Análise mutacional do gene COL7A1 nos pacientes brasileiros e terapia gênica para epidermólise bolhosa distrófica recessiva com macrófagos modificados com COL7A1 em modelo murino

• Ano de defesa: 2016
• Autor(a) principal: Martin, Priscila Keiko Matsumoto [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Recessive dystrophic epidermolysis bullosa; Macrophages; Collagen vii; Gene sequencing; Gene therapy; Epidermólise bolhosa distrófica recessiva; Macrófagos; Colágeno vii; Sequenciamento gênico; Terapia gênica
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3487251; http://repositorio.unifesp.br/handle/11600/47686

Recessive Dystrophic Epidermolysis BulIosa (RDEB) is a very aggressrve monogenic disease whose lack of colIagen VII cause blisters and lesions in all epithelia. There is no effective treatment to date and diagnosis of the disease is still only clinic in Brazil. Due to the large variability sets of mutations and various types of epidermolysis described, sequencing the COL7AI gene would be essential to complete the diagnosis. However, the sequencing is not included in the diagnostic criteria and not available to all patients RDEB, since the variability of change complicates the analysis ofthis very large gene by simple and inexpensive techniques. Using the next generation sequencing (NGS) to analyze the COL7AI gene, we identified the mutations in 26 patient's gene and 22 were RDEB patients. Six novel mutations were identified (c.8047-I G>A, c.l758_1758delC, c.2783_2784insGACAC, c.2903C>T, c.3649G>A e c.8191G>T), being two of them were found in more than one genetic allele (c.l758_I758deIC, c.2783 _2784insGACAC). The most frequent mutation and more frequently alIelic (25%) was the c.5047 C>T. Ten patients had homozygous mutations and nine are resulting from inbreeding. The splice site mutations c. 830~+ 1 G>A e c.8047-I G>A were identified through the introns sequencing, and can be exon skiping therapy or gene edition target, avoiding the entire COL7AI gene insertion, a laborious processo As RDEB patients suffering exclusively from colIagen VII deficiency, " macrophages were modified to express the COL7AI gene under the control of a synthetic specific promoter - the Scavenger promoter. Thinking of a clinical protocol in the future, non-viral Sleeping Beauty system was chosen for gene integration. Initially nuc1eofection was tested in hematopoietic stem cells from mice (HSC) and primary mouse macrophages (PMM). In vitro a high expression of type VII collagen was seen after transfection of pT2-SP-CoI7 vector in HEK293T cells and in PMM but HSC have not remained viable after nucleofection. For in vivo experiments in knockout animaIs for the COL7AI gene (COL7AI - / - mouse), the PMM were nucleofected with pT2-SP-CoI7and pCMVSBIOOX (expresses Sleeping Beauty) and labeled with CM-Dil. Intradermal and intraperitoneal injection was carried out in newbom COL7AI - / - mouse. There was an increase in survival of treated animaIs ranging from 7 to 21 days depending on the type of treatment. None of the animaIs had wounds or blisters during the evaluation period, but had size and weight corresponding to half ofthe WT mouse. The PMM injected were located in the human skin and collagen VII was seen intracellular and in extracellular. matrix. In mice that received the cells intraperitoneally, collagen VII expression and macrophages were seen in spleen, intestine and skin, indicating that there were migration of the PMM to the skin. So macrophage therapy expressing collagen VII proved to be very promising in improve survival oftreated COL7Al -1- mice.