Delivery Of Short Hairpin RNA Research Articles

Pharmacological Inhibition of Histone Deacetylases 1 and 2 (HDAC1/2) Induces Fetal Hemoglobin (HbF) through Activation of Gata2

Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease, and could also be effective in treating beta-thalassemia. Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of the fetal beta-like globin gene (HbG) transcript (Bradner JE, Proc Natl Acad Sci, 2010). We have previously shown that selective chemical inhibitors of HDAC1/2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). In this work, we have utilized our proof of concept molecule ACY-957, a selective inhibitor of HDAC1/2, to discover a novel role for Gata2 in the activation of HbG.To identify genes affected by HDAC1/2 inhibition, CD34+ bone marrow cells undergoing erythroid differentiation were treated with ACY-957 or vehicle, followed by mRNA expression profiling. Among the genes differentially regulated by both pharmacological inhibition and genetic ablation of HDAC1/2 were Bcl11a and Sox6, known HbG repressors, and Gata2, a potential HbG activator. Quantitative real time PCR (QRT-PCR) time course experiments confirmed that ACY-957 treatment leads to a 2-fold and 10-fold decrease in Bcl11A and Sox6, respectively, and an 8-fold increase in Gata2 mRNA. Unlike Bcl11a and Sox6, Gata2 induction by ACY-957 was highly correlated with HbG induction, suggesting a possible role for this transcription factor in the direct activation of HbG.To investigate this possibility, lentiviral infection was utilized to overexpress full length Gata2 transcript in differentiating primary erythroblasts. After 5 days of differentiation, Gata2 overexpression resulted in a 2.5-fold increase in HbG mRNA, while the level of the major adult beta-like globin chain (HbB) mRNA was unaffected. HbG mRNA remained elevated by Gata2 overexpression at day 7 of differentiation, while HbB was reduced by 1.6-fold. Gata2 overexpression appeared to have minimal effect on cell differentiation, as determined by the cell surface markers CD71 and GlycophorinA, a finding consistent with observations in ACY-957 treated cells with elevated Gata2. Furthermore, lentiviral delivery of short hairpin RNA (shRNA) targeting Gata2, attenuated HbG induction by ACY-957. These data suggest that elevated levels of Gata2 resulting from HDAC1/2 inhibition is sufficient to induce HbG at early stages of erythroid cell differentiation.To understand how HDAC1/2 inhibition drives Gata2 activation, chromatin immunoprecipitation coupled with either next generation sequencing (ChIP-seq) or QRT-PCR was performed in ACY-957 and vehicle treated cells. HDAC1 and HDAC2 were present throughout the Gata2 gene body and promoter regions, and HDAC1/2 binding levels were highly correlated, suggesting co-occupancy of these enzymes at this locus. ACY-957 treatment led to elevated histone acetylation at previously described Gata2 gene regulatory regions (Bresnick et. al. 2010, J Biol Chem). Specifically, the -1.8 kb and -2.8 kb regulatory regions showed a 6-fold increase in histone H3K9 and H2BK5 acetylation, while the +9.5 kb and -3.9 kb regions showed a 3-fold increase. The Gata2 protein showed increased binding at these regulatory regions in response to ACY-957 treatment, with a maximum increase of 3-fold at the -1.8 kb region. This finding is consistent with the known positive autoregulation of the Gata2 gene. Taken together, these data suggest that selective inhibition of HDAC1/2 leads to elevated Gata2 through acetylation-induced activation of a positive autoregulatory loop.The tight temporal correlation between Gata2 and HbG activation following HDAC1/2 inhibition argues that Gata2 may affect the beta-globin locus directly. ChIP-seq data across the 70-kb beta-globin locus demonstrated that ACY-957 treatment altered Gata2 binding only at a single region, lying within the promoter for delta globin. This region is suspected in playing a role in switching from fetal to adult globin during development, as naturally occurring deletions of this region are associated with elevated fetal hemoglobin in adults (Sankaran et. al. 2011, NEJM). Whether the change in GATA2 binding to this region is responsible for the increased expression of HbG in cells treated with HDAC1/2-selective inhibitors is under investigation. DisclosuresShearstone:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Golonzhka:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

Knockdown of CRF1 receptors in the ventral tegmental area attenuates cue- and acute food deprivation stress-induced cocaine seeking in mice.

Corticotrophin-releasing factor (CRF) modulates the influence of stress on cocaine reward and reward seeking acting at multiple sites, including the ventral tegmental area (VTA). There is controversy, however, concerning the contribution of CRF receptor type 1 (CRFR1) to this effect and whether CRF within the VTA is involved in other aspects of reward seeking independent of acute stress. Here we examine the role of CRFR1 within the VTA in relation to cocaine and natural reward using viral delivery of short hairpin RNAs (lenti-shCRFR1) and investigate the effect on operant self-administration and motivation to self-administer, as well as stress- and cue-induced reward seeking in mice. While knockdown of CRFR1 in the VTA had no effect on self-administration behavior for either cocaine or sucrose, it effectively blocked acute food deprivation stress-induced reinstatement of cocaine seeking. We also observed reduced cue-induced cocaine seeking assessed in a single extinction session after extended abstinence, but cue-induced sucrose seeking was unaffected, suggesting dissociation between the contribution of CRFR1 in the VTA in cocaine reward and sucrose and cocaine seeking. Further, our data indicate a role for VTA CRFR1 signaling in cocaine seeking associated with, and independent of, stress potentially involving conditioning and/or salience attribution of cocaine reward-related cues. CRFR1 signaling in the VTA therefore presents a target for convergent effects of both cue- and stress-induced cocaine-seeking pathways.

A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca2+ Uptake into the Sarcoplasmic Reticulum

In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

Inhibition of human glioma U251 cells growth in vitro and in vivo by hydroxyapatite nanoparticle-assisted delivery of short hairpin RNAs against SATB1

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be over-expressed in many human tumors and knockdown of SATB1 can inhibit tumor growth. The present study was designed to determine the role of SATB1 in the growth of human glioma U251 cells using the plasmid-based SATB1 short hairpin RNA (shRNA) delivered by hydroxyapatite nanoparticles in vitro and in vivo. The in vitro growth, invasion and angiogenesis assays of human glioma U251 cells were done. U251 cells tumor blocks were transplanted into the nude mice. CaCl2-modified hydroxyapatite nanoparticles carrying shRNA-SATB1 plasmids were injected into the tumors. The apoptosis of the tumor U251 cells was examined with TUNEL assay and flow cytometer (FCM). The tumor growth and immunohistochemistry were measured. The expression level of SATB1 mRNA was investigated by RT-PCR. The expression levels of SATB1, Cyclin D1, MMP-2, VEGF, Bax and Caspase-9 protein were determined by western blot analysis. The results showed that hydroxyapatite nanoparticles-delivered shRNA-SATB1 could significantly inhibit the growth, invasion and angiogenesis of U251 cells in vitro and the growth of U251 cells in vivo. FCM results showed that Nano HAP-shRNA-SATB1-induced apoptosis (up to 67.8%). SATB1 expression was strongly down-regulated in the tumor U251 cells. Cyclin D1, MMP-2 and VEGF were also down-regulated in the tumor tissues that also displayed significant increased in Bax expression and Caspase-9 activity. These results show that Nano HAP-shRNA-SATB1 can inhibit the growth of human glioma U251 cells in vitro and in vivo, and hydroxyapatite nanoparticles can be used for the in vitro and in vivo delivery of plasmid-based shRNAs into U251 cells.

Kir6.2 limits Ca2+ overload and mitochondrial oscillations of ventricular myocytes in response to metabolic stress

ATP-sensitive K(+) (KATP) channels are abundant membrane proteins in cardiac myocytes that are directly gated by intracellular ATP and form a signaling complex with metabolic enzymes, such as creatine kinase. KATP channels are known to be essential for adaption to cardiac stress, such as ischemia; however, how all the molecular components of the stress response interact is not fully understood. We examined the effects of decreasing the KATP current density on Ca(2+) and mitochondrial homeostasis and ischemic preconditioning. Acute knockdown of the pore-forming subunit, Kir6.2, was achieved using adenoviral delivery of short hairpin RNA targeted to Kir6.2. The acute nature of the knockdown of Kir6.2 accurately shows the effects of Kir6.2 depletion without any compensatory effects that may arise in transgenic studies. We also investigated the effect of reducing the KATP current while maintaining KATP channel protein in the sarcolemmal membrane using a nonconducting Kir6.2 construct. Only 50% KATP current remained after Kir6.2 knockdown, yet there were profound effects on myocyte responses to metabolic stress. Kir6.2 was essential for cardiac myocyte Ca(2+) homeostasis under both baseline conditions before any metabolic stress and after metabolic stress. Expression of nonconducting Kir6.2 also resulted in increased Ca(2+) overload, showing the importance of K(+) conductance in the protective response. Both ischemic preconditioning and protection during ischemia were lost when Kir6.2 was knocked down. KATP current density was also important for the mitochondrial membrane potential at rest and prevented mitochondrial membrane potential oscillations during oxidative stress. KATP channel density is important for adaption to metabolic stress.

Abstract 3122: HMGA1 reprograms breast cancer cells by inducing transcriptional networks involved in an epithelial-mesenchymal transition, stemness, and metastatic progression.

Abstract Despite advances in our ability to detect and treat breast cancer, it remains a leading cause of death in women with cancer worldwide, and the incidence is rising. Approximately 15-20% of all cases are classified as triple negative breast cancer (TNBC), a subtype that is frequently associated with rapid progression and poor outcome. TNBC refers to the lack of detectable markers for the estrogen receptor (ER), progesterone receptor (PR), and Her2/neu amplification. These tumors do not respond to our most effective and least toxic therapies, including hormonal therapy (tamoxifen) or trastuzumab. We are studying molecular pathways that lead to tumor progression in TNBC and can be targeted with novel therapies. Our focus is the high mobility group A1 (HMGA1) oncogene. HMGA1 is highly expressed during embryogenesis, with low or undetectable levels in differentiated, adult tissues. HMGA1 is enriched in virtually all high-grade (poorly differentiated) cancers studied to date, including TNBCs, and high expression portends a poor prognosis in breast and other cancers. To investigate the role of HMGA1 in tumor progression in breast cancer, we silenced HMGA1 expression in TNBC cell lines (MDA-MB-231, Hs578T) using lentiviral-mediated delivery of short hairpin RNA. Strikingly, proliferation was markedly impaired, and many cells underwent apoptotic cell death within 5 days following HMGA1 knock-down. Surprisingly, cell morphology also changed dramatically, whereby the fibroblast-like, spindle-shaped cells became cuboidal and epithelial-like, consistent with a mesenchymal-epithelial transition, or MET. E-CADHERIN mRNA was induced, while both SNAIL and VIMENTIN were repressed in the knock-down cells, also consistent with MET. In addition, silencing HMGA1 blocked migration, invasion, and the formation of tumor foci in the lungs following tail vein injection of MDA-MB-231 cells. Moreover, both primary tumorigenesis and metastatic progression to the lungs were markedly inhibited in MDA-MB-231 cells with knock-down of HMGA1 following implantation in mammary fat pads. Furthermore, silencing HMGA1 blocked primary and secondary mammosphere formation, indicating that HMGA1 is required for this stem cell property. Tumorigenesis experiments at limiting dilutions showed that silencing HMGA1 depletes the tumor initiator/cancer stem cell pool. Using global gene expression analysis, we identified an HMGA1 signature of differentially-regulated genes in the control cells compared to knock-down cells. We also found that the HMGA1 signature is highly enriched in embryonic stem cells. Together, these findings indicate that silencing HMGA1 reprograms invasive, mesenchymal TNBCs into non-invasive, epithelial-like cells with slower growth and an altered gene expression signature. Studies are now needed to determine how to target HMGA1 in therapy. Citation Format: Sandeep N. Shah, Leslie Cope, Amy Belton, Weijie Poh, Saraswati Sukumar, David Huso, Linda Resar. HMGA1 reprograms breast cancer cells by inducing transcriptional networks involved in an epithelial-mesenchymal transition, stemness, and metastatic progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3122. doi:10.1158/1538-7445.AM2013-3122

Modulation of μ-opioid receptor signaling by RGS19 in SH-SY5Y cells.

Regulator of G-protein signaling protein 19 (RGS19), also known as Gα-interacting protein (GAIP), acts as a GTPase accelerating protein for Gαz as well as Gαi/o subunits. Interactions with GAIP-interacting protein N-terminus and GAIP-interacting protein C-terminus (GIPC) link RGS19 to a variety of intracellular proteins. Here we show that RGS19 is abundantly expressed in human neuroblastoma SH-SY5Y cells that also express µ- and δ- opioid receptors (MORs and DORs, respectively) and nociceptin receptors (NOPRs). Lentiviral delivery of short hairpin RNA specifically targeted to RGS19 reduced RGS19 protein levels by 69%, with a similar reduction in GIPC. In RGS19-depleted cells, there was an increase in the ability of MOR (morphine) but not of DOR [(4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80)] or NOPR (nociceptin) agonists to inhibit forskolin-stimulated adenylyl cyclase and increase mitogen-activated protein kinase (MAPK) activity. Overnight treatment with either MOR [D-Ala, N-Me-Phe, Gly-ol(5)-enkephalin (DAMGO) or morphine] or DOR (D-Pen(5)-enkephalin or SNC80) agonists increased RGS19 and GIPC protein levels in a time- and concentration-dependent manner. The MOR-induced increase in RGS19 protein was prevented by pretreatment with pertussis toxin or the opioid antagonist naloxone. Protein kinase C (PKC) activation alone increased the level of RGS19 and inhibitors of PKC 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile and mitogen-activated protein kinase kinase 1 2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one, but not protein kinase A (H89), completely blocked DAMGO-induced RGS19 protein accumulation. The findings show that RGS19 and GIPC are jointly regulated, that RGS19 is a GTPase accelerating protein for MOR with selectivity over DOR and NOPR, and that chronic MOR or DOR agonist treatment increases RGS19 levels by a PKC and the MAPK pathway-dependent mechanism.

Cystatin M/E knockdown by lentiviral delivery of shRNA impairs epidermal morphogenesis of human skin equivalents

The protease inhibitor cystatin M/E (CST6) regulates a biochemical pathway involved in stratum corneum homeostasis, and its deficiency in mice causes ichthyosis and neonatal lethality. Cystatin M/E deficiency has not been described in humans so far, and we did not detect disease-causing mutations in the CST6 gene in a large number of patients with autosomal recessive congenital ichthyosis, who were negative for mutations in known ichthyosis-associated genes. To investigate the phenotype of CST6 deficiency in human epidermis, we used lentiviral delivery of short hairpin RNAs that target CST6 in a 3D reconstructed skin model. Surprisingly, CST6 deficiency did not cause an ichthyosis-like phenotype, but prevented the development of a multilayered epidermis. From this study, we conclude that CST6 deficiency may be incompatible with normal human foetal development.

Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord

In this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI). Short hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. Animals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment. The improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI.

Blockade of NFκB activity by Sunitinib increases cell death in Bortezomib-treated endometrial carcinoma cells

Endometrial carcinoma is one of the most common malignancies in the female genital tract, usually treated by surgery and radiotherapy. Chemotherapy is used when endometrial carcinoma is associated with widespread metastasis or when the tumor recurs after radiation therapy. In the present study, we demonstrate that the tyrosine kinase receptor inhibitor Sunitinib reduces cell viability, proliferation, clonogenicity and induces apoptotic cell death in endometrial carcinoma cell lines, which is not due to its action through the most known targets like VEGFR, nor through EGFR as demonstrated in this work. Interestingly, Sunitinib reduces NFκB transcriptional activity either at basal level or activation by EGF or TNF-α. We observed that Sunitinib was able to inhibit the Bortezomib-induced NFκB transcriptional activity which correlates with a decrease of the phosphorylated levels of IKKα and β, p65 and IκBα. We evaluated the nature of the interaction between Sunitinib and Bortezomib by the dose effect method and identified a synergistic effect (combination index < 1). Analogously, silencing of p65 expression by lentiviral-mediated short-hairpin RNA delivery in Bortezomib treated cells leads to a strongly increased sensitivity to Bortezomib apoptotic cell death. Altogether our results suggest that the combination of Sunitinib and Bortezomib could be considered a promising treatment for endometrial carcinoma after failure of surgery and radiation.

Senescence‐escape in melanoma

Cellular senescence is an important defense against uncontrolled cellular replication and is a critical tumor suppressor mechanism. Although initially described as an in vitro phenomenon, in recent years, several lines of evidence have indicated that senescence also occurs in vivo. Disparate cellular stresses can provoke senescence including telomere shortening, activation of certain oncogenes, oxidative stress and stalled replication forks. These stresses are best known to engage the p16INK4a-RB and ARF-p53 pathways to effect growth arrest and prevent neoplasia. Among these, ‘oncogene-induced senescence’ (OIS) is particularly known for the prevention of melanoma, resulting from the expression of mutant B-RAF or N-RAS in this disease.

In Vitro and Ex Vivo Delivery of Short Hairpin RNAs for Control of Hepatitis C Viral Transcript Expression

Recurrent hepatitis C virus (HCV) infection is the most common cause of graft loss and patient death after transplantation for HCV cirrhosis. Transplant surgeons have access to uninfected explanted livers before transplantation and an opportunity to deliver RNA interference-based protective gene therapy to uninfected grafts. Conserved HCV sequences were used to design short interfering RNAs and test their ability to knockdown HCV transcript expression in an in vitro model, both by transfection and when delivered via an adeno-associated viral vector. In a rodent model of liver transplantation, portal venous perfusion of explanted grafts with an adeno-associated viral vector before transplantation produced detectable short hairpin RNA transcript expression after transplantation. The ability to deliver anti-HCV short hairpin RNAs to uninfected livers before transplantation and subsequent exposure to HCV offers hope for the possibility of preventing the currently inevitable subsequent infection of liver grafts with HCV.

Small interfering RNA-mediated knockdown of NF-κBp65 attenuates neuropathic pain following peripheral nerve injury in rats

Recent reports show that the nuclear factor-κB (NF-κB) can control numerous genes encoding inflammatory and nociceptive mediators and play an important role in the development of central pain sensitization. The aim of the present study is to assess the role of NF-κB signal pathway and its downstream pro-inflammatory cytokines in the modulation of neuropathic pain, by using small interfering RNAs (siRNAs) technique, which has been shown to result in potent, long-lasting post-transcriptional silencing of specific genes. We developed a highly efficient method of lentivirus-mediated delivery of short-hairpin RNA (shRNA) targeting NF-κBp65 for gene silencing. This method successfully transduced LV-shNF-κBp65 into cultured spinal cord neurons in vitro and spinal cord cells in vivo, inhibited the expression of NF-κBp65 and pro-inflammatory factors (TNF-α, IL-1β and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia for more than 4weeks in chronic constriction injury (CCI) model of rats. Taken together, our results suggest that siRNA against NF-κBp65 is a potential strategy for analgesia. Furthermore, the lentiviral vector derived shRNA approach shows a great promise for the management of neuropathic pain and the study of functional NF-κBp65 gene expression.

Targeted Gene-Silencing Reveals the Functional Significance of Myocardin Signaling in the Failing Heart

BackgroundMyocardin (MYOCD), a potent transcriptional coactivator of smooth muscle (SM) and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF). However, the molecular and functional consequences of myocd upregulation in HF are still unclear.Methodology/Principal FindingsThe goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox)-induced diastolic HF (DHF) model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV) myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1) a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2) amelioration of impaired diastolic dysfunction, and (3) higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery) led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function.Conclusions/SignificanceThese data provide the first evidence that a moderate inhibition (e.g., normalization) of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.

Skin delivery of short hairpin RNA of indoleamine 2,3 dioxygenase induces antitumor immunity against orthotopic and metastatic liver cancer

Liver cancer is one of the most malignant cancers in the world and has a high rate of metastasis. Therefore, development of a novel therapy for liver cancer is a critical issue. Indoleamine 2,3-dioxygenase (IDO) is known as a negative immune regulator in dendritic cells. Our previous study demonstrated that skin delivery of IDO short hairpin RNA (shRNA) induced antitumor immunity in subcutaneous bladder and colon tumor models. Because the immunological environment is quite different between skin and liver, it is essential to evaluate whether skin delivery of IDO shRNA is an effective treatment in metastatic and orthotopic animal tumor models. In the present study, IDO shRNA inhibited tumor growth in subcutaneous, metastatic and orthotopic liver tumor models. The cytotoxicity of splenocytes was significantly elevated in mice treated with IDO shRNA in the orthotopic and metastatic tumor models. Interleukin (IL)-12 and interferon (IFN)-gamma mRNA expression were upregulated while IL-10 was downregulated in the inguinal lymph nodes, which were collected from IDO shRNA-treated mice. Similar results were observed in the spleens of mice inoculated with IDO shRNA by gene gun. The results indicate that skin administration of IDO shRNA is an effective therapy in orthotopic and metastatic liver cancer animal models. Indoleamine 2,3-dioxygenase shRNA might be a potential new treatment for liver cancer in the future.

Inhibition of xenograft tumor growth in mice by gold nanoparticle-assisted delivery of short hairpin RNAs against Mcl-1L

A prerequisite for the therapeutic use of small RNAs is the development of a method that can deliver them into animals. Previous studies have shown the capability of functionalized gold nanoparticles to serve as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNAs (shRNAs) into cultured human cells. Here, we report the ability of the gold nanoparticle-assisted gene delivery system (AuNP-GDS) to deliver shRNA to a xenograft tumor in a mouse model. AuNP-GDS delivery of in vitro synthesized shRNA targeted to the Mcl-1L gene knocked down levels of Mcl-1L mRNA and protein by ~36% and ~26%, respectively, which were sufficient to induce apoptosis of the xenograft tumor cells and consequently inhibited the development of the tumor. We demonstrated that our lego-like AuNP-GDS, which can easily load and deliver shRNAs targeted to any gene of interest into living systems, can deliver shRNAs into xenograft tumors, leading to antitumor activity in an animal model.

RNA interference-produced autoregulation of inducible nitric oxide synthase expression

Vector-mediated delivery of short-hairpin RNA (shRNA) to regulate gene expression holds a great therapeutic promise. We hypothesize that gene expression can be autoregulated with RNA interference. We used inducible nitric oxide synthase (iNOS) as a gene model to test this hypothesis. Lipopolysaccharide dose-dependently increased iNOS in rat aortic smooth muscle cells and the nitrite production from these cells. These increases were attenuated in cells transfected with plasmids containing code for iNOS shRNA whose expression was controlled by an iNOS promoter. The production of shRNA was lipopolysaccharide dose-dependent. The lipopolysaccharide-induced iNOS expression in rat C6 glioma cells also was attenuated by transfection with plasmids containing the iNOS shRNA code. These results provide proof-of-concept evidence for using RNA interference technique to achieve autoregulation of gene expression.

Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway

Despite the use of multiple therapeutic strategies, metastatic melanoma remains a challenge for oncologists. Thus, new approaches using combinational treatment may be used to try to improve the prognosis of this disease. In this report, we have analyzed the expression of receptor tyrosine kinases (RTKs) in melanoma specimens and in four metastatic melanoma cell lines. Both melanoma specimens and cell lines expressed RTKs, suggesting that they may represent eventual targets for multitargeted tyrosine kinase inhibitor, Suntinib. Sunitinib reduced the proliferation of two melanoma cell lines (M16 and M17) and increased apoptosis in one of them (M16). Moreover, the two metastatic melanoma cell lines harbored an activated receptor (PDGFRα and VEGFR, respectively), and Sunitinib suppressed the phosphorylation of the RTKs and their downstream targets Akt and ribosomal protein S6, in these two cell lines. Similar results were obtained when either PDGFRα or VEGFR2 expression was silenced by lentiviral-mediated short-hairpin RNA delivery in M16 and M17, respectively. To evaluate the interaction between Sunitinib and Bortezomib, median dose effect analysis using MTT assay was performed, and combination index was calculated. Bortezomib synergistically enhanced the Sunitinib-induced growth arrest in Sunitinib-sensitive cells (combination index < 1). Moreover, LY294002, a PI3K inhibitor, sensitized melanoma cells to Bortezomib treatment, suggesting that downregulation of phospho-Akt by Sunitinib mediates the synergy obtained by Bortezomib + Sunitinib cotreatment. Altogether, our results suggest that melanoma cells harboring an activated RTK may be clinically responsive to pharmacologic RTK inhibition by Sunitinib, and a strategy combining Sunitinib and Bortezomib, may provide therapeutic benefit.

Tumor-targeting magnetic lipoplex delivery of short hairpin RNA suppresses IGF-1R overexpression of lung adenocarcinoma A549 cells in vitro and in vivo

Liposomal magnetofection potentiates gene transfection by applying a magnetic field to concentrate magnetic lipoplexes onto target cells. Magnetic lipoplexes are self-assembling ternary complexes of cationic lipids with plasmid DNA associated with superparamagnetic iron oxide nanoparticles (SPIONs). Type1insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently overexpressed in lung cancer and mediates cancer cell proliferation and tumor growth. In this study, we evaluated the transfection efficiency (percentage of transfected cells) and therapeutic potential (potency of IGF-1R knockdown) of liposomal magnetofection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) in A549 cells and in tumor-bearing mice as compared to lipofection using Lipofectamine 2000. Liposomal magnetofection provided a threefold improvement in transgene expression over lipofection and transfected up to 64.1% of A549 cells in vitro. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1 ± 6% and by liposomal magnetofection by 85.1 ± 3%. In vivo delivery efficiency of the pGFPshIGF-1R plasmid into the tumor was significantly higher in the liposomal magnetofection group than in the lipofection group. In vivo IGF-1R specific-shRNA by lipofection inhibited IGF-1R protein by an average of 43.8 ± 5.3%; that by liposomal magnetofection inhibited IGF-1R protein by 43.4 ± 5.7%, 56.3 ± 9.6%, and 72.2 ± 6.8%, at 24, 48, and 72 h, respectively, after pGFPshIGF-1R injection. Our findings indicate that liposomal magnetofection may be a promising method that allows the targeting of gene therapy to lung cancer.

Intrathecal Lentiviral-Mediated RNA Interference Targeting PKCγ Attenuates Chronic Constriction Injury–Induced Neuropathic Pain in Rats

In the spinal cord, protein kinase C isoform γ (PKCγ) plays an important role in the development of central pain sensitization. However, there are currently no specific PKCγ inhibitors available. Therefore, the aim of the present study was to assess the role of PKCγ in the modulation of pain using a more selective experimental tool. Although small interfering RNAs have been used to silence genes in neurons, in vivo delivery of RNA interference (RNAi) remains a major challenge, thus limiting its applications. Here we developed a highly efficient method of lentiviral-mediated delivery of short-hairpin RNAs targeting PKCγ for in vivo gene silencing in the spinal cord of rats. This method decreased the expression of PKCγ mRNA and protein, and additionally attenuated chronic constriction injury-induced mechanical allodynia and thermal hyperalgesia for more than 6 weeks. Our study suggests that PKCγ is a potential RNAi target for neuropathic pain. Furthermore, the lentiviral vector delivery strategy holds great promise as a novel approach for the treatment of neuropathic pain and study of PKCγ gene function.