Delivery of Antisense DNA into Pathogenic Parasite Trypanosoma cruzi Using Virus-Like Protein-Based Nanoparticles.

Trypanosoma cruzi, which causes Chagas disease, is one of the most lacerating parasites in terms of health and social impacts. New approaches for its study and treatment are urgently needed since in more than 50 years only two drugs have been approved. Genetic approaches based on antisense oligonucleotides (AONs) are promising; however, to harness their full potential the development of effective carriers is paramount. Here, we report the use of an engineered virus-like protein C-BK12 to transfect AONs into T. cruzi. Using gel electrophoresis, Dynamic Light Scattering, and atomic force microscopy, we found that C-BK12 binds AONs and forms 10-25 nm nanoparticles (NPs), which are very stable when incubated in biological media, only releasing up to 25% of AON. Fluorescence microscopy and qPCR revealed that the NPs successfully delivered AONs into epimastigotes and reduced the expression of a target gene down to 68%. Importantly, the protein did not show cytotoxicity. The combination of high stability and capability to transfect and knock down gene expression without causing cell damage and death makes the protein C-BK12 a promising starting point for the further development of safe and effective carriers to deliver AONs into T. cruzi for biological studies.

AdeB efflux pump gene knockdown by mRNA mediated peptide nucleic acid in multidrug resistance Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii isolates cause critical problems in health-care environments. AdeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically essential antibiotics in A. baumannii, such as ciprofloxacin. This study aimed to target adeB gene with antisense peptide nucleic acid (PNA) and investigate its effect on resistance to antibiotics. NCBI database was used to design appropriate PNA to target adeB gene, by connecting PNA to mRNA, the translation of mRNA can be prevented. Three clinical isolates and A. baumannii ATCC 17978 were treated with the designed PNA by electroporation and competence procedure. Minimum Inhibitory concentration (MIC) of ciprofloxacin, colistin, and tetracycline were determined by microbroth dilution method. In addition, the expression level of adeB gene was measured by quantitative real-time PCR (qRT-PCR). Isolates used in this study had mutations in gyrA and parC genes corresponding to resistance to ciprofloxacin. MIC of resistance to ciprofloxacin after treatment with PNA was reduced from 32 µg/ml to16 µg/ml in A. baumannii ATCC 17978 isolate. Susceptibility level of tetracycline, in the 2 clinical isolates was decreased from 64 µg/ml to 32 µg/ml and in the other isolate was reduced from 128 µg/ml to 64 µg/ml. The expression level of adeB gene was decreased in A. baumannii ATCC 17978 (P > 0.01) but not in clinical isolate (P = 0.107). Findings of the present study indicate overexpression of adeB efflux pump has extra effect on resistance to antibiotics in isolates with a defined mechanism of resistance. Antisense technology is a feasible technique to suppress the function of these genes, which may be further exploited to control multidrug-resistant isolates.

Lowering Mutant Huntingtin Using Tricyclo-DNA Antisense Oligonucleotides As a Therapeutic Approach for Huntington's Disease.

Huntington's disease is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of huntingtin gene (HTT) encoding for a toxic polyglutamine protein. This disease is characterized by motor, psychiatric, and cognitive impairments. Currently, there is no disease modifying treatment. However, reducing the expression of the huntingtin protein (HTT) using antisense oligonucleotides (ASOs) has been shown as a promising therapeutic strategy. In this study, we explore the therapeutic potential of ASO made of tricyclo-DNA (tcDNA), a conformationally constrained DNA analog, to silence HTT. We used a gapmer ASO, containing central DNA nucleotides flanked by tcDNA modifications on 5' and 3' ends, allowing the recruitment of RNAse H and subsequent degradation of the messenger RNA. After transfection of tcDNA-ASO in patient-derived fibroblast cell lines, we show a strong decrease of HTT mRNA and protein levels. As a control, 2'O-methyl-RNA targeting the same region of HTT was also tested and did not induce a significant effect. tcDNA-ASO were also evaluated in vivo in the YAC128 mice, containing the full-length human HTT gene with 128 CAG repeat expansion. Single intracerebroventricular (ICV) injections of tcDNA induce a significant decrease of HTT messenger and protein levels in the cortex, hippocampus, striatum, and cerebellum of treated mice. tcDNA-ASO were found well distributed in the central nervous system (CNS) and show long lasting effect with protein levels still low, 12 weeks after a single ICV injection. This proof of concept study suggests the therapeutic potential of gapmer tcDNA ASO to downregulate huntingtin in vitro and in vivo.

Complex consisting of antisense DNA and ß-glucan promotes internalization into cell through Dectin-1 and hybridizes with target mRNA in cytosol.

Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the ß-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.

Inhibiting Methicillin-Resistant Staphylococcus aureus by Tetrahedral DNA Nanostructure-Enabled Antisense Peptide Nucleic Acid Delivery.

One of the biggest obstacles for the use of antisense oligonucleotides as antibacterial therapeutics is their limited uptake by bacterial cells without a suitable carrier, especially in multi-drug-resistant bacteria with a drug efflux mechanism. Existing vectors, such as cell-penetrating peptides, are inefficient and nontargeting, and accordingly are not ideal carriers. A noncytotoxic tetrahedral DNA nanostructure (TDN) with a controllable conformation has been developed as a delivery vehicle for antisense oligonucleotides. In this study, antisense peptide nucleic acids (asPNAs) targeting a specific gene ( ftsZ) were efficiently transported into methicillin-resistant Staphylococcus aureus cells by TDNs, and the expression of ftsZ was successfully inhibited in an asPNA-concentration-dependent manner. The delivery system specifically targeted the intended gene. This novel delivery system provides a better platform for future applications of antisense antibacterial therapeutics and provides a basis for the development of a new type of antibacterial drug for multi-drug-resistant bacterial infections.

Chronic Sleep Restriction Induces Cognitive Deficits and Cortical Beta-Amyloid Deposition in Mice via BACE1-Antisense Activation.

AIMS: To clarify the correlation between chronic sleep restriction (CSR) and sporadic Alzheimer disease (AD), we determined in wild-type mice the impact of CSR, on cognitive performance, beta-amyloid (Aß) peptides, and its feed-forward regulators regarding AD pathogenesis. METHODS: Sixteen nine-month-old C57BL/6 male mice were equally divided into the CSR and control groups. CSR was achieved by application of a slowly rotating drum for 2 months. The Morris water maze test was used to assess cognitive impairment. The concentrations of Aß peptides, amyloid precursor protein (APP) and ß-secretase 1 (BACE1), and the mRNA levels of BACE1 and BACE1-antisense (BACE1-AS) were measured. RESULTS: Following CSR, impairments of spatial learning and memory consolidation were observed in the mice, accompanied by Aß plaque deposition and an increased Aß concentration in the prefrontal and temporal lobe cortex. CSR also upregulated the ß-secretase-induced cleavage of APP by increasing the protein and mRNA levels of BACE1, particularly the BACE1-AS. CONCLUSIONS: This study shows that a CSR accelerates AD pathogenesis in wild-type mice. An upregulation of the BACE1 pathway appears to participate in both cortical Aß plaque deposition and memory impairment caused by CSR. BACE1-AS is likely activated to initiate a cascade of events that lead to AD pathogenesis. Our study provides, therefore, a molecular mechanism that links CSR to sporadic AD.

Phosphodiesterase-7 inhibition affects accumbal and hypothalamic thyrotropin-releasing hormone expression, feeding and anxiety behavior of rats.

Thyrotropin-releasing hormone (TRH) has anorexigenic and anxiolytic functions when injected intraventricularly. Nucleus accumbens (NAcc) is a possible brain region involved, since it expresses proTRH. TRH from hypothalamic paraventricular nucleus (PVN) has a food intake-regulating role. TRHergic pathways of NAcc and PVN are implicated in anxiety and feeding. Both behaviors depend on cAMP and phosphorylated-cAMP response element binding protein (pCREB) intracellular levels. Intracellular levels of cAMP are controlled by the degrading activity of phosphodiesterases (PDEs). Since TRH transcription is activated by pCREB, a specific inhibitor of PDE7B may regulate TRH-induced effects on anxiety and feeding. We evaluated the effectiveness of an intra-accumbal and intraperitoneal (i.p.) administration of a PDE7 inhibitor (BRL-50481) on rats' anxiety-like behavior and food intake; also on TRH mRNA and protein expression in NAcc and PVN to define its mediating role on the PDE7 inhibitor-induced behavioral changes. Accumbal injection of 4µg/0.3µL of PDE7 inhibitor decreased rats' anxiety. The i.p. injection of 0.2mg/kg of the inhibitor was able to increase the PVN TRH mRNA expression and to decrease feeding but did not change animals' anxiety levels; in contrast, 2mg/kg b.w inhibitor enhanced accumbal TRH mRNA, induced anxiolysis with no change in food intake. PDE7 inhibitor induced anxiolytic and anorexigenic like behavior depending on the dose used. Results supported hypothalamic TRH mediated feeding-reduction effects, and accumbal TRH mediation of inhibitor-induced anxiolysis. Thus, an i.p dose of this inhibitor might be reducing anxiety with no change in feeding, which could be useful for obese patients.

The Drosophila Receptor Protein Tyrosine Phosphatase LAR Is Required for Development of Circadian Pacemaker Neuron Processes That Support Rhythmic Activity in Constant Darkness But Not during Light/Dark Cycles.

InDrosophila, a transcriptional feedback loop that is activated by CLOCK-CYCLE (CLK-CYC) complexes and repressed by PERIOD-TIMELESS (PER-TIM) complexes keeps circadian time. The timing of CLK-CYC activation and PER-TIM repression is regulated post-translationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Although kinases that control PER, TIM, and CLK levels, activity, and/or subcellular localization have been identified, less is known about phosphatases that control clock protein dephosphorylation. To identify clock-relevant phosphatases, clock-cell-specific RNAi knockdowns ofDrosophilaphosphatases were screened for altered activity rhythms. One phosphatase that was identified, the receptor protein tyrosine phosphatase leukocyte-antigen-related (LAR), abolished activity rhythms in constant darkness (DD) without disrupting the timekeeping mechanism in brain pacemaker neurons. However, expression of the neuropeptide pigment-dispersing factor (PDF), which mediates pacemaker neuron synchrony and output, is eliminated in the dorsal projections from small ventral lateral (sLNv) pacemaker neurons whenLarexpression is knocked down during development, but not in adults. Loss ofLarfunction eliminates sLNvdorsal projections, but PDF expression persists in sLNvand large ventral lateral neuron cell bodies and their remaining projections. In contrast to the defects in lights-on and lights-off anticipatory activity seen in flies that lack PDF,LarRNAi knockdown flies anticipate the lights-on and lights-off transition normally. Our results demonstrate thatLaris required for sLNvdorsal projection development and suggest that PDF expression in LNvcell bodies and their remaining projections mediate anticipation of the lights-on and lights-off transitions during a light/dark cycle. SIGNIFICANCE STATEMENT: In animals, circadian clocks drive daily rhythms in physiology, metabolism, and behavior via transcriptional feedback loops. Because key circadian transcriptional activators and repressors are regulated by phosphorylation, we screened for phosphatases that alter activity rhythms when their expression was reduced. One such phosphatase, leukocyte-antigen-related (LAR), abolishes activity rhythms, but does not disrupt feedback loop function. Rather,Lardisrupts clock output by eliminating axonal processes from clock neurons that release pigment-dispersing factor (PDF) neuropeptide into the dorsal brain, but PDF expression persists in their cell bodies and remaining projections. In contrast to flies that lack PDF, flies that lackLaranticipate lights-on and lights-off transitions normally, which suggests that the remaining PDF expression mediates activity during light/dark cycles.

Optimal sequence of antisense DNA to silence YB-1 in lung cancer by use of a novel polysaccharide drug delivery system.

Silencing Y-box binding protein 1 (YB-1) can be an excellent target for cancer therapy and many lung cancer cells express the polysaccharide-recognition receptor Dectin-1. We designed a Dectin-1 targeting vehicle delivering YB-1-antisense DNA. First, we selected five optimal antisense DNA sequences to silence YB-1 from among 153 candidates. We chose the sequence closest to the start codon (AS014), and attached dA40 to the 3' end; dA40 promotes complex formation with a ß-(1➝3)-d-glucan called schizophyllan (SPG). The resultant complexes were applied to 12 human-oriented lung cancer cell lines, and cell viability was examined. The cell lines exhibited decreased viability and showed strong affinity to bind SPG, suggesting the AS014/SPG complex entered the cells via the Dectin-1 mediated pathway.

Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration.

Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, ~80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive-like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant-like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies.

Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown.

Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into 'gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.

Antisense- and siRNA-mediated inhibition of the anti-apoptotic gene Bcl-xL for chemosensitization of bladder cancer cells.

Bcl-xL is an apoptosis inhibitor that is upregulated in bladder cancer (BCa) and provides an attractive target for molecular therapies. Treatment with specific antisense oligodeoxynucleotides (AS­ODNs) or small interfering RNAs (siRNAs) were able to sensitize BCa cells to conventional chemotherapeutics. Ten new Bcl­xL­targeting AS­ODNs were systematically designed by using predicting software. AS­BX2034 and AS­BX2100 as well as the previously optimized siRNA construct si­BX713 were selected for further detailed in vitro analysis in the BCa cell lines UM­UC­3 and EJ28. Bcl­xL mRNA and protein expression levels, cell viability and apoptosis were examined 72 h after transfection. A single treatment with AS­BX2034 or AS­BX2100 caused only a low inhibition of the Bcl­xL mRNA expression with the highest reduction of ≤20% in UM­UC­3 cells. In contrast, a single treatment with si­BX713 strongly decreased Bcl­xL mRNA expression level by ≤69% in UM­UC­3 cells and by ≤86% in EJ28 cells. Both gene expression inhibitor types induced a low to moderate reduction of viability. Depending on the cell line, a combined treatment with AS­BX2100 or si­BX713 and cisplatin (CDDP) caused an additional inhibition of cell viability by ~33 and 38%, respectively, compared to the respective control construct combined with CDDP. In comparison to the respective control treatment, combinations of AS­BX2100 and CDDP led to a stronger induction of apoptosis by 57% in UM­UC­3 cells and 44% in EJ28 cells, whereas the combination of si­BX713 and CDDP enhanced apoptosis by 38 and 118% in UM­UC­3 and EJ28 cells, respectively. Our comparative studies showed a stronger knockdown of Bcl­xL by the siRNA construct compared to AS­ODN treatment in both BCa cell lines. In combinatory treatments, the Bcl­xL-directed siRNA markedly enhanced the anti-proliferative and apoptotic effects of CDDP and therefore, may serve as suitable tool for chemosensitization of BCa cells.

Carbon tetrachloride-induced liver injury in mice is tissue factor dependent.

Tissue factor (TF) is a membranous glycoprotein that activates the coagulation system when blood vessels or tissues are damaged. TF was up-regulated in monocrotaline (MCT)/lipopolysaccharide (LPS) hepatotoxicity model. The present study aimed to test the hypothesis that TF-dependent fibrin deposition occurs in liver toxicity induced by CCl4 in mice. Pericentral deposition of TF and fibrin is induced after CCl4-induced liver toxicity. The toxicity was evaluated by determination of serum activities of ALT, AST and ALP as well as GSH content and histopathological changes. The results showed that injection of mice with TF-antisense deoxyoligonucleotide (TF-AS) prevented the accumulation of TF and fibrin in the hepatic tissues. Furthermore, it significantly restored blood biochemical parameters, GSH content and distorted histopathological features caused by CCl4. The current study demonstrates that TF activation is associated with CCl4-induced liver injury. Furthermore, administration of TF-AS successfully prevented this type of liver injury.

DNA and RNA derivatives to optimize distribution and delivery.

Synthetic, complementary DNA single strands and short interfering RNA double strands have been found to inhibit the expression of animal, plant, and viral genes in cells, animals, and patients, in a dose dependent and sequence specific manner. DNAs and RNAs, however, are readily digested in biological systems. Hence, chemists are obliged to design and synthesize nuclease-resistant analogs of normal DNA (Fig. 1).

Antisense vimentin cDNA combined with chondroitinase ABC promotes axon regeneration and functional recovery following spinal cord injury in rats.

The formation of glial scar restricts axon regeneration after spinal cord injury (SCI) in adult mammalian. Chondroitin sulfate proteoglycans (CSPGs) are mostly secreted by reactive astrocytes, which form dense scar tissues after SCI. Chondroitinase ABC (ChABC), which can digest CSPGs, is a promising therapeutic strategy for SCI. However, to date ChABC has exhibited only limited success in the treatment of chronic SCI. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. We targeted glial scar in injured spinal cord by sustained infusion of ChABC and antisense vimentin cDNA. Using anterograde tracing, BBB scoring and hind limb placing response, we found that this combined treatment promoted axon regeneration and functional recovery after SCI in rats. Our results indicate that axon regeneration may be promoted by modified physical and biochemical characteristics of intra- and extracellular architecture in glial scar tissues. Theses findings could potentially help us to understand better the composition of glial scar in central nervous system injury.

Dorsal root ganglion myeloid zinc finger protein 1 contributes to neuropathic pain after peripheral nerve trauma.

Peripheral nerve injury-induced changes in gene transcription and translation in primary sensory neurons of the dorsal root ganglion (DRG) are considered to contribute to neuropathic pain genesis. Transcription factors control gene expression. Peripheral nerve injury increases the expression of myeloid zinc finger protein 1 (MZF1), a transcription factor, and promotes its binding to the voltage-gated potassium 1.2 (Kv1.2) antisense (AS) RNA gene in the injured DRG. However, whether DRG MZF1 participates in neuropathic pain is still unknown. Here, we report that blocking the nerve injury-induced increase of DRG MZF1 through microinjection of MZF1 siRNA into the injured DRG attenuated the initiation and maintenance of mechanical, cold, and thermal pain hypersensitivities in rats with chronic constriction injury (CCI) of the sciatic nerve, without affecting locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking the nerve injury-induced increase of DRG MZF1 through microinjection of recombinant adeno-associated virus 5 expressing full-length MZF1 into the DRG produced significant mechanical, cold, and thermal pain hypersensitivities in naive rats. Mechanistically, MZF1 participated in CCI-induced reductions in Kv1.2 mRNA and protein and total Kv current and the CCI-induced increase in neuronal excitability through MZF1-triggered Kv1.2 AS RNA expression in the injured DRG neurons. MZF1 is likely an endogenous trigger of neuropathic pain and might serve as a potential target for preventing and treating this disorder.

Functional correction by antisense therapy of a splicing mutation in the GALT gene.

In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

Targeting essential genes in Salmonella enterica serovar typhimurium with antisense peptide nucleic acid.

We investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF)(3)K cell-penetrating peptide to target possible essential genes (ligA, rpoA, rpoD, engA, tsf, and kdtA) in Salmonella enterica serovar Typhimurium and inhibit bacterial growth in vitro and in cell culture. All targeted PNA-based gene inhibition has shown great potency in gene expression inhibition in a sequence-specific and dose-dependent manner at micromolar concentrations. Among tested PNAs, the anti-rpoA and -rpoD PNAs showed the greatest potency.

Survival motor neuron affects plastin 3 protein levels leading to motor defects.

The actin-binding protein plastin 3 (PLS3) has been identified as a modifier of the human motoneuron disease spinal muscular atrophy (SMA). SMA is caused by decreased levels of the survival motor neuron protein (SMN) and in its most severe form causes death in infants and young children. To understand the mechanism of PLS3 in SMA, we have analyzed pls3 RNA and protein in zebrafish smn mutants. We show that Pls3 protein levels are severely decreased in smn(-/-) mutants without a reduction in pls3 mRNA levels. Moreover, we show that both pls3 mRNA and protein stability are unaffected when Smn is reduced. This indicates that SMN affects PLS3 protein production. We had previously shown that, in smn mutants, the presynaptic protein SV2 is decreased at neuromuscular junctions. Transgenically driving human PLS3 in motoneurons rescues the decrease in SV2 expression. To determine whether PLS3 could also rescue function, we performed behavioral analysis on smn mutants and found that they had a significant decrease in spontaneous swimming and turning. Driving PLS3 transgenically in motoneurons rescued both of these defects. These data show that PLS3 protein levels are dependent on SMN and that PLS3 is able to rescue the neuromuscular defects and corresponding movement phenotypes caused by low levels of Smn suggesting that decreased PLS3 contributes to SMA motor phenotypes.