AlPaCas: allele-specific CRISPR gene editing through a protospacer-adjacent-motif (PAM) approach.

Gene therapy of dominantly inherited genetic diseases requires either the selective disruption of the mutant allele or the editing of the specific mutation. The CRISPR-Cas system holds great potential for the genetic correction of single nucleotide variants (SNVs), including dominant mutations. However, distinguishing between single-nucleotide variations in a pathogenic genomic context remains challenging. The presence of a PAM in the disease-causing allele can guide its precise targeting, preserving the functionality of the wild-type allele. The AlPaCas (Aligning Patients to Cas) webserver is an automated pipeline for sequence-based identification and structural analysis of SNV-derived PAMs that satisfy this demand. When provided with a gene/SNV input, AlPaCas can: (i) identify SNV-derived PAMs; (ii) provide a list of available Cas enzymes recognizing the SNV (s); (iii) propose mutational Cas-engineering to enhance the selectivity towards the SNV-derived PAM. With its ability to identify allele-specific genetic variants that can be targeted using already available or engineered Cas enzymes, AlPaCas is at the forefront of advancements in genome editing. AlPaCas is open to all users without a login requirement and is freely available at https://schubert.bio.uniroma1.it/alpacas.

Cost and availability of novel cell and gene therapies: Can we avoid a catastrophic second valley of death?: Can we avoid a catastrophic second valley of death?

Advanced gene and cellular therapies risk a second "valley of death" due to their high costs and low patient population. As these are life-saving therapies, measures are urgently needed to prevent their withdrawal from the market.

Transgenic Epidermal Cultures for Junctional Epidermolysis Bullosa - 5-Year Outcomes.

Inherited junctional epidermolysis bullosa is a severe genetic skin disease that leads to epidermal loss caused by structural and mechanical fragility of the integuments. There is no established cure for junctional epidermolysis bullosa. We previously reported that genetically corrected autologous epidermal cultures regenerated almost an entire, fully functional epidermis on a child who had a devastating form of junctional epidermolysis bullosa. We now report long-term clinical outcomes in this patient. (Funded by POR FESR 2014-2020 - Regione Emilia-Romagna and others.).

Anakinra-Loaded Sphingomyelin Nanosystems Modulate In Vitro IL-1-Dependent Pro-Tumor Inflammation in Pancreatic Cancer.

Pancreatic cancer is a very aggressive disease with a dismal prognosis. The tumor microenvironment exerts immunosuppressive activities through the secretion of several cytokines, including interleukin (IL)-1. The IL-1/IL-1 receptor (IL-1R) axis is a key regulator in tumor-promoting T helper (Th)2- and Th17-type inflammation. Th2 cells are differentiated by dendritic cells endowed with Th2-polarizing capability by the thymic stromal lymphopoietin (TSLP) that is secreted by IL-1-activated cancer-associated fibroblasts (CAFs). Th17 cells are differentiated in the presence of IL-1 and other IL-1-regulated cytokines. In pancreatic cancer, the use of a recombinant IL-1R antagonist (IL1RA, anakinra, ANK) in in vitro and in vivo models has shown efficacy in targeting the IL-1/IL-1R pathway. In this study, we have developed sphingomyelin nanosystems (SNs) loaded with ANK (ANK-SNs) to compare their ability to inhibit Th2- and Th17-type inflammation with that of the free drug in vitro. We found that ANK-SNs inhibited TSLP and other pro-tumor cytokines released by CAFs at levels similar to ANK. Importantly, inhibition of IL-17 secretion by Th17 cells, but not of interferon-γ, was significantly higher, and at lower concentrations, with ANK-SNs compared to ANK. Collectively, the use of ANK-SNs might be beneficial in reducing the effective dose of the drug and its toxic effects.

Regeneration of the entire human epidermis using transgenic stem cells.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Dissecting CYP1A2 Activation by Arylalkanoic Acid Prodrugs toward the Development of Anti-Inflammatory Agents.

Arylalkane-derived prodrugs of arylacetic acids are a small group of substances that have long been known for their anti-inflammatory action. Despite their ease of synthesis and good potential for the development of new potent and safe anti-inflammatory agents, this group of substances has not received much attention from researchers so far. Therefore, representative arylalkane derivatives were investigated through molecular docking techniques to verify the possible hepatic activation mode toward active metabolites by CYP1A2. In this regard, arylalkanoic acid prodrugs were docked with a crystallographic structure of human CYP1A2, in which the enzyme is co-crystallized with the selective competitive inhibitor α-naphthoflavone BHF. Of note, all the examined compounds proved capable of interacting with the enzyme active site in a manner similar to Nabumetone, thus confirming that a productive metabolic transformation is feasible. On the basis of these findings, it is possible to argue that subtle differences in the way CYP1A2 accommodates the ligands depend on the fine details of their molecular structures. Overall, these data suggest that compounds simply formed by an aromatic moiety bearing an appropriate alkane-derived chain could lead to innovative anti-inflammatory agents.

Multivariate Curve Resolution Methodology Applied to the ATR-FTIR Data for Adulteration Assessment of Virgin Coconut Oil.

Virgin coconut oil (VCO) is a functional food with important health benefits. Its economic interest encourages fraudsters to deliberately adulterate VCO with cheap and low-quality vegetable oils for financial gain, causing health and safety problems for consumers. In this context, there is an urgent need for rapid, accurate, and precise analytical techniques to detect VCO adulteration. In this study, the use of Fourier transform infrared (FTIR) spectroscopy combined with multivariate curve resolution-alternating least squares (MCR-ALS) methodology was evaluated to verify the purity or adulteration of VCO with reference to low-cost commercial oils such as sunflower (SO), maize (MO) and peanut (PO) oils. A two-step analytical procedure was developed, where an initial control chart approach was designed to assess the purity of oil samples using the MCR-ALS score values calculated on a data set of pure and adulterated oils. The pre-treatment of the spectral data by derivatization with the Savitzky-Golay algorithm allowed to obtain the classification limits able to distinguish the pure samples with 100% of correct classifications in the external validation. In the next step, three calibration models were developed using MCR-ALS with correlation constraints for analysis of adulterated coconut oil samples in order to assess the blend composition. Different data pre-treatment strategies were tested to best extract the information contained in the sample fingerprints. The best results were achieved by derivative and standard normal variate procedures obtaining RMSEP and RE% values in the ranges of 1.79-2.66 and 6.48-8.35%, respectively. The models were optimized using a genetic algorithm (GA) to select the most important variables and the final models in the external validations gave satisfactory results in quantifying adulterants, with absolute errors and RMSEP of less than 4.6% and 1.470, respectively.

Curcumin-Sodium Alginate and Curcumin-Chitosan Conjugates as Drug Delivery Systems: An Interesting Rheological Behaviour.

The conjugation of polyphenols is a valuable strategy with which to confer tailored properties to polymeric materials of biomedical interest. Within this investigation, we aim to explore the possibility to use this synthetic approach to increase the viscosity of conjugates, thus allowing the release of a loaded therapeutic to be better controlled over time than in neat polyphenols. Curcumin (CUR) was conjugated to sodium alginate (CA) and chitosan (CS) with functionalisation degrees of 9.2 (SA-CUR) and 15.4 (CS-CUR) mg g-1. Calorimetric analyses showed higher degrees of chain rigidity upon conjugation, with a shift of the degradation peaks to higher temperatures (from 239 to 245 °C and from 296 to 303 °C for SA-CUR and CS-CUR, respectively). Rheological analyses were used to prove the enhanced interconnection between the polymer chains in the conjugates, confirmed by the weak gel parameters, A and z. Moreover, the typical non-Newtonian behaviour of the high-molecular-weight polysaccharides was recorded, together with an enhancement of the activation energy, Ea, in CS-CUR vs. CS (opposite behaviour recorded for SA-CUR vs. SA). The evaluation of the delivery performance (of Doxorubicin as a model drug) showed sustained release profiles, opening opportunities for the development of controlled delivery systems.

The steep uphill path leading to ex vivo gene therapy for genodermatoses.

Cell therapy, gene therapy, and tissue engineering have the potential to revolutionize the field of regenerative medicine. In particular, gene therapy is understood as the therapeutical correction of mutated genes by addition of a correct copy of the gene or site-specific gene modifications. Gene correction of somatic stem cells sustaining renewing tissues is critical to ensure long-term clinical success of ex vivo gene therapy. To date, remarkable clinical outcomes arose from combined ex vivo cell and gene therapy of different genetic diseases, such as immunodeficiencies and genodermatoses. Despite the efforts of researchers around the world, only a few of these advanced approaches have yet made it to routine therapy. In fact, gene therapy poses one of the greatest technical challenges in modern medicine, spanning safety and efficacy issues, regulatory constraints, registration and market access, all of which need to be addressed to make the therapy available to patients with rare disease. In this review, we survey some of the main challenges in the development of combined cell and gene therapy of genetic skin diseases.

Interaction of letrozole and its degradation products with aromatase: chemometric assessment of kinetics and structure-based binding validation.

Letrozole is one of the most prescribed drugs for the treatment of breast cancer in post-menopausal women, and it is endowed with selective peripheral aromatase inhibitory activity. The efficacy of this drug is also a consequence of its long-lasting activity, likely due to its metabolic stability. The reactivity of cyano groups in the letrozole structure could, however, lead to chemical derivatives still endowed with residual biological activity. Herein, the chemical degradation process of the drug was studied by coupling multivariate curve resolution and spectrophotometric methodologies in order to assess a detailed kinetic profile. Three main derivatives were identified after drug exposure to different degradation conditions, consisting of acid-base and oxidative environments and stressing light. Molecular docking confirmed the capability of these compounds to accommodate into the active site of the enzyme, suggesting that the sustained inhibitory activity of letrozole may be at least in part attributed to the degradation compounds.

Anticancer Drugs: Recent Strategies to Improve Stability Profile, Pharmacokinetic and Pharmacodynamic Properties.

In past decades, anticancer research has led to remarkable results despite many of the approved drugs still being characterized by high systemic toxicity mainly due to the lack of tumor selectivity and present pharmacokinetic drawbacks, including low water solubility, that negatively affect the drug circulation time and bioavailability. The stability studies, performed in mild conditions during their development or under stressing exposure to high temperature, hydrolytic medium or light source, have demonstrated the sensitivity of anticancer drugs to many parameters. For this reason, the formation of degradation products is assessed both in pharmaceutical formulations and in the environment as hospital waste. To date, numerous formulations have been developed for achieving tissue-specific drug targeting and reducing toxic side effects, as well as for improving drug stability. The development of prodrugs represents a promising strategy in targeted cancer therapy for improving the selectivity, efficacy and stability of active compounds. Recent studies show that the incorporation of anticancer drugs into vesicular systems, such as polymeric micelles or cyclodextrins, or the use of nanocarriers containing chemotherapeutics that conjugate to monoclonal antibodies can improve solubility, pharmacokinetics, cellular absorption and stability. In this study, we summarize the latest advances in knowledge regarding the development of effective highly stable anticancer drugs formulated as stable prodrugs or entrapped in nanosystems.

Epigenetic and metabolic regulation of epidermal homeostasis.

Continuous exposure of the skin to environmental, mechanical and chemical stress necessitates constant self-renewal of the epidermis to maintain its barrier function. This self-renewal ability is attributed to epidermal stem cells (EPSCs), which are long-lived, multipotent cells located in the basal layer of the epidermis. Epidermal homeostasis - coordinated proliferation and differentiation of EPSCs - relies on fine-tuned adaptations in gene expression which in turn are tightly associated with specific epigenetic signatures and metabolic requirements. In this review, we will briefly summarize basic concepts of EPSC biology and epigenetic regulation with relevance to epidermal homeostasis. We will highlight the intricate interplay between mitochondrial energy metabolism and epigenetic events - including miRNA-mediated mechanisms - and discuss how the loss of epigenetic regulation and epidermal homeostasis manifests in skin disease. Discussion of inherited epidermolysis bullosa (EB) and disorders of cornification will focus on evidence for epigenetic deregulation and failure in epidermal homeostasis, including stem cell exhaustion and signs of premature ageing. We reason that the epigenetic and metabolic component of epidermal homeostasis is significant and warrants close attention. Charting epigenetic and metabolic complexities also represents an important step in the development of future systemic interventions aimed at restoring epidermal homeostasis and ameliorating disease burden in severe skin conditions.

Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin ß3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin ß3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin ß3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin ß3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6ß4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

Photodegradation of Anti-Inflammatory Drugs: Stability Tests and Lipid Nanocarriers for Their Photoprotection.

The present paper provides an updated overview of the methodologies applied in photodegradation studies of non-steroidal anti-inflammatory drugs. Photostability tests, performed according to international standards, have clearly demonstrated the photolability of many drugs belonging to this class, observed during the preparation of commercial forms, administration or when dispersed in the environment. The photodegradation profile of these drugs is usually monitored by spectrophotometric or chromatographic techniques and in many studies the analytical data are processed by chemometric procedures. The application of multivariate analysis in the resolution of often-complex data sets makes it possible to estimate the pure spectra of the species involved in the degradation process and their concentration profiles. Given the wide use of these drugs, several pharmaceutical formulations have been investigated to improve their photostability in solution or gel, as well as the pharmacokinetic profile. The use of lipid nanocarriers as liposomes, niosomes or solid lipid nanoparticles has demonstrated to both minimize photodegradation and improve the controlled release of the entrapped drugs.

Toward Multitasking Pharmacological COX-Targeting Agents: Non-Steroidal Anti-Inflammatory Prodrugs with Antiproliferative Effects.

The antitumor activity of certain anti-inflammatory drugs is often attributed to an indirect effect based on the inhibition of COX enzymes. In the case of anti-inflammatory prodrugs, this property could be attributed to the parent molecules with mechanism other than COX inhibition, particularly through formulations capable of slowing down their metabolic conversion. In this work, a pilot docking study aimed at comparing the interaction of two prodrugs, nabumetone (NB) and its tricyclic analog 7-methoxy-2,3-dihydro-1H-cyclopenta[b]naphthalen-1-one (MC), and their common active metabolite 6-methoxy-2-naphthylacetic acid (MNA) with the COX binding site, was carried out. Cytotoxicity, cytofluorimetry, and protein expression assays on prodrugs were also performed to assess their potential as antiproliferative agents that could help hypothesize an effective use as anticancer therapeutics. Encouraging results suggest that the studied compounds could act not only as precursors of the anti-inflammatory metabolite, but also as direct antiproliferative agents.

Essential Oils of Foeniculum vulgare subsp. piperitum and Their in Vitro Anti-Arthritic Potential.

Wild Foeniculum vulgare subsp. piperitum (C.Presl) Bég. flowers, fruits and leaves were extracted with steam distillation and obtained essential oils (EOs) were characterized using GC/MS. The study was designed to verify the potential effectiveness of fennel EOs in the treatment of inflammation and arthritis. Since tissue proteins denaturation is a major cause of arthritic diseases, fennel EOs and their main constituents were evaluated for their ability to inhibit the heat-induced proteins degradation using bovine serum albumin as a protein model. Moreover, the in vitro inhibitory effects of the three EOs on the pro-inflammatory mediator nitric oxide (NO) production were verified in LPS-stimulated RAW 264.7 cells. Estragole (28.81-33.40 %), anethole (24.16-27.40 %), fenchone (9.76-18.48 %), α-phellandrene (1.63-8.37 %) and limonene (5.54-6.05 %) were the major constituents. All the EOs showed a concentration-dependent biological activity, being the flower EO the most effective in inhibiting NO production (IC50 =232.2±11.3 µg/mL). The leaf EO showed a very good bovine serum albumin (BSA) anti-denaturation activity (IC50 =95.9±2.4 µg/mL). Moreover, four components were proved to be effective in protecting protein from heat-induced degradation, being α-phellandrene the most active compound (IC50 =73.2±1.9 µg/mL).

CCR5/CXCR4 Dual Antagonism for the Improvement of HIV Infection Therapy.

HIV entry in the host cell requires the interaction with the CD4 membrane receptor, and depends on the activation of one or both co-receptors CCR5 and CXCR4. Former selective co-receptor antagonists, acting at early stages of infection, are able to impair the receptor functions, preventing the viral spread toward AIDS. Due to the capability of HIV to develop resistance by switching from CCR5 to CXCR4, dual co-receptor antagonists could represent the next generation of AIDS prophylaxis drugs. We herein present a survey on relevant results published in the last few years on compounds acting simultaneously on both co-receptors, potentially useful as preventing agents or in combination with classical anti-retroviral drugs based therapy.

Photo and thermal stress of linseed oil and stabilization strategies.

The thermal and light stability of linseed oil has been studied by monitoring the concentrations of fatty acids and lignans, as main nutraceutical components. Linseed oil was subjected to stressing light and temperature conditions, in accordance with the ICH international rules, and monitored by UV-vis spectroscopy and HPLC-DAD. The change of UV spectra along the photodegradation tests, setting the irradiation power at 350 W/m2, confirmed a significant overall sensitivity of linseed oil to light. At the same time, the HPLC determination of the major fatty acids showed a marked variation in their concentration up to a residual concentration of 62.3 and 67.2% for α-linolenic and linoleic acid, respectively, after 18 h. In contrast, thermal tests at 60 °C showed some stability, with a concentration of residual fatty acids in the range 82-95% after 48 h. The examined lignans showed significant stability when exposed to both light and heat. Several photoprotection approaches have been also studied to increase the photostability of linseed oil. A significant increase in the stability of fatty acids has been observed using amber glass containers or ascorbic acid or by combining the two protection factors.

Regulation of stem cell therapies under attack in Europe: for whom the bell tolls.

At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context-social, financial, medical, legal-in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of 'translational medicine'. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.

Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine.

Recent breakthroughs in regenerative medicine have generated enthusiasm and many efforts to explore new therapeutic potentials of both somatic and pluripotent stem cells. About 30 years passed since a discovery of a method of producing a great number of human epidermal keratinocytes by cultivation from a small skin biopsy, many possibilities are now envisaged for therapeutic application of different cultured cell types. The importance of stem cell content was proven for many tissues or organs in different pathologies. Ocular burns cause depletion of limbal stem cells, which lead to corneal opacification and visual loss. Most of available treatments are palliative and focused on the relief of the devastating clinical picture. This review is focused on recent developments in cell-based therapy of limbal stem cell deficiency. All findings can provide support for improvement and standardization of the cure for this disabling disease.