The main goal of this study was to evaluate tumor necrosis factor-alpha (TNF-α) gene silencing in peritoneal macrophages upon activation with lipopolysaccharide (LPS), using CD44-targeting hyaluronic ...acid (HA)-based nanoparticles encapsulating TNF-α-specific small interfering RNA (siTNF-α). HA nanoparticles were formulated by blending hyaluronic acid-poly(ethylene imine) (HA-PEI), hyaluronic acid-hexyl fatty acid (HA-C6), and hyaluronic acid-poly(ethylene glycol) (HA-PEG) in 3:2:1 weight ratio, and encapsulating siTNF-α to form spherical particles of 78–90 nm diameter. Following intraperitoneal (IP) administration in LPS-treated C57BL/6 mice, the nanoparticles were actively taken up by macrophages and led to a significant downregulation of peritoneal TNF-α level. Downregulation of peritoneal macrophage-specific TNF-α also had a significant impact on other pro-inflammatory cytokine and chemokine levels in the serum. The C57BL/6 group of mice challenged with 5 mg/kg LPS had a significantly higher survival rate when they were treated with 3 mg/kg siTNF-α, either prior or simultaneously with the LPS administration, as compared to the LPS-challenged mice, which were treated with controls including the scrambled siRNA formulation. Overall, the results of this study demonstrate that CD44 targeting HA nanoparticles can selectively deliver siTNF-α to peritoneal macrophages leading to downregulation of pro-inflammatory cytokines in the peritoneal fluid and in the serum. This RNAi strategy could potentially provide an important therapeutic modality for acute inflammatory diseases, such as septic shock.
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IJS, KILJ, NUK, PNG, UL, UM
2.
Novel RNA interference-based therapies for sepsis Kosovrasti, Verbena Y; Lukashev, Dmitriy; Nechev, Lubomir V ...
Expert opinion on biological therapy,
04/2014, Volume:
14, Issue:
4
Journal Article
Peer reviewed
Introduction:
Sepsis is an extremely fast-paced disease, initiated by an infection that can progress to multiple organ dysfunction and death. The complexity associated with sepsis makes the therapies ...difficult to develop. Moreover, the 'one-fits-all' kind of therapy is far from being realistic.
Areas covered:
This review provides a conspectus of the current results of sepsis therapies and their benefits, focusing on the development of small interfering RNA (siRNA) therapeutics for targeting immune cells and sepsis pathways.
Expert opinion:
The question, 'When will an effective therapy for sepsis be available for patients?' remains unanswered. New RNA interference-mediated therapies are emerging as novel approaches for the treatment of sepsis by downregulating key inflammatory cytokine expression. Strategies that exploit multimodal gene silencing using siRNA and targeted delivery systems are discussed in this review. Some of these strategies have shown positive results in preclinical model of sepsis.
We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid ...1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.
Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct ...CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease.
► Chronic convection enhanced delivery of siRNA targeting Huntingtin in primate brain. ► Widespread drug distribution results in Huntingtin lowering throughout striatum. ► Empirical model of data enables scaling of siRNA delivery. ► Potential as key disease-modifying therapeutic approach for Huntington's disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the ...corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip-mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Ever since the first representation of RNAi phenomenon in 1998, its ride has been quite complicated. If handled carefully, RNAi could have the potential of transforming into a therapeutic which can ...apply in a wide range of diseases from viral to cancer. The use of a powerful therapeutic tool like siRNA therapeutics has faced many barriers, like: molecule stability, specific delivery to desired cells and tissues, triggering of innate immune response, and effects achieved off the desired site. Theoretically, siRNA can be used to silence any gene, therefore is a potential therapeutic to many genetic diseases, and not only. However, given the enormous genetic variation between species RNAi becomes a challenge. Sepsis syndrome is one of those diseases that quickly progresses over time. There is a demanding need in finding a therapy for advanced sepsis syndrome, a fast-running progressive disease. The main mediators of inflammation in general and sepsis in particular like, TNF-α and IL1 play an important role in triggering a spillover of a flood of cytokines leading to sepsis condition. Anti-TNF-α therapies have been applied in inflammatory diseases and in sepsis in a long time, but despite the promising results of the in vivo work the translation to humans has been difficult. From its role prospects, TNF-α gene is a tricky target to modulate, and siRNA therapy offers transitory effect instead of an enduring effect over this target. We developed a nanosized delivery system, made of modified hyaluronic acid polymer encapsulating cholesterol modified siRNA in its core, to enable the siRNA supply in the macrophages` cytoplasm. The particle we developed was characterized and a size of nanometer range was determined, which intact the siRNA through charge and hydrophobic interaction. The main goal of this thesis work was to develop a therapy for sepsis utilizing RNAi to target TNF-α, based on biomarkers identification to follow on the disease progression. One important goal achieved was the creation of a multimodal delivery system, to overcome the long known obstacle of siRNA delivery in cells cytoplasm. More specifically, targeting macrophage cells is a complicated task. Despite their ability to take up quickly, they also possess the high capability of degrading the engulfed materials. Nanotechnology principles are very helpful in generating delivery vehicles with unique physicochemical and biological properties. As a nanosized system, the formulation offered by this thesis work represents a combinatorial approach that contains poly(ethylene imine) (PEI) modified hyaluronic acid polymer in combination with lipid modified polymer. A poly(ethylene glycol) (PEG)-modified polymer is used as a component of the formulation to provide more stability given the negative charge of the siRNA. On one hand, the positively charged PEI plays an important role in encapsulating the siRNA through charge interaction, while on the other hand it contributes a proton sponge effect within the cells that facilitates the siRNA delivery into cytoplasm after escaping the late endosome. The chemical modification of the siRNA, by addition of a cholesterol molecule, and addition of lipid modified hyaluronic acid polymer, lead to improvement of encapsulation efficacy, while enhancing the TNF-α silencing efficacy in peritoneal macrophages. The silencing efficacy was supported by the biodistribution data in vivo, which showed efficacious siRNA uptake preferably in peritoneal macrophages, as compared to other organs tissues in mice. Septic mice treated with anti- TNF-α siRNA nanoparticle showed a significantly improved survival rate, as compared to septic mice treated with scramble siRNA nanoparticle, in a LPS developed model of sepsis. Thus, the novel multifunctional nanoparticle encapsulating siRNA, offers an alternate, safe, and effective siRNA delivery system to treat sepsis, and in more general terms, the inflammatory diseases.
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