Mutation-independent treatment of autosomal dominant Retinitis Pigmentosa (adRP)

Viral-mediated gene therapy holds great promise for the treatment of severe inherited retinal diseases, such as Retintitis Pigmentosa (RP), which is caused by mutations in genes preferentially expressed in photoreceptor cells. The availability of vectors derived from the small adeno-associated virus (AAV) which efficiently and stably transduce the retina of animal models after intraocular administration strongly support the possibility to develop novel strategies for the treatment of such severe retinal degenerations otherwise incurable thus far. The main goals of my PhD project were: - generate artificial transcription repressors (ZFPs) targeted to the human rhodopsin promoter to silence at the transcriptional level the rhodopsin gene; - assess the efficacy of the treatment and the impact on the disease progression in the RP mouse model. Retinitis pigmentosa is by far the most studied inherited retinal disease. It is clinically and genetically heterogeneous recognizing autosomal recessive (arRP), autosomal dominant (adRP), X-linked, and digenic patterns of inheritance. More than 30 diseases genes have been identified so far and 12 of these have been associated with (adRP), representing between 15% and 35% of all cases. Despite recent success of the gene-based complementation approach for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc finger protein (ZFP)-based-repression of transcription may represent a novel and alternative mutation independent therapeutic approach for treating gain-of-function mutations, but proof-of-concept of this use is still lacking. In my PhD project we used a novel strategy to treat adRP based on zinc-finger-based artificial transcription factors (ZF-ATFs). These molecules can be engineered to silence genes carrying gain-of-function mutations that cause toxic effects into the cell where they are expressed. We generated ten artificial transcriptional repressors targeted to the human Rhodopsin which is the gene most commonly associated with adRP (20–30% of cases) with more than 150 mutations identified throughout its sequence, representing the most commonly mutated gene in RP. We characterized in vitro the ability of artificial transcriptional repressors to bind specifically the human rhodopsin promoter in order to exert a specific transcriptional control and we selected two out of ten functional zinc-finger-based repressors of rhodopsin. One of this was selected as the most efficient and was enclosed in an AAV2/8 for in vivo experiments. We demonstrated that the selected artificial zinc-finger-based repressors (ZFRs) resulted in a robust transcriptional repression of hRHO impacting disease progression in a mouse model of adRP over-expressing the P347S mutation. The data obtained support the use of ZFP-mediated silencing as a potentially relevant therapeutic strategy to treat gain of function mutations

Management of a complex dentoalveolar trauma: a case report

This paper describes the case of a 12-year-old male patient who presented a severe lateral luxation of the maxillary central incisors due to a bicycle fall. Treatment involved suture of the soft tissues lacerations, and repositioning and splinting of the injured teeth, followed by endodontic treatment and periodontal surgery. After a 2-year follow-up, clinical and radiographic evaluation revealed that the incisors presented satisfactory esthetic and functional demands.Este artigo apresenta o caso de um paciente de 12 anos de idade que apresentou uma luxação lateral severa dos incisivos centrais superiores decorrente de uma queda de bicicleta. O tratamento envolveu a sutura dos tecidos moles dilacerados e reposicionamento e fixação dos dentes traumatizados, seguidos por tratamento endodôntico e cirurgia periodontal. Após um acompanhamento de 2 anos, a avaliação clinica e radiográfica revelou que os incisivos apresentavam exigências estéticas e funcionais satisfatórias

125. Engineered Nucleases-Mediated In Situ Correction of a Genetic Defect By Homologous Recombination Into the Native Locus

Engineered nucleases specific for genomic targets are extensively used to generate DSBs that increase the rate and efficiency of homologous recombination (HR). We seek to determine the efficacy of nucleases in a clinical relevant genetic defect.The genetic defect we are addressing as model to test the nucleases-mediated genome editing technology is the junctional epidermolysis bullosa (JEB), a family of severe skin adhesion disorders due to autosomal recessive mutations in the LAMB3 gene coding for the laminin-332 heterotrimer, a key component of the dermal-epidermaljunction. Recently, we provided proof of principle that ZFN-mediated, AAVS1-targeted GFP addition can be achieved in human keratinocytes and in long-term repopulating epithelial stem cells in a validated preclinical model of xenotransplantation of human skin equivalents on immunodeficient mice.This project aims at the demonstration of a successful in situ correction of the LAMB3 gene in primary keratinocytes from Herlitz JEB patients. Recently TALEN-based gene correction for dystrophic EB has been reported. Similarly, we have developed a genome editing approach for JEB. In particular we have designed TALENs specific for the second intron of LAMB3 gene and a HR cassette including a splicible LAMB3 cDNA (from exon 3 to the end of the gene). In particular immortalized JEB keratinocytes were transfected with TALEN mRNAs and infected with an IDLV vector carrying the HR cassette. The in situ gene correction has been evaluated by site-specific PCR and knock-in expression of the corrected LAMB3 gene on bulk population. We then assessed targeting efficiency and specificity by extensive molecular analyses of single-cell clones isolated by limiting dilution from the TALENs/IDLV-treated immortalized JEB population. We isolated 256 clones and expanded 69 of them. Sixteen out of 69 clones showed an in vitro adhesion advantage, hosted the HR cassette correctly integrated into the predetermined locus, expressed the corrected LAMB3 gene and produced the laminin-332 protein. In parallel, CRISPR-Cas9 nuclease has been designed on the same locus to compare the transduction efficiency and cleavage activity and to translate the knock-in targeting platform to primary JEB keratinocytes

Vertical alveolar crest bone maintenance around implants in two-stage surgery: an in situ study in dogs

The aim of this study was to evaluate in situ changes in the alveolar crest bone height around immediate implant-supported crowns in comparison to tooth-supported crowns (control) with the cervical margins located at the bone crest level, without occlusal load. In Group I, after extraction of 12 mandibular premolars from 4 adult dogs, implants from Branemark System (MK III TiU RP 4.0 x 11.5 mm) were placed to retain complete acrylic crowns. In Group II, premolars were prepared to receive complete metal crowns. Sixteen weeks after placement of the crowns (38 weeks after tooth extraction), the height of the alveolar bone crest was measured with a digital caliper. Data were analyzed statistically by the Mann-Whitney test at 5% significance level. The in situ analysis showed no statistically significant difference (p=0.880) between the implant-supported and the tooth-supported groups (1.528 + 0.459 mm and 1.570 + 0.263 mm, respectively). Based on the findings of the present study, it may be concluded that initial peri-implant bone loss may result from the remodeling process necessary to establish the biological space, similar to which occurs with tooth-supported crowns.O objetivo do presente estudo foi comparar in situ modificações ocorridas na crista óssea alveolar induzidas por implantes imediatos e às induzidas por dentes naturais, ambos preparados para suportarem dispositivos protéticos situados sobre a margem óssea cervical, sem carga oclusal. No grupo I, após a extração de 12 pré-molares inferiores de 4 cães adultos, foram instalados implantes do Sistema Branemark (MK III TiU RP 4,0 x 11,5 mm) e coroas totais acrílicas. No grupo II, os pré-molares, foram submetidos a preparos convencionais, sendo cimentadas coroas totais metálicas. Após 16 semanas da instalação das coroas (38 semanas após a extração), a reabsorção da crista óssea alveolar foi avaliada in situ, por meio da mensuração das peças, com paquímetro digital. Para obtenção das medidas do grupo I, os pontos de referência foram a plataforma de assentamento do implante e a crista óssea alveolar. No grupo II, a posição do limite cervical da prótese e o início da crista óssea. Os valores obtidos foram comparados pelo teste de Mann-Whitmey com nível de significância de 5%. A média de reabsorção da crista óssea foi de 1,528 + 0,459 mm para o grupo I e de 1,570 + 0,263 mm para o grupo II, não havendo diferença estatisticamente significante (p=0,880) entre os grupos. Os resultados do presente estudo permitiram concluir que a reabsorção perimplantar inicial que ocorre na crista óssea resulta de um padrão de remodelação necessário para o estabelecimento do espaço biológico na área, assim como ocorre nos dentes naturais preparados para receberem próteses convencionais

Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells

The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platfor