Preclinical model of gene therapy for leukocyte adhesion deficiency type I
Author
Mesa Núñez, CristinaEntity
UAM. Departamento de Bioquímica; Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT); Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)Date
2019-06-17Subjects
Terapia genética - Tesis doctorales; Leucocitos - Terapia - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 17-06-2019Esta tesis tiene embargado el acceso al texto completo hasta el 17-12-2020
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
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.
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