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Gene Therapy for Cancer Treatment
Published in Yashwant V. Pathak, Gene Delivery Systems, 2022
Manish P. Patel, Mansi S. Shah, Mansi N. Athalye, Jayvadan K. Patel
Retroviral vectors are primarily used by approved gene transfer protocols [Rothand Cristiano 1997]. Retroviruses have a linear single-stranded RNA of around 7 to 10 kb and have a lipid envelope. It consists of a double0stranded RNA genome, on which there are two long terminal repeats (LTRs), namely 5’ and 3’. Adjacent to the 5’ LTR is the region at which the transfer RNA (tRNA) primer binds and then gives signals for the reverse transcription process [Seth 2005]. Unlike other RNA viruses, these viruses are able to reverse-transcribe their genetic blueprint of positive, single-stranded RNA into dsDNA and insert it into the host cell genome. A major benefit of retroviral vectors for cancer gene therapy is their ability for transgene expression in only dividing cells to prevent undesired expression in non-dividing cells of surrounding tissues. The most notable disadvantages are less ability for cell specificity and the chances of insertional mutagenesis. However, this type of vector has been the most commonly used vehicle for gene transfer in the clinic. The risk of insertional oncogenesis has been seen in a clinical trial of X-linked severe combined immunodeficiency (X-SCID) infants in 2003, which has limited the use of retroviral gene transfer systems in human subjects [Akbulut et al. 2015]. A new technique called non-integrating retrovirus-based CRISPR/Cas9 vectors has been developed for targeted gene knockout. Creating vectors that target specific genes would help in developing therapeutic strategies by avoiding insertional mutagenesis issues [Goswami et al. 2019].
Genome Editing and Gene Therapies: Complex and Expensive Drugs
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2020
All four nucleases cleave DNA in a site-specific manner to form double-strand breaks (DSBs) as a consequence of which endogenous repair mechanisms become active leading to the respective genome modifications. These repair pathways are either the non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is error-prone and tends to introduce a frameshift via insertions or deletions (indels) at the target site of the respective nuclease; the resulting premature stop codon prevents correct translation leading to nonfunctional protein synthesis. Such a gene knockout by disrupting its expression is essential for determining the gene’s function, e.g., in connection with developing disease models. In contrast, HDR (the efficiency of which is significantly lower than for knockout) allows for targeted integration (gene knock-in). This requires co-transfection of the nuclease with a donor plasmid containing the DNA segment of interest that is flanked by homology sequences identical to those within the target region. HDR enables modifications from single nucleotide exchanges to large insertions.
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Published in Michael Hehenberger, Zhi Xia, Huanming Yang, Our Animal Connection, 2020
Michael Hehenberger, Zhi Xia, Huanming Yang
Gene targeting is often used to inactivate single genes. Such gene “knockout” experiments have elucidated the roles of numerous genes in embryonic development, adult physiology, aging, and disease. To date, most mouse genes (approximately half of the genes in the mammalian genome) have been knocked out. “Knockout mice” are now routinely created and distributed by institutions such as the Jackson Lab.
Development of capability for genome-scale CRISPR-Cas9 knockout screens in New Zealand
Published in Journal of the Royal Society of New Zealand, 2018
Francis W. Hunter, Peter Tsai, Purvi M. Kakadia, Stefan K. Bohlander, Cristin G. Print, William R. Wilson
Few New Zealand scientists will be unaware of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) as a remarkable new technology that is transforming many aspects of biological research. This ancient apparatus of adaptive immunity, widely distributed in archaea and bacteria (Bhaya et al. 2011), is based on the recognition of foreign nucleic acids rather than peptides, facilitating its adaptation as a molecular biology tool. CRISPR-Cas systems have been reengineered to seek out and manipulate genes in living cells (including higher eukaryotes) providing techniques for precise re-writing of DNA sequences (gene editing) (Komor et al. 2016; Paquet et al. 2016), inactivating genes (gene knockout) (Kleinstiver et al. 2016), transcriptional or epigenetic modification of levels of gene expression (Larson et al. 2013; Perez-Pinera et al. 2013), dynamic imaging of genomic loci and RNA movement in cells (Chen et al. 2013) and as a diagnostic for facile and extremely sensitive detection of specific nucleic acids (Gootenberg et al. 2017). These tools are already finding widespread and diverse application in the biological and biomedical sciences in New Zealand and may yet play an important role in our biosecurity through, for example, CRISPR-mediated gene drive technology (Hammond et al. 2015).