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Nanoparticle-Mediated Small RNA Deliveries for Molecular Therapies
Published in D. Sakthi Kumar, Aswathy Ravindran Girija, Bionanotechnology in Cancer, 2023
Ramasamy Paulmurugan, Uday Kumar Sukumar, Tarik F. Massoud
SiRNA is dsRNA of 20–25 base pairs long designed against any region of the target mRNA (coding region or 3’ UTR) to achieve gene silencing by mRNA degradation. Sequence-specific post transcriptional gene silencing can be achieved by delivering synthetic siRNAs via RNAi pathway in mammalian cells [93]. To achieve improved silencing effect, many of the currently available siRNAs are designed as pooled molecules, where more than one region of the target mRNAs is used as mixtures. The siRNA technology has rapidly evolved as a powerful tool in selectively silencing gene expression [93, 94]. Even though siRNAs are efficient for gene silencing, their intracellular delivery in intact form is challenging, and more so when considering this approach for in vivo gene silencing. Hence, while developing strategies to synthesize stable RNA molecules using modified bases (2-O-Me, 2’OMe-PS, 2’OMe-PO, 2’MOE, 2’MOE-PS, LNA, FANA, etc.,), enormous efforts have also been spent in developing nanodelivery systems for intact delivery of siRNAs by overcoming nuclease-mediated degradations in the systemic circulation. This can be achieved only by preventing nuclease access to RNAs. Nanoparticles synthesized from various polymers have served as excellent platforms that encapsulate RNAs within the nanoparticles and prevent them from exposure to RNases.
Should Genome Editing Replace Embryo Selection Following PGT?
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
An improvement came in the early 1990s with the introduction of zinc finger nucleases (ZFNs) and transcription-activator-like effector nucleases (TALENs), which could be engineered in order to induce DSBs at specified sites. ZFNs utilize zinc finger protein domains that bind to a 3-bp motif in a modular manner, making them ideal as building units for the creation of sequence-specific DNA binding nucleases (19). TALENs, on the other hand, recognize a single base in each repeat domain, allowing up to four different domains to be mixed and matched to generate a novel DNA binding protein. However, both of these programmable nucleases are associated with an appreciable incidence of “off-target” effects, defined as non-specific cleavage of the DNA at locations other than the intended target site, potentially resulting in cytotoxicity (19,21). Furthermore, since the target specificity is determined by modification of the DNA binding domain, the application of these nucleases is limited to cases where successful engineering of binding domains is possible, at significant cost of time and resources.
Biological reactions to reconstructive materials
Published in Steven J. Kronowitz, John R. Benson, Maurizio B. Nava, Oncoplastic and Reconstructive Management of the Breast, 2020
Steven J. Kronowitz, John R. Benson, Maurizio B. Nava
Each method of decellarization has advantages and limitations with respect to these two competing aims. Purely physical methods such as the use of multiple freeze-thaw cycles have the advantage of avoiding chemical agents that can be retained in the matrix, though may not result in sufficient decellularization for thicker tissues such as dermis.7 Agitation is commonly employed in combination with a chemical agent or detergent to achieve superior decellularization. A drawback of detergent use is that it may weaken the collagen network of the matrix and denature growth factors, and residual detergent left in the ADM can be cytotoxic, inhibiting cell migration and proliferation.9 Addition of an enzymatic nuclease (DNAse or RNAase) to the above protocols is common in order to remove residual nucleic acids.
Current developments in gene therapy for epidermolysis bullosa
Published in Expert Opinion on Biological Therapy, 2022
Thomas Kocher, Igor Petkovic, Johannes Bischof, Ulrich Koller
Besides efficiency of gene editing in general, recent observations of large chromosomal truncations, translations, and rearrangements at CRISPR/Cas9-generated DSB sites [90,148–150] suggest to lay the focus also on safety, especially when an in vivo application is considered. Actual methods to deal with these issues include guide RNA optimization, as well as the engineering of novel Cas9 variants that exhibit reduced off-target effects while maintaining editing efficiency. Aiming at the reduction of general Cas9 – DNA interactions the reliance on sgRNA:DNA heteroduplex formation is increased to direct the specificity of DSB induction [151]. The use of Cas9 nickases is currently one of the safest gene editing approaches, as SSBs are normally repaired in a traceless manner via the cell’s base excision repair pathway (BER) [56]. Furthermore, immunological consequences upon in vivo treatment using nucleases have to be addressed in future studies.
Developments in reading frame restoring therapy approaches for Duchenne muscular dystrophy
Published in Expert Opinion on Biological Therapy, 2021
Anne-Fleur E. Schneider, Annemieke Aartsma-Rus
The first attempt at gene editing in the DMD gene used meganucleases. Proof of principle was shown by insertion of meganuclease target sites in the middle of a dog microdystrophin plasmid containing a frame-shift mutation that was transfected into 293 FT cells [91]. Overexpression of this meganuclease was able to induce small indels at these target sites, which restored the reading frame and microdystrophin. This same approach was applied later in rag/mdx mouse and human myoblasts where restoration of micro-dystrophin in the muscle fibers and myoblasts was observed [92]. Later a meganuclease was designed to cleave within intron 44 in patient-derived myoblasts, just upstream of the deletion hotspot. Repair matrixes carrying exon 45–52 were co-transduced into patient-derived myoblasts carrying a deletion of exon 45–52. This resulted in the production of full-length dystrophin mRNA [93]. However, an important drawback of meganucleases is that the recognition site has to be introduced in the desired gene. Whilst this does ensure low off-target nuclease activity, first having to genetically modify the target gene has its own risk of off-target effects and is very impractical in a clinical setting.
DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology
Published in Journal of Extracellular Vesicles, 2019
Elisa Lázaro-Ibáñez, Cecilia Lässer, Ganesh Vilas Shelke, Rossella Crescitelli, Su Chul Jang, Aleksander Cvjetkovic, Anaís García-Rodríguez, Jan Lötvall
The release of cellular DNA in many different structural forms such as apoptotic blebs, histone/DNA complexes or nucleosomes, DNA/RNA-lipoprotein complexes or virtosomes, DNA traps, etc., has been well documented [7–10]. Such extracellular structures, classified in umbrella terms such as circulating DNA or cell-free DNA, largely serve to protect the DNA from nucleases that are present in, for example, the circulation and to reduce the likelihood of DNA being seen as a danger signal by the immune system [11]. As nucleases are essential enzymes that control DNA repair and therefore, genomic stability, their defects or absence are associated with diseases in which the sensing of self-nucleic acids is critical [12]. For instance, the knockout of the DNase I and II family members are linked to severe autoimmune and metabolic diseases [13].