MA (Fine Art), Faculty of the Humanities, University of the Witwatersrand, 2011

Farber disease is a rare lysosomal storage disease (LSD) caused by a deficiency of acid ceramidase (AC). Patients show a classic triad of symptoms including subcutaneous granulomas, laryngeal involvement and painful swollen joints. The most common and severe form has neurological manifestations and patients typically die by the age of two. Current treatment consists of symptomatic supportive care and allogeneic bone marrow transplantation (BMT). However, BMT has shown limited success. Gene therapy has previously been shown to be a promising treatment strategy for monogenetic diseases and has the potential to treat the underlying cause of the disease. Presented here is the first report of in vivo testing of retrovirus-mediated gene therapy strategies for the treatment of Farber disease. Retroviral vectors were engineered to overexpress AC and a cell surface marker, human CD25. Transduction with these viral vectors corrected the enzymatic defect in Farber patient cells and in vivo administration of the lentiviral vector led to long-term expression of the marking transgene as well as increased AC expression in the liver. To determine the effect of over-expression of AC, human CD34+ cells were transduced and transplanted into NOD/SCID animals. It was found that transgene-expressing cells could reconstitute the host. To address the neurological manifestations of Farber disease, vascular endothelial growth factor (VEGF) was investigated as an agent to transiently open the blood brain barrier for entry of lentivirus. It was found that in addition to increasing the amount of therapeutic virus in the brain, VEGF treatment also increased transduction in other organs. Further, to address the concerns of insertional mutagenesis associated with using integrating vectors, an immunotoxin-based strategy was developed as a safety system to clear transduced cells. It was found that a CD25-targeted immunotoxin could eliminate both transduced hematopoietic cells as well as tumor cells over-expressing CD25. This strategy can be employed following gene therapy should an unwanted proliferative event occur. Together, these studies represent considerable advances towards the development of a cure for Farber disease, demonstrating both therapeutic potential and also containing a built-in safety system.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Farber disease is a rare lysosomal storage disease (LSD) caused by a deficiency of acid ceramidase (AC). Patients show a classic triad of symptoms including subcutaneous granulomas, laryngeal involvement and painful swollen joints. The most common and severe form has neurological manifestations and patients typically die by the age of two. Current treatment consists of symptomatic supportive care and allogeneic bone marrow transplantation (BMT). However, BMT has shown limited success. Gene therapy has previously been shown to be a promising treatment strategy for monogenetic diseases and has the potential to treat the underlying cause of the disease. Presented here is the first report of in vivo testing of retrovirus-mediated gene therapy strategies for the treatment of Farber disease. Retroviral vectors were engineered to overexpress AC and a cell surface marker, human CD25. Transduction with these viral vectors corrected the enzymatic defect in Farber patient cells and in vivo administration of the lentiviral vector led to long-term expression of the marking transgene as well as increased AC expression in the liver. To determine the effect of over-expression of AC, human CD34+ cells were transduced and transplanted into NOD/SCID animals. It was found that transgene-expressing cells could reconstitute the host. To address the neurological manifestations of Farber disease, vascular endothelial growth factor (VEGF) was investigated as an agent to transiently open the blood brain barrier for entry of lentivirus. It was found that in addition to increasing the amount of therapeutic virus in the brain, VEGF treatment also increased transduction in other organs. Further, to address the concerns of insertional mutagenesis associated with using integrating vectors, an immunotoxin-based strategy was developed as a safety system to clear transduced cells. It was found that a CD25-targeted immunotoxin could eliminate both transduced hematopoietic cells as well as tumor cells over-expressing CD25. This strategy can be employed following gene therapy should an unwanted proliferative event occur. Together, these studies represent considerable advances towards the development of a cure for Farber disease, demonstrating both therapeutic potential and also containing a built-in safety system.

L’ostéonécrose (ON) et les fractures (FR) sont des complications qui prennent de plus en plus place dans le traitement pédiatrique de la leucémie aiguë lymphoblastique (LAL). L’ON peut être causée par différents facteurs, dont principalement l’utilisation de glucocorticoïdes. Les glucocorticoïdes sont administrés lors du traitement de la leucémie dans le but d’initier l’apoptose des cellules malignes tout en ayant un effet anti-inflammatoire. Cependant, l’utilisation de ces corticostéroïdes comprend des effets secondaires sérieux, notamment le développement d’ostéonécrose. Des variantes génétiques peuvent mettre certains patients plus à risque que d’autres. Plusieurs gènes ont déjà été signalés comme régulés par les actions glucocorticoïdes (GC). Les variations génétiques présentes dans les régions régulatrices de ces gènes peuvent affecter leur fonctionnement normal et, en fin de compte, de déterminer un risque accru de développer l’ON associé au traitement contre la leucémie. Pour cette raison, plusieurs polymorphismes ont été identifiés et étudiés dans la cohorte QcALL de Ste-Justine, concernant les gènes suivants : ABCB1, ACP1, BCL2L11, NFKB1, PARP1, et SHMT1. Ces gènes jouent majoritairement un rôle dans les mécanismes d’action des glucocorticoïdes, mais quelques-uns ont plutôt un effet direct sur le développement d’ostéonécrose. Nos recherches ont démontré une corrélation entre ces polymorphismes et l’apparition d’ostéonécrose chez les patients de la cohorte QcALL, traités aux glucocorticoïdes. L'incidence cumulative de l'ostéonécrose a été évaluée rétrospectivement chez 305 enfants atteints de la leucémie qui ont subi un traitement à l’hôpital Ste-Justine selon les protocoles DFCI de Boston (87-01, 91-01, 95-01 et 2000-01). Parmi les huit polymorphismes de BCL2L11 étudiés, les 891T> G (rs2241843) et 29201C> T (rs724710) ont été significativement associés à ON (p = 0.01 et p = 0.03, respectivement). L'association du polymorphisme 891T> G a été modulée par le type de corticostéroïde (CS), l’âge, le sexe et le groupe à risque (p ≤ 0,05). Le polymorphisme 29201C> T était particulièrement apparent chez les patients à haut risque (p = 0,003). La même étude était conduite en parallèle sur des patients de la cohorte DFCI de Boston (N = 192), et montrait des résultats significatifs pour les polymorphismes étudiés. En conclusion, les résultats de cette étude permettront de confirmer l’association de ces polymorphismes au développement d’ON chez les patients de LLA traités aux GC. / Osteonecrosis (ON) and fractures (FR) are complications that take place in the treatment of children acute lymphoblastic leukemia (cALL). They can be caused by various factors, mainly using glucocorticoids. The corticosteroids, dexamethasone (DXM) and prednisone (PDN) are administered during the treatment of leukemia to initiate apoptosis of malignant cells; while having an anti-inflammatory effect. However, the use of these corticosteroids has severe side effects, including the development of osteonecrosis. Moreover, some patients develop resistance to treatment, and are at risk of developing side effects. The genetic variants predispose some patients at higher risk than others. Several genes have been previously reported as up- or down regulated by the GCs actions. The genetic variations present in gene coding or regulatory regions can affect their function and ultimately determine an increased risk of developing ON associated to ALL therapy. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs) in six candidate genes: BCL2L11, NFKB1, PARP1, ABCB1, ACP1, and SHMT1. These genes play a role in the mechanisms of action of glucocorticoids, but some have more of a direct effect on the development of osteonecrosis. Our research has shown a correlation between these polymorphisms and the occurrence of osteonecrosis in patients in the QCALL cohort, treated with glucocorticoids. Cumulative incidence of osteonecrosis was assessed retrospectively in 305 children with ALL who underwent treatment with DFCI protocols (87-01, 91-01, 95-01 and 2000-01) in childhood ALL cohort from Quebec (QcALL). Among the eight tag BCL2L11 polymorphisms studied the 891T>G (rs2241843) and 29201C>T (rs724710) were significantly associated with ON (p = 0.01 and p = 0.03, respectively). Association of 891T>G polymorphism was modulated by type of corticosteroid (CS), age, sex and risk group (p ≤ 0.05 and that of 29201C>T was particularly apparent among high risk (p = 0.003) patients. These polymorphisms have shown significant ON association in several QcALL risk groups, mainly in corticosteroid groups, age < 10 years, and high risk (HR) group. Furthermore, the same study was conducted in parallel with patients in the replication (DFCI) cohort (N = 192), and we showed significant genetic association results for all studied polymorphisms. In conclusion, this study identifies that some ALL children have a high incidence of ON during the treatment that is highly associated with polymorphisms in different genes regulated by corticosteroids and ALL prognostic factors.