9^th International Conference and Exhibition on Advanced Cell and Gene Therapy March 21-22, 2019 Hotel: Holiday Inn Rome Aurelia, Rome, Italy

Enio Aparecido Zacarias obtained his Biology degree from the Paulista University (2015) and is now a Post-graduate student (MS) at the Biotechnology Center of the Nuclear and Energy Research Institute, located in the University of São Paulo Campus

Our group has been working with gene therapy models for growth hormone deficiency. We are using an in vivo approach in which expression vectors containing the growth hormone (GH) gene are administered in mice, followed by electrotransference. In previous studies, elevated levels of human GH (hGH) in mice serum (~20 ng/mL) and high growth approximation to normal mice (catch-up growth) of ~70% for body weight and of ~80% for femur length were obtained, using a plasmid containing the genomic sequence (gDNA) of GH with the ubiquitin-C promoter. On the other hand, we had an indication that the complementary sequence (cDNA) may have an advantage over gDNA in gene therapy protocols. Our objective is to carry out a comparative study between vectors containing the hGH gDNA or cDNA sequences. First, the two vectors were analyzed for in vitro expression levels by transfecting HEK-293 cells. Expression levels reached 250+50 ng hGH/mL for gDNA and 20+9.4 ng hGH/mL for cDNA transfected cells. Although in vitro expression of cDNA-containing vector was lower than that containing gDNA, we believe the cDNA vector may have better expression in vivo, due to a possible better incorporation by the muscle cells in electrotransference. Then, bioassays will be performed administering these vectors into dwarf mice, via electrotransference in the muscle. This will verify the expression profile of GH in vivo, concerning levels and durability, as well as body weight, total body, tail and femur length, mouse insulin-like growth factor-1 levels and catch-up growth.

Alissandra de Moura Gomes is a Biologist and MSc student working at Biotechnology Center of the Nuclear and Energy Research Institute, located in the University of São Paulo Campus under the guidance of Prof. Dr. Cibele Nunes Peroni, and with a scholarship by the National Council for Scientific and Technological Development.

Osteogenesis imperfecta (OI) is an inherited connective tissue disease characterized by fragility, deformity and low bone density, as well as by other clinical manifestations. Type I OI is the mildest and most common form of the disease, caused by mutation in the COL1A1 gene, resulting in the production of only ~50% of normal collagen. The corresponding animal model is the oim mouse, presenting a phenotype very similar to human type I OI. We aim, for the first time, at evaluating the electrotransference of mouse growth hormone (mGH) gene, encoding a protein that already showed therapeutic effects, together with the administration of murine mesenchymal stem cells (MSCs), for improving heterozygous oim mice phenotype. We already prepared and evaluated two populations of MSCs (bone marrow and adipose tissue) that emit red fluorescence. These are administrated in different amounts and via three routes (intravenously, intraperitoneally or locally into the femoral condyles) using the in vivo imaging system and histological sections of femur, liver and kidneys, a methodology we recently set up and adapted to our specific conditions. Then, we will administer the most efficient MSCs population combined with mGH gene electrotransfer. In this bioassay, we are ready to analyse different parameters: body weight, total body, tail and femur length, bone mineral density and femur fragility by a biomechanical flexion test. The results will indicate if the administration of GH by gene therapy, together with MSCs infusion, can be a promising treatment for improving type I OI phenotype.

Osteogenesis imperfecta (OI) is a congenital dysplasia of connective tissue characterized mainly by fragility and low bone density. The type I OI, a mild form of the disease, is associated with the quantitative decrease of type I collagen in the extracellular matrix, a characteristic also observed in the oim mice used in pre-clinical OI research. Previous studies have shown the efficiency of recombinant human growth hormone (GH) treatment for OI, both in animal and human models, reducing bone fragility, increasing bone density and stimulating α 1 and α 2 procollagen synthesis. This work aimed at using plasmid containing the murine GH (mGH) gene to treat oim heterozygous mice. Serological quantification of mGH by ELISA was carried out to evaluate the plasmid expression, bone density by DEXA and three-point flexion test were performed on the femurs to assess bone quality. In a short-term (3-day) trial, mGH levels of treated animals were 20.6±6.6 ng/mL, versus 3.3±2.2 ng/mL for those receiving saline and 2.3±1.3 ng/mL for the untreated wild-type group. A 90-day assay with plasmid applications at 0, 30 and 60 days was performed in parallel. The results of bone density showed greater effectiveness of the hormone during the first month of treatment, with a 34% increase in comparison with the saline group, whereas in the second month there was no significant statistical difference (p>0.05). The three-point bending test and the final analysis of bone density are currently being carried out.

Gustavo Protasio Pacheco de Jesus holds a Bachelor's degree in Biomedicine from Lusíada University Center - Santos (2010) and a Master's degree in Pathology from the Federal University of São Paulo (2013). He has published 7 articles and a book chapter and is currently a PhD student at the Biotechnology Center of the Nuclear and Energy Research Institute, located in the University of São Paulo campus and Professor of Pathology, Immunology, Microbiology and Environmental Analysis at Lusíada University Center - Santos

Meshari Alhadlaq has received his BS and MS in Molecular Biology from Qassim and Bangor Universities in Saudi Arabia and United Kingdom in 2007 and 2013 respectively. Then he joined the Molecular Biology and Biotechnology Department at the University of Sheffield as a PhD candidate in 2015, to study the structure and biochemistry of protein. Since then his studies focus on the characterisation and role of WhiB proteins of Kytococcus sedentarius.

Kytococcus sedentarius (Ks) is an opportunistic bacterium involved in pitted keratolysis, cerebral cyst infections, endocarditis and bacteraemia. WhiB-like (Wbl) proteins are a family of proteins that are only located in actinomycetes and play important role in developmental processes. The C-terminal regions are rich in positively charged amino acids, suggesting a role in DNA-binding. The N-terminal regions possess four conserved cysteine residues that act as anchors for iron-sulfur clusters, which respond to redox stress. This study shows that: (i) the cluster can be isolated in three forms, (ii) the cluster is important to structure the protein and, (iii) the cluster is sensitive to spermine nitric oxide (NO) but not oxygen (O2). The significance is the iron-sulfur cluster of WhiB1 is a key factor in the protein function. The cluster modulates the conformation of the protein, changes the DNA-binding properties and allows the protein to respond to NO but not O2. These facts suggest that WhiB1 has a role as an NO-responsive gene regulator that could be important for survival and persistence in human macrophages.