Attenuated diphtheria toxin mediates siRNA delivery

Arnold, Amy E.; Smith, Laura J.; Beilhartz, Greg L.; Bahlmann, Laura C.; Jameson, Emma; Melnyk, Roman A.; Shoichet, Molly S.

doi:10.1126/sciadv.aaz4848

Cited by 17 publications

(24 citation statements)

References 49 publications

(54 reference statements)

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“…Utilizing bacterial toxin-based systems to deliver cargoes into the cytosol of cells has been explored with various toxins such as DT ( Arnold et al, 2020 ; Auger et al, 2015 ; Vidimar et al, 2020 ), BoNTs ( Bade et al, 2004 ; McNutt et al, 2021 ; Miyashita et al, 2021 ), anthrax toxin ( Milne et al, 1995 ; Young and Collier, 2007 ), Pseudomonas exotoxin A ( Ryou et al, 2016 ; Verdurmen et al, 2015 ), heat-labile enterotoxin ( Chen et al, 2015 ), Shiga toxins ( Ryou et al, 2016 ; Schmit et al, 2019 ), Clostridium botulinum C2 toxin ( Barth et al, 2002 ; Fahrer et al, 2013 ), and Photorhabdus luminescens Tc toxins ( Ng Ang et al, 2019 ; Roderer et al, 2019 ). The mechanisms of membrane translocation differ among these toxins.…”

Section: Discussionmentioning

confidence: 99%

“…This is possibly because the RNA components dissociate from Cas proteins during membrane translocation; there-fore, the crRNA components must be co-delivered by other methods. A recent study showed that a DT-based system can be used to deliver siRNA into cells ( Arnold et al, 2020 ), suggesting that crRNA may be delivered using AB toxin-based systems.…”

Section: Discussionmentioning

confidence: 99%

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Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms

et al. 2022

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms

et al. 2022

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“…However, in its current state, the CRISPA approach needs to be combined with other viral or non-viral methods that are capable of delivering the single guide RNA into cells. Notably, a transient toxin-based siRNA delivery strategy was developed just recently on the basis of an attenuated diphtheria toxin ( Arnold et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin

et al. 2021

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Translating the CRISPR/Cas9 genome editing technology into clinics is still hampered by rather unspecific, unsafe and/or inconvenient approaches for the delivery of its main components - the Cas9 endonuclease and a guide RNA - into cells. Here, we describe the development of a novel transient and non-viral Cas9 delivery strategy based on the translocation machinery of the Bacillus anthracis anthrax toxin, PA (protective antigen). We show that Cas9 variants fused to the N-terminus of the lethal factor or to a hexahistidine tag are shuttled through channels formed by PA into the cytosol of human cells. As proof-of-principle, we applied our new approach, denoted as CRISPA, to knock out lipolysis-stimulated lipoprotein receptor (LSR) in the human colon cancer cell line HCT116 and green-fluorescent protein (GFP) in human embryonic kidney 293T cells stably expressing GFP. Notably, we confirmed that the transporter PA can be adapted to recognize specific host cell-surface receptor proteins and may be optimized for cell type-selective delivery of Cas9. Altogether, CRISPA provides a novel, transient and non-viral way to deliver Cas9 into specific cells. Thus, this system is an additional step towards safe translation of the CRISPR/Cas9 technology into clinics.

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“…First, they conjugated DT with cysteine (Cys); next, the Cys was modified with a maleimide cross-linked to dibenzocyclooctyne (DBCO) group and the siRNA molecules were attached to the DBCO. When the conjugated DT was added to GSCs, significant reductions in the mRNA levels of ITGB1 and eIF-3b were observed ( 127 ). More studies using bacterial toxins for RNAi delivery should be performed in GBM cells and GBM mouse models.…”

Section: Delivery Strategies For Rnai-based Therapies Against Gbmmentioning

confidence: 99%

Targeting Non-coding RNA for Glioblastoma Therapy: The Challenge of Overcomes the Blood-Brain Barrier

Sharma

Calderón

Vivas‐Mejía

2021

Front. Med. Technol.

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Glioblastoma (GBM) is the most malignant form of all primary brain tumors, and it is responsible for around 200,000 deaths each year worldwide. The standard therapy for GBM treatment includes surgical resection followed by temozolomide-based chemotherapy and/or radiotherapy. With this treatment, the median survival rate of GBM patients is only 15 months after its initial diagnosis. Therefore, novel and better treatment modalities for GBM treatment are urgently needed. Mounting evidence indicates that non-coding RNAs (ncRNAs) have critical roles as regulators of gene expression. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are among the most studied ncRNAs in health and disease. Dysregulation of ncRNAs is observed in virtually all tumor types, including GBMs. Several dysregulated miRNAs and lncRNAs have been identified in GBM cell lines and GBM tumor samples. Some of them have been proposed as diagnostic and prognostic markers, and as targets for GBM treatment. Most ncRNA-based therapies use oligonucleotide RNA molecules which are normally of short life in circulation. Nanoparticles (NPs) have been designed to increase the half-life of oligonucleotide RNAs. An additional challenge faced not only by RNA oligonucleotides but for therapies designed for brain-related conditions, is the presence of the blood-brain barrier (BBB). The BBB is the anatomical barrier that protects the brain from undesirable agents. Although some NPs have been derivatized at their surface to cross the BBB, optimal NPs to deliver oligonucleotide RNA into GBM cells in the brain are currently unavailable. In this review, we describe first the current treatments for GBM therapy. Next, we discuss the most relevant miRNAs and lncRNAs suggested as targets for GBM therapy. Then, we compare the current drug delivery systems (nanocarriers/NPs) for RNA oligonucleotide delivery, the challenges faced to send drugs through the BBB, and the strategies to overcome this barrier. Finally, we categorize the critical points where research should be the focus in order to design optimal NPs for drug delivery into the brain; and thus move the Oligonucleotide RNA-based therapies from the bench to the clinical setting.

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Attenuated diphtheria toxin mediates siRNA delivery

Cited by 17 publications

References 49 publications

Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms

Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms

CRISPA: A Non-viral, Transient Cas9 Delivery System Based on Reengineered Anthrax Toxin

Targeting Non-coding RNA for Glioblastoma Therapy: The Challenge of Overcomes the Blood-Brain Barrier

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