Undesirable Off-target Effects Research Articles

Bacteriophage λ exonuclease and a 5'-phosphorylated DNA guide allow PAM-independent targeting of double-stranded nucleic acids.

Sequence-specific recognition of double-stranded nucleic acids is essential for molecular diagnostics and in situ imaging. Clustered regularly interspaced short palindromic repeats and Cas systems rely on protospacer-adjacent motif (PAM)-dependent double-stranded DNA (dsDNA) recognition, limiting the range of targetable sequences and leading to undesired off-target effects. Using single-molecule fluorescence resonance energy transfer analysis, we discover the enzymatic activity of bacteriophage λ exonuclease (λExo). We show binding of 5'-phosphorylated single-stranded DNA (pDNA) to complementary regions on dsDNA and DNA-RNA duplexes, without the need for a PAM-like motif. Upon binding, the λExo-pDNA system catalytically digests the pDNA into nucleotides in the presence of Mg2+. This process is sensitive to mismatches within a wide range of the pDNA-binding region, resulting in exceptional sequence specificity and reduced off-target effects in various applications. The absence of a requirement for a specific motif such as a PAM sequence greatly broadens the range of targets. We demonstrate that the λExo-pDNA system is a versatile tool for molecular diagnostics, DNA computing and gene imaging applications.

Regulatory T Cells Nanoextinguisher to Manipulate Multiple Immune Evasion for Immunotherapy.

Regulatory T cells (Treg) play key roles in inhibiting effective antitumor immunity. However, therapeutic Treg depletion fails to consistently enhance immune responses due to the emergence of a wave of peripherally converted Treg cells postdepletion, along with undesired off-target side effects. Here, we report a nanoextinguisher decorated with functional peptides via tumor microenvironment responsive linkers to selectively block Treg function and maintain Treg levels rather than deplete them. The nanoextinguisher specifically neutralizes TGF-β to inhibit the recruitment of Treg cells and the conversion of naive T cells into Treg cells, thus promoting antitumor immunity. Moreover, the nanoextinguisher can alleviate tumor resistance to immunogenic photodynamic therapy, vaccination therapy, and checkpoint inhibition. The nanoextinguisher showed 30-fold potentiation in antitumor effect compared to standalone photodynamic therapy or vaccination therapy. Overall, utilizing a nanoextinguisher to inhibit Treg function without triggering reconversion represents a generalizable method to reverse immune evasion, yielding antitumor efficacy.

Photocleavable Guide RNA for Photocontrolled CRISPR/Cas9 System

Objective: The development of CRISPR/Cas-based gene-editing systems having a higher efficacy and specificity, and capable of changing activity in response to light irradiation is an urgent problem. A promising approach to this problem is to modify CRISPR/Cas components, in particular guide RNA, by introducing photocleavable linkers. We developed an approach to the synthesis of photocleavable single guide RNA (sgRNA) for the CRISPR/Cas9 system containing linkers on the basis of 1-(2-nitrophenyl)-1,2-ethanediol. Such photomodified guide RNAs are cleaved under UV irradiation, thereby inactivating the CRISPR/Cas9 system. Methods: Automatic solid-phase phosphoramidate method was used for photomodified sgRNA synthesis. Model plasmid was used for designed system testing. Results and Discussion: We obtained three variants of photomodified sgRNA with different photolinker positions. Evidence was obtained showing that the sgRNA with the photolinker introduced in the protein Cas9 site of binding and hairpin formation is able to effectively guide Cas9 nuclease for target DNA cleavage before UV irradiation and lose its activity after irradiation. The conditions of controllable 40% cleavage of a model target DNA were chosen. Conclusions: The work presents the results of photocleavable sgRNA design and usage as a component of photoregulated CRISPR/Cas9 system. The developed approach makes possible specific inactivation of the CRISPR/Cas9 gene editing system in a specific time moment in a definite place. The photoregulation of the gene-editing system not only allows one to reduce undesirable off-target effects, but also forms the basis for genetic disease therapy.

Enhancing osteoporosis treatment using a targeted, sustained-release drug delivery system based on macrocyclic amphiphile

Osteoporosis, a prevalent systemic bone metabolic disorder, primarily affects postmenopausal women and is characterized by increased bone fragility and a heightened risk of fractures. The efficacy of current osteoporosis treatments is often limited by non-specific drug targeting and undesirable off-target skeletal side effects. To address this challenge, we have developed a novel hydroxyapatite-responsive drug delivery system. This system utilizes a self-assembled p-phosphonatocalix[4]arene tetradodecyl ether (PC4A12C), engineered to specifically target and sustain the release of osteoporosis medication at sites of bone remodeling. Our focus centers on icariin (ICA), a drug known for its potent osteogenic properties and minimal adverse effects. In vitro, ICA-loaded PC4A12C (ICA@PC4A12C) demonstrated enhanced proliferation, differentiation, and mineralization in bone marrow mesenchymal stem cells (BMSCs). In vivo, ICA@PC4A12C exhibited superior efficacy in specifically targeting bone tissue, ensuring a controlled and slow release of icariin directly within the bone environment. In an osteoporosis mouse model, treatment with ICA@PC4A12C showed notable enhancement in osteogenic activity and a significant increase in bone density compared to ICA alone. These results demonstrate the potential of PC4A12C as an effective drug carrier in the development of advanced antiosteoporotic drug delivery systems.

A platform to deliver single and bi-specific Cas9/guide RNA to perturb genes in vitro and in vivo

Although CRISPR/Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate DNA oligos (PS), one can efficiently deliver single and bi-specific CRISPR/Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.

Exploring the Theranostic Applications and Prospects of Nanobubbles.

Anticancer medications as well as additional therapeutic compounds, have poor clinical effectiveness due to their diverse distribution, non-selectivity for malignant cells, and undesirable off-target side effects. As a result, ultrasound-based targeted delivery of therapeutic compounds carried in sophisticated nanocarriers has grown in favor of cancer therapy and control. Nanobubbles are nanoscale bubbles that exhibit unique physiochemical properties in both their inner core and outer shell. Manufacturing nanobubbles primarily aims to enhance therapeutic agents' bioavailability, stability, and targeted delivery. The small size of nanobubbles allows for their extravasation from blood vessels into surrounding tissues and site-specific release through ultrasound targeting. Ultrasound technology is widely utilized for therapy due to its speed, safety, and cost-effectiveness, and micro/nanobubbles, as ultrasound contrast agents, have numerous potential applications in disease treatment. Thus, combining ultrasound applications with NBs has recently demonstrated increased localization of anticancer molecules in tumor tissues with triggered release behavior. Consequently, an effective therapeutic concentration of drugs/genes is achieved in target tumor tissues with ultimately increased therapeutic efficacy and minimal side effects on other non-cancerous tissues. This paper provides a brief overview of the production processes for nanobubbles, along with their key characteristics and potential therapeutic uses.

A Platinum-Aluminum Bimetallic Salen Complex for Pro-senescence Cancer Therapy.

Cell senescence is defined as irreversible cell cycle arrest, which can be triggered by telomere shortening or by various types of genotoxic stress. Induction of senescence is emerging as a new strategy for the treatment of cancer, especially when sequentially combined with a second senolytic drug capable of killing the resulting senescent cells, however severely suffering from the undesired off-target side effects from the senolytic drugs. Here, we prepare a bimetalic platinum-aluminum salen complex (Alumiplatin) for cancer therapy-a combination of pro-senesence chemotherapy with in situ senotherapy to avoid the side effects. The aluminum salen moiety, as a G-quadruplex stabilizer, enhances the salen's ability to induce cancer cell senescence and this phenotype is in turn sensitive to the cytotoxic activity of the monofunctional platinum moiety. It exhibits an excellent capability for inducing senescence, a potent cytotoxic activity against cancer cells both in vitro and in vivo, and an improved safety profile compared to cisplatin. Therefore, Alumiplatin may be a good candidate to be further developed into safe and effective anticancer agents. This novel combination of cell senescence inducers with genotoxic drugs revolutionizes the therapy options of designing multi-targeting anticancer agents to improve the efficacy of anticancer therapies.

The effects of the β1-adrenergic receptor antagonist bisoprolol administration on mirabegron-stimulated human brown adipose tissue thermogenesis.

Pharmacological stimulation of human brown adipose tissue (BAT) has been hindered by ineffective activation or undesirable off-target effects. Oral administration of the maximal allowable dose of mirabegron (200 mg), a β3-adrenergic receptor (β3-AR) agonist, has been effective in stimulating BAT thermogenesis and whole-body energy expenditure. However, this has been accompanied by undesirable cardiovascular effects. Therefore, we hypothesized that combining mirabegron with a β1-AR antagonist could suppress these unwanted effects and increase the stimulation of the β3-AR and β2-AR in BAT. We performed a randomized crossover trial (NCT04823442) in 8 lean men. Mirabegron (200 mg) was administered orally with or without the β1-AR antagonist bisoprolol (10 mg). Dynamic [11C]-acetate and 2-deoxy-2-[18F]fluoro-d-glucose PET/CT scans were performed sequentially after oral administration of mirabegron ± bisoprolol. Compared to room temperature, mirabegron alone increased BAT oxidative metabolism (0.84 ± 0.46 vs. 1.79 ± 0.91 min-1, p = 0.0433), but not when combined with bisoprolol. The metabolic rate of glucose in BAT, measured using [18F]FDG PET, was significantly higher with mirabegron than mirabegron with bisoprolol (24 ± 10 vs. 16 ± 8 nmol/g/min, p = 0.0284). Bisoprolol inhibited the mirabegron-induced increase in systolic blood pressure and heart rate. The administration of bisoprolol decreases the adverse cardiovascular effects of mirabegron. However, the provided dose also blunted the mirabegron-stimulated increase in BAT lipolysis, thermogenesis, and glucose uptake. The attenuation in BAT blood flow induced by the large dose of bisoprolol may have limited BAT thermogenesis.

C-terminal binding protein 2 is a novel tumor suppressor targeting the MYC-IRF4 axis in multiple myeloma

AbstractMultiple myeloma (MM) cells are addicted to MYC and its direct transactivation targets IRF4 for proliferation and survival. MYC and IRF4 are still considered “undruggable,” as most small-molecule inhibitors suffer from low potency, suboptimal pharmacokinetic properties, and undesirable off-target effects. Indirect inhibition of MYC/IRF4 emerges as a therapeutic vulnerability in MM. Here, we uncovered an unappreciated tumor-suppressive role of C-terminal binding protein 2 (CTBP2) in MM via strong inhibition of the MYC-IRF4 axis. In contrast to epithelial cancers, CTBP2 is frequently downregulated in MM, in association with shortened survival, hyperproliferative features, and adverse clinical outcomes. Restoration of CTBP2 exhibited potent antitumor effects against MM in vitro and in vivo, with marked repression of the MYC-IRF4 network genes. Mechanistically, CTBP2 impeded the transcription of MYC and IRF4 by histone H3 lysine 27 deacetylation (H3K27ac) and indirectly via activation of the MYC repressor IFIT3. In addition, activation of the interferon gene signature by CTBP2 suggested its concomitant immunomodulatory role in MM. Epigenetic studies have revealed the contribution of polycomb-mediated silencing and DNA methylation to CTBP2 inactivation in MM. Notably, inhibitors of Enhance of zeste homolog 2, histone deacetylase, and DNA methyltransferase, currently under evaluation in clinical trials, were effective in restoring CTBP2 expression in MM. Our findings indicated that the loss of CTBP2 plays an essential role in myelomagenesis and deciphers an additional mechanistic link to MYC-IRF4 dysregulation in MM. We envision that the identification of novel critical regulators will facilitate the development of selective and effective approaches for treating this MYC/IRF4-addicted malignancy.

Development of Phenyl-substituted Isoindolinone- and Benzimidazole-type Cereblon Ligands for Targeted Protein Degradation.

Thalidomide, pomalidomide and lenalidomide, collectively referred to as immunomodulatory imide drugs (IMiDs), are frequently employed in proteolysis-targeting chimeras (PROTACs) as cereblon (CRBN) E3 ligase-recruiting ligands. However, their molecular glue properties that co-opt the CRL4CRBN to degrade its non-natural substrates may lead to undesired off-target effects for the IMiD-based PROTAC degraders. Herein, we reported a small library of potent and cell-permeable CRBN ligands, which exert high selectivity over the well-known CRBN neo-substrates of IMiDs by structure-based design. They were further utilized to construct bromodomain-containing protein 4 (BRD4) degraders, which successfully depleted BRD4 in the tested cells. Overall, we reported a series of functionalized CRBN recruiters that circumvent the promiscuity from traditional IMiDs, and this study is informative to the development of selective CRBN-recruiting PROTACs for many other therapeutic targets.

Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies.

The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.

Pharmacologic Therapies in Beta Hemoglobinopathies: Fetal Globin Gene Induction in the First Molecular Diseases

Beta hemoglobinopathies and thalassemias are caused by diverse molecular mutations of the βA globin chains and modulated by polymorphisms. These are important disorders in medical history, as they became prevalent globally in regions where P. falciparum malaria was endemic. Heterozygous or carrier states conferred a survival advantage; however, doubly heterozygous or homozygous states cause severe hemolytic anemia and multi-organ complications. These disorders are somewhat unique in that all humans have alternate genes for fetal globin chains, which are expressed in fetal life but are silenced on a developmental clock in infancy. An established approach to ameliorating conditions of abnormal adult globin genes is to reactivate, or increase expression, of the endogenous fetal globin genes to replace the missing protein chains in beta thalassemias or inhibit polymerization of hemoglobin S and reduce the red cell abnormalities. This review provides a high-level overview of cell and molecular mechanisms that mediate fetal to adult globin gene switching and pharmacologic therapies that can reactivate fetal globin through different actions. Intermittent or metronomic dosing regimens have overcome challenges of undesirable off-target effects by agents that cause erythroid cell growth arrest. Multiple pharmacologic candidates reactivate or increase the expression of fetal globin protein and proportions of red blood cells containing HbF (F-cells). Hydroxyurea maintains high levels of HbF if begun in infancy, with some decline in mid-childhood. It elicits lower responses in HbF in adult patients, but still has broad clinical benefit in reducing many complications. However, many adult patients do not tolerate optimal hydroxyurea dosing due to cytopenias. Parenterally administered therapies with differing molecular actions, such as demethylating agents and histone deacetylase inhibitors, have shown proof-of-principle in reactivating HbF. Orally bioavailable candidates with complementary molecular mechanisms are in trials. Combining fetal globin-inducing agents with other therapies with complementary mechanisms, such as recombinant erythropoietin that promotes the survival of red blood cells and therapeutics that promote erythroid cell metabolism, should have additive effects. These pharmaceutical candidates offer great clinical potential and global feasibility for ameliorating these serious diseases.

Unveiling neural network potential in forecasting CRISPR effects and off-target prophecies for gene editing

The revolutionary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - associated protein 9 (Cas9) systems has emerged as a groundbreaking gene-editing tool, widely embraced within biomedical research. Nonetheless, the utilization of guide RiboNucleic Acids (gRNAs) in the CRISPR-Cas9 system can inadvertently trigger undesired off-target effects, consequently impinging on the practical implementation of this technique. Existing in silico prediction methods that focus on off-target effects have exhibited constrained predictive accuracy, necessitating further enhancement. To tackle this challenge, we present a Base Editing and Prime Editing approach in this study. This approach aim to enhance the precision and specificity of DeoxyRiboNucleic Acid (DNA) modifications compared to traditional CRISPR-Cas9 methods. Both techniques provide unique approaches to achieve targeted changes in the DNA sequence without inducing double-stranded breaks, which can lead to off-target effects. Base editing is highly specific and allows for the correction of point mutations associated with diseases while Prime Editing allows for a wider range of modifications compared to base editing, including the ability to insert or delete specific sequences. Their ability to achieve specific genetic changes while minimizing off-target effects makes them valuable additions to the gene editing toolkit. The findings of this research contribute to the advancement of precision gene editing, offering an enhanced predictive framework to mitigate off-target effects in the realm of CRISPR-Cas9 technology.

The untapped potential of targeting NRF2 in neurodegenerative disease.

Since its initial discovery almost three decades ago, the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) has been shown to regulate a host of downstream transcriptional responses and play a critical role in preventing or promoting disease progression depending on the context. Critically, while the importance of proper nuclear factor erythroid 2-related factor 2 function has been demonstrated across a variety of pathological settings, the ability to progress NRF2-targeted therapeutics to clinic has remained frustratingly elusive. This is particularly true in the case of age-related pathologies, where nuclear factor erythroid 2-related factor 2 is a well-established mitigator of many of the observed pathogenic effects, yet options to target this pathway remain limited. Along these lines, loss of nuclear factor erythroid 2-related factor 2 function has clearly been shown to enhance neuropathological outcomes, with enhancing nuclear factor erythroid 2-related factor 2 pathway activation to prevent neurodegenerative/neurological disease progression continuing to be an active area of interest. One critical obstacle in generating successful therapeutics for brain-related pathologies is the ability of the compound to cross the blood brain barrier (BBB), which has also hampered the implementation of several promising nuclear factor erythroid 2-related factor 2 inducers. Another limitation is that many nuclear factor erythroid 2-related factor 2 activators have undesirable off-target effects due to their electrophilic nature. Despite these constraints, the field has continued to evolve, and several viable means of targeting nuclear factor erythroid 2-related factor 2 in a neuropathological context have emerged. In this perspective, we will briefly discuss the key findings and promising therapeutic options that have been discovered to date, as well as highlight emerging areas of NRF2-neurodegeneration research that provide hope for successfully targeting this pathway in the future.

Unbiased whole genome detection of ultrarare off-target mutations in genome-edited cell populations by HiFi sequencing.

DNA base editors (BEs) composed of a nuclease-deficient Cas9 fused to a DNA-modifying enzyme can achieve on-target mutagenesis without creating double-strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease-proficient Cas9 systems, which do rely on the creation of DSBs to achieve on-target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off-target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10-8 mutations per base pair. In this proof-of-principle study, we evaluated whether this technique could also detect the on- and off-target mutations generated by a cytosine-to-thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on-target mutant allele fractions ranging from ~7% to ~63%, depending on the single-guide RNA (sgRNA) used, while no on-target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3-fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of C:G>T:A substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on- and off-target mutations in cell populations that have undergone genome editing.

Current trends and risks associated with the use of therapies based on genome editing

Scientific relevance. To date, multiple approaches to genome editing have been developed based on different genome-editing systems (GESs) and genome modifications that result in single- or double-strand DNA breaks, either in vivo or ex vivo, followed by homologous recombination or non-homologous end joining to restore the sequence. However, the use of GESs is associated with a number of potential risks arising from the complex biology of such medicinal products and the fundamental role of their target, i.e. the DNA molecule.Aim. This study analysed the most relevant trends and risks associated with medicinal products based on genome editing, the ways taken to overcome these risks, and the research methods used to identify and control the development of undesirable effects.According to the literature, the adverse effects of GESs may arise both from the methods used to deliver GES components into the cell and from the functional activity of the GES itself, which includes insufficient on-target or undesirable off-target effects. This review indicates the main risks associated with the use of GESs. Preferable strategies to mitigate the risks of using GESs include repairing DNA breaks by homologous recombination, selecting GESs and related endonucleases that have greater specificity and restriction accuracy, increasing guide RNA specificity (for CRISPR/Cas), correcting the activity of the system regulating the cell cycle and apoptosis in a controlled manner, regulating the duration of expression and persistence of GES components in cells, etc.Conclusions. The requirement to include quality, efficacy, and safety data when submitting registration dossiers for advanced therapy medicinal products prompts the discussion of the main risks associated with such products.

Bioorthogonal PROTAC Prodrugs Enabled by On-Target Activation.

Although proteolysis targeting chimeras (PROTACs) have become promising therapeutic modalities, important concerns exist about the potential toxicity of the approach owing to uncontrolled degradation of proteins and undesirable ligase-mediated off-target effects. Precision manipulation of degradation activity of PROTACs could minimize potential toxicity and side effects. As a result, extensive efforts have been devoted to developing cancer biomarker activating prodrugs of PROTACs. In this investigation, we developed a bioorthogonal on-demand prodrug strategy (termed click-release "crPROTACs") that enables on-target activation of PROTAC prodrugs and release of PROTACs in cancer cells selectively. Inactive PROTAC prodrugs TCO-ARV-771 and TCO-DT2216 are rationally designed by conjugating a bioorthogonal trans-cyclooctenes (TCO) group into the ligand of the VHL E3 ubiquitin ligase. The tetrazine (Tz)-modified RGD peptide, c(RGDyK)-Tz, which targets integrin αvβ3 biomarker in cancer cells, serves as the activation component for click-release of the PROTAC prodrugs to achieve targeted degradation of proteins of interest (POIs) in cancer cells versus noncancerous normal cells. The results of studies accessing the viability of this strategy show that the PROTAC prodrugs are selectively activated in an integrin αvβ3-dependent manner to produce PROTACs, which degrade POIs in cancer cells. The crPROTAC strategy might be a general, abiotic approach to induce selective cancer cell death through the ubiquitin-proteasome pathway.

Ionizing Radiation and Inflammatory Reaction. Formation Mechanisms and Implications

Ionizing radiation induces a complex of genetic, biochemical, structural and functional changes in the body. The inflammatory response development is acknowledged as one of the manifestations of systemic bodily response to ionizing radiation exposure, and this response, through the activation of immunity, acts both as protector and leads to the development of undesirable early, delayed and off-target effects. Its underlying molecular and cellular mechanisms are defined by DNA damage, free radical metabolic changes (primarily reactive oxygen and nitrogen species), oxidative stress development, inflammasome activation, “danger signals” release and pro-inflammatory cytokines production. The role of non-apoptotic cell death forms (ferroptosis and pyroptosis) is described in the genesis of post-radiation inflammatory response and subsequent tissue, organ, and system damages. The post-radiation inflammatory reaction’s ability to take form of a time-stable self-sustaining process – that increases the radiation-induced damage severity – due to the presence of a positive feedback between different components of its pathogenesis is noted.

The VEGF/VEGFR Axis Revisited: Implications for Cancer Therapy

The vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) axis is indispensable in the process of angiogenesis and has been implicated as a key driver of tumor vascularization. Consequently, several strategies that target VEGF and its cognate receptors, VEGFR-1 and VEGFR-2, have been designed to treat cancer. While therapies targeting full-length VEGF have resulted in an improvement in both overall survival and progression-free survival in various cancers, these benefits have been modest. In addition, the inhibition of VEGFRs is associated with undesirable off-target effects. Moreover, VEGF splice variants that modulate sprouting and non-sprouting angiogenesis have been identified in recent years. Cues within the tumor microenvironment determine the expression patterns of these variants. Noteworthy is that the mechanisms of action of these variants challenge the established norm of VEGF signaling. Furthermore, the aberrant expression of some of these variants has been observed in several cancers. Herein, developments in the understanding of the VEGF/VEGFR axis and the splice products of these molecules, as well as the environmental cues that regulate these variants are reviewed. Furthermore, strategies that incorporate the targeting of VEGF variants to enhance the effectiveness of antiangiogenic therapies in the clinical setting are discussed.

CRISPR-Cas9 Genome Editing of Primary Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is a common B-cell neoplasia that exhibits a very heterogeneous course. Over the last decade, multiple molecular studies have described the genomic, epigenomic and transcriptional landscape of the disease. These studies have led to the identification of more than 60 recurrently mutated genes as well as a large number of deregulated genes and regulatory elements. While the prognostic significance of some of these alterations is well described, their specific contributions to the pathogenesis of the disease remains largely unknown. Attempts to address this experimentally have employed genetically engineered mouse models and human cell lines. Although mouse models have been helpful to recapitulate disease aspects that can be only properly studied in vivo, significant limitations exist that prevent extrapolation of data from mice to human. Moreover, cell lines are commonly obtained from patients with end-stage, refractory diseases and are frequently EBV-positive, showing great discrepancies with primary cells, including high proliferation rate and absence of spontaneous apoptosis. In addition, they do not faithfully recapitulate the transcriptional and chromatin landscape of CLL primary cases. Thus, there is a need of better cellular models that recapitulate these disease aspects. An attractive solution to this problem would be the use of primary human CLL cells as a platform for ex vivo genetic manipulation, applying CRISPR-Cas9 genome editing technology. However, this is technically challenging, as (i) CLL cells are critically dependent on stimulatory signals coming from the tumor microenvironment, particularly in the lymph nodes (LN), and (ii) primary cells are resistant to most gene transfer methods. In order to overcome these limitations, we took advantage of a cell culture system that is reminiscent of a LN environment and induces vigorous proliferation of CLL primary cells for several weeks, allowing retroviral gene transfer with high efficiency and minimal toxicity (Mangolini, M. et al., 2022, Nat Commun, in press). To avoid undesired off-target effects induced by the constitutive expression of a CRISPR-Cas9 viral plasmid, we used transient expression systems. As an initial proof of concept, we designed sgRNA targeting the CD19 gene, a pan-B cell marker highly expressed in CLL cells, and assessed the transfection efficiency and cell viability when introducing the Cas9-CD19 sgRNA ribonucleoprotein complex using the Neon Transfection System (Thermo Fisher Scientific). Cryopreserved CLL primary cells (from peripheral blood samples) were expanded for 3 days in vitro and were subsequently electroporated with optimized conditions. Cell viability and electroporation efficiency were assessed 24 hours later and demonstrated a remarkable low percentage of apoptotic cells, with only 10% of cell death compared to non-electroporated cells (measured by flow cytometry using the LIVE/DEAD fixable Aqua dead cell stain, n= 6 cases); while high transfection efficiency was accomplished, with over 80% of cells containing the fluorescent Cas9-ATTO550 sgRNA ribonucleoprotein. Finally, 72 hours after electroporation we observed a downregulation greater than 85% of CD19 levels by flow cytometry (n=6 cases). After successful CD19 downregulation, we aimed at targeting the cell cycle regulatory protein Cyclin D2 (CCND2), which is upregulated by CLL cells at proliferation centers and the most upregulated cyclin in our cell culture system. Two independent sgRNAs were electroporated with the Cas9 protein into 3 CLL primary cases, with a minimum effect on cell viability after 24 hours. CCND2 knock-down (KD) efficiency was of 70% and 60% respectively in all cases studied, assessed by qRT-PCR. Interestingly, despite being exposed to cell culture conditions promoting vigorous proliferation, CCND2 KD blocked cell proliferation, and a cell growth reduction of 50-70% was obtained with both sgRNAs compared with non-targeting controls already at 48 hours. Altogether, these results demonstrate the feasibility to genetically modify primary CLL cells using the CRISPR-Cas9 system. This strategy overcomes the current dependency on cell lines or mouse models and represents a powerful tool to unveil the role of candidate genes involved in CLL pathogenesis.