Characterization of a novel fast-growing zebrafish: a new approach to growth hormone transgenesis.

The manipulation of the somatotropic axis, governing growth, has been a focus of numerous transgenic approaches aimed at developing fast-growing fish for research, medicine and aquaculture purposes. However, the excessively high growth hormone (GH) levels in these transgenic fish often result in deformities that impact both fish health and consumer acceptance. In an effort to mitigate these issues and synchronize exogenous GH expression with reproductive processes, we employed a novel transgenic construct driven by a tilapia luteinizing hormone (LH) promoter. This approach was anticipated to induce more localized and lower exogenous GH secretion. In this study, we characterized the growth and reproduction of these transgenic LHp-GH zebrafish using hormonal and physiological parameters. Our findings reveal that LHp-GH fish exhibited accelerated growth in both length and weight, along with a lower feed conversion ratio, indicating more efficient feed utilization, all while maintaining unchanged body proportions. These fish demonstrated higher expression levels of LH and GH in the pituitary and elevated IGF-1 levels in the liver compared to wild-type fish. An examination of reproductive function in LHp-GH fish unveiled lower pituitary LH and FSH contents, smaller follicle diameter in female gonads, and reduced relative fecundity. However, in transgenic males, neither the distribution of spermatogenesis stages nor sperm concentrations differed significantly between the fish lines. These results suggest that coupling exogenous GH expression with endogenous LH expression in females directs resource investment toward somatic growth at the expense of reproductive processes. Consequently, we conclude that incorporating GH under the LH promoter represents a suitable construct for the genetic engineering of commercial fish species, providing accelerated growth while preserving body proportions.

Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.

Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.

Novel Viral Vectors for Gene Therapy.

Viral vectors are gene transfer tools assembled from the backbones of naturally occurring viruses [...].

Safety and Efficacy of IL-12 Plasmid DNA Transfection into Pig Skin: Supportive Data for Human Clinical Trials on Gene Therapy and Vaccination.

Gene electrotransfer (GET) of plasmids encoding interleukin 12 (IL-12) has already been used for the treatment of various types of tumors in human oncology and as an adjuvant in DNA vaccines. In recent years, we have developed a plasmid encoding human IL-12 (phIL12) that is currently in a phase I clinical study. The aim was to confirm the results of a non-clinical study in mice on pharmacokinetic characteristics and safety in a porcine model that better resembled human skin. The GET of phIL12 in the skin was performed on nine pigs using different concentrations of plasmid phIL12 and invasive (needle) or noninvasive (plate) types of electrodes. The results of our study demonstrate that the GET of phIL-12 with needle electrodes induced the highest expression of IL-12 at the protein level on day 7 after the procedure. The plasmid was distributed to all tested organs; however, its amount decreased over time and was at a minimum 28 days after GET. Based on plasmid copy number and expression results, together with blood analysis, we showed that IL-12 GET is safe in a porcine animal model. Furthermore, we demonstrated that pigs are a valuable model for human gene therapy safety studies.

Cryo-shocked tumor cells deliver CRISPR-Cas9 for lung cancer regression by synthetic lethality.

Although CRISPR-mediated genome editing holds promise for cancer therapy, inadequate tumor targeting and potential off-target side effects hamper its outcomes. In this study, we present a strategy using cryo-shocked lung tumor cells as a CRISPR-Cas9 delivery system for cyclin-dependent kinase 4 (CDK4) gene editing, which initiates synthetic lethal in KRAS-mutant non-small cell lung cancer (NSCLC). By rapidly liquid nitrogen shocking, we effectively eliminate the pathogenicity of tumor cells while preserving their structure and surface receptor activity. This delivery system enables the loaded CRISPR-Cas9 to efficiently target to lung through the capture in pulmonary capillaries and interactions with endothelial cells. In a NSCLC-bearing mouse model, the drug accumulation is increased nearly fourfold in lung, and intratumoral CDK4 expression is substantially down-regulated compared to CRISPR-Cas9 lipofectamine nanoparticles administration. Furthermore, CRISPR-Cas9 editing-mediated CDK4 ablation triggers synthetic lethal in KRAS-mutant NSCLC and prolongs the survival of mice.

Engineering aspects of lipid-based delivery systems: In vivo gene delivery, safety criteria, and translation strategies.

Defects in the genome cause genetic diseases and can be treated with gene therapy. Due to the limitations encountered in gene delivery, lipid-based supramolecular colloidal materials have emerged as promising gene carrier systems. In their non-functionalized form, lipid nanoparticles often demonstrate lower transgene expression efficiency, leading to suboptimal therapeutic outcomes, specifically through reduced percentages of cells expressing the transgene. Due to chemically active substituents, the engineering of delivery systems for genetic drugs with specific chemical ligands steps forward as an innovative strategy to tackle the drawbacks and enhance their therapeutic efficacy. Despite intense investigations into functionalization strategies, the clinical outcome of such therapies still needs to be improved. Here, we highlight and comprehensively review engineering aspects for functionalizing lipid-based delivery systems and their therapeutic efficacy for developing novel genetic cargoes to provide a full snapshot of the translation from the bench to the clinics. We outline existing challenges in the delivery and internalization processes and narrate recent advances in the functionalization of lipid-based delivery systems for nucleic acids to enhance their therapeutic efficacy and safety. Moreover, we address clinical trials using these vectors to expand their clinical use and principal safety concerns.

Antimicrobial peptide A9K as a gene delivery vector in cancer cells.

Designed peptides are promising biomaterials for biomedical applications. The amphiphilic cationic antimicrobial peptide (AMP), A9K, can self-assemble into nano-rod structures and has shown cancer cell selectivity and could therefore be a promising candidate for therapeutic delivery into cancer cells. In this paper, we investigate the selectivity of A9K for cancer cell models, examining its effect on two human cancer cell lines, A431 and HCT-116. Little or no activity was observed on the control, human dermal fibroblasts (HDFs). In the cancer cell lines the peptide inhibited cellular growth through changes in mitochondrial morphology and membrane potential while remaining harmless towards HDFs. In addition, the peptide can bind to and protect nucleic acids while transporting them into both 2D cultures and 3D spheroids of cancer cells. A9K showed high efficiency in delivering siRNA molecules into the centre of the spheroids. A9K was also explored in vivo, using a zebrafish (Danio rerio) development toxicity assay, showing that the peptide is safe at low doses. Finally, a high-content imaging screen, using RNA interference (RNAi) targeted towards cellular uptake, in HCT-116 cells was carried out. Our findings suggest that active cellular uptake is involved in peptide internalisation, mediated through clathrin-mediated endocytosis. These new discoveries make A9K attractive for future developments in clinical and biotechnological applications.

Intrathecal Gene Therapy for Giant Axonal Neuropathy.

BACKGROUND: Giant axonal neuropathy is a rare, autosomal recessive, pediatric, polysymptomatic, neurodegenerative disorder caused by biallelic loss-of-function variants in GAN, the gene encoding gigaxonin. METHODS: We conducted an intrathecal dose-escalation study of scAAV9/JeT-GAN (a self-complementary adeno-associated virus-based gene therapy containing the GAN transgene) in children with giant axonal neuropathy. Safety was the primary end point. The key secondary clinical end point was at least a 95% posterior probability of slowing the rate of change (i.e., slope) in the 32-item Motor Function Measure total percent score at 1 year after treatment, as compared with the pretreatment slope. RESULTS: One of four intrathecal doses of scAAV9/JeT-GAN was administered to 14 participants - 3.5×1013 total vector genomes (vg) (in 2 participants), 1.2×1014 vg (in 4), 1.8×1014 vg (in 5), and 3.5×1014 vg (in 3). During a median observation period of 68.7 months (range, 8.6 to 90.5), of 48 serious adverse events that had occurred, 1 (fever) was possibly related to treatment; 129 of 682 adverse events were possibly related to treatment. The mean pretreatment slope in the total cohort was -7.17 percentage points per year (95% credible interval, -8.36 to -5.97). At 1 year after treatment, posterior mean changes in slope were -0.54 percentage points (95% credible interval, -7.48 to 6.28) with the 3.5×1013-vg dose, 3.23 percentage points (95% credible interval, -1.27 to 7.65) with the 1.2×1014-vg dose, 5.32 percentage points (95% credible interval, 1.07 to 9.57) with the 1.8×1014-vg dose, and 3.43 percentage points (95% credible interval, -1.89 to 8.82) with the 3.5×1014-vg dose. The corresponding posterior probabilities for slowing the slope were 44% (95% credible interval, 43 to 44); 92% (95% credible interval, 92 to 93); 99% (95% credible interval, 99 to 99), which was above the efficacy threshold; and 90% (95% credible interval, 89 to 90). Between 6 and 24 months after gene transfer, sensory-nerve action potential amplitudes increased, stopped declining, or became recordable after being absent in 6 participants but remained absent in 8. CONCLUSIONS: Intrathecal gene transfer with scAAV9/JeT-GAN for giant axonal neuropathy was associated with adverse events and resulted in a possible benefit in motor function scores and other measures at some vector doses over a year. Further studies are warranted to determine the safety and efficacy of intrathecal AAV-mediated gene therapy in this disorder. (Funded by the National Institute of Neurological Disorders and Stroke and others; ClinicalTrials.gov number, NCT02362438.).

Nanocarrier-based gene delivery for immune cell engineering.

Clinical advances in genetically modified immune cell therapies, such as chimeric antigen receptor T cell therapies, have raised hope for cancer treatment. The majority of these biotechnologies are based on viral methods for ex vivo genetic modification of the immune cells, while the non-viral methods are still in the developmental phase. Nanocarriers have been emerging as materials of choice for gene delivery to immune cells. This is due to their versatile physicochemical properties such as large surface area and size that can be optimized to overcome several practical barriers to successful gene delivery. The in vivo nanocarrier-based gene delivery can revolutionize cell-based cancer immunotherapies by replacing the current expensive autologous cell manufacturing with an off-the-shelf biomaterial-based platform. The aim of this research is to review current advances and strategies to overcome the challenges in nanoparticle-based gene delivery and their impact on the efficiency, safety, and specificity of the process. The main focus is on polymeric and lipid-based nanocarriers, and their recent preclinical applications for cancer immunotherapy.

VEGF and Other Gene Therapies Improve Flap Survival-A Systematic Review and Meta-Analysis of Preclinical Studies.

Surgical flaps are basic tools in reconstructive surgery. Their use may be limited by ischemia and necrosis. Few therapies address or prevent them. Genetic therapy could improve flap outcomes, but primary studies in this field present conflicting results. This systematic review and meta-analysis aimed to appraise the efficacy of external gene delivery to the flap for its survival in preclinical models. This review was registered with PROSPERO (CRD42022359982). PubMed, Embase, Web of Science, and Scopus were searched to identify studies using animal models reporting flap survival outcomes following any genetic modifications. Random-effects meta-analysis was used to calculate mean differences in flap survival with accompanying 95% CI. The risk of bias was assessed using the SYRCLE tool. Subgroup and sensitivity analyses were performed to ascertain the robustness of primary analyses, and the evidence was assessed using the GRADE approach. The initial search yielded 690 articles; 51 were eventually included, 36 of which with 1576 rats were meta-analyzed. VEGF gene delivery to different flap types significantly improved flap survival area by 15.66% (95% CI 11.80-19.52). Other interventions had smaller or less precise effects: PDGF-13.44% (95% CI 3.53-23.35); VEGF + FGF-8.64% (95% CI 6.94-10.34); HGF-5.61% (95% CI 0.43-10.78); FGF 3.84% (95% CI 1.13-6.55). Despite considerable heterogeneity, moderate risk of bias, and low quality of evidence, the efficacy of VEGF gene therapy remained significant in all sensitivity analyses. Preclinical data indicate that gene therapy is effective for increasing flap survival, but further animal studies are required for successful clinical translation.

CRISPR/Cas gene editing and delivery systems for cancer therapy.

CRISPR/Cas systems stand out because of simplicity, efficiency, and other superiorities, thus becoming attractive and brilliant gene-editing tools in biomedical field including cancer therapy. CRISPR/Cas systems bring promises for cancer therapy through manipulating and engineering on tumor cells or immune cells. However, there have been concerns about how to overcome the numerous physiological barriers and deliver CRISPR components to target cells efficiently and accurately. In this review, we introduced the mechanisms of CRISPR/Cas systems, summarized the current delivery strategies of CRISPR/Cas systems by physical methods, viral vectors, and nonviral vectors, and presented the current application of CRISPR/Cas systems in cancer clinical treatment. Furthermore, we discussed prospects related to delivery approaches of CRISPR/Cas systems. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Nontoxic, Biodegradable Hyperbranched Poly(ß-amino ester)s for Efficient siRNA Delivery and Gene Silencing.

RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(ß-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

A flexible electrode Array for genetic transfection of different layers of the retina by electroporation.

Electroporation (in which the permeability of a cell membrane is increased transiently by exposure to an appropriate electric field) has exhibited great potential of becoming an alternative to adeno-associated virus (AAV)-based retina gene delivery. Electroporation eliminates the safety concerns of employing exogenous viruses and exceeds the limit of AAV cargo size. Unfortunately, several concerns (e.g., relatively high electroporation voltage, poor surgical operability and a lack of spatial selectivity of retina tissue) have prevented electroporation from being approved for clinical application (or even clinical trials). In this study, a flexible micro-electrode array for retina electroporation (FERE) was developed for retina electroporation. A suitably shaped flexible substrate and well-placed micro-electrodes were designed to adapt to the retina curvature and generate an evenly distributed electric field on the retina with a significantly reduced electroporation voltage of 5 V. The FERE provided (for the first time) a capability of controlled gene delivery to the different structural layers of retina tissue by precise control of the distribution of the electrical field. After ensuring the surgical operability of the FERE on rabbit eyeballs, the FERE was verified to be capable of transfecting different layers of retina tissue with satisfactory efficiency and minimum damage. Our method bridges the technical gap between laboratory validation and clinical use of retina electroporation.

Mage transposon: a novel gene delivery system for mammalian cells.

Transposons, as non-viral integration vectors, provide a secure and efficient method for stable gene delivery. In this study, we have discovered Mage (MG), a novel member of the piggyBac(PB) family, which exhibits strong transposability in a variety of mammalian cells and primary T cells. The wild-type MG showed a weaker insertion preference for near genes, transcription start sites (TSS), CpG islands, and DNaseI hypersensitive sites in comparison to PB, approaching the random insertion pattern. Utilizing in silico virtual screening and feasible combinatorial mutagenesis in vitro, we effectively produced the hyperactive MG transposase (hyMagease). This variant boasts a transposition rate 60% greater than its native counterpart without significantly altering its insertion pattern. Furthermore, we applied the hyMagease to efficiently deliver chimeric antigen receptor (CAR) into T cells, leading to stable high-level expression and inducing significant anti-tumor effects both in vitro and in xenograft mice models. These findings provide a compelling tool for gene transfer research, emphasizing its potential and prospects in the domains of genetic engineering and gene therapy.

Enhancing gene transfection of poly(ß-amino ester)s through modulation of amphiphilicity and chain sequence.

Poly(ß-amino ester)s (PAEs) have emerged as a type of highly safe and efficient non-viral DNA delivery vectors. However, the influence of amphiphilicity and chain sequence on DNA transfection efficiency and safety profile remain largely unexplored. In this study, four PAEs with distinct amphiphilicity and chain sequences were synthesized. Results show that both amphiphilicity and chain sequence significantly affect the DNA binding and condensation ability of PAEs, as well as size, zeta potential and cellular uptake of PAE/DNA polyplexes. PAEs with different amphiphilicity and chain sequence exhibit cell type-dependent transfection capabilities: in human bladder transitional cell carcinoma (UM-UC-3), hydrophilic PAE (P-Philic) and amphiphilic PAE random copolymer (R-Amphilic) exhibit relatively higher gene transfection efficiency, while in human bladder epithelial immortalized cells (SV-HUC-1), hydrophobic PAE (P-Phobic), R-Amphilic, and amphiphilic PAE block copolymer (B-Amphilic) demonstrate higher transfection capability. Regardless of cell types, amphiphilic PAE block copolymer (B-Amphilic) always exhibits much lower gene transfection efficiency. In addition, in human colon cancer cells (HCT-116), P-Philic and R-Amphilic achieved superior gene transfection efficiency at high and low polymer/DNA weight ratios, respectively. Importantly, R-Amphilic can effectively deliver the gene encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to human chondrosarcoma cells SW1353 to induce their apoptosis, highlighting its potential application in cancer gene therapy. This study not only establishes a new paradigm for enhancing the gene transfection efficiency of PAEs by modulating their amphiphilicity and chain sequence but also identifies R-Amphilic as a potential candidate for the effective delivery of TRAIL gene in cancer gene therapy.

An optimized polymeric delivery system for piggyBac transposition.

The piggyBac transposon/transposase system has been explored for long-term, stable gene expression to execute genomic integration of therapeutic genes, thus emerging as a strong alternative to viral transduction. Most studies with piggyBac transposition have employed physical methods for successful delivery of the necessary components of the piggyBac system into the cells. Very few studies have explored polymeric gene delivery systems. In this short communication, we report an effective delivery system based on low molecular polyethylenimine polymer with lipid substitution (PEI-L) capable of delivering three components, (i) a piggyBac transposon plasmid DNA carrying a gene encoding green fluorescence protein (PB-GFP), (ii) a piggyBac transposase plasmid DNA or mRNA, and (iii) a 2 kDa polyacrylic acid as additive for transfection enhancement, all in a single complex. We demonstrate an optimized formulation for stable GFP expression in two model cell lines, MDA-MB-231 and SUM149 recorded till day 108 (3.5 months) and day 43 (1.4 months), respectively, following a single treatment with very low cell number as starting material. Moreover, the stability of the transgene (GFP) expression mediated by piggyBac/PEI-L transposition was retained following three consecutive cryopreservation cycles. The success of this study highlights the feasibility and potential of employing a polymeric delivery system to obtain piggyBac-based stable expression of therapeutic genes.

Current Status and Prospects of Viral Vector-Based Gene Therapy to Treat Kidney Diseases.

Inherited kidney diseases are among the leading causes of chronic kidney disease, reducing the quality of life and resulting in substantial socioeconomic impact. The advent of early genetic testing and the growing understanding of the molecular basis and pathophysiology of these disorders have opened avenues for novel treatment strategies. Viral vector-based gene therapies have evolved from experimental treatments for rare diseases to potent platforms that carry the intrinsic potential to provide a cure with a single application. Several gene therapy products have reached the market, and the numbers are only expected to increase. Still, none target inherited kidney diseases. Gene transfer to the kidney has lagged when compared to other tissue-directed therapies such as hepatic, neuromuscular, and ocular tissues. Systemic delivery of genetic information to tackle kidney disease is challenging. The pharma industry is taking steps to take on kidney disease and to translate the current research into the therapeutic arena. In this review, we provide an overview of the current viral vector-based approaches and their potential. We discuss advances in platforms and injection routes that have been explored to enhance gene delivery toward kidney cells in animal models, and how these can fuel the development of viable gene therapy products for humans.