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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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration., Competing Interests: Declaration of interests M.D.L. is co-founder and member of the Board of Directors of Holostem Terapie Avanzate (HTA) s.r.l in liquidation, Modena, Italy, as well as consultants for J-TEC-Japan Tissue Engineering, Ltd., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses., (Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved.)

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects in type-VII collagen (C7), a protein encoded by the COL7A1 gene and essential for anchoring fibril formation at the dermal-epidermal junction. Gene therapy of RDEB is based on transplantation of autologous epidermal grafts generated from gene-corrected keratinocytes sustaining C7 deposition at the dermal-epidermal junction. Transfer of the COL7A1 gene is complicated by its very large size and repetitive sequence. This article reports a gene delivery approach based on the Sleeping beauty transposon, which allows integration of a full-length COL7A1 cDNA and secretion of C7 at physiological levels in RDEB keratinocytes without rearrangements or detrimental effects on their clonogenic potential. Skin equivalents derived from gene-corrected RDEB keratinocytes were tested in a validated preclinical model of xenotransplantation on immunodeficient mice, where they showed normal deposition of C7 at the dermal-epidermal junction and restoration of skin adhesion properties. These results indicate the feasibility and efficacy of a transposon-based gene therapy approach to RDEB., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.

The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2(S)-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer.

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity "sandwich" (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications.

Introduction: Obesity is a risk factor for both cardiovascular disease and cancer development. Leptin, a cytokine produced by adipose tissue, controls different processes in peripheral tissues, including cancer development and thrombotic disorders in patients with a variety of clinical disorders. Tissue factor (TF), the trigger of blood clotting, is abundant in the adipose tissue. Since TF, often expressed by cancer cells, is considered a hallmark of cancer progression, we investigated whether leptin could modulate TF in the human metastatic breast carcinoma cell line MCF-7., Materials and Methods: MCF-7 cells were incubated with or without the different reagents at 37 °C. At the end of incubation, cells were tested for procoagulant activity by a one-stage clotting assay, TF and TNF-α antigen levels and mRNA by ELISA and real-time RT-PCR, respectively. Leptin receptor was studied by FACS., Results: Both TF activity and antigen constitutively expressed by MCF-7 were significantly increased by leptin in a dose-dependent fashion. TF mRNA levels were also enhanced indicating that leptin exerts its effect at the transcription level. The effect of leptin was specific and required binding to its receptor (Ob-R), which was found on the surface of the cells, since antibodies against leptin and Ob-R completely prevented TF expression upregulation. In addition, leptin enhanced both TNF-α mRNA synthesis and secretion from MCF7. An anti-TNF-α MoAb completely abolished the leptin-induced TF expression., Conclusions: These data support the hypothesis that leptin, by its upregulation of TF, possibly mediated by TNF-α synthesis, may contribute to processes underlying both cancer and vascular cell disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A typical pathoadaptive mutation of Shigella and enteroinvasive Escherichia coli (EIEC) is the inactivation of the cad locus which comprises the genes necessary for lysine decarboxylation, an enzyme involved in pH homoeostasis. In E. coli, the cadBA operon, encoding lysine decarboxylase (CadA) and a lysine cadaverine antiporter (CadB), is submitted to the control of CadC, a positive activator whose gene maps upstream the operon, and is transcribed independently from the same strand. CadC is an integral inner membrane protein which acts both, as signal sensor and as transcriptional regulator responding to the low pH and lysine signals. Analysis of the molecular rearrangements responsible for the loss of lysine decarboxylase activity in Shigella and EIEC has revealed that the inactivation of the cadC gene is a common feature. The 3 major adaptive acid resistance (AR) systems - AR1, AR2, and AR3 - are known to be activated at low pH by Shigella and E. coli, allowing them to withstand extremely acid conditions. In this study, evaluating the survival of S. flexneri, S. sonnei, and EIEC strains complemented with a functional cadC gene and challenged at low pH, we present evidence that CadC negatively regulates the expression of the arginine-dependent adaptive acid-resistance system (AR3), encoded by the adi locus while it has no effect on the expression of AR1 and AR2 systems. Moreover, since our results indicate that in enteroinvasive strains the presence of CadC reduces the expression of the arginine decarboxylase encoding gene adiA, it is possible to hypothesize that the loss of functionality of lysine decarboxylase is counterbalanced by a higher expression of the adi system, and that CadC, besides specifically affecting the regulation of the cadBA operon, is also relevant to other systems responding to low pH., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Introduction: Moderate alcohol consumption is protective against cardiovascular disease (CAD). ADHs are major enzymes of alcohol metabolism. A polymorphism in the alcohol dehydrogenases 1C gene (ADH1C) was reportedly associated with the protective effect of alcohol consumption on CAD risk and risk factor levels., Aims: The aim of our study was to investigate whether the association of alcohol consumption with metabolic risk factors for CAD is related to ADH1C variants., Methods: IMMIDIET is a cross-sectional study of 974 healthy male-female pairs living together, randomly recruited in Belgium, Italy and England. The rs698 ADH1C polymorphism was genotyped. A 1-year recall food frequency questionnaire was used to estimate alcohol intake., Results: The intake of alcohol did not vary in relation to ADH1C genotypes. BMI, waist circumference (WC), waist-to-hip ratio, blood pressure, HDL or total cholesterol, triglycerides and FVII:ag levels were positively associated with alcohol intake in men (multivariate ANOVA). Regression coefficient for alcohol and BMI or WC was progressively higher in heterozygotes and gamma 2 homozygotes as compared to gamma 1 homozygotes (p=0.006 and p=0.03 for interaction, respectively). No interaction was found for other risk factors. In women, alcohol intake was positively associated with HDL, LDL and FVII:ag levels but no interaction was found between ADH1C polymorphism and any risk factor., Conclusion: Regulation of ADH1C genotype on the association between alcohol consumption, BMI and WC was found in men from different European countries. In men homozygous for the gamma 2 alleles, intake of alcohol was positively associated with both BMI and WC values.

Objective: The exposure of tissue factor (TF) to blood flow is the initial step in the coagulation process and plays an important role in thrombogenesis. We investigated the role of genetic polymorphisms and haplotypes of the TF gene in the risk of ischemic vascular disease., Methods: Four hundred and twenty-two Italian patients with juvenile myocardial infarction (MI) and 434 controls, 808 US cases with MI and 1005 controls, 267 Italian cases with juvenile ischemic stroke and 209 controls and 148 German cases with juvenile ischemic stroke and 191 controls were studied. rs1361600, rs3917629 (rs3354 in the US population), rs1324214 and rs3917639 Tag single nucleotide polymorphisms were genotyped. Additionally, a meta-analysis of all previous studies on TF loci and the risk of ischemic coronary disease (ICD) was performed., Results: After multivariable analysis none of the SNPs, major SNP haplotypes or haplotype-pairs showed any consistent association with MI. Pooled meta-analysis of six studies also suggested that TF polymorphisms are not associated with CHD. A significant, independent association between SNP rs1324214 (C/T) and juvenile stroke was found in Italian and German populations (OR for TT homozygotes = 0.47, 95% CI 0.24-0.92, in combined analysis). Pooled analysis also showed a significant association for haplotype H3 (OR = 0.76, 95% CI 0.57-1.00) and haplotype-pair H3-H3 (OR = 0.43, 95% CI 0.20-0.92)., Conclusions: TF genetic variations were associated with the risk of ischemic stroke at young age, but did not affect ischemic coronary disease.

Paclitaxel, a microtubule-stabilising compound with potent anti-tumour activity, has been clinically used in a wide variety of malignancies. Tissue factor (TF) is often expressed by tumour-associated endothelial and inflammatory cells, as well as by cancer cells themselves, and it is considered a hallmark of cancer progression. We investigated whether paclitaxel could modulate TF in human mononuclear (MN) cells, human umbilical vein endothelial cells (HUVEC) and the metastatic breast carcinoma cell line MDA-MB-231. Cells were incubated with or without paclitaxel at 37 degrees C. At the end of incubation, cells were disrupted and tested for procoagulant activity by a one-stage clotting assay, for TF antigen levels by ELISA and TF mRNA by real-time RT-PCR. IL-6 and IL-1beta were tested by ELISA in conditioned medium. Both the strong TF activity and antigen constitutively expressed by MDA-MB-231 and the TF induced by LPS, TNF-alpha and IL-1beta in MN cells and HUVEC were significantly reduced by paclitaxel. In the presence of paclitaxel, lower TF mRNA levels were also detected. Since paclitaxel has been shown to induce the expression of inflammatory genes in monocytes and tumour cells, we tested whether paclitaxel could influence IL-6 and IL-1beta release from the cells used in this paper. Neither the constitutive expression of IL-6 and IL-1beta by MDA-MB-231 nor the basal and LPS-induced release from MN cells and HUVEC was affected. Our data support the hypothesis that the anti-tumour effects of paclitaxel may, at least in part, be mediated by the capacity of this drug to modulate the procoagulant potential of cancer and host cells.

The objective was to investigate whether genotypes, haplotypes and haplotype-pairs of interleukin (IL) gene cluster are associated with risk of Myocardial Infarction (MI) at young age and with the release of IL-1B and expression of tissue factor pro-coagulant activity (TFPCA), after stimulation in vitro with lipopolysaccharide (LPS) of human peripheral blood mononuclear cells (PBMCs). Patients with MI at young age, frequency-matched for age, sex and recruitment centre, with healthy population-based controls and PBMCs from healthy volunteers were studied. Five single nucleotide polymorphisms (SNPs), identifying two haplotype-blocks, in IL-1B gene and one SNP in IL-1A and IL-RA genes were genotyped. In multivariate analyses, haplotype A2 (122) and A4 (112) were associated with decreased risk of MI [OR = 0.62 (95% CI = 0.40-0.95), p = 0.01; OR = 0.69 (95% CI = 0.51-0.92), p = 0.03, respectively]. Haplotype-pair A2/A2 showed significant reduction in the risk of MI [OR = 0.38 (95% CI = 0.18-0.81); p = 0.01]. Haplotype A2 and A4 were associated with lower levels of IL-1B (respectively p = 0.01; p = 0.04, multivariate analysis) and haplotype-pair A2/A4 showed decreased levels of IL-1beta (p = 0.02). No association was found between block "B" IL-1B haplotypes or IL-1A and IL-RA polymorphisms and risk of MI. IL-1B haplotypes influence the inflammatory response of human mononuclear cells to LPS and affect the risk of MI at young age.

Interleukin-1beta (IL-1beta) activates inflammatory mediator cascades and has been implicated in the pathogenesis of several diseases. Single nucleotide polymorphisms (SNPs) of the IL1B promoter have been associated with various inflammatory diseases. We recently reported that IL1B gene transcription was influenced by four promoter SNPs, and that individual SNP function in vitro was governed by haplotype context. In the present study we tested the in vivo relevance of this observation by comparing IL1B promoter haplotype-pairs with IL-1beta protein levels in 900 gingival tissue fluid samples. Three SNPs (-511, -1464, -3737) defined four IL1B promoter haplotypes that occurred in the study population and could be assigned unambiguously to each chromosome. The four haplotypes defined ten haplotype-pairs of which four pairs, representing 57% of the population, were associated with 28-52% higher IL-1beta protein levels in vivo. Two of these pairs, characterized by homozygosity for the common allele at -3737, were also associated with raised serum levels of C-reactive protein (p = 0.02). We validated these findings in stimulated peripheral blood mononuclear cells (PBMCs) from a separate population (N = 70). PBMCs with IL1B haplotype-pairs associated with higher in vivo levels of IL-1beta produced 86-287% more IL-1beta in vitro than the reference group. We believe that this is the first demonstration of a relationship between in vivo levels of an inflammatory mediator and gene promoter haplotypes on both chromosomes. These findings may apply to other inducible genes and could provide a logical framework for exploring disease risk related to genetic variability in pathogenic mediators.

The border between health and disease is often set by a complex equilibrium between two elements, genetics on one hand, lifestyle on the other, To know it better, means to give new weapons, often crucial, in the hands of the doctors and their patients. It also means to adjust therapies, to find out which drug is good for a patient and which prevention strategy will work better for him/her. Nutrigenomics is an approach to individualize or personalize food and nutrition, and ultimately health, by tailoring the food to the individual genotype. In this review, we present the interaction between certain genetic polymorphisms and diet and increased cardiovascular or cancer risk. It is, indeed, now clear that a large number of bioactive food components may provide risk or protection at several stages of both atherosclerosis and cancer formation processes. We are giving here few examples of gene-food interactions relevant for both the risk of cardiovascular disease and cancer, since a common soil could exist in the genesis of cardiovascular disease and of some types of cancer (mainly gastrointestinal tract and hormone-dependent).

Introduction: Recent post-mortem studies of suicide victims have implicated brain-derived neurotrophic factor (BDNF) in suicide. Therefore, it was decided to examine the possible role of a gene in the regulation of BDNF activity in relation to suicidal behaviour among depressed patients., Method: A series of 170 depressed patients were evaluated for their history of suicide attempts and genotyped for the BDNF Val66Met polymorphism (SNP ID: rs6265). Depressed patients who had (n = 97) or had not (n = 73) attempted suicide were compared., Results: Depressed patients who carried the BDNF Val66Met polymorphism variant (GA + AA) appeared to show a significantly increased risk of suicidal behaviour. The risk of a suicide attempt was also significantly higher among those reporting higher levels of childhood emotional, physical and sexual abuse. Secondary analyses suggested that depression severity was a significant risk factor only in the wild-type BDNF genotype, and that the risk of suicide attempts was more predictable within the wild-type group., Conclusion: These preliminary data suggest that BDNF may play a role in the suicidal behaviour of depressed patients., (Copyright 2008 S. Karger AG, Basel.)

We describe identification and functional characterization of ISEc11, a new insertion sequence that is widespread in enteroinvasive E. coli (EIEC), in which it is always present on the virulence plasmid (pINV) and very frequently also present on the chromosome. ISEc11 is flanked by subterminal 13-bp inverted repeats (IRs) and is bounded by 3-bp terminal sequences, and it transposes with target specificity without generating duplication of the target site. ISEc11 is characterized by an atypical transposase containing the DEDD motif of the Piv/MooV family of DNA recombinases, and it is closely related to the IS1111 family. Transposition occurs by formation of minicircles through joining of the abutted ends and results in assembly of a junction promoter (P juncC) containing a -10 box in the interstitial sequence and a -35 box upstream of the right IR. A natural variant of ISEc11 (ISEc11p), found on EIEC pINV plasmids, contains a perfect duplication of the outermost 39 bp of the right end. Upon circularization, ISEc11p forms a junction promoter (P juncP) which, despite carrying -10 and -35 boxes identical to those of P juncC, exhibits 30-fold-greater strength in vivo. The discovery of only one starting point in primer extension experiments rules out the possibility that there are alternative promoter sites within the 39-bp duplication. Analysis of in vitro-generated transcripts confirmed that at limiting RNA polymerase concentrations, the activity of P juncP is 20-fold higher than the activity of P juncC. These observations suggest that the 39-bp duplication might host cis-acting elements that facilitate the binding of RNA polymerase to the promoter.

Shigella, the etiological agent of the bacillary dysentery, belongs to the extremely diverse species of Escherichia coli. In the evolutionary route of Shigella from commensal E. coli ancestors towards a pathogenic lifestyle, the critical events have been the acquisition of the pINV plasmid, through horizontal transfer and the inactivation of pre-existing genes. These so-called pathoadaptive mutations affect the expression of genes negatively interfering with the newly acquired functions necessary for the colonization of the host niche. Cadaverine, a small polyamine resulting from decarboxylation of lysine, has been shown to hamper the full expression of Shigella invasiveness mainly by altering the inflammatory response. Recent analysis of the evolution of the Shigella and enteroinvasive E. coli (EIEC) cad region indicates that silencing of the cad locus has been attained with several strategies. The increasing relevance of S. sonnei in both, developing and industrial countries, prompted us to analyze the molecular origin of the LDC- phenotype in these strains. The results obtained on several S. sonnei strains reveal that despite the difference in geographic origin and antibiotic resistance patterns, all the strains have undergone the same modifications. Multiple IS insertions into the cadBA operon have interrupted gene continuity without inducing deletions or inversions of the cadA and cadB genes which are remained entirely conserved. Moreover, by functional analysis we show that all the strains carry a defective cadC gene, thus strengthening the hypothesis that inactivation of the regulatory cadC gene might have been the first step towards a complete lack of the cad locus.

Enteroinvasive E. coli (EIEC), like Shigella, is the etiological agent of bacillary dysentery, a particularly severe syndrome in children in developing countries. All EIEC strains share with Shigella the inability to synthesize lysine decarboxylase (the LDC phenotype). The lack of this function is considered a pathoadaptive mutation whose emergence was necessary to obtain the full expression of invasiveness. Cadaverine, the product of lysine decarboxylation, is a small polyamine which interferes mainly with the inflammatory process induced by dysenteric bacteria. Genes coding for lysine decarboxylase and its transporter constitute a single operon (cadBA) and are expressed at low pH under the positive control of CadC. This regulator is an inner membrane protein that is able to sense pH variation and to respond by transcriptionally activating the cadBA genes. In this study we show that, unlike in Shigella, mutations affecting the cad locus in the EIEC strains we have analyzed are not followed by a novel gene arrangement and that the LCD(-) phenotype is dependent mainly on inactivation of the cadC gene. Introduction of a functional CadC restores cadaverine expression in all EIEC strains harboring either an IS2 element or a defective cadC promoter. Comparative analysis between the cad regions of S. flexneri and EIEC suggests that the LDC(-) phenotype has been attained by different strategies within the E. coli species.