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Review
. 2024 Oct 20;10(21):e39323.
doi: 10.1016/j.heliyon.2024.e39323. eCollection 2024 Nov 15.

Emerging Gene-editing nano-therapeutics for Cancer

Najma Nujoom  1 , Manzoor Koyakutty  1 , Lalitha Biswas  1 , Thangarajan Rajkumar  1 , Shantikumar V Nair  1
Affiliations
Review

Emerging Gene-editing nano-therapeutics for Cancer

Najma Nujoom et al. Heliyon. .

Abstract

Remarkable progress has been made in the field of genome engineering after the discovery of CRISPR/Cas9 in 2012 by Jennifer Doudna and Emmanuelle Charpentier. Compared to any other gene-editing tools, CRISPR/Cas9 attracted the attention of the scientific community because of its simplicity, specificity, and multiplex editing possibilities for which the inventors were awarded the Nobel prize for chemistry in 2020. CRISPR/Cas9 allows targeted alteration of the genomic sequence, gene regulation, and epigenetic modifications using an RNA-guided site-specific endonuclease. Though the impact of CRISPR/Cas9 was undisputed, some of its limitations led to key modifications including the use of miniature-Cas proteins, Cas9 Retron precise Parallel Editing via homologY (CRISPEY), Cas-Clover, or development of alternative methods including retron-recombineering, Obligate Mobile Element Guided Activity(OMEGA), Fanzor, and Argonaute proteins. As cancer is caused by genetic and epigenetic alterations, gene-editing was found to be highly useful for knocking out oncogenes, editing mutations to regain the normal functioning of tumor suppressor genes, knock-out immune checkpoint blockade in CAR-T cells, producing 'off-the-shelf' CAR-T cells, identify novel tumorigenic genes and functional analysis of multiple pathways in cancer, etc. Advancements in nanoparticle-based delivery of guide-RNA and Cas9 complex to the human body further enhanced the potential of CRISPR/Cas9 for clinical translation. Several studies are reported for developing novel delivery methods to enhance the tumor-specific application of CRISPR/Cas9 for anticancer therapy. In this review, we discuss new developments in novel gene editing techniques and recent progress in nanoparticle-based CRISPR/Cas9 delivery specific to cancer applications.

Keywords: Alternatives of CRISPR/Cas9; CRISPR/Cas9; Cancer; Gene-editing; Nanoparticles; Non-viral delivery.

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Conflict of interest statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Shantikumar Nair reports was provided by Amrita Vishwa Vidyapeetham Amrita Centre for Nanosciences and Molecular Medicine. Shantikumar Nair reports a relationship with Amrita Vishwa Vidyapeetham Amrita Centre for Nanosciences and Molecular Medicine that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Schematic of representation of A) mechanism of gene-editing using CRISPR/Cas9 via the double-strand breaks. The Cas9/sgRNA ribonucleoprotein (RNP) complex recognize and binds the complimentary sequence of guide RNA and cleaves the target DNA in the template and non-template strand using RuvC and HNH domains of Cas9 to produce double strand breaks (DSBs). Followed by the formation of DSBs, cell's DNA repair machinery repair the break via either the non-homologous end-joining (NHEJ) pathway or the homology-directed repair (HDR) pathway. B&C) Mechanism of transcriptional and epigenetic regulation by dCas9. Dead Cas9 (dCas9) fused with active domains of other proteins perform different functions such as transcriptional and epigenetic regulation of gene expression. D) Base editors (BEs) and Prime editors (PEs). dCas9 or Cas9 nickase (Cas9n) fused with adenine and cytidine base editors function as sequence specific base editors. Cas9n fused with a reverse transcriptase and prime editing guide RNA (pegRNA) having guide sequence and reverse transcriptase template with specific gene modification are known as prime editors (PEs).
Fig. 2
Fig. 2
Diagrammatic representation of systemic delivery of nanoparticle-based CRISPR/Cas9 gene therapy. A) Different formats of CRISPR cargoes include plasmid DNA, mRNA and Cas9/sgRNA ribonucleoprotein (RNP) complex. B) representative nanoparticles for CRISPR/Cas9 delivery include liposomes, targeted lipid nanoparticles, polymeric NPs, gold NPs and organic silica.
Fig. 3
Fig. 3
Ex-vivo gene editing approach in clinical trials. Target cell population are isolated from blood collected from either a healthy donor or from patients. Cells are gene edited using different methods such as viral vectors, electroporation and using nanoparticles. The edited cells are selected and enriched in the laboratory and infused back to the patient.
Fig. 4
Fig. 4
Schematic representation of the process of retron recombineering inside a cell. Retron plasmids encode sequences of msr/msd sequence with specific mutations, reverse transcriptase (RT) and single strand annealing proteins (SSAP). Inside the cells these plasmids produce multicopy satellite DNA (msd) donor template using reverse transcriptase. These msd donor template with specific mutations anneal to the replicating DNA at the target DNA with sequence complimentary to the msd donor template.
Fig. 5
Fig. 5
Schematic representation of the mechanism of CRISPEY. CRISPEY donor plasmid contains gRNA-msr/msd donor template sequence, and sequence for Cas9-reverse transcriptase (RT). Inside the cells, RT produce gRNA – donor ssDNA, which complex with the cas9 enzyme and perform gene editing (knocking in) at the target sequence.
Fig. 6
Fig. 6
Representative images of A) Cas-CLOVER: mechanism of gRNA/dCas9 guided dimerization of monomeric Clo51 endonuclease at the target dsDNA. Fusion of Cas9 – monomeric Clo51 protein with guide RNA complex binds to the left and right side of the target sequence. Binding of left and right gRNA/Cas9/Clo51 complex result in dimerization of Clo51 endonuclease. Dimerized Clo51 cleaves the double stranded DNA (dsDNA) at the centre and produce DSBs. B) Argonaute protein. Argonaute proteins is multimeric protein with PAZ, PIWI, N and MID domains. They use a single stranded DNA with 5′-phosphate group as the guide DNA and produce DSBs at the target site. and C) Fanzor proteins use a guide RNA known as omega RNA (ωRNA) to produce staggered double strand break at the target DNA.
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