Squalenoyl siRNA PMP22 nanoparticles are effective in treating mouse models of Charcot-Marie-Tooth disease type 1 A.

Charcot-Marie-Tooth disease type 1 A (CMT1A) lacks an effective treatment. We provide a therapy for CMT1A, based on siRNA conjugated to squalene nanoparticles (siRNA PMP22-SQ NPs). Their administration resulted in normalization of Pmp22 protein levels, restored locomotor activity and electrophysiological parameters in two transgenic CMT1A mouse models with different severity of the disease. Pathological studies demonstrated the regeneration of myelinated axons and myelin compaction, one major step in restoring function of myelin sheaths. The normalization of sciatic nerve Krox20, Sox10 and neurofilament levels reflected the regeneration of both myelin and axons. Importantly, the positive effects of siRNA PMP22-SQ NPs lasted for three weeks, and their renewed administration resulted in full functional recovery. Beyond CMT1A, our findings can be considered as a potent therapeutic strategy for inherited peripheral neuropathies. They provide the proof of concept for a new precision medicine based on the normalization of disease gene expression by siRNA.

New Formulation for the Delivery of Oligonucleotides Using "Clickable" siRNA-Polyisoprenoid-Conjugated Nanoparticles: Application to Cancers Harboring Fusion Oncogenes.

The aim of the present study is to take advantage of the unique property of polyisoprenoid chains to adopt a compact molecular conformation and to use these natural and biocompatible lipids as nanocarriers of drugs to deliver siRNA. A new chemical strategy is applied here to conjugate squalene (SQ) and solanesol (SOLA) to siRNA consisting of an activated variant of the azide-alkyne Huisgen cycloaddition also known as copper-free (Cu-free) click chemistry. We conjugated siRNA against TMPRSS2-ERG, a fusion oncogene found in more than 50% of prostate cancers to SQ or SOLA. First, several parameters such as molar ratio, solvents, temperature, incubation time, and the annealing schedule between both siRNA strands were investigated to bioconjugate the SQ or SOLA via Cu-free click chemistry. The best parameters of the new bioconjugation approach allowed us to (i) increase the synthesis yield up to 95%, (ii) avoid the formation of byproducts during the synthesis, and (iii) improve the reproducibility of the bioconjugation. Then, the biological activity of the resulting nanoparticles was assessed. In vitro, all four formulations were able to decrease the corresponding oncogene and oncoprotein expression. In vivo, only two of the four nanoformulations showed anti-neoplastic activity that seems to be tightly related to their dissimilar biodistribution behavior. In conclusion, we performed a new approach easily transposable for pharmaceutical development to synthesize siRNA-SQ and siRNA-SOLA and to obtain efficient siRNA-nanoparticles. The robustness of the process could be extended to several other polyterpenes and likely applied to other siRNA targeting genes whose overexpression results in the development of cancers or other genetic diseases.

Effects of natural environment on reproductive histo-morphometric dynamics of female dromedary camel.

Camel shows a seasonal breeding pattern with enhanced reproductive activity during the period of low climatic temperature, high rainfall and better food conditions. Therefore, the study was conducted to explore the underlying seasonal effects on histological dimensions of reproductive tract in adult female one-humped camel (Camelus dromedarius) kept in the natural environment of Pakistan. A total 25 reproductive tracts were collected during spring, summer, autumn and winter seasons and were analysed for histo-morphometric parameters during different environmental conditions. A significant increase in number (p<0.05) and size (p<0.05) of surface with secondary and tertiary ovarian follicles was observed in winter season. The epithelial height (p<0.05) and luminal diameter (p<0.05) of infundibulum, ampulla and isthmus of uterine tubes were also significantly increased during winter season. Moreover, significantly increased length (p<0.05) and circumference (p<0.05) of uterine cornua, increased number (p<0.001) and diameter (p<0.001) of endometrial glands with enlarged surface and glandular epithelia (p<0.001) were found in winter compared to summer season. Therefore, we concluded that quiescent ovarian follicular and uterine glandular activities are the main reason of camel low breeding during summer season.

Relevance of Fusion Genes in Pediatric Cancers: Toward Precision Medicine.

Pediatric cancers differ from adult tumors, especially by their very low mutational rate. Therefore, their etiology could be explained in part by other oncogenic mechanisms such as chromosomal rearrangements, supporting the possible implication of fusion genes in the development of pediatric cancers. Fusion genes result from chromosomal rearrangements leading to the juxtaposition of two genes. Consequently, an abnormal activation of one or both genes is observed. The detection of fusion genes has generated great interest in basic cancer research and in the clinical setting, since these genes can lead to better comprehension of the biological mechanisms of tumorigenesis and they can also be used as therapeutic targets and diagnostic or prognostic biomarkers. In this review, we discuss the molecular mechanisms of fusion genes and their particularities in pediatric cancers, as well as their relevance in murine models and in the clinical setting. We also point out the difficulties encountered in the discovery of fusion genes. Finally, we discuss future perspectives and priorities for finding new innovative therapies in childhood cancer.

Induction of TTF-1 or PAX-8 expression on proliferation and tumorigenicity in thyroid carcinomas.

TTF-1 and PAX-8 are responsible for thyroid organogenesis and for maintenance of differentiation in thyrocytes. Thus, we hypothesized that the induction of these two transcription factors could affect proliferation and tumorigenicity. Moreover, the ability of various pharmacological agents to modulate expression of the TTF-1 and PAX-8 and their effects on apoptosis were also analysed. For this purpose, cell lines derived from papillary (TPC-1 and BHP 10-3) and anaplastic (ARO) thyroid carcinomas were stably transfected with expression vectors containing TTF-1 or PAX-8 genes. Subsequently, the effects on expression at gene and protein levels, as well as on cell growth, cell cycle, migration and in vivo tumorigenicity were studied. Our results showed that: i) TTF-1 reciprocally induces PAX-8 expression; ii) the basal state of TTF-1 or PAX-8 influences proliferation, migration and tumorigenicity; iii) the induction of TTF-1 acts on cell proliferation more than PAX-8 and mainly affects tumorigenicity; and iv) TTF-1 was found to be more sensitive to epigenetic modulators than PAX-8. Therefore, we postulated that both TTF-1 and PAX-8 when co-expressed have anti-proliferative and anti-tumorigenic properties up to a threshold expression level and beyond that, are able to induce pro-tumorigenic effects. Hence in future, it will be quite interesting to systematically take into account the basal state of expression of TTF-1 and PAX-8. It will also be important to study the two thyroid transcription factors as part of a duo. This could open in the long-term, new therapeutic perspectives for thyroid carcinomas.

Knocking Down TMPRSS2-ERG Fusion Oncogene by siRNA Could be an Alternative Treatment to Flutamide.

Our purpose was to develop a new pharmacological approach for the treatment of prostate cancer (PCa), the most common neoplasia in men. Recently, we developed siRNA against the fusion oncogene TMPRSS2-ERG found in 50% of patients and showed an antitumoral activity in animal model. Herein, we want to compare or combine the developed siRNA to flutamide (FLU), one of the gold-standard treatment of PCa. Therefore, concomitant or subsequent association of FLU to siRNA TMPRSS2-ERG was performed in VCaP cells and in SCID mice bearing xenografted VCaP tumors. ERG, androgen receptor, cleaved-caspase-3 as well as phase 1 and 2 drug-metabolizing enzymes were investigated within tumors. We observed similar results in terms of TMPRSS2-ERG knock-down and cell viability impairment for all distinct schedules of administration. The association of siRNA TMPRSS2-ERG-squalene nanoparticles with flutamide displayed similar tumor growth inhibition as mice treated with siRNA TMPRSS2-ERG-squalene nanoparticles alone and was paralleled with modification of expression of ERG, androgen receptor, and cleaved-caspase-3. Phase 1 and 2 enzymes were essentially affected by FLU and reverted when combined with squalenoylated siRNA. In conclusion, these results confirm the therapeutic effectiveness of squalenoyl siRNA nanomedicine for PCa based on siRNA TMPRSS2-ERG.

Antineoplastic Effects of siRNA against TMPRSS2-ERG Junction Oncogene in Prostate Cancer.

TMPRSS2-ERG junction oncogene is present in more than 50% of patients with prostate cancer and its expression is frequently associated with poor prognosis. Our aim is to achieve gene knockdown by siRNA TMPRSS2-ERG and then to assess the biological consequences of this inhibition. First, we designed siRNAs against the two TMPRSS2-ERG fusion variants (III and IV), most frequently identified in patients' biopsies. Two of the five siRNAs tested were found to efficiently inhibit mRNA of both TMPRSS2-ERG variants and to decrease ERG protein expression. Microarray analysis further confirmed ERG inhibition by both siRNAs TMPRSS2-ERG and revealed one common down-regulated gene, ADRA2A, involved in cell proliferation and migration. The siRNA against TMPRSS2-ERG fusion variant IV showed the highest anti-proliferative effects: Significantly decreased cell viability, increased cleaved caspase-3 and inhibited a cluster of anti-apoptotic proteins. To propose a concrete therapeutic approach, siRNA TMPRSS2-ERG IV was conjugated to squalene, which can self-organize as nanoparticles in water. The nanoparticles of siRNA TMPRSS2-ERG-squalene injected intravenously in SCID mice reduced growth of VCaP xenografted tumours, inhibited oncoprotein expression and partially restored differentiation (decrease in Ki67). In conclusion, this study offers a new prospect of treatment for prostate cancer based on siRNA-squalene nanoparticles targeting TMPRSS2-ERG junction oncogene.

Effects of siRNA on RET/PTC3 junction oncogene in papillary thyroid carcinoma: from molecular and cellular studies to preclinical investigations.

RET/PTC3 junction oncogene is typical of radiation-induced childhood papillary thyroid carcinoma (PTC) with a short latency period. Since, RET/PTC3 is only present in the tumour cells, thus represents an interesting target for specific therapy by small interfering RNA (siRNA). Our aim is to demonstrate in vitro and in vivo molecular and cellular effects of siRNA on RET/PTC3 knockdown for therapeutic application.First, we established a novel cell line stably expressing RET/PTC3 junction oncogene, named RP3 which was found tumorigenic in nude mice compared to NIH/3T3 mouse fibroblasts. Among four siRNAs and five concentrations tested against RET/PTC3, an efficient siRNA RET/PTC3 and an appropriate dose (50 nM) were selected which showed significant inhibition (p<0.001) of gene (RT-qPCR) and protein (Western blot) expressions. This siRNA was found efficient in RP3 cells (harbouring RET/PTC3) but non-efficient in BHP10-3 SCmice cell line (harbouring RET/PTC1) showing that a specific siRNA against fusion sequence is required to target the junction oncogene. In vitro siRNA RET/PTC3 showed significant (p<0.001) inhibitory effects on RP3 cell viability (MTT assay) and on invasion/migration (IncuCyte scratch test) with blockage of cell cycle at G0/G1 phase (flow cytometry) and induced apoptosis by caspase-3 and PARP1 cleavage (WB). After intravenous injection in nude mice, respective squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC3 significantly (p<0.001) reduced RP3 tumour growth, oncogene and oncoprotein expressions, induced apoptosis and partially restored differentiation (decrease in Ki67). Hence, our findings highly support the use of siRNA RET/PTC3-SQ NPs as a new promising treatment for patients affected by PTC expressing RET/PTC3.

Effects of silencing the RET/PTC1 oncogene in papillary thyroid carcinoma by siRNA-squalene nanoparticles with and without fusogenic companion GALA-cholesterol.

BACKGROUND: RET/PTC1 is the most prevalent type of gene rearrangement found in papillary thyroid carcinoma (PTC). Previously, we introduced a new noncationic nanosystem for targeted RET/PTC1 silencing by efficient delivery of small interfering RNA (siRNA) using the "squalenoylation" approach. With the aim of improving these results further, we designed new squalenoyl nanostructures consisting of the fusogenic peptide GALA-cholesterol (GALA-Chol) and squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC1. METHODS: The siRNA RET/PTC1-SQ bioconjugate was synthesized. The corresponding NPs were prepared with or without GALA-Chol by nanoprecipitation and then characterized for their size and zeta potential. The effects of NPs on BHP 10-3 SCmice and TPC-1 cell viability (MTT assay), gene and protein silencing (reverse transcription-quantitative polymerase chain reaction [rt-qPCR], Western blot), and cellular uptake (fluorescent microscopy) were studied. In vivo gene silencing efficiency of siRNA RET/PTC1-SQ NPs was assessed by administration in nude mice via either intratumoral (i.t.) or intravenous (i.v.) routes. Tumor growth was followed for 19 days. Tumors were then collected, and RET/PTC1 gene and protein inhibitions were assessed by RT-qPCR and Western blot. RESULTS: The combination of siRNA RET/PTC1-SQ bioconjugate and GALA-Chol leads to stable NPs of ∼200 nm diameter. In vitro, the results revealed that combining GALA-Chol with siRNA RET/PTC1-SQ NPs decreased cell viability, enhanced cellular internalization, and induced gene silencing efficiency in both human PTC (BHP 10-3 SCmice and TPC-1) cell lines. On the contrary, in vivo, the siRNA RET/PTC1-SQ GALA-Chol NPs were not found to be efficient either in gene silencing or in tumor growth inhibition, compared to siRNA RET/PTC1-SQ NPs both via i.t. and i.v. routes (p<0.001). CONCLUSIONS: Conversely to siRNA RET/PTC1-SQ NPs, the siRNA RET/PTC1-SQ GALA-Chol NPs are efficient in vitro but not in vivo. Finally, NPs of siRNA RET/PTC1-SQ were found to be efficient silencers of the RET/PTC1 fusion oncogene in in vivo applications even at a concentration lower than used in a previously published study.

Significance and applications of nanoparticles in siRNA delivery for cancer therapy.

RNAi is a powerful gene silencing process that holds great promise in cancer therapy by the use of siRNA. The aim of this review is to give an outline on different approaches to deliver siRNA and to describe the advantages and disadvantages of these systems. The prospects for siRNA are to be substantially better than other therapies, as they are easily applicable to any therapeutic target. They also promise potent gene inhibition with exquisite selectivity, down to the level of a single nucleotide polymorphism, and can easily identify offending proteins or variants by screening across a gene sequence. The main obstacle of using RNAi technology in cancer treatment is to protect such a fragile and quickly metabolized biological molecule and to efficiently deliver it in vivo to the target cells. Therefore, there is a requirement for new systems, such as nanoparticles, for siRNA delivery to help the siRNAs reach, and improve their biodistribution in, target tissues.

Anticancer drugs in liposomal nanodevices: a target delivery for a targeted therapy.

For many years, nanocarriers have been investigated to modify pharmacokinetics and biodistribution of various active molecules. In the cancer domain, one of the biggest challenges still remains the improvement of the therapeutic index, often too low, for the majority of antitumor drugs. The application of nanotechnologies for the treatment and the diagnosis of cancers are nowadays currently developed, or under development, and liposomes play an important role in the history of nanodevices. Because of their high degree of biocompatibility, lipid nanosystems have been used to improve pharmacological profiles of various anticancer drugs otherwise discarded because of their low water solubility, poor bioavailability or either fragile and subjected to rapid biotransformations. This review aims at introducing an overview of the last 40 years of liposome researches until the last liposomal formulations commercially available or undergoing clinical trials. Liposome properties will be described, with a particular emphasis over the last generation of carriers appreciated for their active targeting characteristics. Researchers foresee a remarkable impact of nanotechnologies in the field of medicine; this review will try to summarize the main concepts over liposome domain, which can count on encouraging results as target therapy associated with targeted delivery.

Lipid conjugated oligonucleotides: a useful strategy for delivery.

Oligonucleotides, including antisense oligonucleotides and siRNA, are promising therapeutic agents against a variety of diseases. Effective delivery of these molecules is critical in view of their clinical application. Therefore, cation-based nanoplexes have been developed to improve the stability as well as the intracellular penetration of these short fragments of nucleic acids. However, this approach is clearly limited by the strong interaction with proteins after administration and by the inherent toxicity of these positively charged transfection materials. Neutral lipid-oligonucleotide conjugates have become a subject of considerable interest to improve the safe delivery of oligonucleotides. These molecules have been chemically conjugated to hydrophobic moieties such as cholesterol, squalene, or fatty acids to enhance their pharmacokinetic behavior and trans-membrane delivery. The present review gives an account of the main synthetic methods available to conjugate lipids to oligonucleotides and will discuss the pharmacological efficacy of this approach.

Liposomal trichostatin A: therapeutic potential in hormone-dependent and -independent breast cancer xenograft models.

BACKGROUND: Trichostatin A (TSA) is one of the most potent histone deacetylase inhibitors (HDACi) in vitro but it lacks biological activity in vivo when injected intravenously owing to its fast metabolism. MATERIALS AND METHODS: TSA was incorporated into Stealth® liposomes (TSA-lipo) at a high loading and its anticancer activity was evaluated in several types of breast cancer cells and xenografts. RESULTS: In estrogen receptor α (ERα)-positive MCF-7 and T47-D cells, TSA induced a long-term degradation of cyclin A and a proteasome-dependent loss of ERα and cyclin D1, allowed derepression of p21WAF1/CIP1, HDAC1 and RhoB GTPase, concomitantly with blockade in G2/M of the cell cycle and apoptosis induction. In MDA-MB-231 (MDA) and SKBr-3 cells, TSA increased ERα mRNA and p21WAF1/CIP1 protein expression, but decreased cyclin A with a G2/M blockade and cleavage of polyADP-ribose polymerase (PARP). No significant restoration of any ER protein was noticed in any cells. TSA-lipo markedly inhibited tumor growth in MCF-7 and MDA cells xenografts following intravenous injection. Their anticancer effects were characterized by inhibition of Ki-67 labeling, the inhibition of tumor vasculature and an increase of p21WAF1/CIP1 in both tumors. In MCF-7 cell tumors, enhanced RhoB accumulation in the cytoplasm of epithelial cells was noticed, inversely to ERα that was strongly decreased. CONCLUSION: Such anticancer activity of TSA-lipo is exp-lained by the protection provided by HDACi encapsulation and by the strong tumor accumulation of the nanocarriers as revealed by fluorescence confocal microscopy experi-ments. Together with its lack of toxicity, the enhanced stability of TSA-lipo in vivo justifies its development for therapeutic use in the treatment estradiol-dependent and -independent breast cancers.

Liposomes loaded with histone deacetylase inhibitors for breast cancer therapy.

Histone deacetylase (HDAC) inhibitors (HDACi) of the class I trichostatin A (TSA), CG1521 (CG), and PXD101 (PXD) were incorporated at a high rate (approximately 1mM) in liposomes made of egg phosphatidylcholine/cholesterol/distearoylphosphoethanolamine-polyethylenglycol(2000) (64:30:6). Physicochemical parameters (size, zeta potential, loading, stability, release kinetics) of these HDACi-loaded pegylated liposomes were optimized and their cytotoxicity (MTT test) was measured in MCF-7, T47-D, MDA-MB-231 and SkBr3 breast cancer cell lines. In MCF-7 cells, TSA and PXD were efficient inducers of proteasome-mediated estradiol receptor alpha degradation and they both affected estradiol-induced transcription (TSA>PXD) contrary to CG. Moreover, TSA most efficiently altered breast cancer cell viability as compared to the free drug, CG-liposomes being the weakest, while unloaded liposomes had nearly no cytotoxicity. Pegylated liposomes loaded with TSA or PXD remained stable in size, charge and biological activity for one month when stored at 4 degrees C. All HDACi-loaded liposomes released slowly the encapsulated drug in vitro, CG-loaded liposomes showed the slowest release kinetic. These formulations could improve the efficacy of HDACi not only in breast cancers but also in other solid tumors because most of these drugs are poor water soluble and unstable in vivo, and their administration remains a challenge.

Therapeutic potential of new 4-hydroxy-tamoxifen-loaded pH-gradient liposomes in a multiple myeloma experimental model.

PURPOSE: To determine the better liposomal formulation incorporating the active metabolite of tamoxifen, 4-hydroxy-tamoxifen (4HT) and the biological impact of 4HT-pH-gradient liposomes on response to in vivo treatment. METHODS: Several pegylated liposomes were formulated by varying the composition of lipids, increasing external pH from 7.4 to 9.0 and doubling the lipid concentration. Dipalmitoylphosphatidylcholine / cholesterol / distearoylphosphoethanolamine poly(ethylene)glycol liposomes (DL-9 liposomes) were chosen for their physico-chemical properties. Toxicity and release kinetics were assessed in breast cancer MCF-7 as well as in multiple myeloma (MM) cells. In vivo antitumor activity and bio-distribution were measured in the RPMI8226 MM model. RESULTS: Compared to conventional non-pH-gradient liposomes, 4HT-DL-9 liposomes resulted in concentration of up to 1 mM 4HT, greater stability, relative toxicity and slow 4HT release. Intravenous injections of 4HT-DL-9 liposomes at 4 mg/kg/week blocked MM tumor growth. Ki67 and CD34 labeling decreased in treated tumors, concomitantly with increase of activated caspase-3 supporting a cell proliferation arrest, a decrease of tumor vasculature and the induction of tumor cell death. CONCLUSION: This antitumor effect was assumed to be the result of a modified biodistribution of 4HT once trapped in DL-9 liposomes. Such 4HT-containing pH-gradient Stealth nanocarriers could be helpful for MM treatment.