Transplantation of progenitors from induced pluripotent stem cells reprogrammed by lentiviral vectors led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Combined transgene-free reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo.
ObjectiveRecombinant adeno-associated virus (AAV)-based vectors are characterized by their robust and safe transgene delivery. The CRISPR/Cas9 and guide RNA (gRNA) system present a promising genome-editing platform, and a recent development of a shorter Cas9 enzyme from Staphylococcus aureus (SaCas9) allows generation of high titer single AAV vectors which carry both saCas9- and gRNA-expression cassettes. Here, we used two AAV-SaCas9 vectors with distinct GFP-targeted gRNA sequences and determined the impact of AAV-SaCas9-gRNA vector treatment in a single cell clone carrying a GFP-expression cassette.ResultsOur results showed comparable GFP knockout efficiencies (40–50%) upon a single low-dose infection. Three consecutive transductions of 25-fold higher doses of vectors showed 80% GFP knockout efficiency. To analyze the “AAV-SaCas9-resistant cell population”, we sorted the residual GFP-positive cells and assessed their permissiveness to super-infection with two AAV-Cas9-GFP vectors. We found the sorted cells were significantly more resistant to the GFP knockout mediated by the same AAV vector, but not by the other GFP-targeted AAV vector. Our data therefore demonstrate highly efficient genome-editing by the AAV-SaCas9-gRNA vector system. Differential susceptibilities of single cell-derived cells to the AAV-SaCas9-gRNA-mediated genome editing may represent a formidable barrier to achieve 100% genome editing efficiency by this vector system.
Background: A catabolic process that has been preserved throughout evolution, autophagy sends cytoplasmic proteins, organelles, and bacteria to lysosomes for destruction. Within its subtypes, acute myeloid leukaemia (AML) exhibits a significant lot of variety, and autophagy's complex function in the disease's genesis and development is also evident. Chromosome translocations and rearrangements, which are often seen in AML, have been discovered to be related with heterozygous deletions and missense mutations of important autophagy genes. Objective: In this review we aimed to discuss the dual role of autophagy in cancer and focusing on the relation between autophagy and acute myeloid leukemia. Methods: PubMed, Google scholar and Science direct were searched using the following keywords: Autophagy, Cancer, Acute myeloid leukaemia and Chromosomal translocations. The authors also screened references from the relevant literature, including all the identified studies and reviews, only the most recent or complete study was included between December 2001 and May 2022. Documents in a language apart from English have been excluded as sources for interpretation. Papers apart from main scientific studies had been excluded as well as documents unavailable as total written text, conversation, conference abstract papers and dissertations.
Conclusion:It is now understood that the process of autophagy has a dual role in the initiation and progression of cancer. In terms of using autophagy in modern treatment modalities like cancer immunotherapy and precision medicine in AML, this might be advantageous. Additionally, the utilization of autophagy biomarkers for clinical response and therapeutic effectiveness prediction would eventually result in better results and the best possible care for AML patients.
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