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Genome Editing and Gene Therapies: Complex and Expensive Drugs
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2020
Since their discovery CRISPR/Cas9 technologies have been mainly used to generate DNA ds breaks although the majority of genetic diseases are caused by point mutations (Abbasi, 2017). In 2016, Lui and colleagues (Komor et al., 2016) reported about a new genome editing approach that they termed “base editing”; it enabled the programmable conversion of one target DNA base into another base without dsDNA breaks or the need of a donor template. The conversion was achieved by fusing a catalytically inactive CRISPR/dCas9 with a single-strand specific cytidine deaminase (rAPOBEC1, apolipoproteinB mRNA editing enzyme, catalytic polypeptide-like 1) that directly converts cytidine to uridine within an approximately five-nucleotide window, resulting in cytosine (C) to uracil (U) DNA base transformations. The base editor (BE) is further equipped with a uracil DNA glycosylase inhibitor (to prevent uracil excision) and a nickase activity which nicks only the non-edited strand (favoring the repair of the nicked strand resulting in the desired U•A/T•A outcome. The substitution occurs in the ssDNA bubble formed by the interaction with Cas9.
Immune Reconstitution after Hematopoietic Stem Cell Transplantation
Published in Richard K. Burt, Alberto M. Marmont, Stem Cell Therapy for Autoimmune Disease, 2019
Andreas Thiel, Tobias Alexander, Christian A. Schmidt, Falk Hiepe, Renate Arnold, Andreas Radbruch, Larissa Verda, Richard K. Burt
B cells undergo further affinity maturation within lymph node germinal centers by a process of somatic hypermutation (SHM), gene conversion, and class switching recombination (CSR) (Fig. 4). SHM is the term for insertion of point mutations in the vicinity of the variable region exon (Fig. 4) and results in generation of antigen specific high affinity antibodies. Gene conversion is the transfer of a pseudovariable (ipV)gene sequence into the variable region exon (Fig. 4). Both SHM and gene conversion alters the antigen binding site of the immunoglobulin.71-72 CSR involves switching the constant region heavy change (e.g., IgM to IgG) that alters the effector function of the antibody (Fig. 4). The mechanisms involved in DNA SHM, gene conversion, and CSR although incompletely understood probably involve common mechanisms of DNA recognition, targeting, cleavage, and repair.73 The enzyme activation-induced cytidine deaminase (AID) is involved in all three reactions by helping to create the DNA cut or cleavage.65,74-75
Genome Editing for Genetic Lung Diseases
Published in Anthony J. Hickey, Sandro R.P. da Rocha, Pharmaceutical Inhalation Aerosol Technology, 2019
The repair of DSBs by NHEJ is semi-random, resulting in various indel sizes, inversions, or chromosomal translocation.142–145 One strategy to minimize unexpected repair outcome is based on deactivated or nickase version of Cas9.146–152 Rather than generating DSBs, deactivated or nickase Cas9 only binds to target sequence or results a single strand break, respectively.146–152 Fusion of deactivated Cas9 with the transcriptional activation domain VP64 was demonstrated to enhance expression of targeted gene.146,147 Using deactivated Cas9 alone or its fusion with a transcriptional repressor resulted in suppression of gene expression.146 A fusion of Cas proteins with a cytidine deaminase converts cytidine to uridine. Engineered Cas9 base editors can make changes of cytosine (C) to thymine (T) or guanine (G) to adenine (A) substitution within a 5-nt window of the gRNA sequence.21,153,154 Adenine base editors (ABEs) were then created to convert A•T base pairs to G•C in mammalian cells.22 These expanded Cas9 tools provide a programmable introduction of genomic modifications without introducing DSBs. Thus, these therapeutic applications may hold a great potential to reduce the side effects associated with DSBs.
A Diels-Alder polymer platform for thermally enhanced drug release toward efficient local cancer chemotherapy
Published in Science and Technology of Advanced Materials, 2021
Nanami Fujisawa, Masato Takanohashi, Lili Chen, Koichiro Uto, Yoshitaka Matsumoto, Masayuki Takeuchi, Mitsuhiro Ebara
Since the released drug compound L-GEM has a maleimide chain (linker) attached onto it, which differs in structure to the native GEM, the cytotoxicity of L-GEM on MIAPaCa-II cells [32] was evaluated with different L-GEM concentrations (0.01–100 μM) for 24 h at 37°C. Anticancer effect of L-GEM was saturated at the concentration of 0.01 μM, and it was observed that approximately 34% of cells survived post treatment with 100 μM of L-GEM (Figure 6(a)). Figure 6(b) shows the survival numbers of cells after treatment with released L-GEM from the PFMA-L-GEM films. In this study, heating and cell culture experiments were conducted separately to prevent the effects of heating on the cells. First, the films were exposed to AMF for 60 min, and then the released L-GEM (supernatant solution) was collected and applied to MIAPaCa-II cells. For samples wherein heating was not conducted, approximately 28% of the cells were killed. These observations are consistent with the fact that approximately 38 μM of L-GEM was released from the film at 37 °C (Figure 5). On the contrary, approximately 49% of cells were killed, when L-GEM released from the film in the supernatant solution upon heating the cells. Although the number of cells surviving was not significantly decreased by heating, an enhanced cell killing effect was observed by thermally accelerated drug release based on the rDA reaction. Another advantage of using this system is that the structure of L-GEM can avoid deamination of cytidine in the DNA chain by activation-induced (cytidine) deaminase (AID), which enhances the bioavailability and the cytotoxicity effect [33].