Stefano Ferrari

Università degli studi di Parma, Parma, Emilia-Romagna, Italy

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Publications (6)12.42 Total impact

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    ABSTRACT: Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.Gene Therapy advance online publication, 16 January 2014; doi:10.1038/gt.2013.82.
    Gene therapy 01/2014; · 4.75 Impact Factor
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    ABSTRACT: Purpose: To assess the parameters for postmortem retinal tissue recovery and processing that affect the quality of RNA extracted from the retina/retinal pigment epithelium (RPE) complex. Methods: RNA was extracted from retina/RPE samples. The RNA quality was determined based on qualitative/quantitative measurements made with a Bioanalyzer (Agilent) and on the expression of a long retinal gene (RPE65). After a pilot analysis on rats, ocular RNA was extracted from human donor eyeballs (group A) explanted according to conventional procedures for cornea transplantation. In a second experiment, another group of human donor eyeballs (group B) were processed in a much shorter time. The postmortem interval (T) comprised two periods: T1, the time between a donor's death and enucleation, and T2, the time between eyeball explantation and immersion of the excised retina/RPE sample in preservative solution (T = T1 + T2). Results: A short T2 was correlated with good quality of RNA extracted from the retina/RPE complex (p = 0.043) and successful expression of a tissue-specific gene (p = 0.007). No other parameter appeared to influence RNA quality. Conclusions: The time between eyeball explantation and immersion of the retina/RPE sample in preservative solution was the chief parameter affecting the quality of RNA extracted from the retina/RPE complex.
    Ophthalmic Genetics 09/2012; · 1.07 Impact Factor
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    ABSTRACT: Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare autosomal dominant ectodermal dysplasia syndrome. It is caused by heterozygous mutations in TP63, encoding a transcriptional factor of the p53 family. Mutations in TP63, mainly missense in exons 13 and 14 encoding the sterile alpha motif (SAM) and the transactivation inhibitory (TI) domains, account for 99% of mutations in individuals with AEC syndrome. Of these, ≥70% are de novo mutations, present in the affected patient, but not in parents nor in healthy siblings. However, when a mutation appears de novo, it is not possible to differentiate between a sporadic mutation, or germline mosaicism in the parents. In this latter case, there is a risk of having additional affected offspring. We describe two sisters with AEC syndrome, whose parents were unaffected. Both patients carried the heterozygous c.1568T>C substitution in exon 13 of TP63, resulting in a p.L523P change in the SAM domain of the protein. Analyses of DNA from parental blood cells, seminal fluid (from the father) and maternal cells (buccal, vaginal, and cervical) did not reveal the mutation, suggesting that the mosaicism may involve a very low percentage of cells (very low grade somatic mosaicism) or, more likely, maternal gonadal mosaicism. Mosaicism must be considered for the assessment of recurrence risk during genetic counseling in AEC syndrome, and pre-implantation/prenatal genetic diagnosis should be offered to all couples, even when the mutation is apparently de novo.
    American Journal of Medical Genetics Part A 06/2012; · 2.30 Impact Factor
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    ABSTRACT: Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1) can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis.
    Journal of Ophthalmology 01/2012; 2012:594869. · 1.37 Impact Factor
  • Paolo Mora, Luisa Montanini, Stefano Ferrari
    Retina (Philadelphia, Pa.) 10/2010; 30(9):1555; author reply 1556. · 2.93 Impact Factor

Publication Stats

4 Citations
12.42 Total Impact Points

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Institutions

  • 2010
    • Università degli studi di Parma
      Parma, Emilia-Romagna, Italy