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RNA interference is a naturally occurring biological event in cell cytoplasm where the double-stranded (ds) small interference RNA elicits a sequence-specific gene knockdown and subsequent suppression of protein expression. It was first discovered by Andrew Fire and his co-workers during their landmark work to study the effect of exogenous ds-RNA on Unv-22 (encode for myofilament protein) gene expression in C. elegans. [1] The gene-silencing effect of siRNA was further demonstrated in mammalian cells using synthetic siRNA to induce the gene knockdown. [2] RNA interference begins with the breakdown of a long double-stranded RNA by an enzyme called Dicer, forming a 21 to 23 mer small interference RNA. This short strand RNA would then bind with a protein complex, coined as RNA-induced silencing complex (RISC), and split itself into single strands. Following the degradation of the sensing RNA single strand, the antisense RNA will specifically bind with mRNA with complementary sequence and catalytically break down them to achieve gene silence. RNA interference is an important biological process that allows cells to fight against viral attack or gene mutation (or transposon). The RNAi mechanism is also implicated for controlling protein levels to enable to cell to respond to various external stimuli. [3] The RNA interference has offered scientists a fundamental tool to study gene function, but most importantly it ushered in a ground-breaking approach to develop new therapeutics for treating a variety of diseases. Compared to existing drug discovery paradigm involving DNA, protein or small molecule agonists or antagonists, siRNA owns several unique advantages. First it acts on the mRNA during protein translation step, avoiding gene altering event potential occurring with DNA based therapeutics. It also eliminates possible protein over expression issue that typically associated with protein-based treatment. Lastly siRNA is easy to design and can engage a much broader disease targets than existing therapeutic agents, including protein or small molecule-based drugs.
 * Small Interference RNA Therapeutics using Polymers as Delivery Vehicles **
 * 1. Introduction **

The prospect of developing siRNA into clinically viable therapeutics is promising but it is not without challenges. Aside from the toxicities related to off-target gene-silencing effect, the safe and effective delivery of siRNA from systemic circulation to cell plasma still remains the biggest hurdle to overcome. Many promising delivery platforms are designed which include viral or non-viral based delivery systems. The latter one is most attractive owning to better safe profiles. This paper will provide an overview on the non-viral based delivery platform with emphasis on those using polymers as siRNA carriers for target delivery. Key discussion points will include formulation composition, delivery mechanism as well as in vitro/in vivo evaluation of safety and efficacy of the system. [4]    In order to elicit sequence-specific gene knockdown, siRNA needs to be delivered into cell cytoplasm with acceptable safety and efficacy. To impart drug-like properties to siRNA, it has to be assessable to target tissue/cell via systemic circulation (eg. IV injection). Local delivery of naked siRNA is possible with target organs limited to eyes or lung. However to develop siRNA as practical therapeutic agents for a broad applications, a systemic delivery is critically needed. Key barriers to achieving systemic delivery of siRNA are several folds. First is the potential chemical instability of siRNA through nuclease induced hydrolytic degradation in extracecullar environment. Chemical modification of native siRNA with protective groups or physical encapsulation of siRNA into macro assemblies (lipid or polymer aggregates) are utilized as primary solutions. The target delivery of siRNA is achieved through a receptor-mediated endocytosis which typically requires the incorporation of negative charged siRNA into positively charged macromolecules decorated with cell-targeting ligands. Once inside the cell, the intact siRNA will be liberated from the delivery cargo and released into cytoplasm to exert gene-silence function. Accomplishing this complex sequence of events requires elegant design of an integrated delivery system, which typically include the carriers (lipids or polymers), cell targeting ligands and shielding ligands (e.g. PEG) to avoid off-target interaction. |[5], [6] The polymer-based delivery systems include two major categories where the distinction lies in the nature of linkage between the polymer carrier and siRNA. The first one is the nanoparticle-based system where the polymer and siRNA form nano aggregates through electrostatic interaction (non-covalent bonding) while the second one is a more integrated system where the polymer, siRNA and various other functional moieties are brought together through reversible covalent bonds.
 * 2. General considerations for designing siRNA delivery system **
 * 3. siRNA delivery using polymer as carriers **

The nanoparticle-based polymer delivery platform utilizes cationic polymers as carriers to form complex with negatively charged siRNA through electrostatic interaction. The complexation process effectively contract siRNA to prevent its hydrolytic degradation and to facilitate the cell-uptake through endocytosis. In addition to polymers, the delivery systems often contain other components to minimize off-target effect and to improve cell-targeting efficiency. The safe and effective delivery via polymer-siRNA nanoparticles rely on the particle size, its physical stability during extracellular circulation, cellular uptake via endocytosis and efficient release of siRNA from the complex through polymer biodegradation. The polymer includes naturally occurring polycations (e.g Atelocollagen and Chitosam) that are biocompatible/biodegradable as well as the synthetic ones (e.g polyetheleneimine, cyclodetrin and others).
 * 3.1 Nanoparticle-based polymer delivery system **

** 3.1.1 Natural polymers as siRNA carriers ** Natural polymers are among the early candidates explored for siRNA delivery owning to its biocompatible and biodegradable nature. Chitosan is one of most studies natural polymer for cellular delivery. It is a natural co-polymer consisting of GLcNAc (N-acetyl-D-glucosamine) and GLcN (D-glucosamine) building blocks (Figure 1 [|Figure_1.JPG]). The co-polymer carries positive charges under acidic or neutral environment. The decomposition product incurs no toxicity in vivo which makes it a good candidate as polymer carrier for siRNA. [7] The thiamine pyrophosphate (TPP, [|FIgure_2.gif]) salt of Chitosan were shown to form stable nanoparticles with siRNA, effectively shielding siRNA from chemical degradation and promoting efficient cell uptake. The delivery efficient depends on MW of the polymer and the surface charge of polymer – siRNA complex, controlled by the ratio of Chitosan-TPP and siRNA. Polymer carrying more positive charges (high MW or high mass ratio to siRNA) enables a stable complex formation. The % gene-silencing reached maximum with the lowest particle size of the complex. The chitosan salt based delivery system demonstrated no sign of cytotoxicity based MTT assay and the cell viability is ~ 90% of those untreated cell line. [8] Atelocollagen is another natural polymer that was explored for gene and /or siRNA delivery [9]. This natural polymer is prepared through enzymatic digestion of type I collagen of calf dermi to remove telopeptides, known to cause immune response. The delivery formulation is typically a simple binary mixture containing polymer and siRNA. In vivo gene knockdown is promising and the mice that received IV injection of natural polymer Atelocollagen and siRNA mixture showed 80 to 90% in bone tumor metasis. [10] In addition to biodegradable natural polymers, synthetic polymers were also extensively evaluated as potential carriers for siRNA delivery. The delivery system is more complex than those with natural polymer. The system design often time needs careful selection of shielding legands, preventing non-specific interaction between nanoparticles and extracellular proteins, and cell targeting ligands, enabling effective cell update to cytoplasm.
 * 3.1.2 Synthetic polymer as siRNA carriers **

Cyclodetrin (CD) is a synthetic pharmaceutical excipient commonly used for parental formulations to enhance solubilization of poorly soluble drug. The soubilization enhancement is associated with the cavities within CD that allows host-guest interaction (or complexation). CD is safe and well tolerated for human use and a good candicate for gene delivery. [11] A three-component system ([|figure_3.JPG]) was developed for siRNA delivery, which consists of CD-containing linear polymer (CDP), PEG-AD and AD-PEG-Tf (human transferin). [12] The CD containing polymer (carrying polycations) forms nanoparticles with siRNA via electrostatic interaction regardless the size and types of siRNA and it serves as physical barrier to prevent chemical degradation of siRNA. The CD on polymer backbone also functions as host sites for attaching PEG-AD and AD-PEG-Tf functional groups using the AD the anchor. The PEG group functions as stability enhancer to prevent nanoparticle aggregation while the AD-PEG-Tf structural moiety decorated on CD-polymer surface enhances the cell uptake with Tf as the cell targeting ligand. Finally the polymer backbone also linked to amine-containing chemical groups that were designed to facilitate the release of siRNA from the endosome to the cytoplasm. Key variables dictating the delivery properties include the type of CD and charge groups on the polymer backbone as well as the distance between the CD and the charge groups. Studies suggested that the presence of CD regardless the structure is the key to higher delivery efficiency. A longer distance between CD and charge center renders a low toxicity but a low binding affinity with siRNA. Among various charge groups (e.g. amidine, quaternary amine and secondary amine) evaluated on the polymer backbone, the amidine stands out as the best. The formulation preparation is fairly simple which involves mixing a naked siRNA from one vial with pre-mixed CD containing polymer, AD-PEG and AD-PEG-Tf constituents into buffer solution before use. The final mixture yields s stable nanoparticle with size at ~ 70 nm and charge ratio of 1:1. The CD-based delivery platform with siRNA designed for RRM2 gene knockdown showed significant suppression on RRM2 protein. It was shown to be safe and well tolerated based on non-human primate and mice models. The formulation is currently being evaluated for safety in early human clinical trial for treating cancer. The results are expected at the early 2010. 12

Polyethylenimine (PEI) is another popular synthetic polymer often used for DNA or siRNA delivery. [13] It has high density positive charges on polymer backbone under physiologic pH due to the protonated amino groups. The positive charges in PEI enable the formation of stable non-covalent complexes with siRNA bearing negative charges. The complex formation effectively contract siRNA, which achieves two important outcomes: stabilize siRNA against chemical degradation and facilitate endocytosis into the cell membrane. Indeed, a linear PEI with MW of 22KDa demonstrated efficient delivery of siRNA with minimal cytotoxcity. Further studies indicated that the delivery efficiency of PEI system highly depend up the molecular weight and configuration (linear or branch) of the polymer and the mass ratio between PEI and siRNA. [14] The chemical modification of PEI with PEG functionality was shown to improve the transfection efficiency of siRNA while reducing the potential toxicity. The underlining mechanism is that non-ionic PEG group can reduce the potential non-specific interaction between the positive siRNA-polymer complex and extracellular protein or other matrix. In addition, the amino groups on PEI is implicated to engineer the so call "proton sponge effect" [15], where it serves as low pH buffer system in endocytosis /lyosonal system, increasing the osmosis pressure and eventually leading to the breakage the endocytosis membrane to release the siRNA to cytoplasm. PEI is not a biodegradable polymer so the safety profiles is less ideal due to the accumulation of large MW polymer in cells. Bioreduciable polymers such as poly (disulfide amine) were recently reported as an effective carrier to deliver siRNA. [16] The polymer was constructed through co-polymization of low molecular weight cationic monomers containing disulfide groups as reducible linkage. Compared to non-degradable polymer such as PEI, the bioreduciable polymer based delivery platform would be more biological compatible and intrinsically safe and less toxicity. An arginine-modified polydisulfide poly (CBA-DAH-R) was developed to for VEGF gene-silencing siRNA delivery. The polymer forms 200 nm particles once compexing with siRNA and maximal physical stability of the nanoaggegates was achieved at mass ratio from 20 to 1 between polymer and siRNA. The reducible property of the polymer carrier was confirmed by the absence of nano particles in a reductive environment (in solution of DTT: Dithiothretol), where the degradation of polymer disrupted the polymer-siRNA complex. In vitro cytotoxicity assay suggested that the system with Poly (disulfide amine) carrier exhibits ~ 100% cell viability while PEI polymer only with 40% relative cell viability. In vitro transfection efficiency is ~ 80% in human prostate carcinoma PC-3 cell line, and the high cell update indeed correlated with significant suppressed gene expression. The high gene knockdown efficiency associated with Poly (disulfide) is attributed to the reductive nature of the disulfide bond in the polymer backbone. The postulation was confirmed with confocal fluorescence microscopy where the cell lines treated with BSO, an inhibitor to increase the intracellular reducing potential, showed marked decrease in localization of siRNA in cytoplasm. For nanoparticle-based siRNA delivery system, the polymer choice is typically constrained to polycations, where the backbone carries positively charged amine groups. With technology advancement in polymer engineering, it is possible to chemically modify neutral polymer for potential use as siRNA delivery vehicle. Polyethylenoxide and polyester have been extensively used as biomaterial for traditional drug delivery, thus their safety and toxicity were well characterized. The co-polymers of PEO and PE, grafted with a short cationic moiety were recently developed as safe and effective siRNA carrier with promising in vitro results. The system demonstrated efficient cell uptake as well as in vitro gene knockdown encoded for P-glycoprotein. [17] In contrast to the polymer based nano particle delivery system, where the positively charged polymers form a complex with siRNA through electrostatic interaction, the dynamic polymer conjugates chemically linked all foundational groups together through a revisable covalent bonding. The key to the polyconjugate delivery system is to maintain the integrity of the chemical bonds linking siRNA and various functional groups during extracellular circulation. However, once inside the cell the bonds need to self-dissociate to release siRNA to cytoplasm.
 * 3.2 Covalent bond based delivery system: Dynamic polyconjugate **[18]

The first polyconjugate achieving such task is reported by Rozema and the co-workers. The assembling of a negatively charged, non-aggregating siRNA polyconjugate involves a reversible linkage of three main structural motifs onto the polymer backbone (PBAVE-polybutyl amino vinyl ether). The key functional moieties include siRNA, the shielding group (PEG) and the cell-targeting group (NAG). (Figure 4) The disulfide bond links siRNA to the polymer backbone and PEG and NAG groups are attached to the polymer through reversible modification with maleic anhydride derivatives. Polyconjugate enters the hepatocytes through ASGPr receptor-mediated endocytosis. The low pH environment of endosome cleaves the acid labile maleamate bond, releasing PEG and NAG groups. The de-protected free amine groups can disrupt the endosome membrane, releasing polymer-siRNA conjugate into cytoplasm. The disulfide bond linking siRNA and polymer will be subsequently oxidized in cytoplasma, resulting in the release of the free siRNA in the cell. ([|Figure_4.JPG])
 * 3.2.1 Polyvinylether-based polyconjugate **[19]

Both PEG and NEG groups are important for effective siRNA delivery. The dissociation of PEG group from polyconjugate in endosome is the critical step for an effective gene knockdown. When PED group was permanently linked to polymer backbone, the polyconjugate become completely inactive. The NAG functional group is also critical to siRNA cell uptake. The hepatocyte uptake of siRNA was significantly reduced when NAG was replaced with glucose in the polyconjugate assembly, confirming NAG as cell targeting ligand for the liver. The non-covalent bonded polyconjugate, where siRNA and polymer were assembled together through electrostatic interaction, showed a marked decrease in siRNA accumulation in liver, suggesting the importance of covalent bonding between siRNA and polymer backbone.

Dynamic polyconjugates deliver siRNA effectively to liver cell for both in-vitro and in-vivo models. Significant gene knockdown was demonstrated for apoB and pparp genes with maximum efficiency of 80 to 90% at 2.5 mg/kg dose. The time duration of gene knock down is satisfactory. The dose level tested is well tolerated and no toxicity was indicated based on several safety biomarkers. The apoB gene silencing with siRNA polyconjugates effectively elicit a reduction in apoB gene expression and cholesterol level in serum as well as an impairment of triglyceride transport from the liver. However, the fatty liver induced by the gene-knock and its potential acute effect in human should be further investigated prior to advancing apoB siRNA into a viable therapeutic agent.

The PBAVE-based siRNA polyconjugates offer a promising delivery platform for gene-based therapeutic applications with high potency and low toxicity. The reversible covalent bonds used in linking siRNA, cell targeting ligand, and shielding ligand provide added flexibility for targeting variety cell types. The key advantage over the nano-particles based delivery platforms includes minimal accumulation in off-target cell. Particle size reduction of polyconjugate (~ 10nm) compared to nano-particle delivery system eliminates the potential activation of immune cells, results in negligible toxicity effects. Polymer conjugate containing polylysine backbone bonded with PEG and di-methylmaleic anhydride masked malittin (peptide) was recently reported as a potential delivery platform for siRNA therapeutic. The integrated siRNA containing polyconjygate was constructed through a three-step synthesis. ([|Figure_5.JPG]) The first step involves the coupling reaction between PEG and PLL polymer backbone. The modified PLL was then attached with a pH-labile DMMan-Mel group through disulfide bond to impart the endosolmolytic prosperity to the delivery system. Lastly the siRNA was incorporated into the delivery cargo through a reducible covalent bond. Similar to the PBAVE-based system, the PEG group here also functioned as the shielding agent and the methylmaleic anhydride masked malittin is endosolmolytic agent, facilitating the escape of siRNA under weakly acidic endosomal environment, The polyconjugate system forms nanoparticles with size ranging from 80 to 300nm, significantly larger than PBAVE system discussed previously. The invitro gene-silence efficiency was evaluated in cultured Neuro2A cells with stable luciferase expression. The gene-knockdown is ~ 80% at low doses (0.125 ug to 0.25 ug). Despite promising in vitro efficacy, the invivo tox studies suggested unusually high toxicity based on rat model. Lack of PEG shielding in systemic circulation and insufficient cell targeting are the likely the sources. Further optimization of this delivery platform is needed for therapeutic applications.
 * 3.2.2. PolyLycine based system **[20]

siRNA presents an exciting opportunity for fundamental understanding of gene function and disease mechanism. It revolutionized the way future medicines can be developed for treating wide variety of diseases, including cancer. siRNA acts on mRNA during mechanism of action, eliminating the potential toxicity issues associated with gene or protein therapeutics. In spite of the great potential siRNA can offer, the safe and effective cellular delivery of target siRNA remains challenging. Polymer-based delivery system, including nanoparticle of polymer –siRNA complex or polyconjugate, offers promising technology platforms for developing siRNA into novel therapeutics. The future efforts will undoubtedly be focusing on the design and optimization of novel shielding ligands as well as cell-targeting ligands and endosomolytic agents to achieve effective siRNA delivery with high specificity and minimal toxicity.
 * 4. Conclusion **


 * References: **

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