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Choerospondias axillaris (Hog plum)
Published in Mahendra Rai, Shandesh Bhattarai, Chistiane M. Feitosa, Wild Plants, 2020
Dexamethason induces immunodeficiency in the patients who take it for a long time due to thymus atrophy, and as a result, thymocyte apoptosis as well as adenine deaminase deficiency activity is lowered. TFC can promote the immune responses of the body, which provides powerful evidence for the treatment of immunodeficiency. TFC could inhibit dexamethason induced thymocyte apoptosis and promote proliferation differentiation of the thymocytes in different periods. TFC could also facilitate the restoration of adenine deaminase activation of the thymocytes in the thymus atrophy mice (Li et al. 1998).
Recombinant DNA Technology and Gene Therapy Using Viruses
Published in Patricia G. Melloy, Viruses and Society, 2023
Starting 30 years ago, researchers explored the use of gene therapy to treat severe combined immunodeficiency (SCID), a disorder in which the patient’s immune system never fully develops making the person prone to acquiring life-threatening infections. Researchers focused on two forms of SCID, caused by different genes. For adenine deaminase–severe combined immunodeficiency (ADA-SCID), a retroviral vector was used to replace the defective ADA gene, using an ex vivo approach where the patient’s T cells were modified and then transplanted back into the body. It is believed that the therapy worked, but patients were receiving an enzyme-based treatment during the entire gene therapy course. Another form of SCID involving a mutation in a gene on the X chromosome (X-SCID) was also treated using a retroviral vector. However, several children in the trial developed leukemia a few years after treatment, most likely due to a viral integration event affecting cell growth regulation (Minkoff and Baker 2004; Lostroh 2019; Colavito 2007; Kurreck and Stein 2016; Anguela and High 2019; Dunbar et al. 2018). An adverse outcome was also seen with a gene therapy trial to treat a deficiency in the metabolic enzyme ornithine transcarbamylase in 1999. One patient died and the trial was halted (Lostroh 2019; Colavito 2007; Mukherjee 2016; Minkoff and Baker 2004). This tragedy prompted a period of basic research into new viral vector delivery systems and more oversight of gene therapy trials that continues to this day (Collins and Gottlieb 2019). Since that time, new technologies have been used to tackle SCID. Strimvelis is a type of gene therapy product using an updated viral vector delivery system to treat ADA-SCID patients. However, demand for this new therapy is quite low due to the small number of patients with the disease, and it is unclear how long the product will be marketed (Anguela and High 2019; Li and Samulski 2020; Aiuti, Roncarolo, and Naldini 2017; Dunbar et al. 2018). Another gene therapy product, alipogene tiparvovec, also known as Glybera, is used to treat familial lipoprotein lipase deficiency using an AAV vector. It was approved for use in Europe in 2012 (Lostroh 2019; Kurreck and Stein 2016; Wang, Tai, and Gao 2019; Li and Samulski 2020). However, the treatment is quite expensive, costing over a million dollars a treatment (Kurreck and Stein 2016). Glybera is no longer being marketed in Europe (Shahryari et al. 2019). Rexin-G is an anti-cancer gene therapy that works by blocking cell cycle progression by expressing a modified version of a gene called cyclin G using a retroviral vector. It is being tested right now in clinical trials of people with advanced pancreatic cancer (Lostroh 2019; Shahryari et al. 2019). Although the development of gene therapy treatments is a long road with many possible setbacks, several gene therapy products are approved for use in the United States right now, as described next.
CRISPR/Cas9 gene editing therapies for cystic fibrosis
Published in Expert Opinion on Biological Therapy, 2021
Base editing approaches enable precise editing of single nucleotides, particularly cytosine to thymine (C > T) [93], or adenine to guanine (A > G) [94,95]. Adenine base editing utilizes a fused pair of engineered enzymes: an engineered form of Cas9 that forms single-stranded rather than double-stranded DNA breaks (commonly referred to as Cas9 nickase), and an adenosine deaminase obtained by directed evolution which is capable of hydrolyzing adenine into inosine (which is interpreted by replication machinery as guanine) [94]. At present, several versions of this fusion protein exist, but the mechanism of editing is consistent [93,95,96]. The modified Cas9 fusion protein is guided to the target locus by a crRNA, and DNA is bound and ‘opened.’ The adenine deaminase base editor (ABE) hydrolyses adenine residues within a five-nucleotide editing window. Then, the modified Cas9 nicks the non-edited strand, which prompts the cell to repair the non-edited strand based on complementarity to the edited strand, therefore permanently incorporating the desired edit [94]. The lack of DSB formation leads to a lower occurrence of apoptosis and fewer risks of off-target DNA edits [93,94]. However, adenine base editors indiscriminately edit any and all adenines within the editing window, which can lead to unwanted changes if a canonical adenine is present adjacent to the mutated residue.
Liver-directed gene-based therapies for inborn errors of metabolism
Published in Expert Opinion on Biological Therapy, 2021
Pasquale Piccolo, Alessandro Rossi, Nicola Brunetti-Pierri
CRISPR/Cas9 can also be exploited for targeted base editing. The base editor machinery is made of a catalytically inactive Cas9 fused with cytidine or adenine deaminase enzyme and a guide-RNA. Cas9 binding generates local DNA duplex denaturation enabling cytidine deamination into uracil, that pairs as thymidine, or adenine deamination into inosine that is read as guanine by polymerases [99]. Cytosine base editors (CBE) with enhanced editing efficiency have recently been developed and were used to introduce premature stop codon into Pcsk9 or Hpd genes in adult mice and embryos, as a safer alternative to DSB-mediated gene disruption [92,94,100,101]. Nevertheless, correction of disease-causing mutations remains the most attractive goal of base editing. Using dual-AAV split-intein technology to deliver CBE, correction of phenylketonuria in mouse model was achieved after editing of the mutated gene in up to 25% of cells [102]. For the adenosine base editors (ABE) a modified E. coli tRNA adenosine deaminase enzyme combined to a catalytically impaired Cas9 and a sgRNA have been used [103] to correct a A > G splice-site mutation in a HT1 mouse model. Although base editing resulted in correction of a small amount of hepatocytes (about 3%), this was sufficient to achieve partial phenotype correction because of selective growth advantage of corrected hepatocytes [104]. Moreover, DNA base editors can also edit RNA, and they can generate both genomic and transcriptomic off-targets edits [105–107].
Is subretinal AAV gene replacement still the only viable treatment option for choroideremia?
Published in Expert Opinion on Orphan Drugs, 2021
Ruofan Connie Han, Lewis E. Fry, Ariel Kantor, Michelle E. McClements, Kanmin Xue, Robert E. MacLaren
To address the limitations of HDR-mediated gene editing, CRISPR-Cas-mediated single-base pair editing systems (or ‘base editing’ systems) have been devised which allow for targeted restoration of single-base mutations. Two classes of DNA base editors have been described to date: cytosine base editors and adenine base editors [58,59]. DNA base editors encompass two key components. The first is an inactivated Cas enzyme (or Cas nickase, nCas9) which retains its programmable DNA binding ability, but which has lost its ability to generate DSBs. The second is a single-stranded DNA-modifying enzyme (cytidine or adenine deaminase) fused to nCas9 for targeted nucleotide alteration. Collectively, all four transition mutations (A > G, C > T, G > A and T > C) can be installed with the available CRISPR/Cas base editor systems. Recently, Kurt et al. described the engineering of two novel base editor architectures that can efficiently induce targeted C-to-G base transversions [60]. In addition, recent studies report dual-base editor systems for combinatorial editing in human cells. Together, these new base editors expand the range of DNA base editors to transversion mutations and may allow for targeting of more complex compound edits than are currently achievable by a single DNA base editor.