Cystinosis is a metabolic hereditary disease characterized by intracellular accumulation of cystine. Affected individuals typically present with proximal tubulopathy before one year of age and eventually progress to end- stage renal failure. Cystine accumulation leads to multi-organ dysfunction. The drug cysteamine reduces the intracellular cystine content. However, cysteamine does not prevent the proximal tubulopathy nor the end- stage renal failure and only delay the progression of the disease. The long-term objective of this project is to develop a new treatment for cystinosis by transplantation of autologous Hematopoietic Stem and Progenitor Cells (HSPC) genetically modified ex vivo to express a functional CTNS gene. Using the mouse model for cystinosis, the Ctns-/- mice, we showed that transplantation of syngeneic Sca1+ HSPC expressing Ctns resulted in abundant bone marrow-derived cell engraftment and significant reductions of cystine content in all the tissues tested. This treatment also prevented the progression of kidney dysfunction. We obtained the same results with ex vivo transduced HSPC using our lentiviral vector construct, pCCL-CTNS, and established the first proof-of-concept in the Ctns-/- mice that this strategy could work in young patients with cystinosis before significant disease progression. After obtaining the approval from the FDA to move forward to a pre- Investigator New Drug (IND) application, we are now proposing the pharmacology/toxicology studies required to obtain an IND for a phase I a clinical trial for cystinosis. This treatment would represent a life-long theray that may prevent kidney transplantation and long-term complications associated with cystinosis. Lentiviral vectors have proven its efficacy for long-term HSPC transduction in mice but also in humans. All the pharmacology/toxicology studies will be done with a pre-clinical batch of the vector pCCL-CTNS produced under Good Manufacturing Practice.
In Specific aim 1, we propose to optimize the transduction of human CD34+ cells using our vector pCCL-CTNS and to test the capacity of the transduced cells to go through lineage-committed progenitors without becoming leukemic using the Colony-Forming Units (CFU) assay. The other in vitro assay to assess genotoxicity of integrating viral vectors is the In Vitro Immortalization (IVIM) assay using murine lineage-negative bone marrow cells. Vector Copy Numbers (VCN) and Vector Integration Sites (VIS) will be determined in the final colonies and clones for both assays.
In Specific aim 2, we will test the efficacy and safety of this strategy in vivo using murine Sca1+ HSPC ex vivo transduced by pCCL-CTNS and transplanted in primary and secondary recipient Ctns-/- mice. Efficacy will be measured by CTNS expression in blood and tissue samples, tissue cystine levels and renal function. Toxicity will be determined by comprehensive clinical and histological tissue analyses, by assessing VCN and VIS in myeloid and lymphoid cells and detecting potential antibody immunity to CTNS proteins. This work represents the last steps towards a phase I clinical trial for cystinosis and is also a proof of concept to treat other lysosomal storage disorders.

Public Health Relevance

Cystinosis is a hereditary disease characterized by the accumulation of cystine in all the cells of the body leading to cell death and tissue damage to kidneys, liver, eyes, muscle and brain. The long-term objective of this project is to develop a new treatment for cystinosis by using the patient's own bone marrow stem cells for transplantation and to genetically modify them ex vivo to introduce a functional version of the defective gene (CTNS). This proposal represents the pharmacology/toxicology studies required by the FDA to develop a clinical trial for cystinosis and is also a proof of concept to treat other lysosomal storage disorders.

National Institute of Health (NIH)
Research Project (R01)
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Pathobiology of Kidney Disease Study Section (PBKD)
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Moxey-Mims, Marva M
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University of California San Diego
Schools of Medicine
La Jolla
United States
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Naphade, Swati; Sharma, Jay; Gaide Chevronnay, Héloïse P et al. (2015) Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes. Stem Cells 33:301-9