ß-Cyclodextrin-based geometrically frustrated amphiphiles as one-component, cell-specific and organ-specific nucleic acid delivery systems.

We introduce an innovative ß-cyclodextrin (ßCD)-prototype for delivering nucleic acids: "geometrically frustrated amphiphiles (GFAs)." GFAs are designed with cationic centers evenly distributed across the primary O6 and secondary O2 positions of the ßCD scaffold, while hydrophobic tails are anchored at the seven O3 positions. Such distribution of functional elements differs from Janus-type architectures and enlarges the capacity for accessing strictly monodisperse variants. Changes at the molecular level can then be correlated with preferred self-assembly and plasmid DNA (pDNA) co-assembly behaviors. Specifically, GFAs undergo pH-dependent transition between bilayered to monolayered vesicles or individual molecules. GFA-pDNA nanocomplexes exhibit topological and internal order characteristics that are also a function of the GFA molecular architecture. Notably, adjusting the pKa of the cationic heads and the hydrophilic-hydrophobic balance, pupa-like arrangements implying axial alignments of GFA units flanked by quasi-parallel pDNA segments are preferred. In vitro cell transfection studies revealed remarkable differences in relative performances, which corresponded to distinct organ targeting outcomes in vivo. This allowed for preferential delivery to the liver and lung, kidney or spleen. The results collectively highlight cyclodextrin-based GFAs as a promising class of molecular vectors capable of finely tuning cell and organ transfection selectivity.

Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells.

CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. Viral vectors risk genomic integration and immunogenicity while non-viral delivery systems are challenging to adapt to different CRISPR cargos, and many are highly cytotoxic. The arginine-alanine-leucine-alanine (RALA) cell penetrating peptide is an amphiphilic peptide that self-assembles into nanoparticles through electrostatic interactions with negatively charged molecules before delivering them across the cell membrane. This system has been used to deliver DNAs, RNAs, and small anionic molecules to primary cells with lower cytotoxicity compared to alternative non-viral approaches. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA was able to effectively encapsulate and deliver CRISPR in DNA, RNA, and ribonucleic protein (RNP) formats to primary mesenchymal stem cells (MSCs). Comparisons between RALA and commercially available reagents revealed superior cell viability leading to higher numbers of transfected cells and the maintenance of cell proliferative capacity. We then used the RALA peptide for the knock-in and knock-out of reporter genes into the MSC genome as well as for the transcriptional activation of therapeutically relevant genes. In summary, we establish RALA as a powerful tool for safer and effective delivery of CRISPR machinery in multiple cargo formats for a wide range of gene editing strategies.

Development of Non-Viral Targeted RNA Delivery Vehicles - A Key Factor in Success of Therapeutic RNA.

Decade-long efforts in medicinal biotechnology have enabled large-scale in-vitro production of optimized therapeutic RNA constructs for stable in-vivo delivery and modify the expression of disease-related genes. The success of lipid nanoparticle-formulated mRNA vaccines against Severe acute respiratory syndrome Coronavirus-2 (SARS-Cov2) has opened a new era of RNA therapeutics and non-viral drug delivery systems. The major limiting factor in the clinical translation of RNA-based drugs is the availability of suitable delivery vehicles that can protect RNA payloads from degradation, offer controlled release, and pose minimal inherent toxicity. Unwanted immune response, payload size constraints, genome integration, and non-specific tissue targeting limit the application of conventional viral drug-delivery vehicles. This review summarizes current research on nano-sized drug carriers, including lipid nanoparticles, polymer-based formulations, cationic nanoemulsion, and cell-penetrating peptides, for targeted therapeutic RNA delivery. Further, this paper highlights the biomimetic approaches (i.e., mimicking naturally occurring bio-compositions, molecular designs, and systems), including virus-like particles (VLPs), exosomes, and selective endogenous eNcapsidation (SEND) technology being explored as safer and more efficient alternatives.

Gene therapy for hearing loss: Current status and future prospects of non-viral vector delivery systems.

Current therapeutic options for hearing loss rely on hearing aids, ossiculoplasty or cochlear implants. These devices have limitations, particularly in noisy acoustic environments. Therefore, interest in exploring aetiological treatments to improve not only auditory perception but also the quality of life of those affected is increasing. Gene therapy is a promising aetiological treatment that can fully restore auditory function. The success of gene therapy relies on the efficient delivery of therapeutic genes or genetic modifications to the cells of the inner ear that are designed to repair or replace defective genes and restore normal hearing function. Two main strategies for gene therapy involve the use of recombinant viral vectors and nonviral delivery vehicles. Owing to their excellent diffusion properties and compatibility with sensory cells, recombinant viral vectors, particularly adeno-associated viruses (AAVs), have dominated gene therapy in the cochlea. However, recombinant viral vectors have several drawbacks, such as limited transgene size, immunogenicity (particularly in neonates), and potential need for repeat administration. Nonviral vectors, such as cationic lipids and polymeric nanoparticles, are potential attractive alternatives. Nonviral vectors have several advantages, including low immunogenicity and unlimited transgene size. Recent studies have demonstrated significant auditory recovery in vivo using nonviral vectors in murine models. However, nonviral vectors are not as efficient as viral vectors in transferring genetic material. An alternative to nanoparticles is the use of other methods, such as electroporation. The main advantage of electroporation is that it can be used in combination with cochlear implantation and can target surface cells, but this method has a risk of cell damage. The goal of this review is to provide valuable insights into the current state of research on nonviral vectors for inner ear gene therapy and propose the exploration of innovative and effective gene therapy strategies for the treatment of hearing loss.

Optimizing Transfection Efficiency of Spermine Polar Head Cholesterol-based Cationic Lipids with Amino Acid Linker.

In this work, a series of spermine polar head cholesterol-based cationic lipids with various amino acid spacers were synthesized and evaluated as non-viral gene delivery systems. The physicochemical properties of the resulting lipoplexes, formed from these lipids and DOPE, were assessed, including zeta-potential, DNA binding and DNA protection from serum. Transfection efficiency and cytotoxicity were examined under serum-free and 10-40% serum-containing conditions. The results showed that the physicochemical properties of cationic lipids, both with and without amino acid spacers, were not significantly different. Cationic liposomes composed of lipid Sper-Ahx-Chol, which has a 6-aminohexanoic acid spacer, and DOPE exhibited greater transfection efficiency in HeLa cells compared to Lipofectamine3000, both in the absence and presence of 10-40% serum. Additionally, lipid Sper-His-Chol with a histidine spacer and Sper-Ahx-Chol showed higher efficiency than Lipofectamine3000 against HEK293T under 40% serum conditions. These results suggest that the incorporation of amino acids into the cationic lipids can significantly enhance their DNA delivery efficiency. Specifically, certain amino acid modifications improved transfection efficiency while maintaining low cytotoxicity. Our findings highlight the potential of amino acid-tailored cationic lipids as promising vectors for enhanced DNA delivery.

Nanotechnology and CRISPR/Cas-Mediated Gene Therapy Strategies: Potential Role for Treating Genetic Disorders.

Gene therapy has made substantial progress in the treatment of the genetic diseases, focussing on the reduction of characteristics of recessive/dominant disorders, as well as various cancers. Extensive research has been conducted in the past few decades to investigate the application of nanotechnology and CRISPR/Cas technology in gene therapy. Nanotechnology due to attributes such has targeted drug delivery, controlled release, scalability and low toxicity has gained attention of the medical world. CRISPR/Cas9 system is considered as an impactful genome editing tool in the area of next-generation therapeutics and molecular diagnostics. CRISPR technology emphasises on gene editing, gene regulation modulation, and formulation of defined genetic changes. Its applications in treatment of the genetic disorders are extended beyond traditional therapies. These techniques are being explored as treatment of several genetic disorders including Duchenne muscular dystrophy, cystic fibrosis, Alzheimer's disease, Parkinson's disease, and Huntington disease. Despite considerable therapeutic potential of gene therapy, several obstacles must be addressed before it can be widely adopted in clinical practice, particularly in terms of ensuring safety and effectiveness. As research advances in this captivating field, these therapies will become the primary treatments and will have significant beneficial effects on the lives of patients with genetic disorders.

How Advanced are Nanocarriers for Effective Subretinal Injection?

Subretinal injection (SR injection) is a commonly used method of ocular drug delivery and has been mainly applied for the treatment of neovascular age-associated macular degeneration (nAMD) and sub-macular hemorrhage (SMH) caused by nAMD, as well as various types of hereditary retinopathies (IRD) such as Stargardt's disease (STGD), retinitis pigmentosa (RP), and a series of fundus diseases such as Leber's congenital dark haze (LCA), choroidal defects, etc. The commonly used carriers of SR injection are mainly divided into viral and non-viral vectors. Leber's congenital amaurosis (LCA), choroidal agenesis, and a series of other fundus diseases are also commonly treated using SR injection. The commonly used vectors for SR injection are divided into two categories: viral vectors and non-viral vectors. Viral vectors are a traditional class of SR injection drug carriers that have been extensively studied in clinical treatment, but they still have many limitations that cannot be ignored, such as poor reproduction efficiency, small loading genes, and triggering of immune reactions. With the rapid development of nanotechnology in the treatment of ocular diseases, nanovectors have become a research hotspot in the field of non-viral vectors. Nanocarriers have numerous attractive properties such as low immunogenicity, robust loading capacity, stable structure, and easy modification. These valuable features imply greater safety, improved therapeutic efficacy, longer duration, and more flexible indications. In recent years, there has been a growing interest in nanocarriers, which has led to significant advancements in the treatment of ocular diseases. Nanocarriers have not only successfully addressed clinical problems that viral vectors have failed to overcome but have also introduced new therapeutic possibilities for certain classical disease types. Nanocarriers offer undeniable advantages over viral vectors. This review discusses the advantages of subretinal (SR) injection, the current status of research, and the research hotspots of gene therapy with viral vectors. It focuses on the latest progress of nanocarriers in SR injection and enumerates the limitations and future perspectives of nanocarriers in the treatment of fundus lesions. Furthermore, this review also covers the research progress of nanocarriers in the field of subretinal injection and highlights the value of nanocarrier-mediated SR injection in the treatment of fundus disorders. Overall, it provides a theoretical basis for the application of nanocarriers in SR injection.

Advances in CRISPR/Cas systems-based cell and gene therapy.

Cell and gene therapy are innovative biomedical strategies aimed at addressing diseases at their genetic origins. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems have become a groundbreaking tool in cell and gene therapy, offering unprecedented precision and versatility in genome editing. This chapter explores the role of CRISPR in gene editing, tracing its historical development and discussing biomolecular formats such as plasmid, RNA, and protein-based approaches. Next, we discuss CRISPR delivery methods, including viral and non-viral vectors, followed by examining the various engineered CRISPR variants for their potential in gene therapy. Finally, we outline emerging clinical applications, highlighting the advancements in CRISPR for breakthrough medical treatments.

Non-viral approaches in CAR-NK cell engineering: connecting natural killer cell biology and gene delivery.

Natural Killer (NK) cells are exciting candidates for cancer immunotherapy with potent innate cytotoxicity and distinct advantages over T cells for Chimeric Antigen Receptor (CAR) therapy. Concerns regarding the safety, cost, and scalability of viral vectors has ignited research into non-viral alternatives for gene delivery. This review comprehensively analyses recent advancements and challenges with non-viral genetic modification of NK cells for allogeneic CAR-NK therapies. Non-viral alternatives including electroporation and multifunctional nanoparticles are interrogated with respect to CAR expression and translational responses. Crucially, the link between NK cell biology and design of drug delivery technologies are made, which is essential for development of future non-viral approaches. This review provides valuable insights into the current state of non-viral CAR-NK cell engineering, aimed at realising the full potential of NK cell-based immunotherapies.

Polymers as Efficient Non-Viral Gene Delivery Vectors: The Role of the Chemical and Physical Architecture of Macromolecules.

Gene therapy is the technique of inserting foreign genetic elements into host cells to achieve a therapeutic effect. Although gene therapy was initially formulated as a potential remedy for specific genetic problems, it currently offers solutions for many diseases with varying inheritance patterns and acquired diseases. There are two major groups of vectors for gene therapy: viral vector gene therapy and non-viral vector gene therapy. This review examines the role of a macromolecule's chemical and physical architecture in non-viral gene delivery, including their design and synthesis. Polymers can boost circulation, improve delivery, and control cargo release through various methods. The prominent examples discussed include poly-L-lysine, polyethyleneimine, comb polymers, brush polymers, and star polymers, as well as hydrogels and natural polymers and their modifications. While significant progress has been made, challenges still exist in gene stabilization, targeting specificity, and cellular uptake. Overcoming cytotoxicity, improving delivery efficiency, and utilizing natural polymers and hybrid systems are vital factors for prospects. This comprehensive review provides an illuminating overview of the field, guiding the way toward innovative non-viral-based gene delivery solutions.

Advances in Nanoparticles as Non-Viral Vectors for Efficient Delivery of CRISPR/Cas9.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a gene-editing technology. Nanoparticle delivery systems have attracted attention because of the limitations of conventional viral vectors. In this review, we assess the efficiency of various nanoparticles, including lipid-based, polymer-based, inorganic, and extracellular vesicle-based systems, as non-viral vectors for CRISPR/Cas9 delivery. We discuss their advantages, limitations, and current challenges. By summarizing recent advancements and highlighting key strategies, this review aims to provide a comprehensive overview of the role of non-viral delivery systems in advancing CRISPR/Cas9 technology for clinical applications and gene therapy.

Lipid-based Non-viral Vector: Promising Approach for Gene Delivery.

OBJECTIVES: The present review aims to discuss various strategies to overcome intracellular and extracellular barriers involved in gene delivery as well as the advantages, challenges, and mechanisms of gene delivery using non-viral vectors. Additionally, patents, clinical studies, and various formulation approaches related to lipid-based carrier systems are discussed. METHODS: Data were searched and collected from Google Scholar, ScienceDirect, Pubmed, and Springer. RESULTS: In this review, we have investigated the advantages of non-viral vectors over viral vectors. The advantage of using non-viral vectors are that they seek more attention in different fields. They play an important role in delivering the genetic materials. However, few nonviral vector-based carrier systems have been found in clinical settings. Challenges are developing more stable, site-specific gene delivery and conducting thorough safety assessments to minimize the undesired effects. CONCLUSION: In comparison to viral vectors, nonviral vector-based lipid nanocarriers have more advantages for gene delivery. Gene therapy research shows promise in addressing health concerns. Lipid-based nanocarriers can overcome intracellular and extracellular barriers, allowing efficient delivery of genetic materials. Non-viral vectors are more attractive due to their biocompatibility, ease of synthesis, and cost-effectiveness. They can deliver various nucleic acids and have improved gene delivery efficacy by avoiding degradation steps. Despite limited clinical use, many patents have been filed for mRNA vaccine delivery using non-viral vectors.

Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of LbL ELP nucleic acid nanoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

Cationic Serine-Based Gemini Surfactant:Monoolein Aggregates as Viable and Efficacious Agents for DNA Complexation and Compaction: A Cytotoxicity and Physicochemical Assessment.

Cationic gemini surfactants have emerged as potential gene delivery agents as they can co-assemble with DNA due to a strong electrostatic association. Commonly, DNA complexation is enhanced by the inclusion of a helper lipid (HL), which also plays a key role in transfection efficiency. The formation of lipoplexes, used as non-viral vectors for transfection, through electrostatic and hydrophobic interactions is affected by various physicochemical parameters, such as cationic surfactant:HL molar ratio, (+/-) charge ratio, and the morphological structure of the lipoplexes. Herein, we investigated the DNA complexation ability of mixtures of serine-based gemini surfactants, (nSer)2N5, and monoolein (MO) as a helper lipid. The micelle-forming serine surfactants contain long lipophilic chains (12 to 18 C atoms) and a five CH2 spacer, both linked to the nitrogen atoms of the serine residues by amine linkages. The (nSer)2N5:MO aggregates are non-cytotoxic up to 35-90 µM, depending on surfactant and surfactant/MO mixing ratio, and in general, higher MO content and longer surfactant chain length tend to promote higher cell viability. All systems efficaciously complex DNA, but the (18Ser)2N5:MO one clearly stands as the best-performing one. Incorporating MO into the serine surfactant system affects the morphology and size distribution of the formed mixed aggregates. In the low concentration regime, gemini-MO systems aggregate in the form of vesicles, while at high concentrations the formation of a lamellar liquid crystalline phase is observed. This suggests that lipoplexes might share a similar bilayer-based structure.

Non-viral vectors combined delivery of siRNA and anti-cancer drugs to reverse tumor multidrug resistance.

Multidrug resistance (MDR) of tumors is one of the main reasons for the failure of chemotherapy. Multidrug resistance refers to the cross-resistance of tumor cells to multiple antitumor drugs with different structures and mechanisms of action. Current strategies to reverse multidrug resistance in tumors include MDR inhibitors and RNAi technology. siRNA is a small molecule RNA that is widely used in RNAi technology and has the characteristics of being prepared in large quantities and chemically modified. However, siRNA is susceptible to degradation in vivo. The effect of siRNA therapy alone is not ideal, so siRNA and anticancer drugs are administered in combination to reverse the MDR of tumors. Non-viral vectors are now commonly used to deliver siRNA and anticancer drugs to tumor sites. This article will review the progress of siRNA and chemotherapeutic drug delivery systems and their mechanisms for reversing multidrug resistance.

Construction of GSH-responsive polyethyleneimine-based delivery vector for effective gene transfection.

The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.

Non-viral-mediated gene transfer of OX40 ligand for tumor immunotherapy.

Background: Immune checkpoint blockade (ICB) is rapidly becoming a standard of care in the treatment of many cancer types. However, the subset of patients who respond to this type of therapy is limited. Another way to promote antitumoral immunity is the use of immunostimulatory molecules, such as cytokines or T cell co-stimulators. The systemic administration of immunotherapeutics leads to significant immune-related adverse events (irAEs), therefore, the localized antitumoral action is needed. One way to achieve this is intratumoral non-viral gene-immune therapy, which allows for prolonged and localized gene expression, and multiple drug administration. In this study, we combined the previously described non-viral gene delivery system, PEG-PEI-TAT copolymer, PPT, with murine OX40L-encoding plasmid DNA. Methods: The resulting OX40L/PPT nanoparticles were characterized via gel mobility assay, dynamic light scattering analysis and in vitro transfection efficiency evaluation. The antitumoral efficacy of intratumorally (i.t.) administered nanoparticles was estimated using subcutaneously (s.c.) implanted CT26 (colon cancer), B16F0 (melanoma) and 4T1 (breast cancer) tumor models. The dynamics of stromal immune cell populations was analyzed using flow cytometry. Weight loss and cachexia were used as irAE indicators. The effect of combination of i.t. OX40L/PPT with intraperitoneal PD-1 ICB was estimated in s.c. CT26 tumor model. Results: The obtained OX40L/PPT nanoparticles had properties applicable for cell transfection and provided OX40L protein expression in vitro in all three investigated cancer models. We observed that OX40L/PPT treatment successfully inhibited tumor growth in B16F0 and CT26 tumor models and showed a tendency to inhibit 4T1 tumor growth. In B16F0 tumor model, OX40L/PPT treatment led to the increase in antitumoral effector NK and T killer cells and to the decrease in pro-tumoral myeloid cells populations within tumor stroma. No irAE signs were observed in all 3 tumor models, which indicates good treatment tolerability in mice. Combining OX40L/PPT with PD-1 ICB significantly improved treatment efficacy in the CT26 subcutaneous colon cancer model, providing protective immunity against CT26 colon cancer cells. Conclusion: Overall, the anti-tumor efficacy observed with OX40L non-viral gene therapy, whether administered alone or in combination with ICB, highlights its potential to revolutionize cancer gene therapy, thus paving the way for unprecedented advancements in the cancer therapy field.

Branching in poly(amine-co-ester) polyplexes impacts mRNA transfection.

Branching is a key structural parameter of polymers, which can have profound impacts on physicochemical properties. It has been demonstrated that branching is a modulating factor for mRNA delivery and transfection using delivery vehicles built from cationic polymers, but the influence of polymer branching on mRNA delivery remains relatively underexplored compared to other polymer features such as monomer composition, hydrophobicity, pKa, or the type of terminal group. In this study, we examined the impact of branching on the physicochemical properties of poly(amine-co-esters) (PACE) and their efficiency in mRNA transfection in vivo and in vitro under various conditions. PACE polymers were synthesized with various degrees of branching ranging from 0 to 0.66, and their transfection efficiency was systemically evaluated. We observed that branching improves the stability of polyplexes but reduces the pH buffering capacity. Therefore, the degree of branching (DB) must be optimized in a delivery route specific manner due to differences in challenges faced by polyplexes in different physiological compartments. Through a systematic analysis of physicochemical properties and mRNA transfection in vivo and in vitro, this study highlights the influence of polymer branching on nucleic acid delivery.

Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets.

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.

Evaluation of DNA minicircles for delivery of adenine and cytosine base editors using activatable gene on "GO" reporter imaging systems.

Over 30,000 point mutations are associated with debilitating diseases, including many cancer types, underscoring a critical need for targeted genomic solutions. CRISPR base editors, like adenine base editors (ABEs) and cytosine base editors (CBEs), enable precise modifications by converting adenine to guanine and cytosine to thymine, respectively. Challenges in efficiency and safety concerns regarding viral vectors used in delivery limit the scope of base editing. This study introduces non-viral minicircles, bacterial-backbone-free plasmids, as a delivery vehicle for ABEs and CBEs. The research uses cells engineered with the "Gene On" (GO) reporter gene systems for tracking minicircle-delivered ABEs, CBEs, or Cas9 nickase (control), using green fluorescent protein (GFPGO), bioluminescence reporter firefly luciferase (LUCGO), or a highly sensitive Akaluciferase (AkalucGO) designed in this study. The results show that transfection of minicircles expressing CBE or ABE resulted in significantly higher GFP expression and luminescence signals over controls, with minicircles demonstrating the most substantial editing. This study presents minicircles as a new strategy for base editor delivery and develops an enhanced bioluminescence imaging reporter system for tracking ABE activity. Future studies aim to evaluate the use of minicircles in preclinical cancer models, facilitating potential clinical applications.