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Sonogenetics is an innovative technology that integrates ultrasound with genetic editing to precisely modulate cellular activities in a non‐invasive manner. This method entails introducing and activating mechanosensitive channels on the cell membrane of specific cells using gene delivery vectors. When exposed to ultrasound, these channels can be manipulated to open or close, thereby impacting cellular functions. Sonogenetics is currently being used extensively in the treatment of various chronic diseases, including Parkinson's disease, vision restoration, and cancer therapy. This paper provides a comprehensive review of key components of sonogenetics and focuses on evaluating its prospects and potential challenges in the treatment of chronic disease. [ABSTRACT FROM AUTHOR] more...

Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field. more...

Lipoamino acids (LAA) are biocompatible and biodegradable biosurfactants, a promising alternative to viral vectors in gene delivery. LAA are constituted by a polar head, the amino acid, and a hydrocarbon (alkyl) chain usually from a fatty acid or fatty acid derivative, such as a fatty amine or a fatty alcohol. In this work, dodecyl LAA was produced from dodecylamine and natural l‐amino acids cystine (Cys), lysine (Lys), and phenylalanine (Phe) using an enzyme‐based approach with porcine pancreatic lipase. The self‐assembly behavior of LAA solutions, in the absence or presence of DNA, was studied by conductivity and fluorescence regarding the application as transfection agents. Conductivity measurements yielded important system parameters, including critical micelle concentration (CMC) and standard Gibbs energy of micellization (ΔG°mic) for pure LAA systems, and apparent critical aggregation concentration (CACapp) and apparent standard Gibbs energy of aggregation (ΔG°agg) for the mixed LAA‐DNA systems. The CMC increased in the order of decreasing lipophilicity: (C12Cys)2 < C12Phe < C12Lys, CMC values were higher in the presence of DNA, suggesting the formation of a LAA‐DNA complex responsible for hindering the micellization process. Binding of the LAA with DNA was confirmed from fluorescence measurements for the ethidium bromide exclusion assay. Results suggest a weak interaction of the LAA with DNA which can be attributed to their relatively short dodecyl chains and/or the ionic strength of the buffer solution, supporting the role of hydrophobic interactions in complex formation between DNA and the oppositely charged surfactant in combination with electrostatic interactions. The CACapp values decreased with increasing LAA hydrophobicity, reflecting the relevance of hydrophobic interactions in complex coacervation. [ABSTRACT FROM AUTHOR] more...

Recessive dystrophic epidermolysis bullosa (RDEB) is an autosomal monogenic skin disease caused by mutations in COL7A1 gene and lack of functional type VII collagen (C7). Currently, there is no cure for RDEB, and most of the gene therapies under development have been designed as ex vivo strategies because of the shortage of efficient and safe carriers for gene delivery. Herein, we designed, synthesized, and screened a new group of highly branched poly(β amino ester)s (HPAEs) as non-viral carriers for the delivery of plasmids encoding dual single-guide RNA (sgRNA)-guided CRISPR-Cas9 machinery to delete COL7A1 exon 80 containing the c.6527dupC mutation. The selected HPAEs (named PTTA-DATOD) showed robust transfection efficiency, comparable with or surpassing that of leading commercial gene transfection reagents such as Lipofectamine 3000, Xfect, and jetPEI, while maintaining negligible cytotoxicity. Furthermore, CRISPR-Cas9 plasmids delivered by PTTA-DATOD achieved efficient targeted deletion and restored bulk C7 production in RDEB patient keratinocyte polyclones. The non-viral CRISPR-Cas9-based COL7A1 exon deletion approach developed here has great potential to be used as a topical treatment for RDEB patients with mutations in COL7A1 exon 80. Besides, this therapeutic strategy can easily be adapted for mutations in other COL7A1 exons, other epidermolysis bullosa subtypes, and other genetic diseases., Competing Interests: The authors declare no competing interests. W.W. is the founder of Branca Bunús, a UCD startup company that manufactures branched polymers for gene delivery and in which Xianqing W. is involved in collaborative research projects., (© 2023 The Authors.) more...

Gene therapy has emerged as a significant advancement in medicine in recent years. However, the development of effective gene delivery vectors, particularly polymer vectors, remains a significant challenge. Limited understanding of the internal structure of polymer vectors has hindered efforts to enhance their efficiency. This work focuses on investigating the impact of polymer structure on gene delivery, using the well-known polymeric vector poly(β-amino ester) (PAE) as a case study. For the first time, we revealed the distinct characteristics of individual polymer components and their synergistic effects-the appropriate combination of different components within a polymer (high MW and low MW components) on gene delivery. Additionally, artificial intelligence (AI) analysis was employed to decipher the relationship between the polymer component distribution (PCD) and gene transfection performance. Guided by this analysis, a series of highly efficient polymer vectors that outperform current commercial reagents such as jetPEI and Lipo3000 were developed, among which the transfection efficiency of the PAE-B1-based polyplex was approximately 1.5 times that of Lipo3000 and 2 times that of jetPEI in U251 cells. more...

Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field. [ABSTRACT FROM AUTHOR] more...

The world is driving in to the era of transformation of chemical therapeutic molecules to biological genetic material therapeutics, and that is where the biological drugs especially 'genes' come into existence. These genes worked as 'magical bullets' to specifically silence faulty genes responsible for progression of diseases. Viral gene delivery research is far ahead of nonviral gene delivery technique. However, with more advancement in polymer science, new ways are opening for better and efficient nonviral gene delivery. But efficient delivery method is always considered as a bottleneck for gene delivery as success of which will decide the fate of gene in cells. During the past decade, it became evident that extracellular as well as intracellular barriers compromise the transfection efficiency of nonviral vectors. The challenge for gene therapy research is to pinpoint the rate-limiting steps in this complex process and implement strategies to overcome the biological physiochemical and metabolic barriers encountered during targeting. The synergy between studies that investigate the mechanism of breaking in and breaking out of nonviral gene delivery carrier through various extracellular and intracellular barriers with desired characteristics will enable the rational design of vehicles and revolutionize the treatment of various diseases. [ABSTRACT FROM AUTHOR] more...

This review gives representative examples of the various types of synthetic cationic polymers or polyampholytes (chemical structure, architecture, etc) that can be used to complex DNA (forming polyplexes) for their application in gene delivery. In designing polycations for gene delivery, one has to take into account a balance between protection of DNA versus loss of efficiency for DNA condensation and efficient condensation versus hindering of DNA release. Indeed, if the polyplexes are not stable enough, premature dissociation will occur before delivery of the genetic material at the desired place, resulting in low transfection efficiency; on the other hand, a complex that is too stable will not release the DNA, also resulting in low gene expression. The techniques generally used to determine these properties are gel electrophoresis to test the DNA/polymer complexation, ethidium bromide or polyanion displacement to test the affinity of a polymer for DNA, and light scattering to determine the extent of DNA condensation. Moreover, with the development of more precise instruments for physico-chemical characterization and appropriate biochemical and biophysical techniques, a direct link between the physico-chemical characteristics of the polyplexes and their in vitro and in vivo properties can be drawn, thus allowing tremendous progress in the quest towards application of polyplexes for gene therapy, beyond the research laboratory. [ABSTRACT FROM AUTHOR] more...

Introduction: Gene delivery vectors are a crucial determinant for gene therapeutic efficacy. Usually, it is necessary to use an excess of cationic vectors to achieve better transfection efficiency. However, it will cause severe cytotoxicity. In addition, cationic vectors are not resistant to serum, suffering from reduced transfection efficiency by forming large aggregates. Therefore, there is an urgent need to develop optimized gene delivery vectors. Recently, fluorination of vectors has been extensively applied to increase the gene delivery performance because of the unique properties of both hydrophobicity and lipophobicity, and chemical and biological inertness., Areas Covered: This review will discuss the fluorophilic effects that impact gene delivery efficiency, and chemical modification approaches for fluorination. Next, recent advances and applications of fluorinated polymeric and lipidic vectors in gene therapy and gene editing are summarized., Expert Opinion: Fluorinated vectors are a promising candidate for gene delivery. However, it still needs further studies to obtain pure and well-defined fluorinated polymers, guarantee the biosafety, and clarify the detailed mechanism. Apart from the improvements in gene delivery, exploiting other versatility of fluorinated vectors, such as oxygen-carrying ability, high affinity with fluorine-containing drugs, and imaging property upon introducing 19  F , will further facilitate their applications in gene therapy. more...

Abstract: Atomic force microscopy (AFM) has been used to visualize the process of condensation of plasmid DNA by poly-l-ornithine on mica surface. AFM images reveal that the transition of negatively charged DNA to condensed nanoparticles on addition of increasing amounts of positively charged poly-l-ornithine (charge ratio (Z+/Z−) varied between 0.1 and 1) at a wide range of DNA concentrations (3–20 ng/μl) occurs through formation of several distinct morphologies. The nature of the complexes is strongly dependent on both the charge ratio and the DNA concentration. Initiation of condensation when the concentration of DNA is low (∼3–7 ng/μl) occurs possibly through formation of monomolecular complexes which are thick rod-like in shape. On the contrary, when condensation is carried out at DNA concentrations of 13–20 ng/μl, multimolecular structures are also formed even at low charge ratios. This difference in pathway seems to result in differences in the extent of condensation as well as size and aggregation of the nanoparticles formed at the high charge ratios. To the best of our knowledge, this is the first direct single molecule elucidation of the mechanism of DNA condensation by poly-l-ornithine. Cationic poly-aminoacids like poly-l-ornithine are known to be efficient in delivery of plasmid DNA containing therapeutic genes in a variety of mammalian cell lines by forming condensed “nanocarriers” with DNA. Single molecule insight into the mechanism by which such nanocarriers are packaged during the condensation process could be helpful in predicting efficacy of intracellular delivery and release of DNA from them and also provide important inputs for design of new gene delivery vectors. [Copyright &y& Elsevier] more...

Abstract: Tissue engineering is a multifaceted technology developed with a purpose of regenerating complex tissues and organs. Cartilage regeneration continues to challenge engineers and a new wave of efforts focus on developing strategies that provide sustained stimulation to cells by growth factors and other biological molecules to promote their differentiation into chondrocytes. Though significant research is dedicated to developing controlled release systems that deliver growth factors directly, a simpler approach to resolving this dilemma involves converting cells into protein producing factories. This is done through gene delivery. Gene Therapy studies published for articular diseases such as rheumatoid and osteoarthritis provide valuable information regarding different types of cells, gene delivery vectors and genes that can potentially be used to regenerate cartilage. Tissue engineering approaches provide the opportunity to combine two or more strategies used for Gene Therapy thus far and create a cohesive system that addresses both cartilage degeneration and synthesis simultaneously. Adopting gene transfer techniques for tissue engineering is a relatively novel approach, as non-viral gene delivery vectors are continually optimized for therapeutic purposes, and reservations about viral vectors have increasingly dampened their appeal. However, every element involved in gene transfection (i.e., the cell, vector and gene) is a variable which decides the physiological and biomechanical properties of the cartilage produced, and significant work still needs to be done in understanding the contribution of each of these factors to cartilage regeneration. [Copyright &y& Elsevier] more...

Human artificial chromosomes (HACs) are considered promising tools for gene delivery, functional analyses, and gene therapy. HACs have the potential to overcome many of the problems caused by the use of viral-based gene transfer systems, such as limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes. The recently developed alphoid tetO -HAC has an advantage over other HAC vectors because it can be easily eliminated from dividing cells by inactivation of its conditional kinetochore. This provides a unique control mechanism to study phenotypes induced by a gene or genes carried on the HAC. The alphoid tetO -HAC has a single gene acceptor loxP site that allows insertion of an individual gene of interest or a cluster of genes of up to several Mb in size in Chinese hamster ovary (CHO) hybrid cells. The HACs carrying chromosomal copies of genes can then be transferred from these donor CHO cells to different recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we describe a detailed protocol for loading a gene of interest into the alphoid tetO -HAC vector and for the subsequent transfer of the HAC to recipient cells using an improved MMCT protocol. The original MMCT protocol includes treatment of donor cells with colcemid to induce micronucleation, wherein the HAC becomes surrounded with a nuclear membrane. That step is followed by disarrangement of the actin cytoskeleton using cytochalasin B to help induce microcell formation. The updated MMCT protocol, described here, features the replacement of colcemid and cytochalasin B with TN16 + griseofulvin and latrunculin B, respectively, and the use of collagen/laminin surface coating to promote attachment of metaphase cells to plates during micronuclei induction. These modifications increase the efficiency of HAC transfer to recipient cells ten fold. The improved MMCT protocol has been successfully tested on several recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Insertion of a BAC containing a gene of interest into a single loxP loading site of alphoid tetO -HAC in hamster CHO cells Basic Protocol 2: Microcell-mediated chromosome transfer from donor hamster CHO cells to mammalian cells., (© 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.) more...