Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.

The manipulation of animal mitochondrial genomes has long been a challenge due to the lack of an effective transformation method. With the discovery of specific gene editing enzymes, designed to target pathogenic mitochondrial DNA mutations (often heteroplasmic), the selective removal or modification of mutant variants has become a reality. Because mitochondria cannot efficiently import RNAs, CRISPR has not been the first choice for editing mitochondrial genes. However, the last few years witnessed an explosion in novel and optimized non-CRISPR approaches to promote double-strand breaks or base-edit of mtDNA in vivo. Engineered forms of specific nucleases and cytidine/adenine deaminases form the basis for these techniques. I will review the newest developments that constitute the current toolbox for animal mtDNA gene editing in vivo, bringing these approaches not only to the exploration of mitochondrial function, but also closer to clinical use.

Precision-cut lung slices (PCLS), ex vivo 3D lung tissue models, have been widely used for various applications in lung research. PCLS serve as an excellent intermediary between in vitro and in vivo models because they retain all resident cell types within their natural niche while preserving the extracellular matrix environment. This protocol describes the TReATS (TAT-Cre recombinase-mediated floxed allele modification in tissue slices) method that enables rapid and efficient gene modification in PCLS derived from adult floxed animals. Here, we present detailed protocols for the TReATS method, consisting of two simple steps: PCLS generation and incubation in a TAT-Cre recombinase solution. Subsequent validation of gene modification involves live staining and imaging of PCLS, quantitative real-time PCR, and cell viability assessment. This four-day protocol eliminates the need for complex Cre-breeding, circumvents issues with premature lethality related to gene mutation, and significantly reduces the use of animals. The TReATS method offers a simple and reproducible solution for gene modification in complex ex vivo tissue-based models, accelerating the study of gene function, disease mechanisms, and the discovery of drug targets. Key features • Achieve permanent ex vivo gene modifications in complex tissue-based models within four days. • Highly adaptable gene modification method that can be applied to induce gene deletion or activation. • Allows simple Cre dosage testing in a controlled ex vivo setting with the advantage of using PCLS generated from the same animal as true controls. • With optimisation, this method can be applied to precision-cut tissue slices of other organs.

Neurodegenerative diseases (NDs) are a group of debilitating neurological disorders that primarily affect elderly populations and include Alzheimer\'s disease (AD), Parkinson\'s disease (PD), Huntington\'s disease (HD), and amyotrophic lateral sclerosis (ALS). Currently, there are no therapies available that can delay, stop, or reverse the pathological progression of NDs in clinical settings. As the population ages, NDs are imposing a huge burden on public health systems and affected families. Animal models are important tools for preclinical investigations to understand disease pathogenesis and test potential treatments. While numerous rodent models of NDs have been developed to enhance our understanding of disease mechanisms, the limited success of translating findings from animal models to clinical practice suggests that there is still a need to bridge this translation gap. Old World non-human primates (NHPs), such as rhesus, cynomolgus, and vervet monkeys, are phylogenetically, physiologically, biochemically, and behaviorally most relevant to humans. This is particularly evident in the similarity of the structure and function of their central nervous systems, rendering such species uniquely valuable for neuroscience research. Recently, the development of several genetically modified NHP models of NDs has successfully recapitulated key pathologies and revealed novel mechanisms. This review focuses on the efficacy of NHPs in modeling NDs and the novel pathological insights gained, as well as the challenges associated with the generation of such models and the complexities involved in their subsequent analysis.
神经退行性疾病 (Neurodegenerative diseases，ND) 是一类主要影响老年人群的神经疾病，包括阿尔茨海默病 (Alzheimer’s disease，AD)、帕金森病 (Parkinson’s disease，PD)、亨廷顿病 (Huntington’s disease，HD) 和肌萎缩侧索硬化症 (Amyotrophic lateral sclerosis，ALS)。目前，临床上尚无可以延迟、阻止或逆转 ND 病理进展的疗法。随着人口老龄化，ND 给公共卫生系统和患者家庭带来巨大负担。动物模型是临床前研究了解疾病发病机制和测试潜在治疗方法的重要工具。虽然已经开发出许多神经退行性疾病的啮齿类动物模型，并极大地增强了我们对疾病机制的理解，但动物模型的研究结果临床转化效果低下，这表明仍然需要弥合临床前研究和临床研究之间的差距。旧大陆非人类灵长类动物（non-human primates，NHPs），如恒河猴、食蟹猴和黑长尾猴，在系统发育、生理、生化和行为上，特别是在中枢神经系统结构和功能方面与人类最为接近，这使得它们对神经科学的研究具有重要作用。最近，已经有几种神经退行性疾病的转基因 NHP 模型报道，它们复刻了关键病理学指征并揭示了新的分子机制。在这篇综述中，我们描述了 NHP 模拟 ND 的能力以及来自这些 NHP 模型的新病理学见解。我们还讨论了模型制备及后续研究中面临的挑战。.

Transcriptional variation has been studied but post-transcriptional modification due to RNA editing has not been investigated in Plasmodium. We investigated developmental stage-specific RNA editing in selected genes in Plasmodium falciparum 3D7. We detected extensive amination- and deamination-type RNA editing at 8, 16, 24, 32, 40, and 46 h in tightly synchronized Plasmodium. Most of the editing events were observed in 8 and 16 h ring-stage parasites. Extensive A-to-G deamination-type editing was detected more during the 16 h ring stage (25%) than the 8 h ring stage (20%). Extensive U-to-C amination-type editing was detected more during the 16 h ring stage (31%) than the 8 h ring stage (22%). In 28S, rRNA editing converted the loop structure to the stem structure. The hemoglobin binding activity of PF3D7_0216900 was also altered due to RNA editing. Among the expressed 28S rRNA genes, PF3D7_0532000 and PF3D7_0726000 expression was higher. Increased amounts of the transcripts of these two genes were found, particularly PF3D7_0726000 in the ring stage and PF3D7_0532000 in the trophozoite and schizont stages. Adenosine deaminase (ADA) expression did not correlate with the editing level. This first experimental report of RNA editing will help to identify the editing machinery that might be useful for antimalarial drug discovery and malaria control.

Acute myeloid leukemia (AML) is a fatal blood malignancy. With the development of immunotherapy, particularly chimeric antigen receptor T cells (CAR-T), the treatment of AML has undergone a significant change. Despite its advantages, CAR-T still faces a number of limitations and challenges while treating AML. Finding novel targets, altering the structure of CAR to increase efficacy while lowering side effects, and using double-target CAR and logic circuits are typical examples of key to answer these problems. With the advancement of gene editing technology, gene editing of tumor cells or normal cells to create therapeutic effects has grown in popularity. Additionally, the combination of multiple drugs is routinely used to address some of the obstacles and difficulties associated with CAR-T therapy. The review\'s primary goal was to summarize recent strategies and developments of CAR-T therapy for AML.

BACKGROUND: The emergence of SARS-CoV-2 becomes life-threatening for the older and immunocompromised individuals, whereas limited treatment is available on these populations. Mesenchymal stromal cells (MSCs) have been reported to be useful in SARS-CoV-2 treatment and reduce SARS-CoV-2-related sequelae.
RESULTS: In this study, we developed an autonomous cellular machine to secret neutralizing antibody in vivo constantly based on the clinical-grade MSCs, to combat SARS-CoV-2 infections. First, various modified recombinant plasmids were constructed and transfected into clinical-grade MSCs by electroporation, for assembly and expression of neutralizing anti-SARS-CoV-2 antibodies. Second, the stable antibody secreting MSCs clones were screened through pseudovirus neutralization assay. Finally, we investigated the pharmacokinetics and biodistribution of neutralizing antibody secreted by engineered MSCs in vivo. The stable clinical-grade MSCs clones, expressing XGv347-10 and LY-CoV1404-5 neutralizing antibodies, exhibited their feasibility and protective efficacy against SARS-CoV-2 infection. Transplanted engineered clinical-grade MSCs effectively delivered the SARS-CoV-2 antibodies to the lung, and the immune hyperresponsiveness caused by COVID-19 was coordinated by MSC clones through inhibiting the differentiation of CD4 + T cells into Th1 and Th17 subpopulations.
CONCLUSIONS: Our data suggested that engineered clinical-grade MSCs secreting effective neutralizing antibodies as cellular production machines had the potential to combat SARS-CoV-2 infection, which provided a new avenue for effectively treating the older and immunocompromised COVID-19 patients.

Gene-modification therapies are at the forefront of HIV-1 cure strategies. Chimeric antigen receptor (CAR)-T cells pose a potential approach to target infected cells during antiretroviral therapy or following analytical treatment interruption (ATI). However, there are technical challenges in the quantification of HIV-1-infected and CAR-T cells in the setting of lentiviral CAR gene delivery and also in the identification of cells expressing target antigens. First, there is a lack of validated techniques to identify and characterize cells expressing the hypervariable HIV gp120 in both ART-suppressed and viremic individuals. Second, close sequence homology between lentiviral-based CAR-T gene modification vectors and conserved regions of HIV-1 creates quantification challenges of HIV-1 and lentiviral vector levels. Consideration needs to be taken into standardizing HIV-1 DNA/RNA assays in the setting of CAR-T cell and other lentiviral vector-based therapies to avoid these confounding interactions. Lastly, with the introduction of HIV-1 resistance genes in CAR-T cells, there is a need for assays with single-cell resolution to determine the competence of the gene inserts to prevent CAR-T cells from becoming infected in vivo. As novel therapies continue to arise in the HIV-1 cure field, resolving these challenges in CAR-T-cell therapy will be crucial.

The Advanced Therapies Week 2023 conference took place in Miami, FL, USA. Over the course of 4 days packed with talks, panels, company showcases and networking events, a clear message emerged: the future of cell therapy is here. Timely topics covered by speakers and panelists from industry and academia included allogeneic and autologous cell therapies, cell manufacture automation, cell and gene therapy for autoimmune diseases, gene delivery technology, chimeric antigen receptor T-cell therapy in oncology, closed cell therapy manufacturing and how to serve small patient populations. While some challenges remain, this decade will likely witness the US FDA approval of many cell and gene therapies, as well as new devices for their manufacture.

For ischemic cardiomyopathy (ICM) with limited therapeutic options, the induction of arteriogenesis has the potential to improve cardiac function through major restoration of blood flow. We hypothesized that transplantation of a Notch signaling-modified mesenchymal stem cell (SB623 cell) patch would induce angiogenesis and arteriogenesis in ischemic lesions, leading to improvement of left ventricular (LV) function in a rat ICM model. Two weeks after the induction of ischemia, SB623 cell patch transplantation into ICM rats (SB group, n = 10) or a sham operation (no-treatment group, n = 10) was performed. The LV ejection fraction was significantly improved at 6 weeks after SB623 cell patch transplantation (P < 0.001). Histological findings revealed that the number of von Willebrand factor (vWF)-positive capillary vessels (P < 0.01) and alpha smooth muscle actin (αSMA)- and vWF-positive arterioles with a diameter greater than 20 µm (P = 0.002) was significantly increased in the SB group, suggesting the induction of angiogenesis and arteriogenesis. Moreover, rat cardiomyocytes treated with SB623 cell patch transplantation showed upregulation of ephrin-B2 (P = 0.03) and EphB4 (P = 0.01) gene expression, indicating arteriogenesis induction. In conclusion, SB623 cell patch transplantation improved LV function by inducing angiogenesis and arteriogenesis in a rat ICM model.