Preclinical model of gene therapy for leukocyte adhesion deficiency type I

• Autores: Cristina Mesa Núñez
• Directores de la Tesis: Elena Almarza Novoa (dir. tes.), Juan Antonio Bueren Roncero (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2019
• Idioma: inglés
• Número de páginas: 184
• Tribunal Calificador de la Tesis: José María Almendral del Río (presid.), Francisco Martín Molina (secret.), Adrian Cornelis Lankester (voc.), Fernando Larcher Laguzzi (voc.), Jordi Barquinero (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by an impaired neutrophil migration in response to bacterial infections. The genetic causes of LAD-I are mutations in the ITGB2 gene that encodes for CD18, the common subunit of β2 integrins. These heterodimers are involved in the trafficking and extravasation of leukocytes to inflamed areas. The severity of the LAD-I disease is associated with the percentage of CD18 expressing cells. Patients with less than 2% CD18+ cells suffer a severe disease with a very high mortality rate before the age of 2. The current curative treatment of these patients is the allogenic hematopoietic stem cell (HSC) transplantation from healthy donors. The ex vivo gene therapy (GT) of autologous HSCs has been proposed as a good therapeutic option for these patients when non-HLAmatched related donors are available.

    Our laboratory has previously shown that the LV:Chim.hCD18 carrying the human ITGB2 gene under the control of a myeloid Chimeric promoter efficiently expresses the therapeutic human CD18 protein in myeloid cells from CD18HYP mice, and restores the functionality of these cells1. To complete the preclinical studies required for the development of a gene therapy trial in severe LAD-I patients, further preclinical studies have been now developed to confirm the safety and efficacy of the approach.

    The characterization of CD18 expression in human HSCs from healthy donors (HDs) indicated that the absence or low expression of CD18 defines a population with long-term repopulating activity. Significantly, the transduction of HD CD34+ cells with the therapeutic Chim.hCD18- lentiviral vector (LV) did not affect the membrane expression of CD18, and preserved the in vivo repopulating potential of transduced cells. Biodistribution studies in mice showed that there was no mobilization or shedding of the LV, or risks of LV germline transmission after the infusion of transduced HSCs.

    Due to the modest levels of gene marking in human CD34+ cells transduced with the therapeutic LV, different molecules were used to enhance the transduction of these cells.

    The addition of a combination of specific molecules during the transduction protocol revealed a marked increase in the genetic modification of primitive HSCs, without affecting their multi-lineage long-term repopulating potential. Additionally, our optimized transduction protocol demonstrated the phenotypic correction of the neutrophil extravasation defect in a severe mouse model of LAD-I.

    Finally, the transplantation of NSG mice with human CD34+ cells that had been transduced under Good Manufacturing Practice with the therapeutic LV demonstrated the stable gene marking of primitive HSCs with preserved repopulation potential.