Placental dysregulation frequently results in pregnancy complications that impact fetal well-being and potentially predispose the infant to diseases later in life. Thus, efforts to understand the molecular mechanisms underlying placental disorders are crucial to aid the development of effective treatments to restore placental function. Currently, the most common methods used for trophoblast-specific gene modulation in the laboratory are transgenic animals and lentiviral trophectoderm transduction. The generation of transgenic animal lines is costly and requires a considerable amount of time to generate and maintain, while the integration preference of lentiviruses, actively transcribed genes, may result in genotoxicity. Therefore, there is much interest in the development of non-viral in vivo transfection techniques for use in both research and clinical settings. Herein, we describe a non-viral, minimally invasive method for in vivo placental gene modulation through sonoporation, an ultrasound-mediated transfection technique wherein the application of ultrasound on target tissues is used to direct the uptake of DNA vectors. In this method, plasmids are bound to lipid microbubbles, which are then injected into the maternal bloodstream and ultimately delivered to the placenta when subjected to low-frequency ultrasound. Syncytiotrophoblasts are directly exposed to maternal blood and, therefore highly accessible to therapeutic agents in the maternal circulation. This technique can be used to modulate gene expression and, subsequently, the function of the placenta, circumventing the requirement to generate transgenic animals. Sonoporation also offers a safer alternative to existing viral techniques, making it not only an advantageous research tool but also a potentially adaptable technique in clinical settings.

Epithelial cell entrance and trans-epithelial transport are two essential processes that directly affect the efficacy of oral delivery of nanocarriers. Herein, a hydroxyethyl starch-based nanocapsule dual decorated with butyrate and octadecylamine (ODA) was first constructed to enhance transporter-mediated endocytosis and trans-epithelial transport, while reducing exenatide (EXT) loss during absorption. The epithelial barrier was then treated with linoleic acid (LA), which functioned as a cell membrane fluidity regulator. This treatment further improved oral delivery efficiency by lowering the energy cost of endocytosis through fluidizing of the cell membrane and increasing monocarboxylate transporter 1 (MCT1) expression on cell surfaces. The findings revealed that LA upregulated MCT1 expression by 3.26-fold, increased the cellular uptake of nanocapsules co-modified with butyrate and ODA by 4.52-fold, decreased ATP consumption for uptake in LA-pretreated Caco-2 cells to only 18.64 % of that in untreated Caco-2 cells, and increased their transcellular transport by 1.72-fold in a Caco-2/HT29-MTX-E12 co-culture monolayer. Therefore, the oral administration of EXT-loaded Bu-PEG-ODA NCs with LA significantly enhanced the oral bioavailability of EXT (Bu-PEG-ODA NCs group: 10.10 %, Bu-PEG-ODA NCs + LA group: 14.84 %), leading to a significant hypoglycemic effect with a 16.06 % relative pharmacological availability. This dosing strategy exhibited efficacious blood glucose control and pancreatic function recovery capabilities in the type 2 diabetes rat model. This study presents a unique co-optimization strategy based on two key processes in the oral absorption of nanocarriers, yielding significant advancements in the oral bioavailability of nanomedicines and improving their therapeutic efficacy.

The gene therapy approach for retinal disorders has been considered largely over the last decade owing to the favorable outcomes of the US Food and Drug Administration-approved commercial gene therapy, Luxturna. Technological advances in recent years, such as next-generation sequencing, research in molecular pathogenesis of retinal disorders, and precise correlations with their clinical phenotypes, have contributed to the progress of gene therapies for various diseases worldwide, and more recently in India as well. Thus, considerable research is being conducted for the right choice of vectors, transgene engineering, and accessible and cost-effective large-scale vector production. Many retinal disease-specific clinical trials are presently being conducted, thereby necessitating the collation of such information as a ready reference for the scientific and clinical community. In this article, we present an overview of existing gene therapy research, which is derived from an extensive search across PubMed, Google Scholar, and clinicaltrials.gov sources. This contributes to prime the understanding of basic aspects of this cutting-edge technology and information regarding current clinical trials across many different conditions. This information will provide a comprehensive evaluation of therapies in existing use/research for personalized treatment approaches in retinal disorders.

Adeno-associated virus gene therapy has been the subject of intensive investigation for monogenic disease gene addition therapy for more than 25 years, yet few therapies have been approved by regulatory agencies. Most have not progressed beyond phase 1/2 due to toxicity, lack of efficacy, or both. The liver is a natural target for adeno-associated virus since most serotypes have a high degree of tropism for hepatocytes due to cell surface receptors for the virus and the unique liver sinusoidal geometry facilitating high volumes of blood contact with hepatocyte cell surfaces. Recessive monogenic diseases such as hemophilia represent promising targets since the defective proteins are often synthesized in the liver and secreted into the circulation, making them easy to measure, and many do not require precise regulation. Yet, despite initiation of many disease-specific clinical trials, therapeutic windows are often nonexistent, resulting in excess toxicity and insufficient efficacy. Iterative progress built on these attempts is best illustrated by hemophilia, with the first regulatory approvals for factor IX and factor VIII gene therapies eventually achieved 25 years after the first gene therapy studies in humans. Although successful gene transfer may result in the production of sufficient transgenic protein to modify the disease, many emerging questions on durability, predictability, reliability, and variability of response have not been answered. The underlying biology accounting for these heterogeneous responses and the interplay between host and virus is the subject of intense investigation and the subject of this review.

Recent advancements have focused on enhancing factor VIII half-life and refining its delivery methods, despite the well-established knowledge that factor VIII deficiency is the main clotting protein lacking in hemophilia. Consequently, both viral and non-viral delivery systems play a crucial role in enhancing the quality of life for hemophilia patients. The utilization of viral vectors and the manipulation of non-viral vectors through targeted delivery are significant advancements in the field of cellular and molecular therapies for hemophilia. These developments contribute to the progression of treatment strategies and hold great promise for improving the overall well-being of individuals with hemophilia. This review study comprehensively explores the application of viral and non-viral vectors in cellular (specifically T cell) and molecular therapy approaches, such as RNA, monoclonal antibody (mAb), and CRISPR therapeutics, with the aim of addressing the challenges in hemophilia treatment. By examining these innovative strategies, the study aims to shed light on potential solutions to enhance the efficacy and outcomes of hemophilia therapy.

The ability to target the native production site of factor VIII (FVIII)-liver sinusoidal endothelial cells (LSECs)-can improve the outcome of hemophilia A (HA) gene therapy. By testing a matrix of ultrasound-mediated gene delivery (UMGD) parameters for delivering a GFP plasmid into the livers of HA mice, we were able to define specific conditions for targeted gene delivery to different cell types in the liver. Subsequently, two conditions were selected for experiments to treat HA mice via UMGD of an endothelial-specific human FVIII plasmid: low energy (LE; 50 W/cm2, 150 μs pulse duration) to predominantly target endothelial cells or high energy (HE; 110 W/cm2, 150 μs pulse duration) to predominantly target hepatocytes. Both groups of UMGD-treated mice achieved persistent FVIII activity levels of ∼10% over 84 days post treatment; however, half of the HE-treated mice developed low-titer inhibitors while none of the LE mice did. Plasma transaminase levels and histological liver examinations revealed minimal transient liver damage that was lower in the LE group than in the HE group. These results indicate that UMGD can safely target LSECs with a lower-energy condition to achieve persistent FVIII gene expression, demonstrating that this novel technology is highly promising for therapeutic correction of HA.

CRISPR-Cas9-based genome editing technologies, such as base editing, have the potential for clinical translation, but delivering nucleic acids into target cells in vivo is a major obstacle. Viral vectors are widely used but come with safety concerns, while current non-viral methods are limited by low transfection efficiency. Here we describe a new method to deliver CRISPR-Cas9 base editing vectors to the mouse liver using focused ultrasound targeted microbubble destruction (FUTMD). We demonstrate, using the example of cytosine base editing of the Pde3b gene, that FUTMD-mediated delivery of cytosine base editing vectors can introduce stop codons (up to ∼2.5% on-target editing) in mouse liver cells in vivo. However, base editing specificity is less than one might hope with these DNA constructs. Our findings suggest that FUTMD-based gene editing tools can be rapidly and transiently deployed to specific organs and sites, providing a powerful platform for the development of non-viral genome editing therapies. Non-viral delivery also reveals greater off-target base exchange in vivo than in vitro.

Endocytosis plays a crucial role in drug delivery for precision therapy. As a non-invasive and spatiotemporal-controllable stimulus, ultrasound (US) has been utilized for improving drug delivery efficiency due to its ability to enhance cell membrane permeability. When US meets the cell membrane, the well-known cavitation effect generated by US can cause various biophysical effects, facilitating the delivery of various cargoes, especially nanocarriers. The comprehension of recent progress in the biophysical mechanism governing the interaction between ultrasound and cell membranes holds significant implications for the broader scientific community, particularly in drug delivery and nanomedicine. This review will summarize the latest research results on the biological effects and mechanisms of US-enhanced cellular endocytosis. Moreover, the latest achievements in US-related biomedical applications will be discussed. Finally, challenges and opportunities of US-enhanced endocytosis for biomedical applications will be provided.

Hydrodynamics-based gene transfer has been successfully employed for in vivo gene delivery to the liver of small animals by tail vein injection and of large animals using a computer-assisted and image-guided protocol. In an effort to develop a hydrodynamic gene delivery procedure clinically applicable for gene therapy, we have evaluated the safety and effectiveness of a lobe-specific hydrodynamic delivery procedure for hepatic gene delivery in baboons. Reporter plasmid was used to assess the gene delivery efficiency of the lobe-specific hydrodynamic gene delivery, and plasmid-carrying human factor IX gene was used to examine the pattern of long-term gene expression. The results demonstrated liver lobe-specific gene delivery, therapeutic levels of human factor IX gene expression lasting for >100 days, and the efficacy of repeated hydrodynamic gene delivery into the same liver lobes. Other than a transient increase in blood concentration of liver enzymes right after the injection, no significant adverse events were observed in animals during the study period. The results obtained from this first non-human primate study support the clinical applicability of the procedure for lobe-specific hydrodynamic gene delivery to liver.

Gene therapy is a powerful biological tool that is reshaping therapeutic landscapes for several diseases. Researchers are using both non-viral and viral-based gene therapy methods with success in the lab and the clinic. In the cancer biology field, gene therapies are expanding treatment options and the possibility of favorable outcomes for patients. While cellular immunotherapies and oncolytic virotherapies have paved the way in cancer treatments based on genetic engineering, recombinant adeno-associated virus (rAAV), a viral-based module, is also emerging as a potential cancer therapeutic through its malleability, specificity, and broad application to common as well as rare tumor types, tumor microenvironments, and metastatic disease. A wide range of AAV serotypes, promoters, and transgenes have been successful at reducing tumor growth and burden in preclinical studies, suggesting more groundbreaking advances using rAAVs in cancer are on the horizon.