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Molecular Biology and Gene Therapy
Published in R James A England, Eamon Shamil, Rajeev Mathew, Manohar Bance, Pavol Surda, Jemy Jose, Omar Hilmi, Adam J Donne, Scott-Brown's Essential Otorhinolaryngology, 2022
Hereditary information in eukaryotes is stored in the form of double-stranded deoxyribonucleic acid (DNA) and is referred to as the genome. DNA forms a double helix structure bonding complementary pairs of nucleotides, such as adenine (A) with thymine (T) and cytosine (C) with guanine (G). Within the coding part of DNA nucleotides on each strand are grouped in triplets, known as codons. Each codon sequence determines a single specific amino acid. Some codons are ‘stop’ codons, constituting a signal for arrest of translation. The overwhelming majority of DNA (99.9%) exists in the cell nucleus as the nuclear genome and the remaining DNA forms the mitochondrial genome, encoding 37 genes.
Antibiotics: The Need for Innovation
Published in Nathan Keighley, Miraculous Medicines and the Chemistry of Drug Design, 2020
The sequence of bases on the mRNA are organises as discrete triplet codes; three bases code for one amino acid, and different sequences of triplet bases, each called a codon, code for a particular amino acid. For example, GAC codes for the amino acid aspartic acid. The process of translation is not as straightforward as the amino acids lining up along the mRNA strand. A second type of RNA, called transfer-RNA, is involved. The tRNA is a smaller molecule and is responsible for binding free amino acids in the cytoplasm and bringing them to the mRNA template. The tRNA molecules contain an anticodon, which is the opposite sequence to that on the mRNA and is complementary, therefore the tRNA carrying the amino acid can bind to mRNA. Different codons are also present on the mRNA strand to determine where to start and terminate translation into a protein.
Human Bcl-2 Antisense Therapy for Lymphomas
Published in Eric Wickstrom, Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Finbarr E. Cotter, Andrew Webb, Paul Clarke, David Cunningham
The initiating codon of mRNA, the AUG start site, is the point from which protein synthesis is initiated. AO targeted to contain complementary sequences to this area are most frequently used in antisense experiments. Other successful sites include the 5' cap site, the mRNA site that initiates message reading rather than protein synthesis (Daaka and Wickstrom, 1990; Bacon and Wickstrom, 1991) the first splice donor-acceptor site (Daaka and Wickstrom, 1990), the polyadenylation signal, 5' to the polyA tail (Goodchild et al., 1988) and to the junctional sequences of tumor associated chromosomal translocations (Szczylik et al., 1991) Sequences targeted to the RNA loop structures show logarithmically greater hybridization than those in the areas flanking the loop (Lima et al., 1992). The bcl-2 AO were targeted to the AUG start site initially and proved useful, as determined by their ability to downregulate Bcl-2 protein and induce apoptosis in lymphoma cell lines.
Emergence of mRNA vaccines in the management of cancer
Published in Expert Review of Vaccines, 2023
Mohamad Irfan Mohamad Razif, Nabilah Nizar, Nur Hannah Zainal Abidin, Syasya Nasuha Muhammad Ali, Wan Nurul Najihah Wan Zarimi, Junaidi Khotib, Deny Susanti, Muhammad Taufiq Mohd Jailani, Muhammad Taher
After identifying the antigen of choice of the target protein, several steps of gene sequencing, synthesizing, and cloning into the DNA template plasmid are performed [15]. An mRNA-based cancer vaccine production process is initiated by designing a DNA template or pDNA that consists of the ORF, flanking 5’- and 3’-UTRs as well as a primer binding site that contains available RNA polymerase recognition sites to initiate in vitro transcription [14]. Moreover, they also stated that the efficacy of translation of the target protein can be improved by using the codon concurrency that affects the amino acids on the mRNA. The sequence could also be designed in silico producing a variety of antigen sequences that have efficient leader sequences, optimal codon usage, increased neutralization, and reduced cross-reactivity [15]. An interesting method that could be used to increase the efficacy of protein expression or mRNA translation is by substitution of rare codons with regularly used identical codons [16]. The UTRs play an important role in regulating the protein expression, rates of degradation and translation of mRNA by interacting with different RNA-binding proteins [14]. It is also stated that the 5’-UTR initiates the translation and formation of preinitiation complexes and stabilizes the mRNA. However, the efficacy of translation can be improved by shortening the length of 3’-UTR. An optimal template sequence with high stability and translating efficacy is ideal in the formulation of mRNA vaccines.
SCN1A as a therapeutic target for Dravet syndrome
Published in Expert Opinion on Therapeutic Targets, 2023
Ataluren, also known as PTC124, is a drug designed to increase protein expression in patients with nonsense pathogenic variants. A nonsense pathogenic variant is a nucleotide change that results in a ‘stop codon,’ which leads to one of UAA, UAG, or UGA in the mRNA coding sequence. Normally, a stop codon triggers premature termination of translation, and the truncated polypeptide is degraded. However, certain agents, such as aminoglycoside antibiotics, have been shown to promote read-through of nonsense codons, allowing continued translation and ultimately production of a full-length protein product [62]. Gentamicin has been trialed in Duchenne muscular dystrophy and cystic fibrosis due to nonsense pathogenic variants, albeit with limited clinical benefit and concern for side effects such as ototoxicity and nephrotoxicity [63–66].
Targeted next-generation sequencing revealed a novel homozygous mutation in the LRBA gene causes severe haemolysis associated with Inborn Errors of Immunity in an Indian family.
Published in Hematology, 2022
Prabhakar Kedar, Rashmi Dongerdiye, Shanmukhaiah Chandrakala, Umair Ahmed Bargir, Manisha Madkaikar
The LRBA gene is located on chromosome 4 at 4q31.3. It is a 2863 codon spanning into 57 exons. So far, a total of 167 mutations have been reported in the Human Gene Mutation Database (HGMD®) (http://www.biobase-international.com/product/hgmd), which includes 82 missense/nonsense mutations, 18 splicing mutations, 30 Small deletions, 14 Small insertions, 2 Small indels, 15 Gross deletions, 4 Gross insertions, 1 Complex rearrangement and 1 Regulatory modification [10,11]. Mutations in the LRBA are spread throughout the gene, affecting structural rearrangements due to the large numbers of mutations in exons. The discovery of the next-generation sequencing (NGS) technology plays an essential role in diagnosing several unexplained haemolytic anaemias and rare variable immunological disorders [12]. We report an unusual report of a female patient born of a consanguineous marriage, presented with severe anemia and jaundice with a history of multiple blood transfusions of unknown cause till 5yrs of age. Targeted Next-generation sequencing (t-NGS) technology identified a novel mutation in the LRBA gene. Her initial haematological and IEI work-up was normal. However, a timely decision to perform NGS helped diagnose underlying IEI due to LRBA deficiency in the patient who was transfusion-dependent and had a significant family history.