous blood)	PD-1 and PDL-1	12 h before ICH	Mediated microglial polarization to anti-inflammatory phenotype (M2)
Chen et al. [53]	Microglial phenotype polarization	ICH (autologous blood)	Tim-3	48 h before ICH	Promoted anti-inflammatory microglial phenotype (M2) by Tim3/Gal-9/TLR-4 pathway
Alhadidi et al. [26]	Apoptosis	ICH (collagenase model)	CFL1	24 h before ICH	Decreased apoptosis by targeting microglia/oxidative stress pathway
Xu et al. [57]	Apoptosis	ICH (autologous blood)	GATA-4	48 h before ICH	Decreased apoptosis via NF-kB-Bax-caspase-3 pathway

Note: Route of siRNA delivery in all studies was intracerebroventricular.

ICH, intracerebral hemorrhage; PDGF, platelet-derived growth factor;

Figure 1:

Major signaling pathways of primary and secondary injury after intracerebral hemorrhage (ICH) and siRNA-based therapies that target secondary brain injury.

3.1. siRNA targeting inflammation

Before signaling molecules can be targeted, it is imperative to find the role of these molecules in the ICH cascade. Studies have identified many molecules that have either a detrimental or protective function. Additionally, temporal signaling of the molecular cascade must also be taken into consideration. It is well established that after ICH, blood components such as leukocytes enter the brain parenchyma and activate microglial production of pro-inflammatory cytokines that contribute to secondary injury(36). Therefore, attempts have been made to modulate inflammation with siRNA intervention. siRNAs that target mRNA of major inflammatory factors after ICH were effective in secondary injury-related gene silencing treatment (37). Researchers have found that activation of NOD-like receptor family pyrin domain-containing 3 (NLRP3, or NALP3) inflammasome (also called cryopyrin), a key component of the innate system, plays a role in the inflammatory response of central nervous system (CNS) diseases (38). Once NLRP3 is activated, it recruits apoptosis-associated speck-like protein (ASC) and pro-caspase-1 to form an inflammasome, which then activates pro-caspase-1. Activated caspase-1 cleaves pro-IL-1β and pro-IL-18 into their active forms to initiate neuronal damage(39). Compelling evidence suggests that the inflammatory mechanism, particularly the NLRP3 inflammasome, is involved in neuroinflammation and aggravates brain injury after ICH (40). Ma et al. (41) demonstrated that knockdown of NLRP3 with siRNA 24 h before ICH decreased brain edema and myeloperoxidase, and improved neurologic outcomes in a mouse model. In a rat model of ICH, pretreatment with P2X purinoceptor-7 (P2X7R) siRNA (24 h before ICH) and post-treatment with BBG or piroxantrone (ONOO−) catalyst (30 min after ICH) suppressed NLRP3 inflammasome activation and subsequently inhibited caspase-1, IL-1β, and IL-18 release. The authors also reported attenuation of brain edema and neurologic dysfunctions as well as enhancement of NADPH oxidase 2(42). Animal and human studies based on the same hypothesis showed that histone deacetylase 10 (HDAC₁₀), a newly discovered class II histone deacetylase that participates in immune reactions, was upregulated/activated and reached peak levels 24 h after ICH. When the level of HDAC₁₀ in brain tissue around the hematoma was assessed by immunohistochemistry and Western blotting in patients with ICH, it was found to be upregulated compared to that in a normal control group. Silencing HDAC₁₀ after ICH was shown to aggravate brain injury, increase edema, increase neurologic deficits, and increase NLRP3 inflammasome activation via the PTPN₂₂ (protein tyrosine phosphatase nonreceptor type 22) pathway, which is purported to be involved in the inflammatory response. Notably, these effects were significantly reversed by treatment with PTPN₂₂ siRNA. The results suggested that modulating or manipulating NLRP3 inflammasome by siRNA might prove to be a novel strategy to treat ICH(43).

In a separate study, Ma et al. (44) indirectly targeted the cell death pathway by using siRNA against vascular adhesion protein 1 (VAP-1) in a mouse ICH model. VAP-1 is associated with leukocyte migration and infiltration across the BBB into the brain parenchyma during inflammatory conditions. Blocking VAP-1 with siRNA 24 h before ICH inhibited leukocyte migration, decreased cerebral edema, and improved neurobehavioral deficits, suggesting that VAP-1 siRNA could potentially be used to target inflammation after ICH(44). Additionally, Zhang et al. (45) observed that binding of TWEAK (TNF-like weak inducer of apoptosis) to fibroblast growth factor-inducible 14 (Fn14) activates an inflammatory elements such as Janus kinase/transducers and activators of transcription (JAK/STAT). When activated, the JAK/STAT pathway mediates neuroinflammation in neurodegenerative diseases. In a rat model of ICH, overexpression of long non-coding RNA small nucleolar RNA host gene 3 (Snhg3), which is involved in cell proliferation and dysfunction of brain microvascular endothelial cells, increased the expression of TWEAK and Fn14. Knockdown of Snhg3 by siRNA, which was injected 24 h after ICH and every 2 days until day 7, significantly reduced brain edema and cell apoptosis, enhanced BBB integrity, and improved neurobehavioral outcome by suppressing the TWEAK/Fn14/STAT3 pathway(45). Targeting other pathways, Li et al. (46) showed that siRNA-mediated knockdown (24 h before ICH) of fork-head box O-1 (Foxo-1), a transcription factor that regulates signals in cell apoptosis and immune regulation, led to a decrease in the expression of inflammatory cytokines such as TNF-α, IL-1β, IL-18, TLR4, and Nf-κB, eventually mitigating ICH injury and improving neurologic deficits. These results suggest that the Foxo1/TLR4/NF-κB pathway provides a novel strategy to manage the inflammatory response after ICH-related secondary injury(46). It has also been observed that platelet-derived growth factor receptor-β (PDGF-β) mediates neuroinflammation through a phenotypic transformation of vascular smooth muscle around the hematoma region by activating the p38 mitogen-activated kinase pathway after ICH. Knockdown of PDGF-β by siRNA (24 h before ICH) not only prevented plasmin-induced inflammation after ICH but also decreased neutrophil infiltration, intercellular adhesion molecule-1, neurologic deficits, and brain edema(47). These studies targeted neuroinflammation without considering the spatial and temporal signaling of these molecules, as the aim was to achieve complete transfection before ICH. Using pre-treatment paradigms is a major limitation for siRNA therapy. Neuroinflammation is believed to peak within 12 h of ICH and last for 48–72 h. Only one study from the aforementioned reports used a post-treatment paradigm, in which Snhg3 siRNA was injected 24 h after ICH. Based on the timing of the intervention, SnHg3 siRNA administered every two days for 7 days seems to have translational value and warrants further investigation. Given the importance of inflammation in secondary injury, developing siRNA-based therapies that target components of inflammation could be extremely valuable.

3.2. siRNA targeting microglial phenotype polarization

Microglia/macrophages are the primary immune cells of the CNS. As such, they provide the first line of defense against pathogens and during inflammation(48). After ICH, large numbers of microglia and macrophages are activated and amass around the hematoma where they release inflammatory factors. Different phenotypes of microglia/macrophages participate in brain injury and play essential roles, depending on the signal released in the local microenvironment(49, 50). Two main phenotypes of microglia/macrophages, M1 and M2, reflect two opposing effects on the inflammatory response. M1 microglia promote neuroinflammation and produce inflammatory mediators, whereas M2 microglia inhibit inflammatory responses and are involved in tissue repair and tissue debris removal. Which microglial phenotype is predominant after ICH is a key determinant of ICH-induced secondary injury(51, 52). Therefore, siRNA can target the genetic level of early gene expression, which can modulate microglial activation and help attenuate the severity of the M1 phenotype and maintain the M2 phenotype.

Programmed death protein 1 (PD-1) and programmed death-ligand 1 (PDL-1) are reported to play an essential role in mediating the neuroimmune response after CNS disorders. However, no study has described a role for PD-1 and PDL-1 signal transducers and activators in microglial polarization. Wu et al. (48) hypothesized that PD-1 and PDL-1 might regulate microglial polarization during ICH-induced secondary brain injury. They showed that knockdown of PD-1 and PDL-1 by siRNA 12 h before ICH exacerbated inflammation and neuronal death after ICH; however, overexpression of PD-1 and PDL-1 by pDNA inhibited ICH-induced neuronal death and promoted microglial polarization to an anti-inflammatory phenotype. The results suggested that enhancing PD-1 and PDL after ICH can switch microglial polarization to the anti-inflammatory M2 phenotype (48).

In studies to unravel the role of different microglial phenotypes, Chen et al. (53) found that T-cell immunoglobulin-3 (Tim3), mainly expressed in microglia, was involved in the inflammatory response after brain injury. Tim-3 was highly upregulated around the hematoma in a rat model of ICH. Inhibiting Tim-3 by siRNA knockdown (48 h before ICH) significantly decreased inflammatory markers, neuronal cell death, and brain edema and promoted the response of anti-inflammatory M2 microglia. The authors concluded that upregulation of Tim-3 activates TLR-4 and enhances the interaction of Tim-3 and Galectin-9 (Gal-9) after ICH, suggesting that Tim-3 plays a vital role in neuroinflammation and microglial polarization by the Tim3/Gal-9/TLR-4 pathway(53). This study had two major limitations. First, the siRNA was administered before ICH induction. Second, Tim-3 siRNA can also target T lymphocytes and TLR-4–mediated inflammatory pathways and thereby produce off-target effects. Furthermore, targeting all microglia is not suitable because of their biphasic phenotypes. To date, however, no study has directly targeted a microglial-specific gene with siRNA-antibody conjugate to minimize off-target effects. Therefore, future studies should focus on developing antibody-siRNA conjugates that could specifically target genes of interest in specific brain cells to minimize unwanted effects.

3.3. siRNA targeting apoptosis

Promoting neuroprotection after ICH seems to be the favored intervention for restoring the functional activity of neurons. One potential means of achieving this goal is by blocking apoptotic pathways with siRNA to downregulate the expression of cell death mediators and prevent neuronal death.

Thrombin, an important component of secondary brain injury, not only activates microglia but, more importantly, induces apoptosis in neurons and astrocytes(54). Our group demonstrated that cofilin-1 (CFL1) siRNA administered directly into the mouse brain prior to ICH provided neuroprotection and improved neurologic outcomes, possibly through inhibition of apoptosis and oxidative stress(26). Also, microglial activation was reduced in the siRNA-treated mice as compared to that in mice treated with scrambled siRNA. Moreover, overexpression of GATA-binding protein 4 (GATA-4) was observed to mediate neuroinflammation and aging (55) and inhibit astrocyte proliferation(56). Xu et al. (57) showed that GATA-4 was upregulated in the rat brain after ICH and that knocking it down with an siRNA strategy improved secondary brain injury by reducing neuronal apoptosis. As a result, rats exhibited a decrease in neurobehavioral deficits. On the other hand, overexpression of GATA-4 through a plasmid vector exacerbated brain damage by activating NF-κB and increasing the expression of Bax and both caspase-3 and its cleaved form. The net result was an increase in neuronal death. These siRNA-based studies suggest that GATA-4 promotes neuronal apoptosis by activating the NF-κB-Bax-caspase 3 pathway after ICH(57). Although the results of the study were encouraging, the limitations were the same as those for the studies described above: the siRNA was injected 24 h before ICH to achieve complete transfection in the whole brain. Additionally, the study focused on neuronal apoptosis via the GATA-4 pathway without investigating the role of glial cells.

4. Use of siRNA to uncover molecular mechanisms of neurogenesis and angiogenesis

In another line of research, siRNA is being explored as a way to enhance neurogenesis and angiogenesis. Mesenchymal stem cells (MSCs) are important to neurogenesis and angiogenesis because these self-renewing, multipotent cells found in bone marrow are capable of differentiating into many cell types. These cells can be isolated from humans, modified, differentiated in vitro, and then used in stem-cell therapy. Many studies have shown that MSCs have anti-inflammatory (58) and antioxidant (59) properties. Moreover, one study showed that transplantation of MSCs into the brains of mice with ICH significantly restored cognitive and motor function, promoted astrocyte proliferation, and reduced hemorrhagic volume(60). Chen et al. (61) showed that bone marrow MSC transplantation in animal and in vitro models of ICH improved brain injury and protected astrocytes from apoptosis by regulating connexin 43 (Cx43, mainly expressed in astrocytes) and nuclear factor erythroid 2-related factor (Nrf2, a transcription factor that responds to oxidative stress).

In addition, Lei et al. (62) knocked down MMP-9 with siRNA to establish its role in ICH secondary injury. The authors observed that MMP-9 levels increased in the ipsilateral region after collagenase-induced ICH in rats. These higher levels were accompanied by higher expression of nerve growth factor and vascular endothelial growth factor and greater numbers of doublecortin- and 5-bromo-2-deoxyuridine-positive cells(62). Based on these findings, the authors hypothesized that MMP-9 is involved in neurogenesis and angiogenesis after ICH and regulates the expression of growth factors during ICH recovery. Knocking down MMP-9 with siRNA reduced both neurogenesis and angiogenesis, confirming that MMP-9 is involved in both processes during ICH recovery(62). The studies discussed in this section used siRNA as a tool to ascertain the mechanism of action of mesenchymal stem cells and MMP-9 during ICH, rather as a means of therapy. Such studies illustrate that siRNA has broad potential in the field for helping to identify the role of different genes in specific brain cells and enhancing our understanding of molecular mechanisms.

5. Limitations of siRNA as therapy for ICH

Using siRNA-based gene therapy holds exciting promise for treating secondary injury after a hemorrhagic stroke. However, a variety of obstacles stand in the way of its clinical use. One major challenge is that siRNA cannot cross the BBB. Therefore, studies to date have used only intracerebroventricular or intraparenchymal injection as routes of administration(63). Additionally, the poor pharmacokinetics of naked siRNA and the possibilities for induction of an immune response or off-target effects limit its clinical applications.

Moreover, the phosphate backbone of siRNA is vulnerable to degradation by RNases in the systemic circulation. Such degradation would prevent the accumulation of siRNA in the desired target cells and tissue(64). To maximize therapeutic efficacy and minimize the side effects of siRNA, researchers have developed various delivery systems such as viral and non-viral carriers. Currently, no carriers are available that would enable systemic administration for the delivery of siRNA in animal models of ICH. The lack of an efficient delivery system to target and deliver siRNA to the hemorrhagic brain is perhaps the biggest hurdle limiting the therapeutic potential of this approach. Much more research is required to develop an optimal delivery system for siRNA-based therapy that could be used clinically.

Another major limitation of the preclinical models used for siRNA-based research is translating the results to human trials. The siRNA approaches used in animal studies have reduced ICH-related secondary injury. However, because the siRNA was administered prior to the injury, the studies do not reflect real-life scenarios. Therefore, post-treatment studies are needed in which siRNA is delivered immediately or a few hours after ICH to mimic treatment delays that occur clinically. Another important limitation is the biphasic nature of signaling molecules during the hemorrhagic cascade, as their effects vary during injury and recovery phases. Future studies must focus on using siRNA to target the downregulation of molecules that produce detrimental effects during the injury phase. The advantage of siRNA-based deletion therapy is that its effect is transient. It would be ideal to administer one dose of siRNA rather than multiple doses of a small-molecule drug. Although the cost of siRNA therapy will be higher than that of small molecules, given the overall economic burden of ICH, the cost of siRNA can be seen as minimal.

6. siRNA modifications used in clinical studies

In recent years, researchers have made various modifications to siRNA specificity, activity, stability, and toxicity to overcome the limitations of siRNA without compromising its intrinsic activity. Judicious modification approaches can significantly improve siRNA stability and biocompatibility. The overall goal is to maximize the therapeutic benefits while clearing the obstacles to therapeutic use of siRNA. Several strategies have been proposed and applied in preclinical and clinical studies.

Chemical modification can be applied at the sequence or structural level of the nucleotide. For example, patisiran, a chemically modified anti-transthyretin (TTR) siRNA, is formulated in liposomes that include DSPC, DLin-MC3-DMA, cholesterol, and PEG-DMG at a fixed molar ratio of 10/50/38.5/1.5 (64, 65). Modifications that included a combination of 2′-OMe (9 in the sense strand and 3 in the antisense strand) and 2′-deoxy-2′-fluoro (2 in the sense strand and 2 in the antisense strand, all at the 3′ end) were used to facilitate the systemic administration of TTR siRNA and achieve effective gene silencing (66). In addition, a GalNAc–siRNA conjugation has been developed to achieve efficient siRNA delivery to hepatocytes (67). The GalNAc–siRNA conjugate siRNA delivery platform provides greater potency and safety than liposomes for liver-targeted siRNA delivery (68). An enhanced stabilization chemistry platform was used to develop the first GalNAc-conjugate RNAi gene-based therapy worldwide. This strategy includes four phosphorothioate linkages at the 5′-end of the antisense strand and the 3′-end of the sense strand and reduction in 2′-F substitutions. These modifications significantly enhanced siRNA potency and stability, achieving the desired pharmacodynamics at a substantially lower dose (69). As a result, the FDA has approved two RNAi therapeutics, patisiran (70) and givosiran (69), for clinical applications. Two siRNA-based therapeutics have been submitted to the FDA for new drug applications. Several siRNA-based therapeutics are undergoing Phase 3 clinical trials, and more siRNA candidates are in the initial developing stage(34). Additionally, the pharmaceutical company Alnylam has patented a method (WO2019217459A1) for CNS and ocular-targeted delivery by conjugating a lipophilic moiety to the siRNA (64). This milestone in siRNA-based therapy can markedly extend its benefits to other complex diseases.

7. Future improvements of siRNA therapeutics in ICH

Hemorrhagic stroke can cause long-lasting physical and mental symptoms in surviving patients, affecting many aspects of their lives. Unfortunately, after years of effort, no specific treatment has been approved. The goal of therapeutic intervention is to prevent hematoma expansion and restore lost neurologic function in affected patients (71). Now, advances in gene silencing technology are enabling investigators to suppress damaging protein pathways as a way to induce neuroprotection, restore motor function, and reduce inflammation after stroke (72). By silencing harmful gene components, siRNA has the potential to treat diseases with long development periods like the secondary injury phase after ICH.

Many obstacles have limited systemic delivery of siRNA in the CNS, such as enzymatic degradation; the BBB; gene and cell specificity; and off-target effects. Therefore, scientists have begun developing siRNA nanoparticles and siRNA-conjugated molecules to facilitate delivery (73). Nanoparticles have various advantages, including low toxicity, minimum risk of infection, and low possibility of oncogenicity. Many lipid-based systems and cationic polymers are exploited as nanoparticle therapeutic interventions to deliver siRNA (74). Chitosan is a biocompatible cationic polymer that has many functional groups for targeting ligand modification. Chitosan has been used in gene delivery because it can form electrostatic interactions between its own positively charged amino groups and negatively charged nucleic acids such as those in siRNA. A chitosan coating system increases particle stability, controls drug release, and enhances cellular uptake of poly(lactide-co-glycolide) (PLGA) nanoparticles (75). Approved for clinical use by the FDA, PLGA is a polymer that can encapsulate both hydrophobic and hydrophilic drugs, depending on the method used for preparation. PLGA is quickly eliminated from the peripheral circulation before crossing BBB by the reticuloendothelial system (76). A further modification is required to overcome degradation. Coating the PLGA nanoparticle with a hydrophilic polymer such as polyethylene glycol (PEG) improves lifespan in the circulation and enhances targeted delivery (77). For example, Wang et al.(78) demonstrated that encapsulating tissue plasminogen activator into Fe₃O₄-based PLGA nanoparticles coated with cyclic Arg-Gly-Asp (cRGD)-grafted chitosan-PEG led to significantly improved thrombolytic activity. Accordingly, it is possible to hypothesize that chitosan-conjugated antibody coated onto siRNA-loaded PLGA-PEG will preferentially target and deliver siRNA for knockdown of a gene associated with aggravated secondary injury after ICH. Optimizing siRNA formulation to improve its translational application in ICH therapy is likely to make great strides in the coming years as advances in nanoparticle-based siRNA therapeutics raise hopes for the future of ICH treatment.

8. Conclusions and future perspectives

ICH is a complex disease with a multitude of molecular pathways. Thus, it has been challenging to develop drugs that show efficacy in clinical trials. However, myriad molecular cascades also provide numerous opportunities for therapeutic intervention. Gene silencing by siRNA is a novel strategy with possible therapeutic potential for neurologic and neurodegenerative diseases and, in particular, for ICH. Every gene in the human genome that is involved in disease could be amenable to regulation, providing unprecedented opportunities for innovative drug alternatives. siRNA-based therapy might become a highly feasible treatment option for neurologic and neurodegenerative diseases. In this review, we discussed the latest research related to the use of siRNA-based therapy in animal models of hemorrhagic brain injury and modified siRNA in clinical studies for the treatment of rare diseases. Carefully addressing the challenges and difficulties in relation to translational use of siRNA may help to move gene therapy forward for ICH from preclinical studies to clinical trials.