Toxicology studies for gene-modified stem cell transplantation for cystinosis
Description: 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. __SpecificAimsTextDelimiter__ Specific aims Cystinosis is an autosomal metabolic disease that belongs to the family of the Lysosomal Storage disorders. Cystinosis is characterized by a lysosomal accumulation of cystine in all the cells of the body. Three allelic forms of the disease exist: infantile, juvenile and ocular. Patients with the infantile form typically present in their first year of life with symptoms of Fanconi syndrome characterized by severe fluid and electrolyte disturbances (vomiting, poor growth, rickets). The patients will also progress to end-stage renal failure. Cystine accumulation eventually leads to multi-organ dysfunction and patients present with photophobia and blindness, hypothyroidism, hypogonadism, diabetes, myopathy and central nervous system defects. Patients with the juvenile form primarily develop a progressive kidney dysfunction and photophobia around 12 years old while the ocular form consists solely of photophobia in the adults. The only treatment for cystinosis is the drug cysteamine that reduces the intracellular concentration of cystine, and, if used early in the disease and in high doses, can delay the progression of the disease. However, the need for very frequent dosing (every 6 hours orally plus eye drops hourly) and severe side effects such as digestive intolerance and persistent odor, render its chronic administration difficult and compliance is a huge challenge in any pediatric population. Moreover, cysteamine does not prevent the proximal renal tubulopathy nor the end-stage renal failure and only delay the progression of the disease. Using the mouse model for cystinosis, the Ctns-/- mice, we showed that transplantation of syngeneic whole bone marrow cells (BMC) or purified Hematopoietic Stem and Progenitor Cells (HSPC) led to significant decreases of cystine in every tissue tested and the impact correlated with the abundant tissue integration of bone marrow-derived cells [1]. This treatment also successfully protected these animals from the progression of the kidney tissue injury if a relatively high level of donor-derived blood cell engraftment of Ctns-expressing cells was achieved [2]. This work represents the first proof-of-concept for using HSPC transplantation as a therapy for cystinosis. However, due to the risks of mortality associated with allogeneic transplant, our long- term strategy is to develop an autologous transplantation of HSPC ex vivo modified using a lentiviral vector to introduce a functional version of the defective CTNS gene. A key point is that the use of the patient's stem cells would avoid the risks of rejection, graft vs. host disease and the serious complications of immunosuppression. Successful engraftment of stem cells with the functional CTNS gene into the bone marrow compartment will serve as a source of healthy stem cells for any injured tissue for the life of the patient. We have already optimized murine Sca1+ HSPC gene delivery using our vector pCCL-CTNS containing the human version of CTNS, and established the first proof-of-concept in the mouse model of cystinosis that this strategy of gene- modified HSPC transplantation could work in young patients with cystinosis before significant disease progression [3]. While proof-of-concept in older Ctns-/- mice and additional pre-clinical studies still need to be completed, we went forward and submitted our pre-pre-Investigator New Drug (IND) application to start in parallel the studies required by the FDA for the pharmacology/toxicology of our lentiviral vector construct, pCCL-CTNS. The reviews were positive and the FDA allowed us to move forward to a pre-IND application. The pharmacology/toxicology studies will have to be done with a pre-clinical batch of the vector pCCL-CTNS produced under a Good Manufacturing Practice. The studies will involve human CD34+ Peripheral Blood Stem cells (PBSC) and murine HSPC that will be ex vivo transduced by our lentiviral construct pCCL-CTNS and safety and efficacy will be analyzed in vitro and in vivo in the Ctns-/- mice. The objective of this proposal is to perform the pharmacology/toxicology required by the FDA for the transplantation of autologous hematopoietic stem cells genetically modified ex vivo to express a functional CTNS gene using our lentivirus vector pCCL-CTNS in order to obtain an IND for a phase I clinical trial. This work represents the first stem cell and gene therapy treatment strategy for cystinosis. If successful, this treatment could be a life-long therapy that may prevent kidney transplantation and long-term complications associated with cystinosis. Specific aim 1: In vitro safety studies. Insertional mutagenesis screens for pCCL-CTNS will be performed using two different in vitro assays, the Colony Forming Units (CFU) assay using human CD34+ PBSC isolated from healthy donors and cystinotic patients and the In Vitro Immortalization (IVIM) assay using lineage- negative BMC from wild-type C57BL/6 mice. These assays will test the capacity of the transduced cells to differentiate into lineage-committed progenitors, identify leukemic stem cells and assess genotoxicity. Vector Copy Numbers (VCN) and Vector Integration Sites (VIS) will be determined in the final colonies and clones. Specific aim 2: In vivo efficacy and safety studies. Murine Sca1+ HSPC ex vivo transduced by pCCL-CTNS will be 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.
Funding type: research
Funder scheme: R01
Start date: 2013-08-06
End date: 2015-05-31
Investigator: STEPHANIE CHERQUI
Institution: UNIVERSITY OF CALIFORNIA, SAN DIEGO
Subfield: Pathology and Forensic Medicine
Field: Medicine
Domain: Health Sciences
Funded outputs count: 7
Funder award ID: R01DK099338
OpenAlex ID: G562605832