siRNA-based Gene Therapy Research Articles

Extracellular vesicle-packaged lncRNA from cancer-associated fibroblasts promotes immune evasion by downregulating HLA-A in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by immune evasion that contribute to poor prognosis. Cancer-associated fibroblasts (CAFs) play a pivotal role in orchestrating the PDAC tumour microenvironment. We investigated the role of CAF-derived extracellular vesicle (EV)-packaged long non-coding RNAs (lncRNAs) in immune evasion and explored gene therapy using engineered EVs loading small interfering RNAs (siRNAs) as a potential therapeutic strategy. Our findings highlight the significance of EV-packaged lncRNA RP11-161H23.5 from CAF in promoting PDAC immune evasion by downregulating HLA-A expression, a key component of antigen presentation. Mechanistically, RP11-161H23.5 forms a complex with CNOT4, a subunit of the mRNA deadenylase CCR4-NOT complex, enhancing the degradation of HLA-A mRNA by shortening its poly(A) tail. This immune evasion mechanism compromises the anti-tumour immune response. To combat this, we propose an innovative approach utilising engineered EVs as natural and biocompatible nanocarriers for siRNA-based gene therapy and this strategy holds promise for enhancing the effectiveness of immunotherapy in PDAC. Overall, our study sheds light on the critical role of CAF-derived EV-packaged lncRNA RP11-161H23.5/CNOT4/HLA-A axis in PDAC immune evasion and presents a novel avenue for therapeutic intervention.

Cationic Polythiophene as Gene Carrier and Sonosensitizer for Sonodynamic Synergic Gene Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal and highly malignant tumors. Sonodynamic therapy (SDT) is a new cancer treatment method. One of its unique advantages lies in the treatment of deep tumors due to its excellent tissue penetration ability caused by ultrasound (US). However, most sonosensitizers suffer from weak sonodynamic activity and poor tumor-targeting ability. In addition, small interfering RNA (siRNA) is a promising anticancer drug, and the efficacy of siRNA-based gene therapy largely depends on the cell impermeability of the gene carrier. Here, we designed and synthesized a cationic polythiophene derivative (PT2) that can be used as a siRNA carrier for gene therapy. Moreover, PT2 could generate singlet oxygen (1O2) and hydroxyl radicals (O2•-) under US irradiation, which suggests that PT2 could be used for SDT. Our study discovered that NUDT1 promoted HCC proliferation and inhibited intracellular ROS production. Therefore, si-NUDT1 was designed and synthesized. NUDT1 silencing can inhibit the proliferation of tumor cells and increase the production of intracellular ROS to further improve the efficacy of SDT. Then, si-NUDT1 assembled with PT2 and DSPE-PEG-FA to prepare a novel tumor-targeting nanodrug (PT2-siRNA@PEG-FA) for synergic SDT and gene therapy of HCC.

Gint4.T-siHDGF chimera-capped mesoporous silica nanoparticles encapsulating temozolomide for synergistic glioblastoma therapy

Due to glioblastoma (GBM) being the most intractable brain tumor, the continuous improvement of effective treatment methods is indispensable. The combination of siRNA-based gene therapy and chemotherapy for GBM treatment has now manifested great promise. Herein, Gint4.T-siHDGF chimera-capped mesoporous silica nanoparticles (MSN) encapsulating chemotherapy drug temozolomide (TMZ), termed as TMSN@siHDGF-Gint4.T, is developed to co-deliver gene-drug siHDGF and TMZ for synergistic GBM therapy. TMSN@siHDGF-Gint4.T possesses spherical nucleic acid-like architecture that can improve the enzyme resistance of siHDGF and increase the blood-brain barrier (BBB) permeability of the nanovehicle. The aptamer Gint4.T of chimera endows the nanovehicle with GBM cell-specific binding ability. When administered systemically, TMSN@siHDGF-Gint4.T can traverse BBB and enter GBM cells. In the acidic lysosome environment, the cleavage of benzoic-imine bond on MSN surface leads to an initial rapid release of chimera, followed by a slow release of TMZ encapsulated in MSN. The sequential release of siHDGF and TMZ first allows siHDGF to exert its gene-silencing effect, and the downregulation of HDGF expression further enhances the cytotoxicity of TMZ. In vivo experimental results have demonstrated that TMSN@siHDGF-Gint4.T significantly inhibits tumor growth and extends the survival time of GBM-bearing mice. Thus, the as-developed TMSN@siHDGF-Gint4.T affords a potential approach for the combination treatment of GBM.

Emergence of Small Interfering RNA-Based Gene Drugs for Various Diseases.

Small molecule, peptide, and protein-based drugs have been developed over decades to treat various diseases. The importance of gene therapy as an alternative to traditional drugs has increased after the discovery of gene-based drugs such as Gendicine for cancer and Neovasculgen for peripheral artery disease. Since then, the pharma sector is focusing on developing gene-based drugs for various diseases. After the discovery of the RNA interference (RNAi) mechanism, the development of siRNA-based gene therapy has been accelerated immensely. siRNA-based treatment for hereditary transthyretin-mediated amyloidosis (hATTR) using Onpattro and acute hepatic porphyria (AHP) by Givlaari and three more FDA-approved siRNA drugs has set up a milestone and further improved the confidence for the development of gene therapeutics for a spectrum of diseases. siRNA-based gene drugs have more advantages over other gene therapies and are under study to treat different types of diseases such as viral infections, cardiovascular diseases, cancer, and many more. However, there are a few bottlenecks to realizing the full potential of siRNA-based gene therapy. They include chemical instability, nontargeted biodistribution, undesirable innate immune responses, and off-target effects. This review provides a comprehensive view of siRNA-based gene drugs: challenges associated with siRNA delivery, their potential, and future prospects.

Research Status and Prospect of Non-Viral Vectors Based on siRNA: A Review

Gene therapy has attracted much attention because of its unique mechanism of action, non-toxicity, and good tolerance, which can kill cancer cells without damaging healthy tissues. siRNA-based gene therapy can downregulate, enhance, or correct gene expression by introducing some nucleic acid into patient tissues. Routine treatment of hemophilia requires frequent intravenous injections of missing clotting protein. The high cost of combined therapy causes most patients to lack the best treatment resources. siRNA therapy has the potential of lasting treatment and even curing diseases. Compared with traditional surgery and chemotherapy, siRNA has fewer side effects and less damage to normal cells. The available therapies for degenerative diseases can only alleviate the symptoms of patients, while siRNA therapy drugs can upregulate gene expression, modify epigenetic changes, and stop the disease. In addition, siRNA also plays an important role in cardiovascular diseases, gastrointestinal diseases, and hepatitis B. However, free siRNA is easily degraded by nuclease and has a short half-life in the blood. Research has found that siRNA can be delivered to specific cells through appropriate vector selection and design to improve the therapeutic effect. The application of viral vectors is limited because of their high immunogenicity and low capacity, while non-viral vectors are widely used because of their low immunogenicity, low production cost, and high safety. This paper reviews the common non-viral vectors in recent years and introduces their advantages and disadvantages, as well as the latest application examples.

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

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 the 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.

Feasibility of cRGD conjugation at 5′-antisense strand of siRNA by phosphodiester linkage extension

Small interfering RNAs (siRNAs) are widely studied for their highly specific gene silencing activity. However, obstacles remain to the clinical application of siRNAs. Attaching conjugates to siRNAs can improve their stability and broaden their application, and most functional conjugates of siRNAs locate at the 3′-terminus of the sense or antisense strand. In this work, we found that conjugating a group at the 5′-terminus of the antisense strand via phosphodiester was practicable, especially when the group was a flexible moiety such as an alkyl linker. When conjugating a bulky ligand, such as cRGD, the length of the 5′-phosphodiester linker between the ligand and the 5′-terminus of the antisense strand was the key in terms of RNA interference (RNAi). With a relative longer linker, the conjugates showed potency similar to siRNA. A highly efficient transfection system composed of a neutral cytidinyl lipid (DNCA) and a gemini-like cationic lipid (CLD) was employed to deliver siRNAs or their conjugates. The cRGD conjugates showed superior targeting delivery and antitumor efficacy in vivo and also selective cellular uptake in vitro. This unity of encapsulation and conjugation strategy may provide potential strategies for siRNA-based gene therapy.

Tumor targeted delivery of siRNA by a nano-scale quaternary polyplex for cancer treatment

With the capability to specific silence target gene expression, RNA interference (RNAi) technology has emerged as a powerful tool for cancer treatment. Although diversified delivery platforms have been designed for siRNA-based gene therapy, few delivery systems could fulfill all the requirements during the delivery process in vivo. Here, a nano-scale quaternary polyplex (NQP) that can optimize the entire process of tumor-targeted siRNA delivery from intravenous injection to gene silencing inside cells is proposed. NQP is endowed with a core–shell structure composed of an siRNA loaded, ATP-responsive core and a pH-responsive shell. This unique structure enables NQP to achieve optimal efficiency in all essential steps during the siRNA delivery, including blood circulation, tumor accumulation, cell internalization, and controlled release of siRNA. As a result, administration of siSTAT3-loaded NQP to tumor-bearing mice achieved efficient gene silencing and tumor growth inhibition. Since siSTAT3 could be replaced by any siRNA, NQP could become a promising platform for the development of siRNA-based cancer gene therapy.

Preparation Optimization of Bovine Serum Albumin Nanoparticles and Its Application for siRNA Delivery.

BackgroundsiRNA brings hope for cancer therapy. However, there are many obstacles for application of siRNA in clinical. Because of the excellent biocompatibility, non-toxicity and non-immunogenicity of bovine serum albumin (BSA), BSA-based nanoparticles have been widely designed as a drug carrier system.MethodsThe optimal formula for BSA NPs preparation was investigated by central composite design response surface methodology (CCD-RSM), BSA-based survivin-siRNA delivery system (BSA NPs/siRNA) was characterized by dynamic light scattering, atomic force microscope, transmission electron microscope and Bradford method. The in vitro anti-tumor effect and mechanism of BSA NPs were investigated by confocal microscopic imaging, MTT assay, RT-qPCR and ELISA analysis. Moreover, the anti-tumor effect, distribution and biosafety of BSA NPs were studied in vivo.ResultsThe optimal formula for BSA NPs was settled to be 20 mg/mL for BSA concentration, 9 for pH value, 136% for crosslinking degree and 1.6 mL/min for speed of ethanol addition. BSA NPs/siRNA could remain stable at 4°C for 4 weeks and protect siRNA from degradation by RNase A. Besides, BSA NPs/siRNA could maintain a sustained release of siRNA and promote the uptake of siRNA significantly. The survivin-mRNA level and the survivin-protein level were decreased by 55% ± 1.6% and 54% ± 1.6% separately. The in vivo tumor inhibition results suggested that the tumor inhibition rate of BSA NPs/siRNA-treated group was 54% ± 12% and was similar with that of DOX-treated group (57% ± 9.2%, P > 0.05). The biosafety results confirmed that BSA NPs/siRNA could not induce significant damages to the main organs and blood in vivo.ConclusionThese results demonstrated that CCD-RSM was an effective tool for preparation analysis, and the BSA NPs/siRNA was a promising system for siRNA-based gene therapy.

PEI-Coated Fe₃O₄ Nanoparticles Enable Efficient Delivery of Therapeutic siRNA Targeting REST into Glioblastoma Cells.

Glioblastomas (GBM) are the most frequent brain tumors lacking efficient treatment. The increasingly elucidated gene targets make siRNA-based gene therapy a promising anticancer approach, while an efficient delivery system is urgently needed. Here, polyethyleneimine (PEI)-coated Fe3O4 nanoparticles (NPs) have been developed and applied for siRNA delivery into GBM cells to silence repressor element 1-silencing transcription factor (REST). The prepared PEI-coated Fe3O4 NPs were characterized as magnetic nanoparticles with a positive charge, by transmission electronic microscopy, dynamic light-scattering analysis and a magnetometer. By gel retardation assay, the nanoparticles were found to form complexes with siRNA and the interaction proportion of NP to siRNA was 2.8:1. The cellular uptake of NP/siRNA complexes was verified by prussian blue staining, fluorescence labeling and flow cytometry in U-87 and U-251 GBM cells. Furthermore, the REST silencing examined by realtime polymerase chain reaction (PCR) and Western blotting presented significant reduction of REST in transcription and translation levels. Upon the treatment of NP/siRNA targeting REST, the GBM cell viabilities were inhibited and the migration capacities were repressed remarkably, analyzed by cell counting kit-8 and transwell assay separately. In this study, we demonstrated the PEI-coated Fe3O4 nanoparticle as a vehicle for therapeutic siRNA delivery, at an appropriate NP/siRNA weight ratio for REST silencing in GBM cells, inhibiting cell proliferation and migration efficiently. These might represent a novel potential treatment strategy for GBM.

Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing.

Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC). Addition of a cationic engineered supercharged coiled-coil protein (CSP) in a 10:1:1 ratio of DNC:CSP:siRNA produced a stable lipoproteoplex (LPP) nanoparticle, with optimal siRNA complexation, minimal cytotoxicity, and increased transfection efficacy. In a humanized murine diabetic wound healing model, our optimized LPP formulation successfully delivered siRNA targeted against Keap1, key repressor of Nrf2 which is a central regulator of redox mechanisms. Application of LPP complexing siKeap1 restored Nrf2 antioxidant function, accelerated diabetic tissue regeneration, and augmented reduction-oxidation homeostasis in the wound environment. Our topical LPP delivery system can readily be translated into clinical use for the treatment of diabetic wounds and can be extended to other cutaneous diseases with genetic components.

Reductively Responsive Hydrogel Nanoparticles with Uniform Size, Shape, and Tunable Composition for Systemic siRNA Delivery in Vivo.

To achieve the great potential of siRNA based gene therapy, safe and efficient systemic delivery in vivo is essential. Here we report reductively responsive hydrogel nanoparticles with highly uniform size and shape for systemic siRNA delivery in vivo. "Blank" hydrogel nanoparticles with high aspect ratio were prepared using continuous particle fabrication based on PRINT (particle replication in nonwetting templates). Subsequently, siRNA was conjugated to "blank" nanoparticles via a disulfide linker with a high loading ratio of up to 18 wt %, followed by surface modification to enhance transfection. This fabrication process could be easily scaled up to prepare large quantity of hydrogel nanoparticles. By controlling hydrogel composition, surface modification, and siRNA loading ratio, siRNA conjugated nanoparticles were highly tunable to achieve high transfection efficiency in vitro. FVII-siRNA conjugated nanoparticles were further stabilized with surface coating for in vivo siRNA delivery to liver hepatocytes, and successful gene silencing was demonstrated at both mRNA and protein levels.

Enhancing endosomal escape for nanoparticle mediated siRNA delivery.

Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

Poly(ester amine) Synthesized from Trimethylolpropane Triacrylate and Spermine as an Efficient siRNA Carrier

Small interfering RNA (siRNA) is a promising candidate for gene therapy due to the strong and specific gene silencing effects. However, to effectively silence any protein, lack of an efficient delivery system is major obstacle for siRNA therapeutics. In the present study, we report on the development of a novel poly(ester amine) (PEA) as an efficient siRNA carrier. The PEA was prepared by a Michael addition reaction between trimethylolpropane triacrylate (TT) and spermine (SPE). The composition of the PEA was characterized by 1H nuclear magnetic resonance (1H NMR) and the molecular weight was measured using gel permeability chromatography (GPC). This TT-SPE effectively condensed siRNA into a compact nanoscale structure (size: 76 nm) with homogenous size distribution and provided superior protection of siRNA from RNase A enzyme. Furthermore, TT-SPE/ siRNA complexes showed good intracellular uptake and had efficacious gene silencing effect with low toxicity compared to PEI 25K. These findings demonstrated that TT-SPE has potential as an alternative to PEI 25K for siRNA-based gene therapy.

Gene Silencing of Toll-Like Receptor 2 Inhibits Proliferation of Human Liver Cancer Cells and Secretion of Inflammatory Cytokines

BackgroundToll-like receptors (TLRs) are key factors in the innate immune system and initiate the inflammatory response to foreign pathogens such as bacteria, fungi and viruses. In the microenvironment of tumorigenesis, TLRs can promote inflammation and cell survival. Toll-like receptor 2/6 (TLR2/6) signaling in tumor cells is regarded as one of the mechanisms of chronic inflammation but it can also mediate tumor cell immune escape and tumor progression. However, the expression of TLR2 and its biological function in the development and progression of hepatocarcinoma have not been investigated. This study aimed to determine the expression of TLRs 1–10 in the established human hepatocellular carcinoma cell line BLE-7402, to investigate the biological effect of TLR2 on cell growth and survival.MethodsTLR expression in BLE-7402 cells was assayed by RT-PCR, real-time PCR and flow cytometry (FCM). To further investigate the function of TLR2 in hepatocarcinoma growth, BLE-7402 cells were transfected with recombinant plasmids expressing one of three forms of TLR2 siRNA (sh-TLR2 RNAi(A, B and C)). TLR2 knockdown was confirmed using RT-PCR, real-time PCR and fluorescence microscopy. Tumor cell proliferation was monitored by MTT assay and secreted cytokines in the supernatant of transfected cells were measured by bead-based FCM, the function of TLR2 siRNA was also investigated in vivo.ResultsThe BLE-7402 cell line expressed TLRs 2 to 10 at both mRNA and protein levels. TLR2 was the most highly expressed TLR. While all the three siRNAs inhibited TLR2 mRNA and protein expression, sh-TLR2 RNAi(B) had the strongest knockdown effect. TLR2 knockdown with sh-TLR2 RNAi(B) reduced cell proliferation. Furthermore, secretion of IL-6 and IL-8 was also reduced. The result showed a drastic reduction in tumor volume in mice treated with sh-TLR2 RNAi(B).DiscussionThese results suggest that TLR2 knockdown inhibit proliferation of cultured hepatocarcinoma cells and decrease the secretion of cytokines. It is suggested that TLR2 silencing may worth further investigations for siRNA based gene therapy in treatment of hepatocarcinoma.

Abstract 1637: STAT3 as a new molecular target for treating ABC-like DLBCL

Abstract Persistent STAT3 signaling contributes to malignant progression in many diverse human tumors. STAT3 has been found to be constitutively active in activated B cell (ABC)-like diffuse large B cell lymphomas (DLBCL). We investigated the effect of STAT3 silencing in human DLBCL Ly3 cells both in vitro and in vivo. For this purpose, we established stably transduced STAT3 shRNA-expressing lentivirus Ly3 cells and control lentivirus Ly3 cells. The stable expression of STAT3 shRNA resulted in 40-50% reduction of STAT3 protein levels compared to the control cells. In vitro experiments revealed that STAT3 downregulation was associated with a higher percentage of dead cells in STAT3 shRNA cells compared with the control cells, and this percentage was even higher in serum starvation conditions. STAT3 silencing resulted in inhibition of IL-10-inducible upregulation of c-Myc associated with downregulation of IL-10-dependent STAT3 activation and inhibition of IL-10-inducible cell growth. STAT3 inhibition also reduced adhesion to the bone marrow stroma layer and migration toward SDF-1 alpha. Moreover, analysis of inflammatory cytokines and receptors indicated a substantial downregulation of CXCL6 mRNA levels in STAT3 shRNA cells compared to the control cells. Tumors in control Ly3-bearing mice grew progressively, whereas tumors in STAT3 shRNA Ly3-bearing mice regressed 4-5 days after injection. This tumor regression was associated with Caspase-3-dependent apoptosis and significant reduction of STAT3 target genes at the protein level such as Mcl-1, c-Myc and Survivin. Moreover, immunofluorescence analysis of tumor specimens showed that while granulocytes and endothelial cells infiltrated into the control cells-bearing tumors, tumor bearing STAT3 shRNA cells showed less or no infiltration of these cells. We also observed reduction of IL-10 production and the secretion of other cytokines was altered as well. Thus, our results show that even partial down-regulation of STAT3 could achieve complete suppression of the tumorigenesis of ABC-like DLBCL Ly3 cells by affecting also the tumor microenvironment. Furthermore, we tested two STAT3 inhibitors, STATTIC and S31-201, in this system. The viability of Ly3 cells grown in vitro was reduced by both compounds tested in a dose-dependent manner and the antiproliferative effect was associated with inhibition of STAT3 phosphorylation. The antilymphoma activity of STATTIC was also observed in vivo. This is the first demonstration in vivo that direct STAT3 inhibition in ABC-like DLBCL suppresses tumor growth. While developing a small-molecule STAT3 inhibitor as well as siRNA delivery for clinical trials is still a challenge, our studies validate STAT3 as a good target for therapy in DLBCL and also establish that STAT3 siRNA-based gene therapy is a feasible approach for DLBCL. In Sum, our data support continued development of new STAT3 inhibitors as well as STAT3 siRNA delivery strategies for treatment of DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1637. doi:10.1158/1538-7445.AM2011-1637

In Vivo Application of an RNAi Strategy for the Selective Suppression of a Mutant Allele

Gene therapy for dominantly inherited diseases with small interfering RNA (siRNA) requires mutant allele-specific suppression when genes in which mutation causes disease normally have an important role. We previously proposed a strategy for selective suppression of mutant alleles; both mutant and wild-type alleles are inhibited by most effective siRNA, and wild-type protein is restored using mRNA mutated to be resistant to the siRNA. Here, to prove the principle of this strategy in vivo, we applied it to our previously reported anti-copper/zinc superoxide dismutase (SOD1) short hairpin RNA (shRNA) transgenic (Tg) mice, in which the expression of the endogenous wild-type SOD1 gene was inhibited by more than 80%. These shRNA Tg mice showed hepatic lipid accumulation with mild liver dysfunction due to downregulation of endogenous wild-type SOD1. To rescue this side effect, we generated siRNA-resistant SOD1 Tg mice and crossed them with anti-SOD1 shRNA Tg mice, resulting in the disappearance of lipid accumulation in the liver. Furthermore, we also succeeded in mutant SOD1-specific gene suppression in the liver of SOD1(G93A) Tg mice, a model for amyotrophic lateral sclerosis, using intravenously administered viral vectors. Our method may prove useful for siRNA-based gene therapy for dominantly inherited diseases.

SiRNA therapy for cancer and non-lethal diseases such as arthritis and osteoporosis

Importance of the field: Gene silencing mediated by siRNA has been widely investigated as a potential therapeutic approach. The success of these therapies depends on effective systems capable of selectively and efficiently conveying siRNA to targeted cells/organs with minimal toxicity.Areas covered in this review: This review discusses current experimental approaches to siRNA delivery strategies available for arthritis treatment and the management of other musculoskeletal disorders. The review covers literature on the subject from 2000 to 2010.What the reader will gain: In the last decade, extensive improvements have been made to optimize siRNA-based gene therapy and have been tested on several arthritis and orthopedic conditions. However, except for Phase I – II DNA-based gene therapy trials on arthritis, no clinical studies have reported siRNA application in these domains.Take home message: Most musculoskeletal disorders, such as rheumatoid arthritis, osteoarthritis, fracture, aseptic loosening, cartilage and intervertebral disc degeneration are non-fatal and age-related chronic inflammatory conditions, but represent significant morbidity and a socio-economic burden. siRNA-based gene therapy offers treatment opportunities that are less invasive, more effective and less expensive than existing modalities. Future directions for siRNA therapy include the development of safe and more efficient delivery systems and the selection of optimal gene targets for disease control.

SiRNA‐mediated gene silencing in the salivary gland using in vivo microbubble‐enhanced sonoporation

siRNA-induced gene silencing in the salivary gland using microbubble-enhanced sonoporation was used to develop an in vivo gene knockdown technique. siRNA targeting rat glyceraldehyde-3-phosphate dehydrogenas (GAPDH) was mixed with echo-enhanced microbubbles and reverse-injected into rat parotid glands using transdermal ultrasound. To compare direct and transdermal ultrasound efficiencies, an incision was made on the lateral neck to expose the parotid glands for direct application. The efficiency of gene suppression was determined using quantitative reverse transcription-polymerase chain reaction 24-72 h after siRNA delivery. Cytotoxicity was assessed using histological analysis. Expression of rat GAPDH in the parotid glands was silenced 48 h after siRNA was delivered by ultrasound (frequency: 1 MHz; intensity: 2 W cm(-2); exposure time: 2 min). High-intensity ultrasound induced tissue damage and apoptotic change. Echo-enhanced microbubbles significantly improved siRNA-induced gene silencing by 10-50%. Compared with transdermal application, direct-exposure ultrasound was only slightly effective, and no significant difference in gene expression was observed. The results indicate that microbubble-enhanced sonoporation can yield in vivo siRNA gene silencing in the rat parotid gland. This technique could be applied to provide gene knockdown organs for functional genomic analyses and to develop siRNA-based gene therapy.

Inhibition of HIV-1 replication by long-term treatment with a chimeric RNA containing shRNA and TAR decoy RNA

Combinatorial therapies for the treatment of HIV-1 infection are effective for reducing patient viral loads and slowing the progression to AIDS. Our strategy was based on an anti-HIV-1 shRNA vector system in which HIV-1 vif-shRNA was fused to a decoy TAR RNA (mini-TAR RNA) to generate vif-shRNA-decoy TAR RNA under the control of the human U6 Pol III promoter. Upon expression in human cells, the RNA molecule was cleaved into its component parts, which inhibited HIV-1 replication in a synergistic manner. This chimeric RNA expressed a dual RNA moiety and greatly enhanced the inhibition of HIV-1 replication under the production of resistant virus by short interference RNA (siRNA) in long-term culture assays. We suggest that this technique provides a practical basis for the application of siRNA-based gene therapy in the treatment of HIV/AIDS.