a-simplified-technique-for-situ-excision-cornea-evisceration-retinal

A Simplified Technique for <em>In situ</em> Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Fondazione Banca Degli Occhi del Veneto O.N.L.U.S. 

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.

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

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein delta (C/EBPdelta), Bmi1, and DeltaNp63alpha identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPdelta and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPdelta inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27(Kip1) and p57(Kip2). These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBPdelta, but not DeltaNp63alpha, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBPdelta is recruited to the chromatin of positively (p27(Kip1) and p57(Kip2)) and negatively (p16(INK4A) and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.

C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells.

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Article	Year
Biochemical and biophysical research communications\| PubMed ID: 12176036	2002
European journal of human genetics : EJHG\| PubMed ID: 12634859	2003
Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15983386	2005
Microscopy research and technique\| PubMed ID: 16972233	2006
Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nature medicine\| PubMed ID: 17115047	2006
Investigative ophthalmology & visual science\| PubMed ID: 17122109	2006
C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells. The Journal of cell biology\| PubMed ID: 17562792	2007
Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally targeted lentiviral vectors. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 18813277	2008
The British journal of ophthalmology\| PubMed ID: 19740872	2010
Molecular vision\| PubMed ID: 19862337	2009
The British journal of ophthalmology\| PubMed ID: 20494912	2010
Experimental eye research\| PubMed ID: 20537995	2010
The ocular surface\| PubMed ID: 20712971	2010
Clinical & experimental ophthalmology\| PubMed ID: 21668791	2012
Ophthalmology\| PubMed ID: 21959367	2012
The Journal of molecular diagnostics : JMD\| PubMed ID: 22056627	2012
Current genomics\| PubMed ID: 22131869	2011

Enzo Di Iorio

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