Overall Research Objectives
Our research focuses on the use of hematopietic stem cells and gene therapy for the treatment of degenerative multi-systemic disorders and hereditary nephropathies. As a model, we are using cystinosis. We are developing a multi-systemic strategy consisting of transplantation of autologous hematopoietic stem cells genetically modified ex vivo using lentivirus vectors. For hereditary nephropathies, we are developing a kidney-specific strategy using adeno-associated virus (AAV) vectors. The main objectives of the laboratory are the clinical translation of these strategies. We are also investigating the mechanisms of tissue repair by bone marrow-derived stem cells after hematopoietic stem cell transplantation in the context of non-hematopoitic disorders.
Summary of the main Research Projects in the lab
Hematopoietic Stem Cell Gene Therapy for Cystinosis
Cystinosis is an autosomal metabolic disease belonging to the family of lysosomal storage disorders. Mutations in the CTNS gene, encoding a lysosomal cystine transporter, lead to cystine accumulation and multi-organ failure such as blindness, myopathy, diabetes and central nervous system defects. Affected individuals also develop proximal tubulopathy and eventually progress to end stage renal failure. Treatment is available with the drug cysteamine to reduce intracellular cystine content. However, cysteamine only delays the progression of the disease.
In April 2012, the first clinical trial using stem cells for cystinosis was approved as an allogeneic Hematopoietic Stem and Progenitor Cell (HSPC) transplant to be conducted at University of California, Los Angeles. This trial was based on the preclinical studies we performed in the mouse model of cystinosis, the Ctns-/- mice, which showed that transplantation of HSPCs expressing a functional Ctns gene led to the abundant tissue integration of bone marrow-derived cells, a significant decrease of cystine accumulation and kidney preservation. However, allogeneic transplants are associated with high risks of mortality and morbidity. Thus, our long-term goal is to develop an autologous transplantation strategy of HSPCs genetically modified ex vivo to express a functional CTNS gene as a treatment for cystinosis. Preclinical studies using a SIN-lentivirus vector containing CTNS to transduce Ctns-/- HSPCs and transplanted in Ctns-/- mice were promising. Transduced cells were capable of reducing cystine in all tissues and of improving kidney function.
In order reach a phase 1 clinical trial for cystinosis, we are currently performing the preclinical pharmacological and toxicological testing following the FDA requirements. This work represents the first stem cell and gene therapy treatment strategy for cystinosis. If successful, this treatment could be a proof of concept for other degenerative multi-systemic disorders.
Mechanism of Hematopoietic Stem Cell-mediated Therapy in Cystinosis
The mechanisms under investigation for hematopoietic stem cell-based treatment are differentiation, trans-differentiation, cell fusion, cross correction, or a combination of these processes. In this context, we developed a new mouse model for cystinosis, the DsRed Ctns-/- mice, ubiquitously expressing the DsRed reporter gene. We then transplanted GFP-expressing HSPC so the differentiated bone marrow-derived cells (DsRed-GFP+) and the fused cells (DsRed+GFP+) could be unequivocally recognized, quantified and sorted. Using confocal microscopy, flow cytometry and DNA array analysis, we showed that stem cells mainly differentiate in tissue-resident-phagocytic cells such as kupffer cells in the liver, microglia cells in the brain, and dendritic cells in the kidney. We also showed that they could play a role in tissue repair by fusing with or by phagocytosing the apoptotic host cells. Using a lentiviral vector driving the expression of the fusion protein cystinosin-eGFP, we also showed that cystinosin could be transferred from CTNS-expressing cells to Ctns-deficient adjacent cells in vitro and in vivo. This transfer led to cystine decrease in Ctns-deficient cells in vitro.
We optimized in vitro and in vivo functional assays involving macrophages to study these two potential mechanisms in the context of cystinosis. This work will provide new insights into the mechanism of the long-term therapeutic effect of transplanted HSPC in cystinosis. This strategy could potentially be applicable to other genetic diseases for which curative therapy requires the addition of the gene to many cells and multiple tissue compartments, and where the protein involved is an intracellular transmembrane protein.
Kidney-targeted gene delivery using AAV
A wide range of monogenic kidney disorders has been identified and so far no gene therapy approach has been developed to target specifically the kidney whereas renal transplantation is associated with significant morbidity and mortality. Moreover, due to the severe shortage of donor organs, patients may wait three to six years for transplantation. The main goal of our project is to develop an efficient and minimally invasive kidney-targeted gene delivery system using recombinant Adeno-Associated Viruses (rAAV). We recently optimized this strategy via retrograde renal vein injection of rAAV serotype 9 that have the potential of transducing a wide range of renal cells. We now propose to test this approach based on renal vein injection of rAAV-CTNS as a minimally invasive procedure for treating the renal dysfunction in cystinosis. If successful, this strategy may be used in many monogenic hereditary nephropathies.