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Michele De Luca

Publications and source records attributed to Michele De Luca.

17 recordsLinked to original sources

Gene therapy for genodermatoses at the crossroads of innovation and clinical translation.

Inherited genodermatoses are a heterogeneous group of rare monogenic disorders. Among these, epidermolysis bullosa (EB) and ichthyoses represent paradigmatic disorders characterized by severe skin fragility and hyperkeratosis, respectively, and impaired barrier function, often with profound effects on quality of life and systemic health. Current management remains largely palliative, underscoring the urgent need for disease-modifying therapies. Over the past 2 decades, advances in epithelial stem cell biology, vector engineering and genome editing technologies have transformed the therapeutic landscape for genodermatoses. Ex vivo gene therapy has provided the first proof that genetically corrected epidermal stem cells can achieve long-term tissue regeneration in EB skin patients, establishing a new paradigm for regenerative medicine. In parallel, the emergence of programmable genome engineering platforms, including CRISPR/Cas nucleases, base editors and prime editors, have enabled increasingly precise strategies for mutation-specific correction in both recessive and dominant disorders. Furthermore, the development of in vivo topical approaches is expanding the possibility of directly targeting the skin. Despite these advances, substantial translational barriers continue to limit broad clinical implementation. Efficient and durable targeting of epidermal stem cells within a highly regenerative tissue, together with safe delivery across the skin barrier, stringent control of off-target activity, scalable manufacturing and demonstration of long-term safety, remain major challenges for the clinical translation of these approaches. In this Review, we discuss the current state of gene therapy for genodermatoses, highlighting key clinical milestones, emerging genome editing technologies and next-generation delivery systems. We further examine the biological and regulatory challenges that need to be overcome to bridge the gap between experimental innovation and clinically accessible therapies for patients with inherited skin diseases.

epidermolysis bullosa (EB)↗

Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

3T3 Cells↗

A critical study on the application of the zero-crossing derivative spectrophotometry to the photodegradation monitoring of lacidipine.

An extensive study on the application of the "zero-crossing" technique for the analysis of a binary mixture of the photosensitive drug lacidipine and its corresponding by-product by derivative spectrophotometry is described. The technique has been compared to either conventional and recently developed UV spectrophotometric procedures, including chemometric methods. The prediction ability of the different analytical techniques has been checked by using the first-order derivative spectra of drug and photoproduct in binary mixtures. Relative advantages and drawbacks have been discussed. The zero-crossing technique suffers from several limitations, mostly ascribed to the selection of suitable signals along slopes of the spectral curve, giving rise to low accurate and precise results. The mean recovery from the zero-crossing analysis was calculated to lie in the 95.1-98.4% range for lacidipine, and 91.2-118.9% for the photoproduct. Chemometric methods showed a greater prediction ability with a 101.4-103.0% and 96.3-98.4% recovery for drug and degradation product, respectively.

Dihydropyridines↗

Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.

An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.

Albinism, Ocular↗

Q-FIHC: quantification of fluorescence immunohistochemistry to analyse p63 isoforms and cell cycle phases in human limbal stem cells.

Fluorescence microscopy has long been used for qualitative characterization of various parameters such as subcellular distribution of proteins, lipids, nucleic acids, and ions. However, quantification of these parameters is complicated by a variety of optical, biological, and physical factors. In the last decade, the progress achieved with powerful softwares and digital image processing systems has facilitated the development of fluorescence immunohistochemistry (FIHC) into a widely used quantitative assay (quantitative-FIHC or Q-FIHC). We describe here a rapid and sensitive Q-FIHC assay based on the use of a laser scanning confocal microscope and advanced image analysis softwares (Zeiss semi automatic LSM 510 and fully automatic Axiovision 4.4) for the detection and quantification of fluorescent intensity in human corneal tissues and cells obtained from small clinical samples. We have used this methodology to characterize and quantify the gene expression profile of p63 and its DeltaNalpha isoform, specific markers of human limbal stem cells. The validity of this method was evaluated through comparative studies with conventional approaches suggesting no significant differences and providing an alternative technique to traditional methods. Since Q-FIHC requires at least 20-fold less cells than traditional techniques, we have adopted it as the main quality control for our limbal cultures destined to clinical application.

Cell Cycle↗

Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing.

PURPOSE: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS: The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. RESULTS: Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. CONCLUSIONS: The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.

Actins↗

Photo- and thermal-stability studies on benzimidazole anthelmintics by HPLC and GC-MS.

Photo- and thermal-stability of the anthelmintics Albendazole, Mebendazole and Fenbendazole as in solid as in solution form has been investigated, by using a Xenon arc lamp as a radiation source, according to the ICH guideline for the drug stability tests. The degradation process was monitored by a HPLC method. All drugs showed high photosensitivity in solution but a reliable stability in solid form and when exposed to a temperature up to 50 degrees C. Two main degradation products from hydrolysis of the carbamic groups were identified by GC-MS. Validation studies demonstrated high accuracy (recovery 94 to 106%) and precision (RSD under 4.6%) of the HPLC method. The analytical procedure was successfully applied to the control of the drugs in the respective pharmaceutical formulations.

Anthelmintics↗

Gene therapy in combination with tissue engineering to treat epidermolysis bullosa.

In the last 20 years epidermal stem cells have been extensively used for tissue regeneration of epidermis and other epithelial surfaces. The tremendous progress achieved has led to the development of protocols aimed at the correction of rare genetic disorders such as epidermolysis bullosa (EB), a severe, often lethal, blistering disorder of the skin. Approximately 400,000-500,000 people are affected worldwide and no definitive treatments have yet been developed. Gene therapy might represent an alternative therapeutic approach. This paper reviews the different strategies used to genetically modify keratinocytes from EB patients and addresses issues such as the use of in vivo or ex vivo approaches, how to target keratinocytes with stem cell properties in order to have long-term therapeutic gene expression, and which gene transfer agents should be used. The progress made has led the authors' group to submit a request for a Phase I/II ex vivo therapy clinical trial for patients with junctional EB.

Animals↗

Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection.

In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase II or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-beta-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6sbeta(1-->3)gal, accounting for approximately equals 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase II) from Bacillus sp. generates two major products, the monosulfated disaccharide galbeta(1-->4)glcNAc6s ( approximately equals 50% nmol product) and the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s ( approximately equals 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximately equals 108 pmol, 50 ng, to 2,160 pmol, 1,000 ng, for the disaccharide galbeta(1-->4)glcNAc6s, and from 92 pmol, 50 ng, to 1,840 pmol, 1,000 ng, for the disaccharide gal6sbeta(1-->4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80+/-0.34 microg/ml/10(6) cells and composed of approximately equals 71% nmol of disaccharide galbeta(1-->4)glcNAc6s and 18% nmol of the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s having approximately equals 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( approximately 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.

Acetylglucosaminidase↗

Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration.

The p63 gene generates transactivating and N-terminally truncated transcripts (DeltaNp63) initiated by different promoters. Alternative splicing gives rise to three different C termini, designated alpha, beta, and gamma. In the ocular epithelium, the corneal stem cells, which are segregated in the basal layer of the limbus, contain the alpha isoform but not beta or gamma. Holoclones derived from the limbus are rich in alpha, meroclones contain little, and paraclones contain none. In normal resting corneal epithelium, p63 of all isoforms is absent. Upon corneal wounding, cells originating from the limbus and containing alpha migrate progressively through the epithelium of the peripheral and central cornea. In the absence of an attached limbus, no alpha isoform appears in the corneal epithelium. When migrating cells containing the alpha isoform appear in the wounded corneal epithelium, they are confined to the basal layer, but the suprabasal cells, not only of the cornea but of the limbus as well, contain mRNA encoding beta and gamma. These data support the concept that the alpha isoform of p63 is necessary for the maintenance of the proliferative potential of limbal stem cells and their ability to migrate over the cornea. The beta and gamma isoforms, being suprabasal and virtually absent from the resting limbus, are not stem cell markers but are likely to play a role in epithelial differentiation specifically during the process of corneal regeneration.

Blotting, Western↗

Stem cell plasticity: time for a reappraisal?

In recent years an increasing number of publications have claimed that adult mammalian stem cells (SC) may be capable of differentiating across tissue lineage boundaries and that this plasticity may represent a novel therapeutic strategy for tissue regeneration. However, after a first phase of excitement, the issue of somatic SC plasticity remains controversial and the therapeutic perspectives are still elusive. In this review, we examine the general mechanisms which govern the function of SC, the identification and functional characterization of adult SC of different tissues and their putative capacity to transdifferentiate into mature cells of different origin. The potential clinical applications of adult SC for regenerative medicine are also discussed in each chapter. The method employed for preparing this review was the informal consensus development. Members of the Working Group on SC met four times and discussed the single points, previously assigned by the Chairman (S.T.), in order to achieve an agreement on different opinions and approve the final manuscript. All the authors of the present review have been working in the field of SC and have contributed original papers to peer-reviewed journals. In addition to the authors' own work, the present review examines articles published in journals covered by the Science Citation Index and Medline.

Adult↗

Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes.

The human ocular surface is covered by the conjunctival, corneal and limbal stratified epithelia. While conjunctival stem cells are distributed in bulbar and forniceal conjunctiva, corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone between the cornea and the bulbar conjunctiva. Keratinocyte stem and transient amplifying (TA) cells when isolated in culture give rise to holoclones and paraclones, respectively. Keratinocyte replicative senescence ensues when all holoclones have generated paraclones which express high levels of p16(INK4a). In the present study, we show that enforced telomerase activity induces the bypass of replicative senescence in limbal and conjunctival keratinocytes, without the inactivation of the p16(INK4a)/Rb pathway or the abrogation of p53 expression. hTERT-transduced limbal and conjunctival keratinocytes are capable to respond to both growth inhibitory and differentiation stimuli, since they undergo growth arrest in response to phorbol esters, and activate p53 upon DNA damage. Following a sustained PKC stimulation, occasional clones of p16(INK4a)-negative cells emerge and resume ability to proliferate. Telomerase activity, however, is unable to induce the bypass of senescence in corneal TA keratinocytes cultured under the same conditions. These data support the notion that telomere-dependent replicative senescence is a general property of all human somatic cells, including keratinocytes, and suggest that telomerase activity is sufficient to extend the lifespan only of keratinocytes endowed with high proliferative potentials (which include stem cells), but not of TA keratinocytes.

Cell Proliferation↗

sAPP as a regulator of dendrite motility and melanin release in epidermal melanocytes and melanoma cells.

Numerous factors including ultraviolet (UV) radiation and growth factors regulate the specific function of epidermal melanocytes. A recently discovered epidermal growth factor is sAPP, the soluble N-terminal ectodomain of the beta-amyloid precursor protein (APP). Using whole mount preparations of isolated human epidermis, we detected a small population of basal cells, which expressed exceptionally high levels of APP. These cells were identified as melanocytes, which, similar to keratinocytes and neuronal cells, expressed the three APP isoforms 695, 751, and 770. They differed in their expression pattern from that of neuronal cells by expressing only low levels of APP 695. Melanocytes and melanoma cells in vitro released, in addition to keratinocytes, large quantities of sAPP. Because of its growth factor function, we studied possible effects of sAPP on melanocytes. Recombinant sAPP strongly increased lamellipodia activity at dendritic tips, an effect that coincided with increased release of melanin particles. Our observations point to the possible use of APP as an immunocytochemical marker for melanocytes. They suggest that sAPP derived from keratinocytes and/or melanocytes belongs to a family of factors operating in the paracrine and/or autocrine regulation of melanocyte function.

Amyloid beta-Protein Precursor↗

Erbium:YAG laser and cultured epidermis in the surgical therapy of stable vitiligo.

OBJECTIVE: To induce complete and reproducible repigmentation of large "stable" vitiligo lesions by means of autologous cultured epidermal grafts using a rapid, simple, and minimally invasive surgical procedure. DESIGN: Achromic epidermis was removed by means of appropriately settled erbium:YAG laser, and autologous epidermal grafts were applied onto the recipient bed. Melanocyte content was evaluated by dopa reaction. The percentage of repigmentation was calculated using a semiautomatic image analysis system. SETTING: A biosafety level 3-type cell culture facility, a surgical ambulatory department, and a dermatological department in a hospital. PATIENTS: Twenty-one patients with different types of vitiligo were admitted to the study and treated with autologous cultured epidermal grafts. Inclusion criteria were failure of at least 2 standard medical approaches; no therapy for at least 12 months; no progression of old lesions or appearance of new lesions; no Koebner phenomenon within the past 18 months; and no autoimmune disorders. RESULTS: The average percentage of repigmentation in 21 patients was 75.9% (1759.7 cm2 repigmented/2315.8 cm2 transplanted). Three patients showed a reactivation of their vitiligo and did not show repigmentation. The remaining 18 patients, with 43 distinct lesions, showed an average percentage of repigmentation of 90% (1759.7 cm2 repigmented/1953.4 cm2 transplanted). CONCLUSIONS: Under appropriate conditions, cultured epidermal grafts induce complete repigmentation of stable vitiligo lesions. Erbium:YAG laser surgery can supply a fast and precise tool for disepithelialization, hence allowing treatment of large vitiligo lesions during a single surgical operation.

Adolescent↗

Effective retrovirus-mediated gene transfer in normal and mutant human melanocytes.

Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.

Biomarkers↗

A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status.

With increasing frequency during serial passage in culture, primary human keratinocytes express p16(INK4A) (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4(R24C)), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4(R24C) or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16(+/-) individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4(R24C) and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.

Cell Division↗

Gene therapy approaches for epidermolysis bullosa.

Human epidermis consists of a stratified epithelium mainly composed of keratinocytes and relies on a stem cell compartment to undergo constant regeneration. Genetic mutations affecting the capacity of basal keratinocytes to adhere firmly to the epidermal basement membrane lead to severe, and very often lethal, blistering disorders known as epidermolysis bullosa. Gene therapy represents a promising potential treatment for these devastating inherited disorders. Human epidermal stem cells can be cultivated ex vivo and stably transduced with integrating gene transfer vectors, allowing genetic and, more important, phenotypic correction of the adhesion properties of keratinocytes. Here we will review some of the issues that need to be addressed to make gene therapy a realistic treatment for these disorders, such as (1) which cells should be targeted, (2) which approach (in vivo or ex vivo) should be chosen, and (3) which gene transfer vector (retrovirus, lentivirus, or integrating nonviral strategies) should be used for stable gene correction. In the last 10 years, many reports have shown that gene transfer approaches to target epidermal stem cells are feasible and able to restore the adhesion properties of primary keratinocytes from patients with epidermolysis bullosa. In addition, tremendous progress has been achieved in culturing epidermal stem cells and generating sheets of stratified epithelium for permanent coverage of full-thickness burns. Gene modification of stem cells in combination with advanced tissue-engineering techniques could therefore represent a realistic option for patients with epidermolysis bullosa.

Epidermolysis Bullosa↗