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.

During the last two decades, there has been broad interest in RNA-based technologies for the development of prophylactic and therapeutic vaccines. Preclinical and clinical trials have shown that mRNA vaccines provide a safe and long-lasting immune response in animal models and humans. In this review, we summarize current research progress on mRNA vaccines, which have the potential to be quick-manufactured and to become powerful tools against infectious disease and we highlight the bright future of their design and applications.

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IntroductionPioneering clinical studies have shown that transplantation of genetically modified hematopoietic stem cells may cure severe genetic diseases such as severe combined immunodeficiencies (SCID), 1,2 chronic granulomatous disease (CGD), 3 and lysosomal storage disorders. 4 Unfortunately, some of these studies showed also the limitations of retroviral gene transfer technology, which may cause severe and sometimes fatal adverse effects. In particular, insertional activation of proto-oncogenes by vectors derived from the Moloney murine leukemia virus (MLV) caused T-cell lymphoproliferative disorders in patients with X-linked SCID 5,6 and premalignant expansion of myeloid progenitors in patients with CGD. 3 Preclinical studies showed that HIV-derived lentiviral vectors are less likely to cause insertional gene activation, 7,8 although they can still interfere with normal gene expression at the posttranscriptional level, as observed in a clinical trial of gene therapy for ␤-thalassemia. 9 The molecular bases of vector-induced genotoxicity and the influence of vector design, transduction protocols, and the patient's genetic background in inducing severe adverse effects are still poorly understood. A better understanding of the interactions between retroviral vectors and the human genome may provide new cues to explain these phenomena and a rational basis for predicting genotoxic risks in gene therapy.A large number of studies have focused on the molecular mechanisms by which mammalian retroviruses choose their integration sites in the target cell genome. After entering a cell, retroviral RNA genomes are reverse transcribed into double-stranded DNA and assembled in preintegration complexes (PICs) containing viral and cellular proteins. PICs translocate to the nucleus and associate with the host cell chromatin, where the viral integrase mediates proviral insertion in genomic DNA. Integration is a nonrandom process, whereby PICs of different viruses recognize components or features of the host cell chromatin in a specific fashion. 10 For HIV and other lentiviruses, the LEDGF/p75 protein has been identified as the main factor tethering PICs to active chromatin, 11 whereas mechanisms underlying integration site selection of other retroviruses remain largely unknown. We recently showed that MLV-derived vectors integrate preferentially in hot spots near genes controlling growth and development of hematopoietic cells and flanked by defined subsets of transcription factor binding sites (TFBSs) and suggested that MLV PICs are tethered to active regulatory regions by basal components of the transcriptional machinery. 12,13 The MLV integrase and long terminal repeat enhancer are the main determinants of the selection of TFBS-rich regions of the genome. 13,14 We used ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing to build a genomewide, high-definition map of Ͼ 32 000 MLV integration sites in the genome of human CD34 ϩ hematopoietic progenitor cells (HPCs) and used gene expression profiling, chroma...

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/ deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å 2 on fHbp, including >20 fHbp residues distributed on both N-and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.meningococcus | structure | surface plasmon resonance | structural mass spectrometry | antigen-antibody complex