Genome Editing for Genetic Lung Diseases
Anthony J. Hickey, Sandro R.P. da Rocha in 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.
Immune Reconstitution after Hematopoietic Stem Cell Transplantation
Richard K. Burt, Alberto M. Marmont in Stem Cell Therapy for Autoimmune Disease, 2019
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
The Immunoglobulin Variable-Region Gene Repertoire and Its Analysis
Cliburn Chan, Michael G. Hudgens, Shein-Chung Chow in Quantitative Methods for HIV/AIDS Research, 2017
Centroblasts undergo somatic hypermutation (SHM) of their IgVRG, accumulating point mutations at a rate of 10−4–10−3 mutations per nucleotide per cell division (orders of magnitude higher than is observed in typical genome replication) [29–31]. The enzyme activation-induced cytidine deaminase (AID) is responsible for the initial lesions in the DNA, which are followed by error-prone repair by the enzyme polymerase eta (polη). Centroblasts eventually differentiate into centrocytes and migrate to the light zone, where they interact with FDC and T cells and receive signals to survive, divide, or differentiate. Some surviving cells leave the GC as memory B cells, while others return to the dark zone and undergo further rounds of proliferation and mutation. At this stage, B cells may also undergo class switch recombination, which swaps out the constant region genes (IGHC) encoding the Fc portion of the antibody, changing the effector function of the antibody [29,30].
Recent advances in delivering RNA-based therapeutics to mitochondria
Published in Expert Opinion on Biological Therapy, 2022
Yuma Yamada, Sen Ishizuka, Manae Arai, Minako Maruyama, Hideyoshi Harashima
In order to use DddA for mitochondrial genome editing, several challenges needed to be overcome. The most important of these was that cytidine deaminase is toxic to mammalian cells. Mok et al. split the toxin domain of DddA (DddAtox) into two inactive parts (split-DddAtox half), and then fused each DddAtox half with a TALE protein resulting in a preparation that binds to a specific DNA sequence. When the two TALEs bind to mtDNA, the two split-DddAtox half bound to produce an active form. They achieved this by modifying each construct with a targeting signal (Figure 2(b)) [65]. Another challenge arises from the fact that cytidine deaminase converts C to U rather than to thymine (T). The U base is normally replaced by C with the help of uracil glycosylase. Mok et al. fused an uracil glycosylase inhibitor (UGI) to the TALE-split-DddAtox half to prevent this conversion with the help of the enzyme (Figure 2(b)). They investigated the therapeutic potential of DdCBE by evaluating mitochondrial function in cells carrying the G11922A mutation in the mtDNA coding the ND4. Treatment with the DdCBE that recognizes ND4 resulted in mitochondrial gene editing [65].
Characterising acquired resistance to erlotinib in non-small cell lung cancer patients
Published in Expert Review of Respiratory Medicine, 2019
Niki Karachaliou, Jordi Codony-Servat, Jillian Wilhelmina Paulina Bracht, Masaoki Ito, Martyna Filipska, Carlos Pedraz, Imane Chaib, Jordi Bertran-Alamillo, Andres Felipe Cardona, Miguel Angel Molina, Rafael Rosell
All EGFR TKIs, including erlotinib or osimertinib, activate the NFĸB pathway. NFĸB subsequently induces the expression of the activation-induced cytidine deaminase (AICDA). AICDA is implicated in the development of the T790M mutation after treatment with EGFR TKIs of any generation (Figure 2) [34]. Specifically, AICDA methylates cytosine at position c.2369 and the deamination of 5-methylcytosine to thymine generates the T790M mutation [34]. In the PC9 cell line, there was a significant increase at the mRNA levels of AICDA upon treatment with any type of EGFR TKI [34]. AICDA is expressed in germinal center B lymphocytes and induced upon antigen exposure, causing cytosine deamination and point mutations in immunoglobulin genes. These acquired mutation processes are known as somatic hypermutations. Targeting AICDA activity directly (developing specific inhibitors) or indirectly with NFĸB inhibitors, or using DNA repair inhibitors, could delay the emergence of the T790M resistant mutation or fully prevent it (Table 1) [34].
Emerging DNA methylation inhibitors for cancer therapy: challenges and prospects
Published in Expert Review of Precision Medicine and Drug Development, 2019
Aurora Gonzalez-Fierro, Alfonso Dueñas-González
Since DNA methylation is dynamic, mammalian cells also possess the ability to remove these marks. Passive DNA demethylation was the first to be described. As it is passive, it depends on DNA replication and cell division plus the subsequent lack of action of DNA methylation maintenance pathways. On the contrary, active DNA demethylation is replication-independent and occurs through the active enzymatic removal of the methylcytosine [27]. Among DNA demethylases, the enzyme activation-induced cytidine deaminase (AID) deaminate 5-mC yielding thymidine that is replaced by an unmethylated cytosine by the base-excision repair (BER) pathway. Thus, AID may promote aberrant gene expression by decreasing the promoter DNA methylation of specific genes [28,29]. The family of tet1, tet2, and tet3 (ten-eleven translocation) proteins are also considered active DNA demethylases. These enzymes carry out the hydroxylation of 5-mC to 5-hmC [30], 5-hmC, in turn, is replaced with an unmethylated cytosine by the BER pathway [31]. Recent data demonstrate that several proteins bind to 5-hmC, revealing the possibility that specific proteins may be able to interpret the 5-hmC epigenetic mark and subsequently influence chromatin structure and gene expression [32,33]. Taken together, the establishment and maintenance model of DNA methylation is likely an oversimplification of what actually occurs and all DNMTs in concert with tet enzymes, regulate DNA methylation levels through a dynamic equilibrium of site-specific gain and loss of methylation during development and health and disease conditions.
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