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Intraperitoneal nonviral nucleic acid delivery in the treatment of peritoneal cancer
Published in Wim P. Ceelen, Edward A. Levine, Intraperitoneal Cancer Therapy, 2015
George R. Dakwar, Stefaan S.C. De Smedt, Katrien Remaut
The last two decades have witnessed a revolution in genomics and proteomics during which tens of molecular path-ways involved in proliferation of cancer cells were identified. These include immune evasion, angiogenesis, and metastasis. Theoretically, knockdown of every gene should be possible; however, for successful and specific gene knockdown, it is important to screen targets that are overexpressed by cancer cells. As we will discuss later in the section “Current in vivo use of plasmid DNA for IP carcinomatosis,” RNAi opens the opportunity for designing personalized medicine [29], since it can influence the expression or knockdown of a specific gene in a particular cancer patient. To date, the most common RNAi targets that are related to cancer are the multidrug resistance (MDR) proteins [5]. MDR is a situation in which cancer cells develop resistance against anticancer drugs by several mechanisms such as decreased drug uptake, increased drug efflux, and induction of DNA repair mechanisms. For instance, P-glycoprotein (P-gp) is a transporter protein encoded by the MDR-1 gene that pumps drugs into the extracellular space before reaching the target. It has been shown that P-gp is overexpressed in several cancer types following chemotherapeutic treatment, making it an attractive target for RNAi [44]. Another common example of MDR-encoded protein is survivin, a member of the inhibitor of apoptosis (IAP) protein family, since it is upregulated in solid tumors and has been involved with drug resistance [2,79]. Recently, codelivery of conventional anticancer chemotherapeutics and siRNA has received tremendous attention to overcome cancer resistance [12].
Nonclinical Safety Evaluation of Advanced Therapies
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Timothy K. MacLachlan, Kendall S. Frazier, Mercedes Serabian
Recent advances in ASO chemistries and new delivery systems have allowed better tissue penetration, enhanced intracellular targeting, and less-frequent dosing than second generation ASOs (Fiset and Gounni 2001, Akinc et al. 2010). The class of RNA-targeting therapeutic drugs is increasingly diverse and the recent development of chemical variants, novel delivery systems, and N-acetylated galactosamine-conjugated (GalNac) moieties has made some longstanding preconceptions about class-wide toxicity of ASOs less relevant. For instance, specific and targeted cellular uptake by GalNac conjugated ASOs in the liver has resulted in the potential for fewer systemic preclinical and clinical toxicities (Akinc et al. 2010, Wang et al. 2010, Choksi, Wang et al. 2010, Frank-Kamenetsky et al. 2008). The GalNac conjugated ASO or siRNA multivalent molecule binds with high affinity to the asialoglycoprotein receptor (ASGPR), a c-type lectin expressed on hepatocytes (Geuze et al. 1983), and gets rapidly distributed into the cytoplasm, limiting toxicity in other organs. Other strategies to localize gene knockdown therapies into the correct subcellular compartment (e.g., via the conjugation of siRNAs to basic peptides or carrier proteins or to modulate membrane permeability and increase cytoplasmic uptake) may help avoid toxicities associated with lysosomal accumulation (Jarver et al. 2012, Henke et al. 2008, Prakash et al. 2014). Other types have incorporated peptide linkers for directed cellular targeting and even direct transport to the nucleus (Jarver et al. 2012, Henke et al. 2008). Classwide ASO toxicities related to accumulation may still be present with conjugated molecules but appear to be less severe. This is also the case with the new generation, constrained ethyl modified ASOs (cEt), which represents a means to potently target difficult coding and noncoding RNA pathways and in a set of tissues that otherwise are hard to reach in sufficient quantities (Seth et al. 2010). To date, only a few cases of hepatic toxicity have been noted with the cEt ASOs as a class, and they appear to lack many other toxicities noted more commonly with the second-generation ASOs. Recent innovations by other companies have included the so-called gene-silencing oligonucleotides with two free 3′-ends on their nucleic acid sequence. Like the cEt ASOs, this platform reportedly has decreased tissue accumulation and less immune activation than PS ASOs, but long-term, preclinical studies have yet to be performed. With newer generation mRNA expression knockdown therapies entering pharmaceutical development, dogma about “typical toxicities” with this class of compounds are likely to change dramatically in the near future.
Comparison of tyrosine-modified low molecular weight branched and linear polyethylenimines for siRNA delivery
Published in Nanotoxicology, 2022
Małgorzata Kubczak, Sylwia Michlewska, Michael Karimov, Alexander Ewe, Achim Aigner, Maria Bryszewska, Maksim Ionov
Studies on complex activity in vitro showed that tyrosine-modified polymers have a great potential for siRNA delivery. Linear, non-modified polymers are practically unable to bind siRNA, as opposed to branched PEIs which are also known as nanovectors for plasmid DNA (Kwok and Hart 2011). Addition of tyrosine allowed PEIs to complex smaller, more rigid nucleic acids like siRNA. Biologically active complexes can be formed even at low polymer/siRNA ratios. This allows for efficient gene knockdown with little toxic effects. Knockdown efficacy mainly relies on complex stability, charge and hydrophobicity. Tyrosine-modified polymers form complexes that are more stable compared to their non-modified counterparts, which can be explained by the tyrosines’ contribution to electrostatic and π-π interactions (Dougherty 1996; Huang et al. 2015; Plyte and Kneale 1991). Notably, complexes based on LP10Y also mediated quite efficient GADPH gene knockdown in the ex vivo tissue slice model, indicating penetration of the LP10Y/siGAPDH complexes into deeper cell layers of the tissue slices. This indicates that results obtained in traditional 2 D cell culture are not always confirmed in more advanced culture systems (Karimov, Appelhans et al. 2021; Merz et al. 2017). LP10Y seems to form complexes which are readily taken up by the cells, while simultaneously also being able to reach deeper parts of the tissue.
Genetic and epigenetic strategies for advancing ovarian cancer immunotherapy
Published in Expert Opinion on Biological Therapy, 2019
Youngwoo Cho, Lara Milane, Mansoor M. Amiji
Altering gene expression can change disease-related pathways to improve cancer survival, and RNA interference (RNAi) is best suited for this purpose [56]. RNAi is an oligonucleotide-based therapy for gene knockdown via the delivery of small RNA duplexes, including microRNA (miRNA) mimics, short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and Dicer substrate RNAs (dsiRNAs) [57]. As opposed to protein inhibition which suffers toxic off-target effects, gene knockdown offers the special advantage of target specificity with improved potency and toxicity characteristics [58]. Due to its immense therapeutic potential, RNAi has been the focus of extensive research for many years. From the first discovery of gene silencing in C. elegans in 1988, RNAi has evolved at a surprisingly rapid pace, achieving systemic application in humans within the span of only 12 years [59,60]. To date, there are approximately 20 miRNA- and siRNA-based therapeutics that have entered clinical trials, and around 6,500 US patents are granted for ‘siRNA and cancer.’ [61]
zHSF1 modulates zper2 expression in zebrafish embryos
Published in Chronobiology International, 2018
Lucas Mennetrier, Tatiana Lopez, Benoist Pruvot, Nadhir Yousfi, Olivier Armant, Hanae Hazhaz, Vincent Lhuissiez, Carmen Garrido, Johanna Chluba
Gene knockdown experiments were performed using morpholino-modified antisense oligonucleotides (Gene Tools, LLC; Philomath OR, USA). All morpholinos were labeled with Lissamine or Fluorescein to facilitate screening of successfully injected eggs. For the hsf1-mo, we used the sequence: 5′-CACGGAGAGTTTAGTGATGATTTCT-3′. The specificity of this morpholino was already demonstrated by other groups (Evans et al. 2007; Etard et al. 2015; Tucker et al. 2011). As control, we used the standard nonsense control morpholino (control-mo) with the sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′. The morpholinos were injected into one to two cell stage embryos at 250 µM concentration. This was the lowest effective concentration and was determined by titration experiments followed by morphological examination. After injection, only eggs with uniform fluorescence were used for the experiments. The embryos were raised up to 48 hpf at 28 °C.