Human induced pluripotent stem cell (hiPSC)-derived liver cell therapies such as hepatocyte-like cells and liver organoids could provide unlimited therapeutic cells for clinical transplantation, but an inadequate understanding of their in vivo fate impedes translation. Whole body in vivo imaging could enable monitoring of transplanted cell survival and/or expansion non-invasively over time, permitting robust comparisons between emerging therapies to identify those most effective. The human sodium iodide symporter (hNIS) is a radionuclide reporter gene facilitating whole body in vivo cell tracking by positron emission tomography (PET). We gene-edited a clinical Good Manufacturing Practice-compliant hiPSC line at the AAVS1 safe harbor locus enabling constitutive expression of a hNIS-monomeric(m)GFP fusion reporter in hiPSCs and their differentiated progeny. We confirmed reporter integration did not impact pluripotency or differentiation capacity, and radiotracer uptake capacity was retained post-differentiation. In vivo trackable liver bud (LB) organoids were generated from traceable hNIS fused to monomeric GFP (hNIS-mGFP)-hiPSCs and transplanted into healthy and liver-injured mice. LB were imaged quantitatively by 18FBF4 --PET with imaging results confirmed histologically. We report, for the first time, hNIS-mGFP-hiPSC progeny retain differentiated function and PET trackability in vivo using LB. In vivo monitoring could accelerate regenerative cell therapy development by identifying efficacious candidate cells, successful engraftment/survival strategies and addressing safety concerns.

Peptide nucleic acids (PNA), synthetic molecules comprising a peptide-like backbone and natural and unnatural nucleobases, have garnered significant attention for their potential applications in gene editing and other biomedical fields. The unique properties of PNA, particularly enhanced stability/specificity/affinity towards targeted DNA and RNA sequences, achieved significant attention recently for gene silencing, gene correction, antisense therapy, drug delivery, biosensing and other various diagnostic aspects. This review explores the structure, properties, and potential of PNA in transforming genetic engineering including potent biomedical challenges. In Addition, we explore future perspectives and potential limitations of PNA-based technologies, highlighting the need for further research and development to fully realize their therapeutic and biotechnological potential.

Available treatment for chronic hepatitis B virus (HBV) infection offers modest functional curative efficacy. The viral replicative intermediate comprising covalently closed circular DNA (cccDNA) is responsible for persistent chronic HBV infection. Hence, current efforts have focused on developing therapies that disable cccDNA. Employing gene editing tools has emerged as an attractive strategy, with the end goal of establishing permanently inactivated cccDNA. Although anti-HBV designer nucleases are effective in vivo, none has yet progressed to clinical trial. Lack of safe and efficient delivery systems remains the limiting factor. Several vectors may be used to deliver anti-HBV gene editor-encoding sequences, with viral vectors being at the forefront. Despite the challenges associated with packaging large gene editor-encoding sequences into viral vectors, advancement in the field is overcoming such limitations. Translation of viral vector-mediated gene editing against HBV to clinical application is within reach. This review discusses the prospects of delivering HBV targeted designer nucleases using viral vectors.

Remarkable progress has been made in the field of genome engineering after the discovery of CRISPR/Cas9 in 2012 by Jennifer Doudna and Emmanuelle Charpentier. Compared to any other gene-editing tools, CRISPR/Cas9 attracted the attention of the scientific community because of its simplicity, specificity, and multiplex editing possibilities for which the inventors were awarded the Nobel prize for chemistry in 2020. CRISPR/Cas9 allows targeted alteration of the genomic sequence, gene regulation, and epigenetic modifications using an RNA-guided site-specific endonuclease. Though the impact of CRISPR/Cas9 was undisputed, some of its limitations led to key modifications including the use of miniature-Cas proteins, Cas9 Retron precise Parallel Editing via homologY (CRISPEY), Cas-Clover, or development of alternative methods including retron-recombineering, Obligate Mobile Element Guided Activity(OMEGA), Fanzor, and Argonaute proteins. As cancer is caused by genetic and epigenetic alterations, gene-editing was found to be highly useful for knocking out oncogenes, editing mutations to regain the normal functioning of tumor suppressor genes, knock-out immune checkpoint blockade in CAR-T cells, producing 'off-the-shelf' CAR-T cells, identify novel tumorigenic genes and functional analysis of multiple pathways in cancer, etc. Advancements in nanoparticle-based delivery of guide-RNA and Cas9 complex to the human body further enhanced the potential of CRISPR/Cas9 for clinical translation. Several studies are reported for developing novel delivery methods to enhance the tumor-specific application of CRISPR/Cas9 for anticancer therapy. In this review, we discuss new developments in novel gene editing techniques and recent progress in nanoparticle-based CRISPR/Cas9 delivery specific to cancer applications.

Labeling and tracking existing and emerging cell-based immunotherapies using nuclear imaging is widely used to guide the preclinical phases of development and testing of existing and new emerging off-the-shelf cell-based immunotherapies. In fact, advancing our knowledge about their mechanism of action and limitations could provide preclinical support and justification for moving towards clinical experimentation of newly generated products and expedite their approval by the Food and Drug Administration (FDA).Here we provide the reader with a ready to use protocol describing the labeling methodologies and practical procedures to render different candidate cell therapies in vivo traceable by nuclear-based imaging. The protocol includes sufficient practical details to aid researchers at all career stages and from different fields in familiarizing with the described concepts and incorporating them into their work.

Metabolic syndrome (MetS) represents a significant public health concern due to its association with an increased risk of cardiovascular disease, type 2 diabetes, and other serious health conditions. Despite extensive research, the underlying molecular mechanisms contributing to MetS pathogenesis remain elusive. This review aims to provide a comprehensive overview of the molecular mechanisms linking MetS and cluster of differentiation (CD) markers, which play critical roles in immune regulation and cellular signaling. Through an extensive literature review with a systematic approach, we examine the involvement of various CD markers in MetS development and progression, including their roles in adipose tissue inflammation, insulin resistance, dyslipidemia, and hypertension. Additionally, we discuss potential therapeutic strategies targeting CD markers for the management of MetS. By synthesizing current evidence, this review contributes to a deeper understanding of the complex interplay between immune dysregulation and metabolic dysfunction in MetS, paving the way for the development of novel therapeutic interventions.

Targeted modifications of the human epigenome, epigenome editing (EE), are around the corner. For EE, techniques similar to genome editing (GE) techniques are used. While in GE the genetic information is changed by directly modifying DNA, intervening in the epigenome requires modifying the configuration of DNA, for example, how it is folded. This does not come with alterations in the base sequence ('genetic code'). To date, there is almost no ethical debate about EE, whereas the discussions about GE are voluminous. Our article introduces EE into bioethics by translating knowledge from science to ethics and by comparing the risks of EE with those of GE. We, first (I), make the case that a broader ethical debate on EE is due, provide scientific background on EE, compile potential use-cases and recap previous debates. We then (II) compare EE and GE and suggest that the severity of risks of novel gene technologies depends on three factors: (i) the choice of an ex vivo versus an in vivo editing approach, (ii) the time of intervention and intervention windows and (iii) the targeted diseases. Moreover, we show why germline EE is not effective and reject the position of strong epigenetic determinism. We conclude that EE is not always ethically preferable to GE in terms of risks, and end with suggestions for next steps in the current ethical debate on EE by briefly introducing ethical challenges of new areas of preventive applications of EE (III).

Cell therapies are emerging as promising treatments for a range of liver diseases but translational bottlenecks still remain including: securing and assessing the safe and effective delivery of cells to the disease site; ensuring successful cell engraftment and function; and preventing immunogenic responses. Here we highlight three therapies, each utilising a different cell type, at different stages in their clinical translation journey: transplantation of multipotent mesenchymal stromal/signalling cells, hepatocytes and macrophages. To overcome bottlenecks impeding clinical progression, we advocate for wider use of mechanistic in silico modelling approaches. We discuss how in silico approaches, alongside complementary experimental approaches, can enhance our understanding of the mechanisms underlying successful cell delivery and engraftment. Furthermore, such combined theoretical-experimental approaches can be exploited to develop novel therapies, address safety and efficacy challenges, bridge the gap between in vitro and in vivo model systems, and compensate for the inherent differences between animal model systems and humans. We also highlight how in silico model development can result in fewer and more targeted in vivo experiments, thereby reducing preclinical costs and experimental animal numbers and potentially accelerating translation to the clinic. The development of biologically-accurate in silico models that capture the mechanisms underpinning the behaviour of these complex systems must be reinforced by quantitative methods to assess cell survival post-transplant, and we argue that non-invasive in vivo imaging strategies should be routinely integrated into transplant studies.

Noninvasive long-term imaging of therapeutic cells in preclinical models can be achieved through introducing a reporter gene into the cells of interest. Despite important recent developments such as gene editing, cell engineering based on lentiviruses remains a mainstream tool for gene transfer applicable to a variety of different cell types.In this chapter, we describe how to use lentivirus-based genetic engineering to render different candidate cell therapies in vivo traceable by radionuclide imaging. We illustrate this reporter gene technology using the sodium iodide symporter (NIS), which is compatible with both positron emission tomography (PET) and single-photon emission computed tomography (SPECT). For preclinical experimentation, we fused NIS with a suitable fluorescent protein such as monomeric GFP or RFP to streamline cell line generation and downstream analyses of ex vivo tissue samples. We present protocols for reporter gene engineering of human cardiac progenitor cells, regulatory T cells, and effector T cells as well as for the characterization experiments required to validate NIS-fluorescent protein reporter function in these candidate therapeutic cells.

The rate of neurodegenerative disorders (NDDs) is rising rapidly as the world's population ages. Conditions such as Alzheimer's disease (AD), Parkinson's disease (PD), and dementia are becoming more prevalent and are now the fourth leading cause of death, following heart disease, cancer, and stroke. Although modern diagnostic techniques for detecting NDDs are varied, scientists are continuously seeking new and improved methods to enable early and precise detection. In addition to that, the present treatment options are limited to symptomatic therapy, which is effective in reducing the progression of neurodegeneration but lacks the ability to target the root cause-progressive loss of neuronal functioning. As a result, medical researchers continue to explore new treatments for these conditions. Here, we present a comprehensive summary of the key features of NDDs and an overview of the underlying mechanisms of neuroimmune dysfunction. Additionally, we dive into the cutting-edge treatment options that gene therapy provides in the quest to treat these disorders.

Genetically engineered bacteria (GEB) have shown significant promise to revolutionize modern medicine. These engineered bacteria with unique properties such as enhanced targeting, versatility, biofilm disruption, reduced drug resistance, self-amplification capabilities, and biodegradability represent a highly promising approach for targeted drug delivery and cancer theranostics. This innovative approach involves modifying bacterial strains to function as drug carriers, capable of delivering therapeutic agents directly to specific cells or tissues. Unlike synthetic drug delivery systems, GEB are inherently biodegradable and can be naturally eliminated from the body, reducing potential long-term side effects or complications associated with residual foreign constituents. However, several pivotal challenges such as safety and controllability need to be addressed. Researchers have explored novel tactics to improve their capabilities and overcome existing challenges, including synthetic biology tools (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) and bioinformatics-driven design), microbiome engineering, combination therapies, immune system interaction, and biocontainment strategies. Because of the remarkable advantages and tangible progress in this field, GEB may emerge as vital tools in personalized medicine, providing precise and controlled drug delivery for various diseases (especially cancer). In this context, future directions include the integration of nanotechnology with GEB, the focus on microbiota-targeted therapies, the incorporation of programmable behaviors, the enhancement in immunotherapy treatments, and the discovery of non-medical applications. In this way, careful ethical considerations and regulatory frameworks are necessary for developing GEB-based systems for targeted drug delivery. By addressing safety concerns, ensuring informed consent, promoting equitable access, understanding long-term effects, mitigating dual-use risks, and fostering public engagement, these engineered bacteria can be employed as promising delivery vehicles in bio- and nanomedicine. In this review, recent advances related to the application of GEB in targeted drug delivery and cancer therapy are discussed, covering crucial challenging issues and future perspectives.

We present the design and synthesis of artificial specific nucleobases, each one recognizing a single base pair within the major groove of duplex DNA. Computational calculations indicate that PNAs modified with these nucleobases enable the formation of highly stable triple helices with no sequence restrictions through multiple hydrogen bonding and π⋅⋅⋅π stacking interactions, without significantly widening the DNA double helix. New synthetic routes were developed to the structures of these fused heterocycles which have rarely been described in the literature. NMR titration experiments indicate specific hydrogen bonding at the Hoogsteen sites. The new building blocks allow the construction of four PNA monomers for each canonic base pair and their covalent connection to PNA oligomers. These can be designed complementary to any given DNA sequence. With high efficiency and relative simplicity of operation, the described methodologies and strategies hence form the basis for a new supramolecular ligand system targeting double-stranded DNA without strand invasion.

The recent developments in the study of clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system have revolutionized the art of genome-editing and its applications for cellular differentiation and immune response behavior. This technology has further helped in understanding the mysteries of cancer progression and possible designing of novel antitumor immunotherapies. CRISPR/Cas9-based genome-editing is now often used to engineer universal T-cells, equipped with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR). In addition, this technology is used in cytokine stimulation, antibody designing, natural killer (NK) cell transfer, and to overcome immune checkpoints. The innovative potential of CRISPR/Cas9 in preparing the building blocks of adoptive cell transfer (ACT) immunotherapy has opened a new window of antitumor immunotherapy and some of them have gained FDA approval. The manipulation of immunogenetic regulators has opened a new interface for designing, implementation and interpretation of CRISPR/Cas9-based screening in immuno-oncology. Several cancers like lymphoma, melanoma, lung, and liver malignancies have been treated with this strategy, once thought to be impossible. The safe and efficient delivery of CRISPR/Cas9 system within the immune cells for the genome-editing strategy is a challenging task which needs to be sorted out for efficient immunotherapy. Several targeting approaches like virus-mediated, electroporation, microinjection and nanoformulation-based methods have been used, but each procedure offers some limitations. Here, we elaborate the recent updates of cancer management through immunotherapy in partnership with CRISPR/Cas9 technology. Further, some innovative methods of targeting this genome-editing system within the immune system cells for reprogramming them, as a novel strategy of anticancer immunotherapy is elaborated. In addition, future prospects and clinical trials are also discussed.

Small cell lung cancer (SCLC) is a lethal human malignancy, and previous studies support the contribution of microRNA to cancer progression. The prognostic value of miR-219-5p in patients with SCLC remains unclear. This study aimed to evaluate the predictive value of miR-219-5p with respect to mortality in patients with SCLC and to incorporate miR-219-5p level into a prediction model and nomogram for mortality.

Retrospective observational cohort study.

Our main cohort included data from 133 patients with SCLC between 1 March 2010 and 1 June 2015 from the Suzhou Xiangcheng People's Hospital. Data from 86 patients with non-SCLC at Sichuan Cancer Hospital and the First Affiliated Hospital of Soochow University were used for external validation.

Tissue samples were taken during admission and stored, and miR-219-5p levels were measured at a later date. A Cox proportional hazard model was used for survival analyses and for analysing risk factors to create a nomogram for mortality prediction. The accuracy of the model was evaluated by C-index and calibration curve.

Mortality in patients with a high level of miR-219-5p (≥1.50) (n=67) was 74.6%, while mortality in the low-level group (n=66) was 100.0%. Based on univariate analysis, we included significant factors (p<0.05) in a multivariate regression model: patients with high level of miR-219-5p (HR 0.39, 95% CI 0.26-0.59, p<0.001), immunotherapy (HR 0.44, 95% CI 0.23-0.84, p<0.001) and prognostic nutritional index score >47.9 (HR=0.45, 95% CI 0.24-0.83, p=0.01) remained statistically significant factors for improved overall survival. The nomogram had good accuracy in estimating the risk, with a bootstrap-corrected C-index of 0.691. External validation indicated an area under the curve of 0.749 (0.709-0.788).

The miR-219-5p level was associated with a reduced risk of mortality in patients with SCLC. A nomogram incorporating MiR-219-5p level and clinical factors demonstrated good accuracy in estimating the risk of overall mortality. Prospective validation of the prognostic nomogram is needed.

The efficient delivery of CRISPR-Cas components is still a key and unsolved problem. CRISPR-Cas delivery in the form of a Cas protein+sgRNA (ribonucleoprotein complex, RNP complex), has proven to be extremely effective, since it allows to increase on-target activity, while reducing nonspecific activity. The key point for in vivo genome editing is the direct delivery of artificial nucleases and donor DNA molecules into the somatic cells of an adult organism. At the same time, control of the dose of artificial nucleases is impossible, which affects the efficiency of genome editing in the affected cells. Poor delivery efficiency and low editing efficacy reduce the overall potency of the in vivo genome editing process. Here we review how this problem is currently being solved in scientific works and what types of in vivo delivery methods of Cas9/sgRNA RNPs have been developed.

Chimeric antigen receptor (CAR) T-cells are considered "living drugs" and offer a compelling alternative to conventional anticancer therapies. Briefly, T-cells are redirected, using gene engineering technology, toward a specific cancer cell surface target antigen via a synthetic chimeric antigen receptor (CAR) protein. CARs have a modular design comprising four main structures: an antigen-binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains for T-cell activation. A major challenge in the CAR T-cell manufacturing field is balancing product quality with scalability and cost-effectiveness, especially when transitioning from an academic clinical trial into a marketed product, to be implemented across many collection, manufacturing, and treatment sites. Achieving product consistency while circumnavigating the intrinsic variability associated with autologous products is an additional barrier. To overcome these limitations, a robust understanding of the product and its biological actions is crucial to establish a target product profile with a defined list of critical quality attributes to be assessed for each batch prior to product certification. Additional challenges arise as the field progresses, such as new safety considerations associated with the use of allogenic T-cells and genome editing tools. In this chapter, we will discuss the release and potency assays required for CAR T-cell manufacturing, covering their relevance, current challenges, and future perspectives.

Treatment of genetic disorders by genomic manipulation has been the unreachable goal of researchers for many decades. Although our understanding of the genetic basis of genetic diseases has advanced tremendously in the last few decades, the tools developed for genomic editing were not efficient and practical for their use in the clinical setting until now. The recent advancements in the research of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems offered an easy and efficient way to edit the genome and accelerated the research on their potential use in the treatment of genetic disorders. In this review, we summarize the clinical trials that evaluate the CRISPR/Cas systems for treating different genetic diseases and highlight promising preclinical research on CRISPR/Cas mediated treatment of a great diversity of genetic disorders. Ultimately, we discuss the future of CRISPR/Cas mediated genome editing in genetic diseases.

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC, components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor-engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12-deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers-most notably for STAT and AP-1 transcription factors-and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

Cellular immunotherapies have emerged as a successful therapeutic approach to fight a wide range of human diseases, including cancer. However, responses are limited to few patients and tumor types. An in-depth understanding of the complexity and dynamics of cellular immunotherapeutics, including what is behind their success and failure in a patient, the role of other immune cell types and molecular biomarkers in determining a response, is now paramount. As the cellular immunotherapy arsenal expands, whole-body non-invasive molecular imaging can shed a light on their in vivo fate and contribute to the reliable assessment of treatment outcome and prediction of therapeutic response. In this review, we outline the non-invasive strategies that can be tailored toward the molecular imaging of cellular immunotherapies and immune-related components, with a focus on those that have been extensively tested preclinically and are currently under clinical development or have already entered the clinical trial phase. We also provide a critical appraisal on the current role and consolidation of molecular imaging into clinical practice.

T cells engineered to express chimeric antigen receptors (CARs) have shown remarkable success in treating B-cell malignancies, reflected by multiple US Food and Drug Administration-approved CAR-T cell products currently on the market. However, various obstacles have thus far limited the use of approved products and constrained the efficacy of CAR-T cell therapy against solid tumors. Overcoming these obstacles will necessitate multidimensional CAR-T cell engineering approaches and better understanding of the intricate tumor microenvironment (TME). Key challenges include treatment-related toxicity, antigen escape and heterogeneity, and the highly immunosuppressive profile of the TME. Notably, the hypoxic and nutrient-deprived nature of the TME severely attenuates CAR-T cell fitness and efficacy, highlighting the need for more sophisticated engineering strategies. In this review, we examine recent advances in protein- and cell-engineering strategies to improve CAR-T cell safety and efficacy, with an emphasis on overcoming immunosuppression induced by tumor metabolism and hypoxia.

Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4⁺ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4⁺ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4⁺ T cell and up to 88% of CD34⁺ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4⁺ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.

Tumor dormancy is a major contributor to the lethality of metastatic disease, especially for cancer patients who develop metastases years-to-decades after initial diagnosis. Indeed, tumor cells can disseminate during early disease stages and persist in new microenvironments at distal sites for months, years, or even decades before initiating metastatic outgrowth. This delay between primary tumor remission and metastatic relapse is known as "dormancy," during which disseminated tumor cells (DTCs) acquire quiescent states in response to intrinsic (i.e., cellular) and extrinsic (i.e., microenvironmental) signals. Maintaining dormancy-associated phenotypes requires DTCs to activate transcriptional, translational, and post-translational mechanisms that engender cellular plasticity. RNA processing is emerging as an essential facet of cellular plasticity, particularly with respect to the initiation, maintenance, and reversal of dormancy-associated phenotypes. Moreover, dysregulated RNA processing, particularly that associated with alternative RNA splicing and expression of noncoding RNAs (ncRNAs), can occur in DTCs to mediate intrinsic and extrinsic metastatic dormancy. Here we review the pathophysiological impact of alternative RNA splicing and ncRNAs in promoting metastatic dormancy and disease recurrence in human cancers.

"Be careful what you wish for": This adage guides both how this project came to life, and how the topic covered in this review continues to unfold. What began as talks between two friends on shared interests in military history led to a 4-year discussion about how our science curriculum does little to introduce our students to societal and ethical impacts of the science they are taught. What emerged was a curricular idea centered on how "good intentions" of some were developed and twisted by others to result in disastrous consequences of state-sanctioned eugenics. In this article, we take the reader (as we did our students) through the long and soiled history of eugenic thought, from its genesis to the present. Though our focus is on European and American eugenics, we will show how the interfaces and interactions between science and society have evolved over time but have remained ever constant. Four critical 'case studies' will also be employed here for deep, thoughtful exploration on a particular eugenic issue. The goal of the review, as it is with our course, is not to paint humanity with a single evil brush. Instead, our ambition is to introduce our students/readers to the potential for harm through the misapplication and misappropriation of science and scientific technology, and to provide them with the tools to ask the appropriate questions of their scientists, physicians, and politicians.

Precise and site specific genome editing through application of emerging and modern gene engineering techniques, namely zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) have swiftly progressed the application and use of the stem cell technology in the sphere of in-vitro disease modelling and regenerative medicine. Genome editing tools facilitate the manipulating of any gene in various types of cells with target specific nucleases. These tools aid in elucidating the genetics and etiology behind different diseases and have immense promise as novel therapeutics for correcting the genetic mutations, make alterations and cure diseases permanently that are not responding and resistant to traditional therapies. These genome engineering tools have evolved in the field of biomedical research and have also shown to have a significant improvement in clinical trials. However, their widespread use in research revealed potential safety issues, which need to be addressed before implementing such techniques in clinical purposes. Significant and valiant attempts are being made in order to surpass those hurdles. The current review outlines the advancements of several genome engineering tools and describes suitable strategies for their application towards regenerative medicine.

Over the last decade, immunotherapy has established itself as an important novel approach in the treatment of cancer, resulting in a growing importance in oncology. Engineered T cell therapies, namely chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) T cell therapies, are platform technologies that have enabled the development of products with remarkable efficacy in several hematological malignancies and are thus the focus of intense research and development activity. While engineered T cell therapies offer promise in addressing currently intractable cancers, they also present unique challenges, including their nonclinical safety assessment. A workshop organized by HESI and the US Food and Drug Administration (FDA) was held to provide an interdisciplinary forum for representatives of industry, academia and regulatory authorities to share information and debate on current practices for the nonclinical safety evaluation of engineered T cell therapies. This manuscript leverages what was discussed at this workshop to provide an overview of the current important nonclinical safety assessment considerations for the development of these therapeutic modalities (cytokine release syndrome, neurotoxicity, on-target/off-tumor toxicities, off-target effects, gene editing or vector integration-associated genomic injury). The manuscript also discusses approaches used for hazard identification or risk assessment and provides a regulatory perspective on such aspects.

Drug delivery technologies have enabled the development of many pharmaceutical products that improve patient health by enhancing the delivery of a therapeutic to its target site, minimizing off-target accumulation and facilitating patient compliance. As therapeutic modalities expanded beyond small molecules to include nucleic acids, peptides, proteins and antibodies, drug delivery technologies were adapted to address the challenges that emerged. In this Review Article, we discuss seminal approaches that led to the development of successful therapeutic products involving small molecules and macromolecules, identify three drug delivery paradigms that form the basis of contemporary drug delivery and discuss how they have aided the initial clinical successes of each class of therapeutic. We also outline how the paradigms will contribute to the delivery of live-cell therapies.

Cancer immunotherapy has gained attention as the supreme therapeutic modality for the treatment of various malignancies. Adoptive T-cell therapy (ACT) is one of the most distinctive modalities of this therapeutic approach, which seeks to harness the potential of combating cancer cells by using autologous or allogenic tumor-specific T-cells. However, a plethora of circumstances must be optimized to produce functional, durable, and efficient T-cells. Recently, the potential of ACT has been further realized by the introduction of novel gene-editing platforms such as the CRISPR/Cas9 system; this technique has been utilized to create T-cells furnished with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR) that have precise tumor antigen recognition, minimal side effects and treatment-related toxicities, robust proliferation and cytotoxicity, and nominal exhaustion. Here, we aim to review and categorize the recent breakthroughs of genetically modified TCR/CAR T-cells through CRISPR/Cas9 technology and address the pearls and pitfalls of each method. In addition, we investigate the latest ongoing clinical trials that are applying CRISPR-associated TCR/CAR T-cells for the treatment of cancers.

Clinically, intrauterine hypoxia is the foremost cause of perinatal morbidity and developmental plasticity in the fetus and newborn infant. Under hypoxia, deviations occur in the lung cell epigenome. Epigenetic mechanisms (e.g., DNA methylation, histone modification, and miRNA expression) control phenotypic programming and are associated with physiological responses and the risk of developmental disorders, such as bronchopulmonary dysplasia. This developmental disorder is the most frequent chronic pulmonary complication in preterm labor. The pathogenesis of this disease involves many factors, including aberrant oxygen conditions and mechanical ventilation-mediated lung injury, infection/inflammation, and epigenetic/genetic risk factors. This review is focused on various aspects related to intrauterine hypoxia and epigenetic programming in lung development and disease, summarizes our current knowledge of hypoxia-induced epigenetic programming and discusses potential therapeutic interventions for lung disease.

Mitochondrial dysfunction contributes to the imbalance of cellular homeostasis and the development of diseases, which is regulated by mitochondria-associated factors. The present review aims to explore the process of the mitochondrial quality control system as a new source of the potential diagnostic biomarkers and/or therapeutic targets for diseases, including mitophagy, mitochondrial dynamics, interactions between mitochondria and other organelles (lipid droplets, endoplasmic reticulum, endosomes, and lysosomes), as well as the regulation and posttranscriptional modifications of mitochondrial DNA/RNA (mtDNA/mtRNA). The direct and indirect influencing factors were especially illustrated in understanding the interactions among regulators of mitochondrial dynamics. In addition, mtDNA/mtRNAs and proteomic profiles of mitochondria in various lung diseases were also discussed as an example. Thus, alternations of mitochondria-associated regulators can be a new category of biomarkers and targets for disease diagnosis and therapy.

Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change, insert, or remove a genomic sequence of interest. These advanced molecular tools include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and RNA-guided engineered nucleases (RGENs), which create double-strand breaks at specific target sites in the genome, and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism. A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype, without the need for the reengineering of the specific enzyme when targeting different sequences. CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function. RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes, as summarized and exemplified in this manuscript.

Nucleic acid therapeutics (NATs) hold promise in treating undruggable diseases and are recognized as the third major category of therapeutics in addition to small molecules and antibodies. Despite the milestones that NATs have made in clinical translation over the past decade, one important challenge pertains to increasing the specificity of this class of drugs. Activating NATs exclusively in disease-causing cells is highly desirable because it will safely broaden the application of NATs to a wider range of clinical indications. Smart NATs are triggered through a photo-uncaging reaction or a specific molecular input such as a transcript, protein, or small molecule, thus complementing the current strategy of targeting cells and tissues with receptor-specific ligands to enhance specificity. This review summarizes the programmable modalities that have been incorporated into NATs to build in responsive behaviors. We discuss the various inputs, transduction mechanisms, and output response functions that have been demonstrated to date.

The development of genome-editing technologies in 1970s has discerned a new beginning in the field of science. Out of different genome-editing approaches such as Zing-finger nucleases, TALENs, and meganucleases, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR/Cas9) is a recent and versatile technology that has the ability of making changes to the genome of different organisms with high specificity. Cancer is a complex process that is characterized by multiple genetic and epigenetic changes resulting in abnormal cell growth and proliferation. As cancer is one of the leading causes of deaths worldwide, a large number of studies are done to understand the molecular mechanisms underlying the development of cancer. Because of its high efficiency and specificity, CRISPR/Cas9 has emerged as a novel and powerful tool in the field of cancer research. CRISPR/Cas9 has the potential to accelerate cancer research by dissecting tumorigenesis process, generating animal and cellular models, and identify drug targets for chemotherapeutic approaches. However, despite having tremendous potential, there are certain challenges associated with CRISPR/Cas9 such as safe delivery to the target, potential off-target effects and its efficacy which needs to be addressed prior to its clinical application. In this review, we give a gist of different genome-editing technologies with a special focus on CRISPR/Cas9 development, its mechanism of action and its applications, especially in different type of cancers. We also highlight the importance of CRISPR/Cas9 in generating animal models of different cancers. Finally, we present an overview of the clinical trials and discuss the challenges associated with translating CRISPR/Cas9 in clinical use.

The recently introduced genome editing technology has had a remarkable impact on genetic medicine. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas nucleases are the three major platforms used for priming of stem cells or correction of mutated genes. Among these nucleases, CRISPR/Cas is the most easily applicable. Various CRISPR/Cas variants such as base editors, prime editors, mad7 nucleases, RESCUE, REPAIR, digenome sequencing, and SHERLOCK are being developed and considered as a promising tool for gene therapy and drug discovery. These advances in the CRISPR/Cas platform have enabled the correction of gene mutations from DNA to RNA level and validation of the safety of genome editing performance at a very precise level by allowing the detection of one base-pair mismatch. These promising alternatives of the CRISPR/Cas system can benefit millions of patients with intractable diseases. Although the therapeutic effects of stem cells have been confirmed in a wide range of disease models, their safety still remains an issue. Hence, scientists are concentrating on generating functionally improved stem cells by using programmable nucleases such as CRISPR. Therefore, in this chapter, we have summarized the applicable options of the CRISPR/Cas platforms by weighing their advantages and limitations in drug discovery and gene therapy.

Around 77 new oncology drugs were approved by the FDA in the past five years; however, most cancers remain untreated. Small molecules and antibodies are dominant therapeutic modalities in oncology. Antibody-drug conjugates, bispecific antibodies, peptides, cell, and gene-therapies are emerging to address the unmet patient need. Advancement in the discovery and development platforms, identification of novel targets, and emergence of new technologies have greatly expanded the treatment options for patients. Here, we provide an overview of various therapeutic modalities and the current treatment options in oncology, and an in-depth discussion of the therapeutics in the preclinical stage for the treatment of breast cancer, lung cancer, and multiple myeloma.

Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.

Genome-edited human induced pluripotent stem cells (iPSCs) hold great promise for therapeutic applications. However, low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair (HDR)-edited iPSC lines, particularly for silent genes. This is partially due to chromatin compaction, inevitably limiting Cas9 access to the target DNA. Among the six HDAC inhibitors we examined, vorinostat, or suberoylanilide hydroxamic acid (SAHA), led to the highest HDR efficiency at both open and closed loci, with acceptable toxicity. HDAC inhibitors equally increased non-homologous end joining (NHEJ) editing efficiencies (∼50%) at both open and closed loci, due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA expression. However, we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin (2.8 vs. 1.7-fold change). These studies provide a new strategy for HDR-editing of silent genes in iPSCs.

Over the past 3 decades, there has been significant progress in refining gene therapy technologies and procedures. Transduction of hematopoietic stem cells ex vivo using lentiviral vectors can now create a highly effective therapeutic product, capable of reconstituting many different immune system dysfunctions when reinfused into patients. Here, we review the key developments in the gene therapy landscape for primary immune deficiency, from an experimental therapy where clinical efficacy was marred by adverse events, to a commercialized product with enhanced safety and efficacy. We also discuss progress being made in preclinical studies for challenging disease targets and emerging gene editing technologies that are showing promising results, particularly for conditions where gene regulation is important for efficacy.

HIV-1/AIDS remains a global public health problem. The world health organization (WHO) reported at the end of 2019 that 38 million people were living with HIV-1 worldwide, of which only 67% were accessing antiretroviral therapy (ART). Despite great success in the clinical management of HIV-1 infection, ART does not eliminate the virus from the host genome. Instead, HIV-1 remains latent as a viral reservoir in any tissue containing resting memory CD4⁺ T cells. The elimination of these residual proviruses that can reseed full-blown infection upon treatment interruption remains the major barrier towards curing HIV-1. Novel approaches have recently been developed to excise or disrupt the virus from the host cells (e.g., gene editing with the CRISPR-Cas system) to permanently shut off transcription of the virus (block-and-lock and RNA interference strategies), or to reactivate the virus from cell reservoirs so that it can be eliminated by the immune system or cytopathic effects (shock-and-kill strategy). Here, we will review each of these approaches, with the major focus placed on the block-and-lock strategy.

Cancer is the second cause of death worldwide, surpassed only by cardiovascular diseases, due to the lack of early diagnosis, and high relapse rate after conventional therapies. Chemotherapy inhibits the rapid growth of cancer cells, but it also affects normal cells with fast proliferation rate. Therefore, it is imperative to develop other safe and more effective treatment strategies, such as gene therapy, in order to significantly improve the survival rate and life expectancy of patients with cancer. The aim of gene therapy is to transfect a therapeutic gene into the host cells to express itself and cause a beneficial biological effect. However, the efficacy of the proposed strategies has been insufficient for delivering the full potential of gene therapy in the clinic. The type of delivery vehicle (viral or non viral) chosen depends on the desired specificity of the gene therapy. The first gene therapy trials were performed with therapeutic genes driven by viral promoters such as the CMV promoter, which induces non-specific toxicity in normal cells and tissues, in addition to cancer cells. The use of tumor-specific promoters over-expressed in the tumor, induces specific expression of therapeutic genes in a given tumor, increasing their localized activity. Several cancer- and/or tumor-specific promoters systems have been developed to target cancer cells. This review aims to provide up-to-date information concerning targeting gene therapy with cancer- and/or tumor-specific promoters including cancer suppressor genes, suicide genes, anti-tumor angiogenesis, gene silencing, and gene-editing technology, as well as the type of delivery vehicle employed. Gene therapy can be used to complement traditional therapies to provide more effective treatments.

Since tumor-specific T cells were first utilized to treat melanoma patients in 1986 [...].

The development of novel genome editing tools has unlocked new opportunities that were not previously possible in basic and biomedical research. During the last two decades, several new genome editing methods have been developed that can be customized to modify specific regions of the genome. However, in the past couple of years, many newer and more exciting genome editing techniques have been developed that are more efficient, precise, and easier to use. These genome editing tools have helped to improve our understanding of genetic disorders by modeling them in cells and animal models, in addition to correcting the disease-causing mutations. Among the genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has proven to be the most popular one due to its versatility and has been successfully used in a wide variety of laboratory animal models and plants. In this review, we summarize the customizable nucleases currently used for genome editing and their uses beyond the modification of genome. We also discuss the potential future applications of gene editing tools for both basic research and clinical purposes.

The main role of salivary glands (SG) is the production and secretion of saliva, in which aquaporins (AQPs) play a key role by ensuring water flow. The AQPs are transmembrane channel proteins permeable to water to allow water transport across cell membranes according to osmotic gradient. This review gives an insight into SG AQPs. Indeed, it gives a summary of the expression and localization of AQPs in adult human, rat and mouse SG, as well as of their physiological role in SG function. Furthermore, the review provides a comprehensive view of the involvement of AQPs in pathological conditions affecting SG, including Sjögren's syndrome, diabetes, agedness, head and neck cancer radiotherapy and SG cancer. These conditions are characterized by salivary hypofunction resulting in xerostomia. A specific focus is given on current and future therapeutic strategies aiming at AQPs to treat xerostomia. A deeper understanding of the AQPs involvement in molecular mechanisms of saliva secretion and diseases offered new avenues for therapeutic approaches, including drugs, gene therapy and tissue engineering. As such, AQP5 represents a potential therapeutic target in different strategies for the treatment of xerostomia.

Cancer is a major health problem. Most of the treatments exhibit systemic toxicity, as they are not targeted or specific to cancerous cells and tumors. Adenoviruses are very promising gene delivery vectors and have immense potential to deliver targeted therapy. Here, we review a wide range of strategies that have been tried, tested, and demonstrated to enhance the specificity of oncolytic viruses towards specific cancer cells. A combination of these strategies and other conventional therapies may be more effective than any of those strategies alone.