siRNA Conjugates Research Articles

Structure Tuning of Cationic Oligospermine–siRNA Conjugates for Carrier-Free Gene Silencing

Oligospermine-siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphoramidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine-siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA-tris(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3'-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5'-end of the antisense strand abolished the activity. 2'-O-Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3'-end of both strands and at the 5'-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine-siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5'-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine-siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine-siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5'-end of the sense strand.

Gene regulation with carbon-based siRNA conjugates for cancer therapy.

We report fluorescent carbon nanoparticle (FCN)-based small interfering RNA (siRNA) conjugates (C-siRNA) for gene regulation and cancer therapy. The C-siRNA has a core of chitosan-derived FCN and a shell of siRNA, and can down-regulate the expression of polo-like kinase-1 (Plk1), a master regulator of mitosis, via siRNA targeting Plk1 (siPlk1), for cancer therapy. The required amount of the FCNs is only ∼1/30 of that of the gold nanoparticles in delivering equal amount of siRNA. The C-siPlk1 led to ∼80% knockdown of cellular Plk1 mRNA in A375cells, and induced apoptosis of the A375cells (31.9%) and MCF-7cells (20.33%), much higher than those by commercial nonviral gene delivery vectors, such as Lipofectamine 2000 in both cell lines (apoptosis rate<10%). After the C-siPlk1 was administrated to A375 tumor-bearing mice intravenously, the tumor volume was less than 1/11 of the control groups. The C-siRNA can thus be powerful tools for gene delivery and gene therapy.

Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles

Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab′ fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1μg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.

Target specific siRNA-conjugate therapy for experimental autoimmune myasthenia gravis

Abstract Myasthenia gravis (MG) is an autoimmune disease of muscle weakness caused by Acetylcholine receptor- autoantibody and complement mediated damage of neuro-muscular junction (NMJ). We have recently shown a greater advantage of using siRNA over other conventional therapeutic tools in an experimental mouse model of MG (EAMG). However, the major drawbacks of siRNA therapy is reduced efficacy due to the lack of target restricted delivery, where it is necessary to suppress pathogenic response arising predominantly from a particular cell/tissue type. In this study, we used a novel strategy of cell specific new formulation of siRNA or siRNA conjugate delivery to pathogenic cells in EAMG. Our preliminary work shows successful preparation of siRNA conjugate and its functional efficacy over siRNA only treatment in an in vitro rodent cell line. Successful completion of the study may provide a solution for the siRNA or monoclonal antibody therapy hurdle and a new direction to ultimately explore a highly effective and safe therapy for patients with MG.

Targeted nanoconjugate co-delivering siRNA and tyrosine kinase inhibitor to Kras mutant NSCLC reveals Gab1 assisted survival pathway post oncogene knockdown

NSCLC is diagnosed in an estimated 220,000 patients each year with five-year overall survival rates of 16 percent. A recent report confirmed that 16 percent of NSCLC patients carry oncogenic KRAS mutation. Patients with KRAS mutation often harbored in wild-type EGFR tumors, are resistant to Tyrosine Kinase Inhibitors (TKI). A potent drug targeted against KRAS mutation has not yet been developed and the objective response rate with the current standard of care is a meager three percent. Interestingly, KRAS knockdown using siRNA sensitizes tumor to TKI with a good response rate. Retroviral vectors, liposomes, polymeric particles and metallic nanoparticles have been used as carriers to deliver siRNA within cancer-site. However, protecting siRNA from serum degradation and cytoplasmic delivery are two major issues. Also, in most cases, a mere siRNA mediated oncogene knockdown does not have significant impact on the cancer cell apoptosis since the cells adapt to another effector survival pathway. To overcome these challenges and understand the adopted effector downstream mechanism post oncogene knockdown, we hierarchically created a well-defined 200nm tri-block nanocomplex with each sublayer contributing to a definite function. The nanocomplex comprises of an enzymatically cleavable protein (gelatin) nanoparticle encapsulated with a TKI (gefitinib) and surface functionalized with an antibody (Cetuximab)-siRNA (Kras G12C) conjugate. Detailed characterization revealed each nanoparticle of the tri-block nanocomplex comprised of ∼400 antibodies and ∼800 siRNA. We protected 14KDa siRNA within 150KDa cetuximab and sandwiched it between antibody and gelatin nanoparticle to protect from serum degradation, confirmed using SDS-PAGE. To investigate cellular activity, we incubated the nanocomplex in drug resistant Kras G12C mutant NSCLC (H23 cells). The nanocomplex, when delivered to cytoplasm of the drug resistant H23 cells for oncogene knockdown, sensitized the affected cells to the co-delivered TKI. Knockdown of the oncogene was confirmed by monitoring the PI3K and MAPK downstream protein expression levels. Western blot results indicated abrogation of activated PI3K and MAPK pathway proteins. In vitro assays revealed 95% toxicity for the nanocomplex containing 5μM gefitinib as against 0-10% toxicity for 5μM stand-alone gefitinib. rtPCR showed downregulation of DUSP6, a known effect for H23 cells with knocked down oncogene. Flow cytometry results showed 2 fold higher internalization of the nanocomplex compared to transfected siRNA. However, in the absence of TKI, the nanocomplex showed no toxicity suggesting the cells adapt to a parallel effector pathway for survival, although phosphorylated Mek, Erk and Akt were downregulated leading us to investigate a possible survival mechanism. We hypothesized that the downstream signaling is governed by Gab1 assisted pathway. In H23 cells, activated ERK results in phosphorylation of Gab1 on serine and threonine residues and forms Gab1-p85 PI3K complex that are adjacent to p85 PI3K binding sites. Knocking down the oncogene dephosphorylated Erk, and negated the complex formation. This further cascaded in tyrosine phosphorylation at Tyr627 domain of Gab1, which is known to associate with downstream of EGFR but upstream of Ras, to regulate EGFR signaling through several positive feedback loops. We found that TKI binds to this specific phosphotyrosine domain of Gab1, the domain that is responsible for Gab1-Egfr association. In the absence of a TKI, the feedback loop mediated via Gab1 provides a route for survival but is sensitized by abrogation of Gab1-Egfr complex formation post oncogene knockdown when exposed to a TKI. The outcome of this study provides a potential solution for treating patients harboring KRAS mutation.

Clustering siRNA conjugates for MMP-responsive therapeutics in chronic wounds of diabetic animals.

The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was efficiently endocytosed by cells. An in vivo fluorescence resonance energy transfer (FRET) study also revealed that the clusters were effectively dissociated in MMP-rich environments of dorsal wounds in diabetic animals. In addition, diabetic ulcers treated with the clusters showed a faster wound closure rate and the recovered tissue expressed a larger amount of cytokeratin along with a lower expression level of MMP-2 compared to the other groups.

Reducing Toxicity of Immune Therapy Using Aptamer-Targeted Drug Delivery.

Modulating the function of immune receptors with antibodies is ushering in a new era in cancer immunotherapy. With the notable exception of PD-1 blockade used as monotherapy, immune modulation can be associated with significant toxicities that are expected to escalate with the development of increasingly potent immune therapies. A general way to reduce toxicity is to target immune potentiating drugs to the tumor or immune cells of the patient. This Crossroads article discusses a new class of nucleic acid-based immune-modulatory drugs that are targeted to the tumor or to the immune system by conjugation to oligonucleotide aptamer ligands. Cell-free chemically synthesized short oligonucleotide aptamers represent a novel and emerging platform technology for generating ligands with desired specificity that offer exceptional versatility and feasibility in terms of development, manufacture, and conjugation to an oligonucleotide cargo. In proof-of-concept studies, aptamer ligands were used to target immune-modulatory siRNAs or aptamers to induce neoantigens in the tumor cells, limit costimulation to the tumor lesion, or enhance the persistence of vaccine-induced immunity. Using increasingly relevant murine models, the aptamer-targeted immune-modulatory drugs engendered protective antitumor immunity that was superior to that of current "gold-standard" therapies in terms of efficacy and lack of toxicity or reduced toxicity. To overcome immune exhaustion aptamer-targeted siRNA conjugates could be used to downregulate intracellular mediators of exhaustion that integrate signals from multiple inhibitory receptors. Recent advances in aptamer development and second-generation aptamer-drug conjugates suggest that we have only scratched the surface.

Cell-penetrating peptide-siRNA conjugate loaded YSA-modified nanobubbles for ultrasound triggered siRNA delivery

Due to the absence of effective in vivo delivery systems, the employment of small interference RNA (siRNA) in the clinic has been hindered. In this paper, a new siRNA targeting system for EphA2-positive tumors was developed, based on ultrasound-sensitive nanobubbles (NBs) and cell-permeable peptides (CPPs). Here, a CPP–siRNA conjugate (CPP–siRNA) was entrapped in an ephrin mimetic peptide (YSA peptide)-modified NB (CPP–siRNA/YSA-NB) and the penetration of the CPP–siRNA was temporally masked; local ultrasound stimulation triggered the release of CPP–siRNA from the NBs and activated its penetration. Subsequent research demonstrated that the CPP–siRNA/YSA-NBs had particle sizes of approximately 200nm and a siRNA entrapment efficiency of more than 85%. The in vitro release results showed that over 90% of the encapsulated CPP–siRNA released from the NBs in the presence of ultrasound, while less than 1.5% of that (30min) released without ultrasound. Cell experiments showed a the higher CPP–siRNA cellular uptake of CPP–siRNA/YSA-NB among the various formulations in human breast adenocarcinoma cells (MCF-7, EphA2 positive cells). Additionally, after systemic administration in mice, CPP–siRNA/YSA-NB accumulated in the tumor, augmented c-Myc silencing and delayed tumor progression. In conclusion, the application of CPP–siRNA/YSA-NB with ultrasound may provide a strategy for the selective and efficient delivery of siRNA.

Exosomes: Natural Carriers for siRNA Delivery.

Various cells of the human physiological system have the capability to release extracellular vesicles (EVs) involved in intercellular transport of proteins and nucleic acids. Exosomes are a subtype of extracellular vesicles having their origin through endocytic pathway. While being involved in intercellular transport of macromolecules, exosomes, due to their presence in several body fluids, can also be utilized as a system to commute RNA molecules and proteins in the body. Recent advances in gene therapy have provided a new outlook in disease therapeutics by modulation of gene expression using oligonucleotide based approach and exosomes have been reported a potential carrier for nucleic acid based therapeutic moieties. In recent years, small interfering RNA (siRNA) has emerged as promising therapeutic alternative for diseases with gene-based pathophysiology, however poor bioavailability limits its therapeutic potential. For effective delivery and enhancement of bioavailability of siRNA, several carriers including dendrimers, liposomes, siRNA conjugates, and siRNA aptamer chimeras, to name a few, have been explored. Exosomes can be considered a promising carrier for effective delivery of siRNA due to their existence in body's endogenous system and high tolerance. The present review focuses on delivering knowledge about exosomes, siRNA, and capability of exosomes to act as natural carriers for siRNA delivery.

Abstract PR11: Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes

Abstract Recent studies in mice, nonhuman primates, and clinical trials in human patients have emphasized the importance of the persistence of the vaccine-induced immune response, immunological memory, in mediating protective immunity against infectious diseases and cancer. Inhibition of mediators of effector differentiation like mTOR, Blimp-1, CD25, glycolysis, or GSK3β, using genetic means or whenever available pharmacological agents, not only prevented the accumulation of the short-lived effectors but also redirected the activated T cells to differentiate along the memory pathway. For example, pharmacological inhibition of mTOR, or GSK3β with rapamycin, or TSW119, respectively, led to the differentiation of antigen activated CD8+ T cells into long lasting memory cells that exhibited enhanced antiviral and antitumor immunity. Nevertheless, pharmacological agents often exhibit undesirable effects reflecting the broad distribution of their targets. For example, rapamycin inhibition of mTOR promotes the development of Treg, and GSK3β inhibition polarizes dendritic cells to a tolerogenic state, arguably counterproductive in the setting of vaccination. In addition, development of pharmacological agents to modulate the function of intracellular targets that are not accessible to antibodies (undruggable targets) is highly challenging, and their availability especially for clinical use is limited. We are developing a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. RNAi is used to downregulate intracellular mediators of effector differentiation, that are targeted to CD8+ T cells by conjugation to an oligonucleotide aptamer ligand. We have shown that a CD8+ T cell targeted 4-1BB aptamer-raptor siRNA conjugate administered to mice by tail vein injection downregulated mTORC1, while preserving mTORC2, activity in at least 60% of adoptively transferred OVA-specific transgenic OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced memory response in mice. Whereas the aptamer-raptor siRNA generated CTL exhibited normal cytotoxic effector functions and enhanced vaccine-induce protective antitumor immunity in both prophylactic and therapeutic tumor models, the rapamycin generated CTL were defective in their cytotoxic effector functions and failed to elicit protective immunity against a tumor challenge. Underscoring the lack of cell specificity of rapamycin action, and providing a potential mechanisms underlying the effector defect of rapamycin generated memory CD8+ T cells, dendritic cells from mice treated with rapamycin, but not with aptamer-raptor siRNA conjugate, exhibited reduced alloMLR activity, consistent with the known suppressive effects of rapamycin induced mTOR inhibition on DC. Given that inhibition of mTORC1 may not be the optimal way of enhancing memory, we have used the aptamer targeting platform to inhibit other mediators of effector differentiation in activated CD8+ T cells. In preliminary studies 4-1BB aptamer-targeted inhibition of Axin-1, to promote wnt signaling, or reducing IL-2 signaling by downregulating CD25 expression, enhanced vaccine-induced protective antitumor immunity in tumor-bearing mice. Using the OT-I adoptive transfer model, in preliminary experiments we have shown that 4-1BB aptamer targeted co-inhibition of both raptor and PD-1 increased the formation of memory OT-I cells; while not superior to that of inhibiting raptor alone, the OT-I cells expressed significantly less PD-1 on cell surface, and were, therefore, resistant to PD-1-mediated immune suppression. Overall, these studies demonstrate the feasibility and efficiency of aptamer targeted delivery of siRNAs to immune cells, and underscore the potential advantages of aptamer-targeted siRNA delivery over nontargeted administration of pharmacological agents. This abstract is also presented as Poster A23. Citation Format: Alexey Berezhnoy, Rajagopalan Anugraha, Thomas Malek, Eli Gilboa. Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr PR11.

Comparative analysis of polymers for short interfering RNA delivery in vascular smooth muscle cells

The use of short interfering RNA (siRNA) to degrade messenger RNA in the cell cytoplasm and transiently attenuate intracellular proteins shows promise in the inhibition of vascular pathogenesis. However, a critical obstacle for therapeutic application is a safe and effective delivery system. Biodegradable polymers are promising alternative molecular carriers for genetic material. Here, we aim to perform a comparative analysis of poly(B-amino ester) (PBAE) and polyethylenimine (PEI) polymers in their efficacy for vascular smooth muscle cell transfection using siRNA against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene as our test target. MethodsHuman aortic smooth muscle cells (HASMC) were transfected in vitro with polymers conjugated to GAPDH or negative control (NC) siRNAs. Increasing siRNA:polymer ratios were tested for optimal transfection efficiency. DharmaFECT2 chemical transfection complexes were used for comparative analysis. Live/dead dual stain was used to measure cell viability, and GAPDH gene silencing was measured by quantitative polymerase chain reaction normalized to 18S. ResultsThe highest rate of PEI-mediated silencing was achieved with a 9μL polymer:220 pmol/mL siRNA conjugate (16 ± 2% expression versus NC; n = 6). Comparable PBAE-mediated silencing could be achieved with a 1.95μL polymer:100 pmol/mL siRNA conjugate (10 ± 1% expression versus NC; n = 5). Transfection using PEIs resulted in silencing equivalent to other methods but with less efficiency and increased cell toxicity at 24h polymer exposure. Decreasing PEI exposure time to 4 h resulted in similar silencing efficacy (21 ± 9% expression versus NC, n = 6) with an improved toxicity profile. ConclusionsPolymeric bioconjugates transfected HASMCs in a manner similar to chemical complexes, with comparable cell toxicity and silencing efficiency. PEI bioconjugates demonstrated silencing equivalent to PBAE bioconjugates, although less efficient in terms of required polymer concentrations. Given the cost-to-benefit difference between the assayed polymers, and PEI's ability to transfect HASMCs within a short duration of exposure with an improved toxicity profile, this study shows that PEI bioconjugates are a potential transfection agent for vascular tissue. Future studies will expand on this method of gene therapy to validate delivery of gene-specific inhibitors aimed at attenuating smooth muscle cell proliferation, adhesion, and migration. These studies will lay the framework for our future experimental plans to expand on this method of gene therapy for in vivo transfection in animal models of vascular disease.

Inhibition of skin squamous cell carcinoma proliferation and promote apoptosis by dual silencing of NET-1 and survivin.

The simultaneous silencing of multiple upregulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. In the present study, skin squamous cell carcinoma (SSCC) tissue microarray was constructed and the expression of NET-1 and survivin was identified. The high expression of NET-1 and survivin gene in SSCC was confirmed as an important event for the formation and development of the cancer. A total of 100 primary SSCC patients were included in the present study. Expression of NET-1 and survivin in cancer cells was evaluated immunohistochemically in tissue microarrays. The interaction between NET-1 and survivin in SSCC by co-immunoprecipitation was subsequently verified by producing the siRNA sequence targeting the single gene (siRNA-NET-1 and siRNA-survivin) as well as NET-1 and survivin gene (one-chain-double-target siRNA). The levels of NET-1 and survivin mRNA and protein expression in A431 cells were detected by RT-qPCR and western blotting, and the expression of related genes including vascular endothelial growth factor (VEGF), cortactin, Bcl-2, caspase-3 and -8 was identified using RT-qPCR. The protein localization and expression of NET-1 and survivin in A431 cells were documented by immunohistochemistry and immuno-fluorescence staining. The proliferation and apoptosis of A431 cells were detected by CCK-8 assay and flow cytometry (FCM). The tissue microarray showed that NET-1 and survivin were highly expressed in SSCC, while the correlation analysis showed NET-1 expression was positively associated with survivin. In addition, we reported that using the one-chain-double-target siRNA conjugate composed of NET-1 and survivin siRNA sequences in the same backbone inhibited proliferation and promoted apoptosis of SSCC. The one-chain-double-target siRNA showed further downregulation on NET-1 and survivin mRNA and protein levels compared with NET-1 siRNA or survivin siRNA. It also exhibited greater suppression on proliferation and triggering of apoptosis in A431 cells than NET-1 siRNA or survivin siRNA. This result may be explained by the significant downregulation of VEGF, cortactin and Bcl-2, and upregulation of caspase-3 and -8. NET-1 and survivin were overexpressed in SSCC and an interaction between NET-1 and survivin was identified. The one-chain-double-target siRNA appears to be superior in inhibiting cell proliferation and promoting apoptosis compared with the single target siRNA. NET-1 and survivin may have correlative signaling pathways with VEGF, cortactin, Bcl-2, caspase-3 and -8. Simultaneous silencing of NET-1 and survivin using one-chain-double-target siRNA thus provides an advantageous alternative in the development of therapeutics for SSCC.

A New Approach for the Treatment of Arthritis in Mice with a Novel Conjugate of an Anti-C5aR1 Antibody and C5 Small Interfering RNA.

Rheumatoid arthritis (RA) is an inflammatory autoimmune joint disease in which the complement system plays an important role. Of the several components of complement, current evidence points to C5 as the most important inducer of inflammation. Several groups generated Abs or small interfering RNAs (siRNAs) or small molecule inhibitors against C5 and C5aR1 (CD88) that have showed some efficacy in RA in animal models. However, none of these candidate therapeutics has moved from bench to bedside. In this study, we test in collagen Ab-induced arthritis (CAIA) a new therapeutic strategy using a novel anti-C5ab-C5 siRNA conjugate. We first demonstrate that although C5aR2 or C5L2 (GPR77) plays no role in CAIA, C5aR1 contributes to pathogenesis. We demonstrate that injection of siRNAs blocking C5, C5aR1, or the combination decreased clinical disease activity in mice with CAIA by 45%, 51%, and 58%, respectively. Anti-C5 Ab (BB5.1) has only limited efficacy, but significantly reduced arthritis up to 66%. We then generated a novel anti-C5aR1 Ab-protamine-C5 siRNA conjugate. To our knowledge, we show for the first time that whereas unconjugated Ab plus siRNAs reduce arthritis by 19%, our anti-C5aR1 Ab-protamine-C5 siRNA conjugate was effective in reducing arthritis by 83% along with a parallel decrease in histopathology, C3 deposition, neutrophils, and macrophages in the joints of mice with CAIA. These data suggest that by targeting anti-C5 siRNAs to the receptor for its C5a activation fragment (C5aR1), a striking clinical effect can be realized.

Aptamer-targeted siRNA inhibition of raptor promotes the persistence of vaccine-induced CD8+ T cell responses and enhances protective immunity against influenza virus (IRM14P.452)

Abstract Inhibition of mediators of effector differentiation redirects activated T cells to differentiate along the memory pathway. For example, pharmacological inhibition of mTOR with rapamycin led to the enhenced differentiation of antigen activated CD8+ T cells into memory cells. Nevertheless, pharmacological agents are not available for many “undrugable” targets and often exhibit undesirable effects reflecting the broad distribution of their targets. We are developing a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. RNAi is used to downregulate intracellular mediators of effector differentiation that are targeted to CD8+ T cells by conjugation to an oligonucleotide aptamer ligand. We have shown that a CD8+ T cell targeted 4-1BB aptamer-raptor siRNA conjugate administered to mice downregulated mTORC1, but not mTORC2, activity in vaccine-activated CD8+ T cells. In OT-I model, CD8+ memory cells generated with 4-1BB-raptor exhibited normal cytotoxic effector functions while rapamycin generated CTL were defective in their cytotoxic effector functions. Prophylactic immunization with DNA-based vaccine in combination with 4-1BB raptor increased persistence of NP-specific memory CD8+ T cells and protected animals from influenza challenge. Overall, these studies demonstrate the feasibility and efficiency of aptamer-targeted delivery of siRNAs to immune cells.

273. MMPs-Responsive Release of siRNA from 4-Arm PEG siRNA Conjugate

Small interfering RNA (siRNA) has drawn much attention for its unique and effective gene silencing mechanism although its poor cellular membrane permeability, limited stability and low flexibility still remain major concerns. In particular, when siRNAs are complexed with the cationic carriers, the resultant complexes shows much lower physical stability than those prepared with plasmid DNA due to the low charge density of siRNA. Thus, we conjugated siRNAs to 4-arm polyethylene glycol (PEG) via a matrix metalloproteinases (MMPs)-cleavable peptide to enhance complexation with linear polyethyleneimine (LPEI). In addition, release of siRNA/LPEI complex from 4-arm PEG-siRNA/LPEI and subsequent gene silencing efficacy were investigated in diabetic ulcers. After synthesis of 4PEG-siRNA conjugate, the conjugation amount of siRNA per 4-arm PEG was 2.6 (mol/mol). According to gel retardation assay, 4PEG-siRNA/LPEI showed superior association to siRNA/LPEI at the same N/P ratio, which suggests the flexible structure of 4PEG-siRNA conjugate is more favorable for complexation with LPEI than naked siRNA. The particle size and zeta potential of 4PEG-siRNA/LPEI was 1415.5±73.1nm and 13.8±1.9mV, respectively. However, those values were changed similarly to those of siRNA/LPEI in the presence of MMP-2 because the MMPs-cleavable linker between 4-arm PEG and siRNA was digested by MMP-2. In addition, 4PEG-siRNA/LPEI complexes could not be uptaken by cells before dissociation by MMP-2 due to their huge size. In diabetic ulcers, 4PEG-siRNA/LPEI was rapidly cleaved and separated to siRNA/LPEI fraction and showed higher gene silencing of MMP-2 for 14 days. Thus, wound recovery was subsequently enhanced by inhibiting MMP-2 expression in diabetic ulcers. View Large Image | Download PowerPoint Slide

Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo.

We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.

SiRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.

Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3' end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA-GalNAc conjugation strategies.

Hyaluronic acid-fabricated nanogold delivery of the inhibitor of apoptosis protein-2 siRNAs inhibits benzo[a]pyrene-induced oncogenic properties of lung cancer A549 cells

Benzo[a]pyrene (BaP), a component of cooking oil fumes (COF), promotes lung cancer cell proliferation and survival via the induction of inhibitor of apoptosis protein-2 (IAP-2) proteins. Thus knockdown of IAP-2 would be a promising way to battle against lung cancer caused by COF. Functionalized gold nanoparticle (AuNP) is an effective delivery system for bio-active materials. Here, biocompatible hyaluronic acid (HA) was fabricated into nanoparticles to increase the target specificity by binding to CD44-over-expressed cancer cells. IAP-2-specific small-interfering RNA (siRNAs) or fluorescein isothiocyanate (FITC) were then incorporated into AuNP-HA. Conjugation of IAP-2 siRNA into AuNPs-HA was verified by the UV–vis spectrometer and Fourier transform infrared spectrometer. Further studies showed that AuNP-HA/FITC were effectively taken up by A549 cells through CD44-mediated endocytosis. Incubation of BaP-challenged cells with AuNP-HA-IAP-2 siRNAs silenced the expression of IAP-2, decreased cell proliferation and triggered pronounced cell apoptosis by the decrease in Bcl-2 protein and the increase in Bax protein as well as the active form of caspases-3. The BaP-elicited cell migration and enzymatic activity of the secreted matrix metalloproteinase-2 were also substantially suppressed by treatment with AuNP-HA-IAP-2 siRNAs. These results indicated that IAP-2 siRNAs can be efficiently delivered into A549 cells by functionalized AuNP-HA to repress the IAP-2 expression and BaP-induced oncogenic events, suggesting the potential therapeutic application of IAP-2 siRNA or other siRNA-conjugated AuNP-HA composites to COF-induced lung cancer and other gene-caused diseases in the future.

Detection of GNAQ mutations and reduction of cell viability in uveal melanoma cells with functionalized gold nanoparticles.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Early treatment may improve any chances of preventing metastatic disease, but diagnosis of small UM is challenging. Up to 95 % of all UMs carry somatic mutations in the G-coupled proteins GNAQ and GNA11 promoting anchorage-independent growth and proliferation. About 50 % of UMs are fatal. Once metastatic, patients have limited options for successful therapy. We have developed functionalized gold nanoparticles (AuNPs) to visualize transcripts of mutant GNAQ mRNA in living cells. In addition to their suitability as a specific tool for GNAQ mutation detection, we have developed a novel linker that enables conjugation of siRNAs to AuNPs allowing for greater and more rapid intracellular release of siRNAs compared to previously described approaches. Binding of modified AuNPs to matching target mRNA leads to conformational changes, resulting in a detectable fluorescent signal that can be used for mutation detection in living cells. Knockdown of GNAQ with siRNA-AuNPs effectively reduced downstream signals and decreased cell viability in GNAQ mutant uveal melanoma cells. AuNPs may in future be developed to serve as sensors for mutations of vital importance. The new release system for siRNA-AuNP improves previous systems, which conceivably will be useful for future therapeutic gene regulatory approaches.

Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB-siRNA conjugates.

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody–siRNA complexes provide a possible solution. However, initial reports of antibody–siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody–siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.