Research

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18 pages, 2558 KiB

Open AccessArticle

Bioimaging Nucleic-Acid Aptamers with Different Specificities in Human Glioblastoma Tissues Highlights Tumoral Heterogeneity

by Elisabete Cruz Da Silva, Sophie Foppolo, Benoît Lhermitte, Marina Ingremeau, Hélène Justiniano, Lorraine Klein, Marie-Pierre Chenard, Romain Vauchelles, Basma Abdallah, Maxime Lehmann, Nelly Etienne-Selloum, Monique Dontenwill and Laurence Choulier

Pharmaceutics 2022, 14(10), 1980; https://doi.org/10.3390/pharmaceutics14101980 - 20 Sep 2022

Cited by 4 | Viewed by 1662

Abstract

Nucleic-acid aptamers are of strong interest for diagnosis and therapy. Compared with antibodies, they are smaller, stable upon variations in temperature, easy to modify, and have higher tissue-penetration abilities. However, they have been little described as detection probes in histology studies of human [...] Read more.

Nucleic-acid aptamers are of strong interest for diagnosis and therapy. Compared with antibodies, they are smaller, stable upon variations in temperature, easy to modify, and have higher tissue-penetration abilities. However, they have been little described as detection probes in histology studies of human tissue sections. In this study, we performed fluorescence imaging with two aptamers targeting cell-surface receptors EGFR and integrin α5β1, both involved in the aggressiveness of glioblastoma. The aptamers’ cell-binding specificities were confirmed using confocal imaging. The affinities of aptamers for glioblastoma cells expressing these receptors were in the 100–300 nM range. The two aptamers were then used to detect EGFR and integrin α5β1 in human glioblastoma tissues and compared with antibody labeling. Our aptafluorescence assays proved to be able to very easily reveal, in a one-step process, not only inter-tumoral glioblastoma heterogeneity (differences observed at the population level) but also intra-tumoral heterogeneity (differences among cells within individual tumors) when aptamers with different specificities were used simultaneously in multiplexing labeling experiments. The discussion also addresses the strengths and limitations of nucleic-acid aptamers for biomarker detection in histology. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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18 pages, 6024 KiB

Open AccessArticle

NanoSIMS Imaging Reveals the Impact of Ligand-ASO Conjugate Stability on ASO Subcellular Distribution

by Emma Kay, Rouven Stulz, Cécile Becquart, Jelena Lovric, Carolina Tängemo, Aurélien Thomen, Dženita Baždarević, Neda Najafinobar, Anders Dahlén, Anna Pielach, Julia Fernandez-Rodriguez, Roger Strömberg, Carina Ämmälä, Shalini Andersson and Michael Kurczy

Pharmaceutics 2022, 14(2), 463; https://doi.org/10.3390/pharmaceutics14020463 - 21 Feb 2022

Cited by 3 | Viewed by 4089

Abstract

The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted endocytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to [...] Read more.

The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted endocytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to efficient target knockdown, specifically in pancreatic β-cells. It is presumed that ASOs dissociate from GLP1 intracellularly to enable an ASO interaction with its target RNA. It is unknown where or when this happens following GLP1-ASO binding to GLP1R and endocytosis. Here, we use correlative nanoscale secondary ion mass spectroscopy (NanoSIMS) and transmission electron microscopy to explore GLP1-ASO subcellular trafficking in GLP1R overexpressing HEK293 cells. We isotopically label both eGLP1 and ASO, which do not affect the eGLP1-ASO conjugate function. We found that the eGLP1 peptide and ASO are not detected at the same level in the same endosomes, within 30 min of GLP1R-HEK293 cell exposure to eGLP1-ASO. When we utilized different linker chemistry to stabilize the GLP1-ASO conjugate, we observed more ASO located with GLP1 compared to cell incubation with the less stable conjugate. Overall, our work suggests that the ASO separates from GLP1 relatively early in the endocytic pathway, and that linker chemistry might impact the GLP1-ASO function. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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17 pages, 14199 KiB

Open AccessArticle

Oligonucleotide Solid Nucleolipid Nanoparticles against Antibiotic Resistance of ESBL-Producing Bacteria

by Phuoc Vinh Nguyen, Clémentine Aubry, Narimane Boudaoud, Alexandra Gaubert, Marie-Hélène Langlois, Mathieu Marchivie, Karen Gaudin, Corinne Arpin, Philippe Barthélémy and Tina Kauss

Pharmaceutics 2022, 14(2), 299; https://doi.org/10.3390/pharmaceutics14020299 - 27 Jan 2022

Cited by 3 | Viewed by 1845 | Correction

Abstract

Antibiotic resistance has become a major issue in the global healthcare system, notably in the case of Gram-negative bacteria. Recent advances in technology with oligonucleotides have an enormous potential for tackling this problem, providing their efficient intrabacterial delivery. The current work aimed to [...] Read more.

Antibiotic resistance has become a major issue in the global healthcare system, notably in the case of Gram-negative bacteria. Recent advances in technology with oligonucleotides have an enormous potential for tackling this problem, providing their efficient intrabacterial delivery. The current work aimed to apply this strategy by using a novel nanoformulation consisting of DOTAU, a nucleolipid carrier, in an attempt to simultaneously deliver antibiotic and anti-resistance oligonucleotides. Ceftriaxone, a third-generation cephalosporin, was formulated with DOTAU to form an ion pair, and was then nanoprecipitated. The obtained solid nanocapsules were characterized using FT-IR, XRD, HPLC, TEM and DLS techniques and further functionalized by the anti-resistance ONα sequence. To obtain an optimal anti-resistance activity and encapsulation yield, both the formulation protocol and the concentration of ONα were optimized. As a result, monodispersed negatively charged nanoparticles of CFX–DOTAU-ONα with a molar ratio of 10:24:1 were obtained. The minimum inhibitory concentration of these nanoparticles on the resistant Escherichia coli strain was significantly reduced (by 75%) in comparison with that of non-vectorized ONα. All aforementioned results reveal that our nanoformulation can be considered as an efficient and relevant strategy for oligonucleotide intrabacterial delivery in the fight against antibiotic resistance. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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16 pages, 3844 KiB

Open AccessArticle

Efficient Treatment of Rheumatoid Arthritis by Degradable LPCE Nano-Conjugate-Delivered p65 siRNA

by Xiaohua Chen, Bailing Zhou, Yan Gao, Kaiyu Wang, Jieping Wu, Ming Shuai, Ke Men and Xingmei Duan

Pharmaceutics 2022, 14(1), 162; https://doi.org/10.3390/pharmaceutics14010162 - 11 Jan 2022

Cited by 9 | Viewed by 2120

Abstract

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases worldwide, causing severe cartilage damage and disability. Despite the recent progress made in RA treatment, limitations remain in achieving early and efficient therapeutic intervention. Advanced therapeutic strategies are in high demand, and [...] Read more.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases worldwide, causing severe cartilage damage and disability. Despite the recent progress made in RA treatment, limitations remain in achieving early and efficient therapeutic intervention. Advanced therapeutic strategies are in high demand, and siRNA-based therapeutic technology with a gene-silencing ability represents a new approach for RA treatment. In this study, we created a cationic delivery micelle consisting of low-molecular-weight (LMW) polyethylenimine (PEI)–cholesterol–polyethylene glycol (PEG) (LPCE) for small interfering RNA (siRNA)-based RA gene therapy. The carrier is based on LMW PEI and modified with cholesterol and PEG. With these two modifications, the LPCE micelle becomes multifunctional, and it efficiently delivered siRNA to macrophages with a high efficiency greater than 70%. The synthesized LPCE exhibits strong siRNA protection ability and high safety. By delivering nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 siRNA, the p65 siRNA/LPCE complex efficiently inhibited macrophage-based cytokine release in vitro. Local administration of the p65 siRNA/LPCE complex exhibited a fast and potent anti-inflammatory effect against RA in a mouse model. According to the results of this study, the functionalized LPCE micelle that we prepared has potential gene therapeutic implications for RA. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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19 pages, 3435 KiB

Open AccessArticle

Impact of circ-0000221 in the Pathogenesis of Hepatocellular via Modulation of miR-661–PTPN11 mRNA Axis

by Marwa Matboli, Mohmed Kamal Hassan, Mahmoud A. Ali, Mohamed Tarek Mansour, Waheba Elsayed, Reham Atteya, Hebatallah Said Aly, Mahmoud El Meteini, Hesham Elghazaly, Sherif El-Khamisy and Sara H. A. Agwa

Pharmaceutics 2022, 14(1), 138; https://doi.org/10.3390/pharmaceutics14010138 - 06 Jan 2022

Cited by 1 | Viewed by 1640

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death in Egypt. A deep understanding of the molecular events occurring in HCC can facilitate the development of novel diagnostic and/or therapeutic approaches. In the present study, we describe a novel axis of hsa-circ-0000221 [...] Read more.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death in Egypt. A deep understanding of the molecular events occurring in HCC can facilitate the development of novel diagnostic and/or therapeutic approaches. In the present study, we describe a novel axis of hsa-circ-0000221–miR-661–PTPN11 mRNA proposed by in silico and in vitro analysis and its role in HCC pathogenesis. We observe a reduction in the expression levels of hsa-circ-0000221 and PTPN11 mRNA in HCC patients’ sera tested compared with control subjects. The reduction occurs with a concomitant increase in the expression of miR-661. Furthermore, the introduction of exogenous hsa-circ-0000221 into Hep-G2 or SNU449 cell lines results in detectable decrease in cellular viability and an increase in apoptotic manifestations that is associated with G1 accumulation and CCDN1 overexpression. Altogether, these findings indicate the tumor-suppressive role of hsa-circ-0000221 in HCC, which acts through miR-661 inhibition, along with a subsequent PTPN11 mRNA increase, where PTPN11 is known to inhibit cell proliferation in many forms of cancer. Our study encourages further investigation of the role of circRNAs in cancer and their potential use as molecular biomarkers. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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16 pages, 1202 KiB

Open AccessArticle

Polyamine–Oligonucleotide Conjugates: 2′-OMe-Triazole-Linked 1,4,7,10-Tetraazacyclododecane and Intercalating Dyes and Their Effect on the Thermal Stability of DNA Duplexes

by Mateusz D. Tomczyk, Mariusz Zalewski, Per T. Jørgensen, Jesper Wengel and Krzysztof Walczak

Pharmaceutics 2022, 14(1), 66; https://doi.org/10.3390/pharmaceutics14010066 - 28 Dec 2021

Cited by 1 | Viewed by 1604

Abstract

Oligonucleotides with the sequences 5′-GTG AU^PA TGC, 5′-GCA TAU^P CAC and 5′-GU^PG ATA U^PGC, where U^P is 2′-O-propargyl uridine, were subjected to post-synthetic Cu(I)-catalyzed azide–alkyne cycloaddition to attach 1,4,7,10-tetraazacyclododecane (cyclen) and two well-known [...] Read more.

Oligonucleotides with the sequences 5′-GTG AU^PA TGC, 5′-GCA TAU^P CAC and 5′-GU^PG ATA U^PGC, where U^P is 2′-O-propargyl uridine, were subjected to post-synthetic Cu(I)-catalyzed azide–alkyne cycloaddition to attach 1,4,7,10-tetraazacyclododecane (cyclen) and two well-known DNA intercalating dyes: thioxanthone and 1,8-naphthalimide. We propose a convenient cyclen protection–deprotection strategy that allows efficient separation of the resulting polyamine–oligonucleotide conjugates from the starting materials by RP-HPLC to obtain high-purity products. In this paper, we present hitherto unknown macrocyclic polyamine–oligonucleotide conjugates and their hybridization properties reflected in the thermal stability of thirty-two DNA duplexes containing combinations of labeled strands, their unmodified complementary strands, and strands with single base pair mismatches. Circular dichroism measurements showed that the B-conformation is retained for all dsDNAs consisting of unmodified and modified oligonucleotides. An additive and destabilizing effect of cyclen moieties attached to dsDNAs was observed. T_m measurements indicate that placing the hydrophobic dye opposite to the cyclen moiety can reduce its destabilizing effect and increase the thermal stability of the duplex. Interestingly, the cyclen-modified U showed significant selectivity for TT mismatch, which resulted in stabilization of the duplex. We conclude the paper with a brief review and discussion in which we compare our results with several examples of oligonucleotides labeled with polyamines at internal strand positions known in the literature. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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Review

Jump to: Research

23 pages, 1454 KiB

Open AccessEditor’s ChoiceReview

Non-Viral Delivery of CRISPR/Cas Cargo to the Retina Using Nanoparticles: Current Possibilities, Challenges, and Limitations

by Ahmed Salman, Ariel Kantor, Michelle E. McClements, Gemma Marfany, Sonia Trigueros and Robert E. MacLaren

Pharmaceutics 2022, 14(9), 1842; https://doi.org/10.3390/pharmaceutics14091842 - 01 Sep 2022

Cited by 15 | Viewed by 3453

Abstract

The discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool have revolutionized the field of molecular biology and generated excitement for its potential to treat a wide range of human diseases. As a gene therapy target, the retina [...] Read more.

The discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool have revolutionized the field of molecular biology and generated excitement for its potential to treat a wide range of human diseases. As a gene therapy target, the retina offers many advantages over other tissues because of its surgical accessibility and relative immunity privilege due to its blood–retinal barrier. These features explain the large advances made in ocular gene therapy over the past decade, including the first in vivo clinical trial using CRISPR gene-editing reagents. Although viral vector-mediated therapeutic approaches have been successful, they have several shortcomings, including packaging constraints, pre-existing anti-capsid immunity and vector-induced immunogenicity, therapeutic potency and persistence, and potential genotoxicity. The use of nanomaterials in the delivery of therapeutic agents has revolutionized the way genetic materials are delivered to cells, tissues, and organs, and presents an appealing alternative to bypass the limitations of viral delivery systems. In this review, we explore the potential use of non-viral vectors as tools for gene therapy, exploring the latest advancements in nanotechnology in medicine and focusing on the nanoparticle-mediated delivery of CRIPSR genetic cargo to the retina. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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22 pages, 1817 KiB

Open AccessEditor’s ChoiceReview

Towards Personalized Allele-Specific Antisense Oligonucleotide Therapies for Toxic Gain-of-Function Neurodegenerative Diseases

by Jacob Helm, Ludger Schöls and Stefan Hauser

Pharmaceutics 2022, 14(8), 1708; https://doi.org/10.3390/pharmaceutics14081708 - 16 Aug 2022

Cited by 10 | Viewed by 3231

Abstract

Antisense oligonucleotides (ASOs) are single-stranded nucleic acid strings that can be used to selectively modify protein synthesis by binding complementary (pre-)mRNA sequences. By specific arrangements of DNA and RNA into a chain of nucleic acids and additional modifications of the backbone, sugar, and [...] Read more.

Antisense oligonucleotides (ASOs) are single-stranded nucleic acid strings that can be used to selectively modify protein synthesis by binding complementary (pre-)mRNA sequences. By specific arrangements of DNA and RNA into a chain of nucleic acids and additional modifications of the backbone, sugar, and base, the specificity and functionality of the designed ASOs can be adjusted. Thereby cellular uptake, toxicity, and nuclease resistance, as well as binding affinity and specificity to its target (pre-)mRNA, can be modified. Several neurodegenerative diseases are caused by autosomal dominant toxic gain-of-function mutations, which lead to toxic protein products driving disease progression. ASOs targeting such mutations—or even more comprehensively, associated variants, such as single nucleotide polymorphisms (SNPs)—promise a selective degradation of the mutant (pre-)mRNA while sparing the wild type allele. By this approach, protein expression from the wild type strand is preserved, and side effects from an unselective knockdown of both alleles can be prevented. This makes allele-specific targeting strategies a focus for future personalized therapies. Here, we provide an overview of current strategies to develop personalized, allele-specific ASO therapies for the treatment of neurodegenerative diseases, such Huntington’s disease (HD) and spinocerebellar ataxia type 3 (SCA3/MJD). Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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24 pages, 1150 KiB

Open AccessEditor’s ChoiceReview

Oligonucleotide Therapeutics: From Discovery and Development to Patentability

by Lara Moumné, Anne-Céline Marie and Nicolas Crouvezier

Pharmaceutics 2022, 14(2), 260; https://doi.org/10.3390/pharmaceutics14020260 - 22 Jan 2022

Cited by 53 | Viewed by 8921

Abstract

Following the first proof of concept of using small nucleic acids to modulate gene expression, a long period of maturation led, at the end of the last century, to the first marketing authorization of an oligonucleotide-based therapy. Since then, 12 more compounds have [...] Read more.

Following the first proof of concept of using small nucleic acids to modulate gene expression, a long period of maturation led, at the end of the last century, to the first marketing authorization of an oligonucleotide-based therapy. Since then, 12 more compounds have hit the market and many more are in late clinical development. Many companies were founded to exploit their therapeutic potential and Big Pharma was quickly convinced that oligonucleotides could represent credible alternatives to protein-targeting products. Many technologies have been developed to improve oligonucleotide pharmacokinetics and pharmacodynamics. Initially targeting rare diseases and niche markets, oligonucleotides are now able to benefit large patient populations. However, there is still room for oligonucleotide improvement and further breakthroughs are likely to emerge in the coming years. In this review we provide an overview of therapeutic oligonucleotides. We present in particular the different types of oligonucleotides and their modes of action, the tissues they target and the routes by which they are administered to patients, and the therapeutic areas in which they are used. In addition, we present the different ways of patenting oligonucleotides. We finally discuss future challenges and opportunities for this drug-discovery platform. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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18 pages, 1591 KiB

Open AccessEditor’s ChoiceReview

Making Sense of Antisense Oligonucleotide Therapeutics Targeting Bcl-2

by Maria Gagliardi and Ana Tari Ashizawa

Pharmaceutics 2022, 14(1), 97; https://doi.org/10.3390/pharmaceutics14010097 - 01 Jan 2022

Cited by 10 | Viewed by 2673

Abstract

The B-cell lymphoma 2 (Bcl-2) family, comprised of pro- and anti-apoptotic proteins, regulates the delicate balance between programmed cell death and cell survival. The Bcl-2 family is essential in the maintenance of tissue homeostasis, but also a key culprit in tumorigenesis. Anti-apoptotic Bcl-2, [...] Read more.

The B-cell lymphoma 2 (Bcl-2) family, comprised of pro- and anti-apoptotic proteins, regulates the delicate balance between programmed cell death and cell survival. The Bcl-2 family is essential in the maintenance of tissue homeostasis, but also a key culprit in tumorigenesis. Anti-apoptotic Bcl-2, the founding member of this family, was discovered due to its dysregulated expression in non-Hodgkin’s lymphoma. Bcl-2 is a central protagonist in a wide range of human cancers, promoting cell survival, angiogenesis and chemotherapy resistance; this has prompted the development of Bcl-2-targeting drugs. Antisense oligonucleotides (ASO) are highly specific nucleic acid polymers used to modulate target gene expression. Over the past 25 years several Bcl-2 ASO have been developed in preclinical studies and explored in clinical trials. This review will describe the history and development of Bcl-2-targeted ASO; from initial attempts, optimizations, clinical trials undertaken and the promising candidates at hand. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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15 pages, 313 KiB

Open AccessReview

CpG Oligodeoxynucleotides for Anticancer Monotherapy from Preclinical Stages to Clinical Trials

by Zhongkun Zhang, Jimmy Chun-Tien Kuo, Siyu Yao, Chi Zhang, Hira Khan and Robert J. Lee

Pharmaceutics 2022, 14(1), 73; https://doi.org/10.3390/pharmaceutics14010073 - 28 Dec 2021

Cited by 22 | Viewed by 2892

Abstract

CpG oligodeoxynucleotides (CpG ODNs), the artificial versions of unmethylated CpG motifs that were originally discovered in bacterial DNA, are demonstrated not only as potent immunoadjuvants but also as anticancer agents by triggering toll-like receptor 9 (TLR9) activation in immune cells. TLR9 activation triggered [...] Read more.

CpG oligodeoxynucleotides (CpG ODNs), the artificial versions of unmethylated CpG motifs that were originally discovered in bacterial DNA, are demonstrated not only as potent immunoadjuvants but also as anticancer agents by triggering toll-like receptor 9 (TLR9) activation in immune cells. TLR9 activation triggered by CpG ODN has been shown to activate plasmacytoid dendritic cells (pDCs) and cytotoxic T lymphocytes (CTLs), enhancing T cell-mediated antitumor immunity. However, the extent of antitumor immunity carried by TLR agonists has not been optimized individually or in combinations with cancer vaccines, resulting in a decreased preference for TLR agonists as adjuvants in clinical trials. Although various combination therapies involving CpG ODNs have been applied in clinical trials, none of the CpG ODN-based drugs have been approved by the FDA, owing to the short half-life of CpG ODNs in serum that leads to low activation of natural killer cells (NK cells) and CTLs, along with increases of pro-inflammatory cytokine productions. This review summarized the current innovation on CpG ODNs that are under clinical investigation and explored the future direction for CpG ODN-based nanomedicine as an anticancer monotherapy. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

23 pages, 5348 KiB

Open AccessReview

CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications

by Hao Cheng, Feng Zhang and Yang Ding

Pharmaceutics 2021, 13(10), 1649; https://doi.org/10.3390/pharmaceutics13101649 - 09 Oct 2021

Cited by 32 | Viewed by 5626

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered [...] Read more.

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing. Full article

(This article belongs to the Special Issue Oligonucleotides and Nucleic-Acid-Based Strategies to Address Therapeutic Challenges: From the Bench to the Bedside)

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