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Gene therapy is a fast-growing field showing great potential to treat genetic diseases and cancer. With accelerating gene therapy development and approval, their environment risk assessment (ERA) becomes increasingly important. An ERA is an assessment of the risks to human health and the environment upon exposure to a medicinal product as the result of its release during clinical development or after entering the market. Because ERA is an important component of regulatory submission, drug developers must perform a robust assessment to ensure the safety of unintended persons, animal, plants, microorganisms and environment at large. Global regulations on gene therapy ERA continue to evolve. Gene therapy ERAs are carried out according to general principles as provided in regulatory guidelines for application of clinical trials and marketing authorizations. The current review intends to summarize regulations and content requirements on gene therapy ERA in European Union, the USA and Japan. The approved gene therapy products by EMA and US Food and Drug Administration are analyzed for the critical aspects of their ERAs to provide the current status and practice of gene therapy ERAs by drug developers. For this purpose, the main contents of these gene therapy ERAs are summarized. Critical safety factors of gene therapy ERAs are described. With more experience and knowledge to be accumulated, gene therapy ERAs are expected to be less challenging with commonly used viral vectors. [ABSTRACT FROM AUTHOR]

Metachromatic leukodystrophy (MLD) is a fatal, progressive neurodegenerative disorder caused by biallelic pathogenic mutations in the ARSA (Arylsulfatase A) gene. With the advent of presymptomatic diagnosis and the availability of therapies with a narrow window for intervention, it is critical to define a standardized approach to diagnosis, presymptomatic monitoring, and clinical care. To meet the needs of the MLD community, a panel of MLD experts was established to develop disease-specific guidelines based on healthcare resources in the United States. This group developed a consensus opinion for best-practice recommendations, as follows: (i) Diagnosis should include both genetic and biochemical testing; (ii) Early diagnosis and treatment for MLD is associated with improved clinical outcomes; (iii) The panel supported the development of newborn screening to accelerate the time to diagnosis and treatment; (iv) Clinical management of MLD should include specialists familiar with the disease who are able to follow patients longitudinally; (v) In early onset MLD, including late infantile and early juvenile subtypes, ex vivo gene therapy should be considered for presymptomatic patients where available; (vi) In late-onset MLD, including late juvenile and adult subtypes, hematopoietic cell transplant (HCT) should be considered for patients with no or minimal disease involvement. This document summarizes current guidance on the presymptomatic monitoring of children affected by MLD as well as the clinical management of symptomatic patients. Future data-driven evidence and evolution of these recommendations will be important to stratify clinical treatment options and improve clinical care. [ABSTRACT FROM AUTHOR]

To better understand the attitudes and behaviors of investors involved in funding cell and gene therapy (CGT) businesses, the Business Development and Finance) subcommittee of International Society for Cell and Gene Therapy, in collaboration with Truist Securities, conducted a broad survey of the investment community in late 2021. This survey follows a similar study that this group executed in 2018, and the longitudinal comparisons between the two time periods provide insights into how investor behavior in the CGT field has evolved. The vast majority of investor respondents are specialist biotech investors who are primarily active in deploying capital in North America and Europe. There was a notable increase in the proportion of investors actively deploying capital in China and Japan between 2018 and 2021. The percentage of respondents' portfolios dedicated to CGT companies has also increased in this period, reflecting a noteworthy trend in the therapeutic landscape. Clinically significant data remain the dominant force behind investment decisions, whereas competition from other drug modalities has now emerged as the most-cited barrier to making a CGT investment, eclipsing safety concerns as the most significant barrier to investment in 2018. Concerns around manufacturing and scale-up have also increased in prominence amongst the investment community. Gene-editing technologies are attracting investors as the most compelling new CGT technology. This survey also revealed that most investors expect to increase their level of investment in allogeneic technologies relative to autologous products in the coming years. [ABSTRACT FROM AUTHOR]

Cell and gene therapy poses evolving challenges. The current article summarizes the discussions held by European Regional Committee of the International Society for Cell & Gene Therapy and the European Society for Blood and Marrow Transplantation (EBMT) on the current challenges in this field, focusing on the European setting. This article emphasizes the imperative assessment of real-world cell and gene therapy activity, advocating for expanded registries beyond hematopoietic transplantation and chimeric antigen receptor–T-cell therapy. Accreditation's role in ensuring standardized procedures, as exemplified by JACIE (The Joint Accreditation Committee of ISCT-Europe and EBMT), is crucial for safety. Access to commercial products and reimbursement variations among countries underscore the need for uniform access to advanced therapy medical products (ATMPs). Academic product development and point-of-care manufacturing face barriers to patient access. Hospital Exemption's potential, demonstrated by some initial experiences, may increase patient accessibility in individual situations. Regulatory challenges, including the ongoing European ATMPs legislation review, necessitate standardized criteria for Hospital Exemption and mandatory reporting within registries. Efforts to combat unproven therapies and fraud involve collaboration between scientific societies, regulatory bodies and patient groups. Finally, is important to highlight the vital role of education and workforce development in meeting the escalating demand for specialized professionals in the ATMP field. Collaboration among scientific societies, academic institutions, industry, regulatory bodies and patient groups is crucial for overcoming all these challenges to increase gene and cell therapy activity in Europe. [ABSTRACT FROM AUTHOR]

Gene replacement therapy (onasemnogene abeparvovec) is associated with an improvement of the prognosis of children with spinal muscular atrophy, but information on long-term respiratory outcome is scarce. The aim of this study was to report the polysomnography findings and respiratory muscle function of infants with treatment-naive spinal muscular atrophy type 1 and 2 up to 24 months after onasemnogene abeparvovec monotherapy. A clinical and motor evaluation, respiratory muscle function testing, and polysomnography were performed repeatedly. Fifteen spinal muscular atrophy patients (1 presymptomatic, 7 type 1b, 6 type 1c, and 1 type 2) were included at a median age of 8.6 months (range 3.8–12.6) and followed for 24 months. The thoracic over head circumference ratio was close to normal at baseline (median 1.00 (range 0.90–1.05)) and increased significantly over time. All polysomnography and nocturnal gas exchange parameters were within normal ranges at baseline (median apnea-hypopnea index 2.5 events/hour (range 0.4–5.3)) and follow-up. The inspiratory muscle strength was normal at baseline but tended to slightly decrease over time and the expiratory muscle strength was low at any time especially for patients with recurrent respiratory infections (median (range) at baseline in cmH 2 O: crying esophageal pressure 54 (30–110), crying transdiaphragmatic pressure 65 (35–107), gastric pressure during maximal cough 26 (10–130), esophageal pressure during maximal cough 61 (38–150)). Only 3 patients required noninvasive ventilation. A continuous respiratory monitoring of spinal muscular atrophy patients during the first years of life following onasemnogene abeparvovec monotherapy seems recommended despite the normality of polysomnography parameters. • Fifteen patients received gene replacement therapy as first and only treatment. • Thoracic over head circumference was close to normal and increased over time. • All polysomnography parameters were within normal ranges at all time. • The inspiratory muscle strength was normal but the expiratory muscles were weak. • We recommend a continuous respiratory monitoring after gene therapy. [ABSTRACT FROM AUTHOR]

• Two image analysis methods have been developed for viral vector characterization, and their performances were compared. • A combination of cryo-electron microscopy and image analysis enables the accurate assessment of the viral vector population. • Convolutional neural networks and morphological analysis quantified exiguous impurities in viral vector products with 90% purity. • The utility of convolutional neural networks to characterize wider adeno-associated viral products was demonstrated. • Image analysis methods based on cryo-electron micrographs might evaluate the slight difference in the viral vector population. Adeno-associated viruses (AAVs) are effective vectors for gene therapy. However, AAV drug products are inevitably contaminated with empty particles (EP), which lack a genome, owing to limitations of the purification steps. EP contamination can reduce the transduction efficiency and induce immunogenicity. Therefore, it is important to remove EPs and to determine the ratio of full genome-containing AAV particles to empty particles (F/E ratio). However, most of the existing methods fail to reliably evaluate F/E ratios that are greater than 90 %. In this study, we developed two approaches based on the image analysis of cryo-electron micrographs to determine the F/E ratios of various AAV products. Using our developed convolutional neural network (CNN) and morphological analysis, we successfully calculated the F/E ratios of various AAV products and determined the slight differences in the F/E ratios of highly purified AAV products (purity > 95 %). In addition, the F/E ratios calculated by analyzing more than 1000 AAV particles had good correlations with theoretical F/E ratios. Furthermore, the CNN reliably determined the F/E ratio with a smaller number of AAV particles than morphological analysis. Therefore, combining 100 keV cryo-EM with the developed image analysis methods enables the assessment of a wide range of AAV products. [ABSTRACT FROM AUTHOR]

Adeno-associated viruses (AAVs) have become the delivery medium of choice for a variety of genomic medicine applications i.e., gene therapy, gene editing/regulation, and ex-vivo cell therapy. AAVs are protein-DNA complexes which have unique stability characteristics that are susceptible to various stress exposure conditions commonly seen in the drug product (DP) life cycle. This review takes a comprehensive look at AAV DP formulation and process development considerations that could impact critical quality attributes (CQAs) during manufacturing, packaging, shipping, and clinical use. Additional aspects related to AAV development reviewed herein are: (1) Different AAV serotypes with unique protein sequences and charge characteristics potentially leading to discrete stability profiles; (2) Manufacturing process challenges and optimization efforts to improve yield, recovery and purity especially during early development activities; and (3) Defining and identifying CQAs with analytical methods which are constantly evolving and present unique characterization challenges for AAV-based products. [Display omitted] [ABSTRACT FROM AUTHOR]

Arrhythmia detection is essential when assessing the safety of novel drugs and therapies in preclinical studies. Many short-term arrhythmia monitoring methods exist, including non-invasive ECG and Holter. However, there are no reliable, long-term, non-invasive, or minimally invasive methods for cardiac arrhythmia follow-up in large animals that allows free movement with littermates. A long follow-up time is needed when estimating the impact of long-lasting drugs or therapies, such as gene therapy. We evaluated the feasibility and performance of insertable cardiac monitors (ICMs) in pigs for minimally invasive, long-term monitoring of cardiac arrhythmias that allows free movement and species-specific behavior. Multiple implantation sites were tested to assess signal quality. ICMs recognized reliably many different arrhythmias but failed to detect single extrasystoles. They also over-diagnosed T-waves, resulting in oversensing. Muscle activity and natural startles of the animals caused noise, leading to a heterogeneous signal requiring post-recording evaluation. In spite of these shortcomings, the ICMs showed to be very useful for minimally invasive long-term monitoring of cardiac rhythm in pigs. [ABSTRACT FROM AUTHOR]

[Display omitted] • IGF-1 gene therapy improves cognitive deficits caused by 6-hydroxydopamine. • IGF-1 gene therapy increases the expression level of tyrosine hydroxylase in the caudate-putamen. • IGF-1 gene therapy modifies parameters of microglia and astrocyte phenotypes. Cognitive impairment in Parkinson's disease is considered an indicator of the prodromal stages of this condition, occurring prior to the onset of classic and pathognomonic motor symptoms. Among other factors, neuroinflammation is increasingly recognized as a potential mediator of this neurodegenerative process, and glial cells are directly involved. However, the use of neurotrophic factors is associated with neuroprotection and cognitive improvements. Among all those factors, insulin-like growth factor 1 (IGF-1) has attracted considerable attention. In this study, we aimed to investigate the effect of IGF-1 gene therapy in an early animal model of 6-hydroxidopamine (6-OHDA)- induced parkinsonism. For this purpose, we employed male Wistar rats. The animals were first divided into two groups according to the bilateral injection into de Caudate Putamen unit (CPu):(a) VEH group (vehicle solution) and (b) 6-OHDA group (neurotoxic solution). After that, the animals in each group were divided, according to the bilateral injection into the dorsal hippocampus, in a control group (who received a control virus RAd-DSRed) and an experimental group (who received a therapeutic virus (RAd-IGF1). After three weeks of exposure to 6-OHDA, our study showed that IGF-1 gene therapy improved cognitive deficits related to short-term and spatial working memory, it also increased expression levels of tyrosine hydroxylase in the CPu. In addition, the therapy resulted in significant changes in several parameters (area, perimeter, roundness, ramification, and skeleton ́s analyses) related to microglia and astrocyte phenotypes, particularly in the CPu and dorsal hippocampal areas. Our data support the use of IGF-1 as a therapeutic molecule for future gene transfer interventions, that will contribute to a better understanding of the mechanisms correlating cognitive function and inflammatory process. [ABSTRACT FROM AUTHOR]

Gene delivery has revolutionized conventional medical approaches to vaccination, cancer, and autoimmune diseases. However, current gene delivery methods are limited to either intravenous administration or direct local injections, failing to achieve well biosafety, tissue targeting, drug retention, and transfection efficiency for desired therapeutic outcomes. Transdermal drug delivery based on various delivery strategies can offer improved therapeutic potential and superior patient experiences. Recently, there has been increased foundational and clinical research focusing on the role of the transdermal route in gene delivery and exploring its impact on the efficiency of gene delivery. This review introduces the recent advances in transdermal gene delivery approaches facilitated by drug formulations and medical devices, as well as discusses their prospects. [Display omitted] [ABSTRACT FROM AUTHOR]

Autosomal dominant osteopetrosis type 2 (ADO2) is a rare inherited bone disorder characterised by dense but brittle bones. It displays striking phenotypic variability, with the most severe symptoms, including blindness and bone marrow failure. Disease management largely relies on symptomatic treatment since there is no safe and effective treatment. Most ADO2 cases are caused by heterozygous loss-of-function mutations in the CLCN7 gene, which encodes an essential Cl−/H+ antiporter for proper bone resorption by osteoclasts. Thus, siRNA-mediated silencing of the mutant allele is a promising therapeutic approach, but targeting bone for first-in-human translation remains challenging. Here, we demonstrate the utility of silicon-stabilised hybrid lipid nanoparticles (sshLNPs) as a next-generation nucleic acid nanocarrier capable of delivering allele-specific siRNA to bone. Using a Clcn7G213R knock-in mouse model recapitulating one of the most common human ADO2 mutations and based on the 129S genetic background (which produces the most severe disease phenotype amongst current models), we show substantial knockdown of the mutant allele in femur when siRNA targeting the pathogenic variant is delivered by sshLNPs. We observed lower areal bone mineral density in femur and reduced trabecular thickness in femur and tibia, when siRNA-loaded sshLNPs were administered subcutaneously (representing the most relevant administration route for clinical adoption and patient adherence). Importantly, sshLNPs have improved stability over conventional LNPs and enable 'post hoc loading' for point-of-care formulation. The treatment was well tolerated, suggesting that sshLNP-enabled gene therapy might allow successful clinical translation of essential new treatments for ADO2 and potentially other rare genetic bone diseases. [ABSTRACT FROM AUTHOR]

The management of hemophilia A has undergone a remarkable revolution, in line with technological advancement. In the recent past, the primary concern associated with Factor VIII (FVIII) concentrates was the risk of infections, which is now almost resolved by advanced blood screening and viral inactivation methods. Improving patients' compliance with prophylaxis has become a key focus, as it can lead to improved health outcomes and reduced health care costs in the long term. Recent bioengineering research is directed toward prolonging the recombinant FVIII (rFVIII) coagulant activity and synthesising higher FVIII yields. As an outcome, B-domain deleted, polyethylene glycolated, single-chain, Fc-fused rFVIII, and rFVIIIFc-von Willebrand Factor-XTEN are available for patients. Moreover, emicizumab, a bispecific antibody, is commercially available, whereas fitusiran and tissue factor pathway inhibitor are in clinical trial stages as alternative strategies for patients with inhibitors. With these advancements, noninfectious complications, such as inhibitor development, allergic reactions, and thrombosis, are emerging concerns requiring careful management. In addition, the recent approval of gene therapy is a major milestone toward a permanent cure for hemophilia A. The vast array of treatment options at our disposal today empowers patients and providers alike, to tailor therapeutic regimens to the unique needs of each individual. Despite significant progress in modern treatment options, these highly effective therapies are markedly more expensive than conventional replacement therapy, limiting their access for patients in developing countries. [ABSTRACT FROM AUTHOR]

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by a full mutation (> 200 CGG repeats) in the FMR1 gene. FXS is the leading cause of inherited intellectual disabilities and the most commonly known genetic cause of autism spectrum disorder. Children with FXS experience behavioral and sleep problems, anxiety, inattention, learning difficulties, and speech and language delays. There are no approved medications for FXS; however, there are several interventions and treatments aimed at managing the symptoms and improving the quality of life of individuals with FXS. A combination of non‐pharmacological therapies and pharmacotherapy is currently the most effective treatment for FXS. Currently, several targeted treatments, such as metformin, sertraline, and cannabidiol, can be used by clinicians to treat FXS. Gene therapy is rapidly developing and holds potential as a prospective treatment option. Soon its efficacy and safety in patients with FXS will be demonstrated. What this paper adds: Targeted treatment of fragile X syndrome (FXS) is the best current therapeutic approach.Gene therapy holds potential as a prospective treatment for FXS in the future. [ABSTRACT FROM AUTHOR]

ABSTRACT Recombinant adeno‐associated virus (AAV) has become a popular platform for many gene therapy applications. The strength of AAV‐based products is a critical quality attribute that affects the efficacy of the drug and is measured as the concentration of vector genomes, or physical titer. Because the dosing of patients is based on the titer measurement, it is critical for manufacturers to ensure that the measured titer of the drug product is close to the actual concentration of the batch. Historically, dosing calculations have been performed using the measured titer, which is reported on the drug product label. However, due to recent regulatory guidance, sponsors are now expected to label the drug product with nominal or “target” titer. This new expectation for gene therapy products can pose a challenge in the presence of process and analytical variability. In particular, the manufacturer must decide if a dilution of the drug substance is warranted at the drug product stage to bring the strength in line with the nominal value. In this paper, we present two straightforward statistical methods to aid the manufacturer in the dilution decision. These approaches use the understanding of process and analytical variability to compute probabilities of achieving the desired drug product titer. We also provide an approach for determining an optimal assay replication strategy for achieving the desired probability of meeting drug product release specifications. [ABSTRACT FROM AUTHOR]

The microvascular network plays an important role in providing nutrients to the injured tissue and exchanging various metabolites. However, how to achieve efficient penetration of the injured tissue is an important bottleneck restricting the reconstruction of microvascular network. Herein, the hydrogel precursor solution can efficiently penetrate the damaged tissue area, and ultrasound triggers the release of thrombin from liposomes in the solution to hydrolyze fibrinogen, forming a fibrin solid hydrogel network in situ with calcium ions and transglutaminase as catalysts, effectively solving the penetration impedance bottleneck of damaged tissues and ultimately significantly promoting the formation of microvascular networks within tissues. First, the fibrinogen complex solution is effectively permeated into the injured tissue. Second, ultrasound triggered the release of calcium ions and thrombin, activates transglutaminase, and hydrolyzes fibrinogen. Third, fibrin monomers are catalyzed to form fibrin hydrogels in situ in the damaged tissue area. In vitro studies have shown that the fibrinogen complex solution effectively penetrated the artificial bone tissue within 15 s after ultrasonic triggering, and formed a hydrogel after continuous triggering for 30 s. Overall, this innovative strategy effectively solved the problem of penetration resistance of ultrasound‐triggered hydrogels in the injured tissues, and finally activates in situ microvascular networks regeneration. [ABSTRACT FROM AUTHOR]

Inherited metabolic disorders (IMDs) are a growing group of genetic diseases caused by defects in enzymes that mediate cellular metabolism, often resulting in the accumulation of toxic substrates. The liver is a highly metabolically active organ that hosts several thousands of chemical reactions. As such, it is an organ frequently affected in IMDs. In this article, we review current approaches for liver‐directed gene‐based therapy aimed at metabolite detoxification in a variety of IMDs. Moreover, we discuss current unresolved challenges in gene‐based therapies for IMDs. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy. Author summary: Adeno-associated virus (AAV) is a viral vector that is universally applied in human gene therapy. Despite their versatile applications in gene therapy, the origin and evolution of AAV serotypes have not been extensively studied. In this study, positive selection upon diversification of AAV capsid genes was carefully investigated by evolutionary analysis. Numerous diversifying selected sites were dispersed among the AAV lineages, and evolutionary pathways were inferred for two selected sites of the AAV2 capsid gene. Furthermore, the putative immunological drives underlying the selection of AAV2 capsid genes were experimentally dissected. This work provides a novel interpretation of the evolution and diversity of AAV serotypes and might be helpful for their engineering and application in gene therapy. [ABSTRACT FROM AUTHOR]

The article outlines practical operational considerations for health-system pharmacy leaders when planning to onboard new gene therapy approvals. Topics discussed include site preparedness and biosafety recommendations, common gene therapy clinical and operational checklist, and importance of clinical practice guidelines for adverse effect management.

[Display omitted] Combination therapies demand co-delivery platforms with efficient entrapment of distinct payloads and specific delivery to cells and possibly organelles. Herein, we introduce the combination of two therapeutic modalities, gene and photodynamic therapy, in a purely peptidic platform. The simultaneous formation and cargo loading of the multi-micellar platform is governed by self-assembly at the nanoscale. The multi-micellar architecture of the nanocarrier and the positive charge of its constituent micelles offer controlled dual loading capacity with distinct locations for a hydrophobic photosensitizer (PS) and negatively charged antisense oligonucleotides (ASOs). Moreover, the nuclear localization signal (NLS) sequence built-in the peptide targets PS + ASO-loaded nanocarriers to the nucleus. Breast cancer cells treated with nanocarriers demonstrated photo-triggered enhancement of radical oxygen species (ROS) associated with increased cell death. Besides, delivery of ASO payloads resulted in up to 90 % knockdown of Bcl-2, an inhibitor of apoptosis that is overexpressed in more than half of all human cancers. Simultaneous delivery of PS and ASO elicited synergistic apoptosis to an extent that could not be reached by singly loaded nanocarriers or the free form of the drugs. Both, the distinct location of loaded compounds that prevents them from interfering with each other, and the highly efficient cellular delivery support the great potential of this versatile peptide platform in combination therapy. [ABSTRACT FROM AUTHOR]

Background: Biallelic pathogenic variants in USH2A lead to Usher syndrome or non-syndromic retinitis pigmentosa, and shown to have geographical and ethnical distribution in previous studies. This study provided a deeper understanding of the detailed clinical features using multimodal imaging, genetic spectrum, and genotype–phenotype correlations of USH2A-related retinal dystrophies in Taiwan. Results: In our cohort, the mean age at first visit was 47.66 ± 13.54 years, and the mean age at symptom onset, which was referred to the onset of nyctalopia and/or visual field constriction, was 31.21 ± 15.24 years. Among the variants identified, 23 (50%) were missense, 10 (22%) were splicing variants, 8 (17%) were nonsense, and 5 (11%) were frameshift mutations. The most predominant variant was c.2802T>G, which accounted for 21% of patients, and was located in exon 13. Patients with truncated alleles had significantly earlier symptom onset and seemly poorer disease progression regarding visual acuity, ellipsoid zone line length, and hypofluorescent lesions in the macula than those who had the complete gene. However, the clinical presentation revealed similar progression between patients with and without the c.2802T>G variant. During long-term follow-up, the patients had different ellipsoid zone line progression rates and were almost evenly distributed in the fast, moderate, and slow progression subgroups. Although a younger onset age and a smaller baseline intact macular area was observed in the fast progression subgroup, the results showed no significant difference. Conclusions: This is the first cohort study to provide detailed genetic and longitudinal clinical analyses of patients with USH2A-related retinal dystrophies in Taiwan. The mutated allele frequency in exon 13 was high in Taiwan due to the predominant c.2802T>G variant. Moreover, truncated variants greatly impacted disease progression and determined the length of therapeutic windows. These findings provide insight into the characteristics of candidates for future gene therapies. [ABSTRACT FROM AUTHOR]

Background: Fibrosis is a significant pathological feature of chronic skeletal muscle injury, profoundly affecting muscle regeneration. Fibro-adipogenic progenitors (FAPs) have the ability to differentiate into myofibroblasts, acting as a primary source of extracellular matrix (ECM). the process by which FAPs differentiate into myofibroblasts during chronic skeletal muscle injury remains inadequately explored. Method: mouse model with sciatic nerve denervated was constructed and miRNA expression profiles between the mouse model and uninjured mouse were analyzed. qRT/PCR and immunofluorescence elucidated the effect of miR-27b-3p on fibrosis in vivo and in vitro. Dual-luciferase reporter identified the target gene of miR-27b-3p, and finally knocked down or overexpressed the target gene and phosphorylation inhibition of Smad verified the influence of downstream molecules on the abundance of miR-27b-3p and fibrogenic differentiation of FAPs. Result: FAPs derived from a mouse model with sciatic nerves denervated exhibited a progressively worsening fibrotic phenotype over time. Introducing agomiR-27b-3p effectively suppressed fibrosis both in vitro and in vivo. MiR-27b-3p targeted Transforming Growth Factor Beta Receptor 1 (TGF-βR1) and the abundance of miR-27b-3p was negatively regulated by TGF-βR1/Smad. Conclusion: miR-27b-3p targeting the TGF-βR1/Smad pathway is a novel mechanism for regulating fibrogenic differentiation of FAPs. Increasing abundance of miR-27b-3p, suppressing expression of TGF-βR1 and inhibiting phosphorylation of smad3 presented potential strategies for treating fibrosis in chronic skeletal muscle injury. [ABSTRACT FROM AUTHOR]

Gene therapy aims to modify or manipulate gene expression and change the biological characteristics of living cells to achieve the purpose of treating diseases. The safe, efficient, and stable expression of exogenous genes in cells is crucial for the success of gene therapy, which is closely related to the vectors used in gene therapy. Currently, gene therapy vectors are mainly divided into two categories: viral vectors and non-viral vectors. Viral vectors are widely used due to the advantages of persistent and stable expression, high transfection efficiency, but they also have certain issues such as infectivity, high immunological rejection, randomness of insertion mutation, carcinogenicity, and limited vector capacity. Non-viral vectors have the advantages of non-infectivity, controllable chemical structure, and unlimited vector capacity, but the transfection efficiency is low. With the rapid development of nanotechnology, the unique physicochemical properties of nanomaterials have attracted increasing attention in the field of drug and gene delivery. Among many nanomaterials, iron-based nanomaterials have attracted much attention due to their superior physicochemical properties, such as Fenton reaction, magnetic resonance imaging, magnetothermal therapy, photothermal therapy, gene delivery, magnetically-assisted drug delivery, cell and tissue targeting, and so on. In this paper, the research progress of iron-based nanomaterials in gene delivery and tumor gene therapy is reviewed, and the future application direction of iron-based nanomaterials is further prospected. [ABSTRACT FROM AUTHOR]

Niemann–Pick disease (NPD) is another type of metabolic disorder that is classified as lysosomal storage diseases (LSDs). The main cause of the disease is mutation in the SMPD1 (type A and B) or NPC1 or NPC2 (type C) genes, which lead to the accumulation of lipid substrates in the lysosomes of the liver, brain, spleen, lung, and bone marrow cells. This is followed by multiple cell damage, dysfunction of lysosomes, and finally dysfunction of body organs. So far, about 346, 575, and 30 mutations have been reported in SMPD1, NPC1, and NPC2 genes, respectively. Depending on the type of mutation and the clinical symptoms of the disease, the treatment will be different. The general aim of the current study is to review the clinical and molecular characteristics of patients with NPD and study various treatment methods for this disease with a focus on gene therapy approaches. Significance statement: This work concerns an overview of Niemann–Pick disease (types A, B, and C) of clinical, molecular, and therapeutic features (focus on therapy approaches especially gene therapy). [ABSTRACT FROM AUTHOR]

Background and Objective: Gene therapies for sickle cell disease (SCD) may offer meaningful benefits for patients and society. This study evaluated the cost-effectiveness of lovotibeglogene autotemcel (lovo-cel), a one-time gene therapy administered via autologous hematopoietic stem cell transplantation, compared with common care for patients in the United States (US) with SCD aged ≥ 12 years with ≥ 4 vaso-occlusive events (VOEs) in the past 24 months. Methods: We developed a patient-level simulation model accounting for lovo-cel and SCD-related events, complications, and mortality over a lifetime time horizon. The pivotal phase 1/2 HGB-206 clinical trial (NCT02140554) served as the basis for lovo-cel efficacy and safety. Cost, quality-of-life, and other clinical data were sourced from HGB-206 data and the literature. Analyses were conducted from US societal and third-party payer perspectives. Uncertainty was assessed through probabilistic sensitivity analysis and extensive scenario analyses. Results: Patients treated with lovo-cel were predicted to survive 23.84 years longer on average (standard deviation [SD], 12.80) versus common care (life expectancy, 62.24 versus 38.40 years), with associated discounted patient quality-adjusted life-year (QALY) gains of 10.20 (SD, 4.10) and direct costs avoided of $1,329,201 (SD, $1,346,446) per patient. Predicted societal benefits included discounted caregiver QALY losses avoided of 1.19 (SD, 1.38) and indirect costs avoided of $540,416 (SD, $262,353) per patient. Including lovo-cel costs ($3,282,009 [SD, $29,690] per patient) resulted in incremental cost-effectiveness ratios of $191,519 and $124,051 per QALY gained from third-party payer and societal perspectives, respectively. In scenario analyses, the predicted cost-effectiveness of lovo-cel also was sensitive to baseline age and VOE frequency and to the proportion of patients achieving and maintaining complete resolution of VOEs. Conclusions: Our analysis of lovo-cel gene therapy compared with common care for patients in the US with SCD with recurrent VOEs estimated meaningful improvements in survival, quality of life, and other clinical outcomes accompanied by increased overall costs for the health care system and for broader society. The predicted economic value of lovo-cel gene therapy was influenced by uncertainty in long-term clinical effects and by positive spillover effects on patient productivity and caregiver burden. [ABSTRACT FROM AUTHOR]

Each month, subscribers to The Formulary Monograph Service receive 5 to 6 well-documented monographs on drugs that are newly released or are in late phase 3 trials. The monographs are targeted to Pharmacy & Therapeutics Committees. Subscribers also receive monthly 1-page summary monographs on agents that are useful for agendas and pharmacy/nursing in-services. A comprehensive target drug utilization evaluation/medication use evaluation (DUE/MUE) is also provided each month. With a subscription, the monographs are available online to subscribers. Monographs can be customized to meet the needs of a facility. Through the cooperation of The Formulary, Hospital Pharmacy publishes selected reviews in this column. For more information about The Formulary Monograph Service, contact Wolters Kluwer customer service at 866-397-3433. [ABSTRACT FROM AUTHOR]

The article discusses a Phase I trial of gene therapy for Giant Axonal Neuropathy (GAN), an ultrarare childhood neurodegenerative disease. The study assesses safety and efficacy through intrathecal injection of a gene editing approach targeting motor neuron bodies and sensory neurons, showing promise in stabilizing disease progression at one year. Topics include disease pathology, trial methodology, and safety outcomes, highlighting the potential of intrathecal AAV gene therapy for GAN.

The tumor microenvironment is importantly shaped by various cytokines, where interleukins (ILs) and interferons (IFNs) shape the balance of immune activity within tumor niche and associated lymphoid organs. Their importance in activation and tuning of both innate and adaptive immune responses prompted their use in several clinical trials, albeit with limited therapeutic efficacy and risk of toxicity due to systemic administration. Increasing preclinical evidence suggests that local delivery of ILs and IFNs could significantly increase their effectiveness, while simultaneously attenuate the known side effects and issues related to their biological activity. A prominent way to achieve this is to use cell-based delivery vehicles. For this purpose, mesenchymal stromal stem cells (MSCs) are considered an almost ideal candidate. Namely, MSCs can be obtained in large quantities and from obtainable sources (e.g. umbilical cord or adipose tissue), their ex vivo expansion is relatively straightforward compared to other cell types and they possess very low immunogenicity making them suitable for allogeneic use. Importantly, MSCs have shown an intrinsic capacity to respond to tumor-directed chemotaxis. This review provides a focused and detailed discussion on MSC-based gene therapy using ILs and IFNs, engineering techniques and insights on potential future advancements. [Display omitted] • MSCs represent one of the most ideal candidates for intratumoral cytokine delivery. • Both ILs and IFNs importantly shape the tumor microenvironment. • Gene delivery of ILs and IFNs can favourably shape the tumor immune contexture. • MSCs can be modified to gain synergistic anti-tumor effects and to inhibit tumor promoting activities of certain cytokines. [ABSTRACT FROM AUTHOR]

• DMD is caused by DMD gene mutations that result in a lack of dystrophin protein. • BMD is also caused by DMD mutations, but symptoms are milder compared with DMD. • We present a case study of a patient with BMD and his affected relatives. • The patient could walk at age 61 years, despite 46% of his DMD gene being missing. • These findings informed minigene constructs, a promising therapy option for DMD. Duchenne muscular dystrophy is a neuromuscular disease caused by DMD gene mutations that result in an absence of functional dystrophin protein. Patients with Duchenne experience progressive muscle weakness, are typically wheelchair dependent by their early teens, and develop respiratory and cardiac complications that lead to death in their twenties or thirties. Becker muscular dystrophy is also caused by DMD gene mutations, but symptoms are less severe and progression is slower compared with Duchenne. We describe a case study of a patient with Becker muscular dystrophy who was still ambulant at age 61 years and had a milder phenotype than Duchenne, despite 46% of his DMD gene being missing. His affected relatives had similarly mild phenotypes and clinical courses. These data guided the understanding of the criticality of various regions of dystrophin and informed the development of micro-dystrophin constructs to compensate for the absence of functional dystrophin in Duchenne. [ABSTRACT FROM AUTHOR]

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides (ASOs) are short, synthetic, single‐stranded deoxynucleotide sequences composed of phosphate backbone‐connected sugar rings. Designing of those strands is based on Watson‐Crick hydrogen bonding mechanism. Thanks to rapidly advancing medicine and technology, evolving of the gene therapy area and ASO approaches gain attention. Considering the genetic basis of diseases, it is promising that gene therapy approaches offer more specific and effective options compared to conventional treatments. The objective of this review is to explain the mechanism of ASOs and discuss the characteristics and safety profiles of therapeutic agents in this field. Pharmacovigilance for gene therapy products is complex, requiring accurate assessment of benefit‐risk balance and evaluation of adverse effects. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is a type of hemoglobinopathy due to an autosomal recessive genetic defect, causing significant red cell sickling, multi‐organ damage and long‐term severe morbidities. Due to its complicated care and the impact on quality of life, a curative treatment for SCD is highly desirable. In recent years, gene therapy is emerging as a curative option for SCD, where autologous haematopoietic stem cells are collected from SCD patients and genetically modified ex vivo to reduce its sickling tendency before reinfusion. Although still largely investigational, a limited number of gene therapy options have been recently granted approval for SCD patients. Published data are still currently limited, but early studies have so far demonstrated the intended outcomes of less vaso‐occlusive crisis and haemolysis. Nonetheless, despite its curative potential, larger clinical trials and longer follow‐up period are still necessary to evaluate the safety of this treatment option, especially the risk of unintended genetic modifications. Furthermore, SCD patients frequently have limited access to specialty care; hence, the issues of affordability and accessibility to SCD gene therapy must also be addressed for it to benefit the appropriate patient population. [ABSTRACT FROM AUTHOR]

Diamond–Blackfan anemia (DBA) is a rare genetic disorder affecting the bone marrow's ability to produce red blood cells, leading to severe anemia and various physical abnormalities. Approximately 75% of DBA cases involve heterozygous mutations in ribosomal protein (RP) genes, classifying it as a ribosomopathy, with RPS19 being the most frequently mutated gene. Non-RP mutations, such as in GATA1, have also been identified. Current treatments include glucocorticosteroids, blood transfusions, and hematopoietic stem cell transplantation (HSCT), with HSCT being the only curative option, albeit with challenges like donor availability and immunological complications. Gene therapy, particularly using lentiviral vectors and CRISPR/Cas9 technology, emerges as a promising alternative. This review explores the potential of gene therapy, focusing on lentiviral vectors and CRISPR/Cas9 technology in combination with non-integrating lentiviral vectors, as a curative solution for DBA. It highlights the transformative advancements in the treatment landscape of DBA, offering hope for individuals affected by this condition. [ABSTRACT FROM AUTHOR]

The author identified the genes and proteins of human enzymes involved in the biosynthesis of catecholamines (dopamine, norepinephrine, epinephrine) and tetrahydrobiopterin (BH4): tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), and GTP cyclohydrolase I (GCH1). In Parkinson's disease (PD), the activities and levels of mRNA and protein of all catecholamine-synthesizing enzymes are decreased, especially in dopamine neurons in the substantia nigra. Hereditary GCH1 deficiency results in reductions in the levels of BH4 and the activities of TH, causing decreases in dopamine levels. Severe deficiencies in GCH1 or TH cause severe decreases in dopamine levels leading to severe neurological symptoms, whereas mild decreases in TH activity in mild GCH1 deficiency or in mild TH deficiency result in only modest reductions in dopamine levels and symptoms of DOPA-responsive dystonia (DRD, Segawa disease) or juvenile Parkinsonism. DRD is a treatable disease and small doses of L-DOPA can halt progression. The death of dopamine neurons in PD in the substantia nigra may be related to (i) inflammatory effect of extra neuronal neuromelanin, (ii) inflammatory cytokines which are produced by activated microglia, (iii) decreased levels of BDNF, and/or (iv) increased levels of apoptosis-related factors. This review also discusses progress in gene therapies for the treatment of PD, and of GCH1, TH and AADC deficiencies, by transfection of TH, AADC, and GCH1 via adeno-associated virus (AAV) vectors. [ABSTRACT FROM AUTHOR]

OBJECTIVE The aim of this study was to assess the efficacy and safety of a third-generation lentivirus-based vector encoding the feline erythropoietin (EPO) (feEPO) gene in vitro and in rodent models in vivo. This vector incorporates a genetic mechanism to facilitate the termination of the therapeutic effect in the event of supraphysiologic polycythemia, the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene." ANIMALS CFRK cells and replication-defective lentiviral vectors encoding feEPO were used for in vitro experiments. Eight Fischer rats were enrolled in the pilot in vivo study, 24 EPO-deficient mice were used in the initial mouse study, and 15 EPO-deficient mice were enrolled in the final mouse study. METHODS Efficacy of a third-generation lentivirus encoding feEPO was determined in vitro using western blot assays. Subsequently, in a series of rodent experiments, animals were administered the viral vector in progressively increasing inoculation doses with serial measurements of blood packed cell volume (PCV) over time. RESULTS We documented production of feEPO protein in transduced CRFK cells with subsequent cessation of production when treated with the HSV-TK substrate ganciclovir. In vivo, we demonstrated variably persistent elevated PCV values in treated rats and mice with eventual return to baseline values over time. CLINICAL RELEVANCE These results provide justification for a lentiviral gene therapy approach to the treatment of nonregenerative anemia associated with chronic renal disease in cats. [ABSTRACT FROM AUTHOR]

It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20–42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice. AAV mediated gene editing using CRISPR/Cas9 facilitates integration of a bypass coagulation factor VIIa and rescues bleeding phenotype in hemophilia B mice. Image created using Biorender.com. [Display omitted] • CRISPR/Cas9 mediated targeting of Rosa26 locus was found to have 20-42% efficiency in vitro. • Successful insertion of murine activated factor VII (mFVIIa) into the Rosa26 locus was achieved by CRISPR/Cas9 gene editing. • AAV mediated knock-in of bypass clotting factor mFVIIa using CRISPR/Cas9 mitigated bleeding phenotype in hemophilia B mice. • Increased FVIIa activity and shortened prothrombin time was attained after gene editing therapy in hemophilia B mice. [ABSTRACT FROM AUTHOR]

Workforce education and development are key cornerstones in advancing and maturing the Cell & Gene Therapy sector. A skilled worker shortage can significantly impact and delay progress as well as the quality of output for any developer, thereby negatively impacting a patient's access to life-saving treatments. Several roundtable discussions were held at the International Society for Cell & Gene Therapy (ISCT) 2023 Annual Meeting to dive deeper into the current state of workforce development and solutions to address this bottleneck. One roundtable discussion was co-hosted by the Alliance for Regenerative Medicine (ARM) and ISCT, which focused on the gap analysis provided for the United States Cell & Gene Therapy (CGT) sector, highlighting the lack of skilled workers in manufacturing and quality control. In this manuscript, the roundtable participants continue this conversation, review the roles and staffing requirements in both academic and industry as well as small and large company settings. The adoption of increased manufacturing automation is one promising solution to propel the sector forward. However, automation alone won't replace on-site staff, but will lower the bar to entry for a larger pool of people and require different training. This paper also addresses the workforce development and training paradigm shift as advanced manufacturing techniques are implemented, which will differ considerably based on the type of manufacturing efforts, thus emphasizing the need for a well-thought-out strategy to up-skill and re-skill the technical workforce to adapt to these advancements. Organizations such as ISCT and ARM have a role to play in propelling the field forward, providing awareness and education to stakeholders at all levels, as well as acting as a convener and participating as a key stakeholder in discussions and partnerships between academia and industry towards solutions for training the best personnel for CGT manufacturing. This scope includes novel digital tools and technologies to simplify training to increase access to new talent pools interested in careers in a rapidly advancing sector. [ABSTRACT FROM AUTHOR]

Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). After HSPCs were transduced with an LV encoding the therapeutic β-globin (βA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10−3 ng and 5 × 10−6 ng of target copy per reaction. We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of βA-T87Q protein detected by reverse-phase high-performance liquid chromatography. Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting. [ABSTRACT FROM AUTHOR]

Allogeneic hemopoietic cell transplantation remains the goal of therapy for high-risk acute myeloid leukemia (AML). However, treatment failure in the form of leukemia relapse or severe graft-versus-host disease remains a critical area of unmet need. Recently, significant progress has been made in the cell therapy-based interventions both before and after transplant. In this review, the Stem Cell Engineering Committee of the International Society for Cell and Gene Therapy summarizes the literature regarding the identification of high risk in AML, treatment approaches before transplant, optimal transplant platforms and measures that may be taken after transplant to ideally prevent, or, if need be, treat AML relapse. Although some strategies remain in the early phases of clinical investigation, they are built on progress in pre-clinical research and cellular engineering techniques that are already improving outcomes for children and adults with high-risk malignancies. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cell (HSC) gene therapy has shown potential as a therapeutic approach for thalassemia in recent years. However, a comparison of the varying gene therapy methods of HSC gene therapy in thalassemia has never been reviewed. This study aims to evaluate the utilization of HSC gene therapy approaches in animal models of thalassemia. A systematic review was conducted in five databases: PubMed, EBSCOHost, Science Direct, SCOPUS, and Proquest using a combination of the terms hematopoietic stem cell or hematopoietic stem cell or HSC, thalassemia, genetic therapy or gene therapy and animal model. Only journals published in English between 2008 and 2023 were included. This literature included six studies analyzing the use of HSC gene therapy in thalassemic mice models. The three outcomes being assessed in this review were globin levels, hematological parameters, and red blood cell (RBC) phenotypes. Gene therapy approaches for thalassemia using HSC showed significant improvement in β‐globin levels and RBC phenotypes. Phenotypic improvements were also observed. These outcomes indicate good efficacy in gene therapy for thalassemia in mice models. Furthermore, more studies assessing the efficacy of HSC gene therapy in the human model should be done in future studies. [ABSTRACT FROM AUTHOR]

[Display omitted] Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently. Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Although clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing holds immense potential for genetic manipulation, its clinical application is hindered by the absence of an efficient heart-targeted drug delivery system. Herein, we developed CRISPR-Cas9 ribonucleoprotein (RNP)-loaded extracellular vesicles (EVs) conjugated with cardiac-targeting peptide (T) for precise cardiac-specific genome editing. RNP complexes containing Cas9 and single guide RNA targeting miR-34a, an MI-associated molecular target, were loaded into EVs (EV@RNP). Gene editing by EV@RNP attenuated hydrogen peroxide-induced apoptosis in cardiomyocytes via miR-34a inhibition, evidenced by increased B-cell lymphoma 2 levels, decreased Bcl-2-associated X protein levels, and the cleavage of caspase-3. Additionally, to improve cardiac targeting in vivo , we used click chemistry to form functional T-EV@RNP by conjugating T peptides to EV@RNP. Consequently, T-EV@RNP-mediated miR-34a genome editing might exert a protective effect against MI, reducing apoptosis, ameliorating MI injury, and facilitating the recovery of cardiac function. In conclusion, the genome editing delivery system established by loading CRISPR/Cas9 RNP with cardiac-targeting EVs is a powerful approach for precise and tissue-specific gene therapy for cardiovascular disease. [Display omitted] [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a progressive autoimmune disease and drug therapy has been restricted due to poor therapeutic efficacy and adverse effects. In RA synovium, dendritic cells present self-antigens to activate cascade immune pathway. Furthermore, downstream macrophages secrete high levels of pro-inflammatory cytokines; Hyperplasia of activated synovial fibroblasts (FLS) is responsible for hypoxic synovium microenvironment, secretion of cytokines/chemokines and erosion of bone/cartilage tissues. Positive feedback loop of inflammation between macrophages and FLS independent of antigen-presentation is constructed. Herein, an injectable pH-sensitive peptide hydrogel encapsulating siRNA/Methotrexate-polyethyleneimine (siMP, including sip65MP, sip38MP, siCD86MP) and Bismuthene nanosheet/Methotrexate-polyethyleneimine (BiMP) is successfully developed. Among them, siCD86MP reduces protein level of co-stimulatory molecule CD86 while sip65MP and sip38MP separately inhibit NF-κB and MAPK-p38 pathways of macrophages and FLS to suppress secretion of cytokines and MMPs. Meanwhile, reduction in anti-apoptotic property of FLS induced by inhibition of NF-κB pathway has a synergistic effect with photodynamic therapy (PDT) and photothermal therapy (PTT) mediated by BiMP for FLS elimination, effectively ameliorating hypoxic synovium microenvironment. After being injected into synovium, hydrogel responds to acidic microenvironment and serves as a reservoir for sustained drug release and inherent retention capacity of which enables cationic nanoparticles to bypass tissue barrier for precise synovium targeting. This brand-new drug delivery system combines modulating cascade immune pathway from beginning to end by RNAi and eliminating FLS for improving synovium microenvironment by phototherapy together, providing a robust strategy for clinical RA treatment. In RA synovium, dendritic cells present self-antigens to activate the whole inflammatory pathway. Inhibiting antigen-presentation could effectively block the initiation of entire pathway. However, the downstream macrophages secrete high levels of pro-inflammatory cytokines, while excessively proliferated synovial fibroblasts (FLS) are responsible for hypoxia microenvironment, cytokine/chemokine release, bone/cartilage erosion, thus constructing an immune cycle independent of antigen-presentation together with macrophages. In this timely research, we successfully developed an implantable and pH-sensitive peptide hydrogel loading siRNA/methotrexate-polyethyleneimine (siMP: siCD86MP, sip65MP, sip38MP) and bismuthene nanosheet/methotrexate-polyethyleneimine (BiMP). siCD86MP can reduce the protein level of co-stimulatory molecule CD86 to block the initiation of inflammatory pathway. sip65MP and sip38MP can inhibit NF-κB and MAPK-p38 pathways in macrophages and FLS to ameliorate secretion of cytokines and MMPs, fundamentally blocking inflammatory circulation. Meanwhile, reduction in anti-apoptosis property of FLS induced by siMP had the synergistic effect with PDT and PTT of BiMP for FLS elimination, effectively ameliorating synovium microenvironment. After injection into synovium, the hydrogel responded to the acidic microenvironment and served as a drug reservoir for sustained drug release, while reducing systematic toxicity of cationic nanoparticles. The inherent retention capacity of hydrogel enabled nanoparticles to bypass the tissue barrier and achieve precise targeting. This brand-new drug delivery system combines modulating inflammation pathways from beginning to end by RNAi and eliminating FLS by phototherapy for improving synovium microenvironment together, providing a robust strategy for RA treatment. [Display omitted] • siRNA modulated cascade immune activation through inhibiting upstream antigen-presentation and downstream inflammatory circulation. • Bismuthene nanosheets exerted efficient PDT&PTT therapy to eliminate FLS for alleviating hypoxic synovium microenvironment. • Modulation of cascade immune activation and alleviating hypoxia microenvironment can synergistically treat RA. • Nap Hydrogel possessed excellent biocompatibility, injectability, self-healing, pH responsiveness, synovium targeting and retention. [ABSTRACT FROM AUTHOR]

Adoptive cell therapy with tumor-infiltrating T cells (TILs) has generated exciting clinical trial results for the treatment of unresectable solid tumors. However, solid tumors remain difficult targets for adoptively transferred T cells, due in part to poor migration of TILs to the tumor, physical barriers to infiltration, and active suppression of TILs by the tumor. Furthermore, a highly skilled team is required to obtain tumor tissue, isolate and expand the TILs ex vivo, and reinfuse them into the patient, which drives up costs and limits patient access. Here, we describe a cell-free polymer implant designed to recruit, genetically reprogram and expand host T cells at tumor lesions in situ. Importantly, the scaffold can be fabricated on a large scale and is stable to lyophilization. Using a mouse breast cancer model, we show that the implants quickly and efficiently amass cancer-specific host lymphocytes at the tumor site in quantities sufficient to bring about long-term tumor regression. Given that surgical care is the mainstay of cancer treatment for many patients, this technology could be easily implemented in a clinical setting as an add-on to surgery for solid tumors. Furthermore, the approach could be broadened to recruit and genetically reprogram other therapeutically desirable host cells, such as macrophages, natural killer cells or dendritic cells, potentially boosting the antitumor effectiveness of the implant even more. Our group developed an implantable device that (1) recruits T cells in situ, (2) genetically equips them with tumor-specific receptors, and (iii) rapidly expands and disperses them into solid tumor lesions. More specifically, the implant is based on a porous collagen scaffold (inherently supporting rapid T-cell migration), which is functionalized with a potent chemoattractant (CCL21) to recruit host T cells. To efficiently reprogram and expand recruited T cells within the scaffold, vectors encoding a tumor-specific receptor (chimeric antigen receptor in our study) and stimulatory anti- CD3/CD28 antibodies were co-encapsulated. All these components can be manufactured at large-scale and without the need to prepare a cellular product for each individual patient, and the final device can be frozen and stored until use. [Display omitted] • Rationally designed biomaterial scaffolds recruit, reprogram, expand and release human T cells. • Biomaterial scaffolds can efficiently transduce host T cells with tumor-specific CAR transgenes. • Scaffold-mediated in situ programming of CAR T-cells can improve survival of mice with established disease. [ABSTRACT FROM AUTHOR]

Delivering functional gene into targeted skin cells or tissues to modulate the genes expression, has the potential to treat various hereditary cutaneous disorders. Nevertheless, the lack of safe and effective gene delivery vehicles greatly limits the clinical translation of gene therapy for inherited skin diseases. Herein, we developed a facile elution fractionation strategy to isolate eight HPAEs with M w ranging from 7.6 to 131.8 kg/mol and Đ < 2.0 from the one crude HPAE 23.7k , and investigated the expression efficiency for TGM1 and COL7A1 plasmids. Gene transfection results revealed that the intermediate MW HPAEs, HPAE 20.6k , exhibited the highest gene transfection efficiency (46.4%) and the strongest mean fluorescence intensity (143,032 RLU), compared to other isolated components and the crude product. Importantly, best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. Our study establishes a straightforward step-by-step elution fractionation strategy for the development of HPAEs gene delivery vectors, expediting their clinical translation in inherited skin diseases. HPAEs crude polymer is fractionated to eight HPAEs with M w ranging from 7.6 to 131.8 kg/mol and Đ < 2.0. The best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. [Display omitted] • We establish a straightforward step-by-step elution fractionation strategy to isolate HPAEs with different molecular weight. • Isolated intermediate molecular weight HPAEs exhibit high transfection efficiency and biocompatibility in diverse cell types. • The top-performing HPAEs efficiently deliver TGM and COL7A1 DNA to potentially rescue hereditary cutaneous disorders. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IDD) is widely recognized as the primary culprit of chronic low back pain. Restoring deteriorated intervertebral disc (IVD) and alleviating IDD‐induced low back pain are remaining enormous challenges. There is a genetic susceptibility to IDD, and gene therapy has some therapeutic potential. However, traditional gene therapy still has certain drawbacks, including host immunity, temporary release, and suppression of gene medication function. Although regenerative materials can effectively improve the local microenvironment, they cannot treat IDD from the root. Gene‐functionalized regenerative material (GRM) is constructed based on physical embedding or forming chemical bonds by introducing particular genes, such as pDNA, siRNA, mRNA, and miRNA, into the regenerated materials. The findings demonstrate that GRM not only enhances the safety and controllability of gene therapy, but also effectively repairs IDD by overcoming the constraint that simple regenerative materials cannot reverse the disease's progression from the root. This article provided a brief overview of the physiological and pathological features of the IVD, genetic susceptibility to IDD, available treatment options, and their limitations. Then, the significance of GRM‐based treatment of IDD is proposed, and the future challenges and development in this field are finally prospected. [ABSTRACT FROM AUTHOR]

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches. [ABSTRACT FROM AUTHOR]

Mutations in the Microrchidia CW-type zinc finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high CNS transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss-of-function characteristics due to protein synthesis defects in Morc2a p.S87L were also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy. [ABSTRACT FROM AUTHOR]

Background: X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS. Methods: hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy. Results: ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation. Conclusions: The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy. [ABSTRACT FROM AUTHOR]

Human brain experimental models recapitulating age- and disease-related characteristics are lacking. There is urgent need for human-specific tools that model the complex molecular and cellular interplay between different cell types to assess underlying disease mechanisms and test therapies. Here we present an adapted ex vivo organotypic slice culture method using human post-mortem brain tissue cultured at an air-liquid interface to also study brain white matter. We assessed whether these human post-mortem brain slices recapitulate the in vivo neuropathology and if they are suitable for pathophysiological, experimental and pre-clinical treatment development purposes, specifically regarding leukodystrophies. Human post-mortem brain tissue and cerebrospinal fluid were obtained from control, psychiatric and leukodystrophy donors. Slices were cultured up to six weeks, in culture medium with or without human cerebrospinal fluid. Human post-mortem organotypic brain slice cultures remained viable for at least six weeks ex vivo and maintained tissue structure and diversity of (neural) cell types. Supplementation with cerebrospinal fluid could improve slice recovery. Patient-derived organotypic slice cultures recapitulated and maintained known in vivo neuropathology. The cultures also showed physiologic multicellular responses to lysolecithin-induced demyelination ex vivo, indicating their suitability to study intrinsic repair mechanisms upon injury. The slice cultures were applicable for various experimental studies, as multi-electrode neuronal recordings. Finally, the cultures showed successful cell-type dependent transduction with gene therapy vectors. These human post-mortem organotypic brain slice cultures represent an adapted ex vivo model suitable for multifaceted studies of brain disease mechanisms, boosting translation from human ex vivo to in vivo. This model also allows for assessing potential treatment options, including gene therapy applications. Human post-mortem brain slice cultures are thus a valuable tool in preclinical research to study the pathomechanisms of a wide variety of brain diseases in living human tissue. [ABSTRACT FROM AUTHOR]

Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy. In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy. [ABSTRACT FROM AUTHOR]