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Open Access Publications from the University of California

Scholarly Works (38 results)

  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2014)
Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The defective gene is CTNS, which encodes the lysosomal cystine transporter, cystinosin. Cystine accumulates in all tissues and leads to organ damage including end-stage renal disease. In this review, we outline the studies that support that genetic rescue of cystinosis could be an achievable goal, even though cystinosis is a multi-compartmental disease and cystinosin an intracellular transmembrane protein. Using the mouse model of cystinosis, the Ctns(-/-) mice, we showed that transplanted hematopoietic stem cells (HSCs) were able to act as vehicles for the delivery of a functional Ctns gene to the different organs and led to the significant decrease of the tissue cystine content and tissue preservation. Ex vivo gene-modified Ctns(-/-) HSC transplantation using a lentiviral vector containing CTNS complementary DNA (cDNA) was also successful in the Ctns(-/-) mice and built the foundations for a clinical trial for autologous HSC transplantation for cystinosis. The capacity of HSCs for rescuing non-hematopoietic disease is controversial, and new insights into regenerative medicine could be gained from unraveling the underlying mechanism of action.
    Cover page: Is genetic rescue of cystinosis an achievable treatment goal?
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2021)
    Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The gene involved is the CTNS gene that encodes cystinosin, a seven-transmembrane domain lysosomal protein, which is a proton-driven cystine transporter. Cystinosis is characterized by the lysosomal accumulation of cystine, a dimer of cysteine, in all the cells of the body leading to multi-organ failure, including the failure of the kidney, eye, thyroid, muscle, and pancreas, and eventually causing premature death in early adulthood. The current treatment is the drug cysteamine, which is onerous and expensive, and only delays the progression of the disease. Employing the mouse model of cystinosis, using Ctns-/- mice, we first showed that the transplantation of syngeneic wild-type murine hematopoietic stem and progenitor cells (HSPCs) led to abundant tissue integration of bone marrow-derived cells, a significant decrease in tissue cystine accumulation, and long-term kidney, eye and thyroid preservation. To translate this result to a potential human therapeutic treatment, given the risks of mortality and morbidity associated with allogeneic HSPC transplantation, we developed an autologous transplantation approach of HSPCs modified ex vivo using a self-inactivated lentiviral vector to introduce a functional version of the CTNS cDNA, pCCL-CTNS, and showed its efficacy in Ctns-/- mice. Based on these promising results, we held a pre-IND meeting with the Food and Drug Administration (FDA) to carry out the FDA agreed-upon pharmacological and toxicological studies for our therapeutic candidate, manufacturing development, production of the GMP lentiviral vector, design Phase 1/2 of the clinical trial, and filing of an IND application. Our IND was cleared by the FDA on 19 December 2018, to proceed to the clinical trial using CD34+ HSPCs from the G-CSF/plerixafor-mobilized peripheral blood stem cells of patients with cystinosis, modified by ex vivo transduction using the pCCL-CTNS vector (investigational product name: CTNS-RD-04). The clinical trial evaluated the safety and efficacy of CTNS-RD-04 and takes place at the University of California, San Diego (UCSD) and will include up to six patients affected with cystinosis. Following leukapheresis and cell manufacturing, the subjects undergo myeloablation before HSPC infusion. Patients also undergo comprehensive assessments before and after treatment to evaluate the impact of CTNS-RD-04 on the clinical outcomes and cystine and cystine crystal levels in the blood and tissues for 2 years. If successful, this treatment could be a one-time therapy that may eliminate or reduce renal deterioration as well as the long-term complications associated with cystinosis. In this review, we will describe the long path from bench-to-bedside for autologous HSPC gene therapy used to treat cystinosis.
      Cover page: Hematopoietic Stem Cell Gene Therapy for Cystinosis: From Bench-to-Bedside
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2023)
      Cystinosis is an autosomal recessive metabolic disease characterized by lysosomal accumulation of cystine in all the cells of the body. Infantile cystinosis begins in infancy by a renal Fanconi syndrome and eventually leads to multi-organ failure, including the kidney, eye, thyroid, muscle, and pancreas, eventually causing premature death in early adulthood. The current treatment is the drug cysteamine that only delays the progression of the disease. We identified the gene involved, CTNS, and showed that the encoded protein, cystinosin, is a proton-driven cystine transporter. We generated a mouse model of cystinosis, the Ctns-/- mice, that recapitulates the main disease complications. The goal was next to develop a gene therapy approach for cystinosis. We used bone marrow stem cells as a vehicle to bring the healthy CTNS gene to tissues, and we showed that wild-type hematopoietic stem and progenitor cell (HSPC) transplantation led to abundant tissue integration of bone marrow-derived cells, significant decrease of tissue cystine accumulation and long-term kidney, eye and thyroid preservation. We then developed an autologous transplantation approach of HSPCs modified ex vivo using a lentiviral vector to introduce a functional CTNS cDNA, and showed its efficacy in Ctns-/- mice. We conducted the pharmacology/toxicology studies, developed the manufacturing process using human CD34+ cells, and design the clinical trial. We received Food and Drug Administration (FDA)-clearance to start a phase 1/2 clinical trial for cystinosis in December 2018. Six patients have been treated so far. In this review, we describe the path to go from the gene to a gene therapy approach for cystinosis.
        Cover page: Cystinosis: From the gene identification to the first gene therapy clinical trial
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2022)
        Friedreich's ataxia (FRDA) is an inherited, multisystemic disorder predominantly caused by GAA hyper expansion in intron 1 of frataxin (FXN) gene. This expansion mutation transcriptionally represses FXN, a mitochondrial protein that is required for iron metabolism and mitochondrial homeostasis, leading to neurodegerative and cardiac dysfunction. Current therapeutic options for FRDA are focused on improving mitochondrial function and increasing frataxin expression through pharmacological interventions but are not effective in delaying or preventing the neurodegeneration in clinical trials. Recent research on in vivo and ex vivo gene therapy methods in FRDA animal and cell models showcase its promise as a one-time therapy for FRDA. In this review, we provide an overview on the current and emerging prospects of gene therapy for FRDA, with specific focus on advantages of CRISPR/Cas9-mediated gene editing of FXN as a viable option to restore endogenous frataxin expression. We also assess the potential of ex vivo gene editing in hematopoietic stem and progenitor cells as a potential autologous transplantation therapeutic option and discuss its advantages in tackling FRDA-specific safety aspects for clinical translation.
          Cover page: Advantages and Limitations of Gene Therapy and Gene Editing for Friedreich’s Ataxia
          • Thesis
          • Peer Reviewed
          UC San Diego Electronic Theses and Dissertations (2020)

          Cystinosis is a multisystem genetic lysosomal storage disorder caused by mutations in the CTNS gene leading to cystine crystal accumulation within lysosomes. Current treatments merely delay disease progression so we have pioneered hematopoietic stem and progenitor cell (HSPC) transplantation in mice. Our group previously discovered that HSPC-derived macrophages are important effectors of this therapy by facilitating the delivery of functional lysosomes to diseased tissue through membranous protrusions called tunneling nanotubes (TNTs). This transplantation approach is being translated into human cystinosis patients in an ongoing Phase I/II clinical trial.

          To understand the mechanistic underpinnings of this therapy, we investigated macrophage polarization and TNT-mediated intercellular trafficking. Employing an in vitro polarization co-culture system and automated image analysis workflows, we found that pro-inflammatory stimulation reduced TNT-like protrusion formation and lysosomal transport. However, we found that co-culture of macrophages with diseased cystinotic fibroblasts increased TNT formation and trafficking, signaling we further explored via metabolomic analysis of culture media.

          Next, we addressed if SHPK, a metabolic gene involved in macrophage polarization that is frequently eliminated in cystinosis, is required for optimal HSPC therapy. We generated novel knockouts using CRISPR-Cas9, confirmed that Shpk expression was ablated and described a hepatic and urinary metabolic phenotype. We then transplanted Shpk-/- HSPCs into cystinotic mice and determined that they are equally efficacious as wildtype (WT) donors. Based on these findings, SHPK-/- patients will be enrolled as future subjects in the clinical trial.

          Finally, we developed a novel clinical endpoint for the clinical trial by quantitating cystine crystals from intradermal confocal images of patients. We created an automated image analysis methodology to measure crystal density in both 2D and 3D. We found that patients have significantly higher normalized confocal crystal volume (nCCV) than healthy controls, and that this new metric correlates with patient age and several clinical outcomes. This methodology presents the potential to become a biomarker to monitor long-term disease trend, compliance with treatment, and anticipatory guidance for potential complications.

          Overall, this work serves to highlight the potential of HSPC-based regenerative therapies to treat previously incurable genetic diseases. This work highlights how an understanding of basic molecular mechanisms can be directly translated into developing novel regenerative therapies for humans.

          • 1 supplemental PDF
          • 13 supplemental videos
          • 3 supplemental ZIPs
          Cover page: Mechanisms of Cystinosis Regenerative Therapies and Pathogenesis
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2019)
          Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders (LSDs). Initial symptoms of cystinosis correspond to the renal Fanconi syndrome. Patients then develop chronic kidney disease and multi-organ failure due to accumulation of cystine in all tissue compartments. LSDs are commonly characterized by a defective activity of lysosomal enzymes. Hematopoietic stem and progenitor cell (HSPC) transplantation is a treatment option for several LSDs based on the premise that their progeny will integrate in the affected tissues and secrete the functional enzyme, which will be recaptured by the surrounding deficient cells and restore physiological activity. However, in the case of cystinosis, the defective protein is a transmembrane lysosomal protein, cystinosin. Thus, cystinosin cannot be secreted, and yet, we showed that HSPC transplantation can rescue disease phenotype in the mouse model of cystinosis. In this review, we are describing a different mechanism by which HSPC-derived cells provide cystinosin to diseased cells within tissues, and how HSPC transplantation could be an effective one-time treatment to treat cystinosis but also other LSDs associated with a lysosomal transmembrane protein dysfunction.
            Cover page: Potential use of stem cells as a therapy for cystinosis
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2017)
            Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. It is caused by a defect in the lysosomal cystine transporter, cystinosin, which results in an accumulation of cystine in all organs. Despite the ubiquitous expression of cystinosin, a renal Fanconi syndrome is often the first manifestation of cystinosis, usually presenting within the first year of life and characterized by the early and severe dysfunction of proximal tubule cells, highlighting the unique vulnerability of this cell type. The current therapy for cystinosis, cysteamine, facilitates lysosomal cystine clearance and greatly delays progression to kidney failure but is unable to correct the Fanconi syndrome. This Review summarizes decades of studies that have fostered a better understanding of the pathogenesis of the renal Fanconi syndrome associated with cystinosis. These studies have unraveled some of the early molecular changes that occur before the onset of tubular atrophy and identified a role for cystinosin beyond cystine transport, in endolysosomal trafficking and proteolysis, lysosomal clearance, autophagy and the regulation of energy balance. These studies have also led to the identification of new potential therapeutic targets and here, we outline the potential role of stem cell therapy for cystinosis and provide insights into the mechanism of haematopoietic stem cell-mediated kidney protection.
              Cover page: The renal Fanconi syndrome in cystinosis: pathogenic insights and therapeutic perspectives
              • Thesis
              • Peer Reviewed
              UC San Diego Electronic Theses and Dissertations (2020)

              Cystinosis is an early onset multisystemic lysosomal storage disorder characterized by deleterious CTNS gene mutations causing defective export and crystallization of amino acid dimer cysteine within lysosomes. Previous studies found that wild-type hematopoietic stem and progenitor cell (HSPC) transplantation into cystinotic (Ctns-/-) mice prevents disease progression via HSPC-derived macrophage-mediated TNT delivery of functional Ctns-carrying lysosomes to diseased cells. While Phase I/II clinical trials for ex vivo lentivirally gene-corrected autologous HSPC transplantation is ongoing at UC San Diego, this approach does not yet extend to 40% of patients harboring homozygous 57 kilobase pair mutations eliminating both CTNS and neighboring pentose phosphate pathway SHPK genes. With roles in both metabolism and macrophage polarization, understanding the clinical relevance of SHPK to our macrophage-based gene therapy is essential to elucidate if SHPK-deficient patients are likely to benefit from the therapy. In this pursuit, we examined the role of polarization conditions mediating macrophage-derived TNT intracellular trafficking in robust in vitro and in vivo systems. We generated novel Shpk knockout (Shpk-/-) mice models which upheld mild metabolic phenotypes as a consequence of isolated Shpk-deficiency. Furthermore, we found that Shpk-/-/Ctns+/+ HSPC transplantation into lethally-irradiated cystinotic Ctns-/-/Shpk+/+ mice resulted in normal macrophage tissue integration, restored Ctns mRNA expression, reduced cystine content, and improved renal function, inducing widespread disease rescue. Ultimately, these findings provide insight into the pathogenic role of SHPK in cystinosis, validate the utility of HSPC transplantations as gene-modifying therapies for disorders of similar cellular/molecular underpinnings, and confirms eligibility of patients with SHPK-deficiency for cystinosis clinical trials.

              • 5 supplemental videos
              • 2 supplemental ZIPs
              Cover page: Cellular and Biochemical Underpinnings of Stem Cell Gene Therapies for Cystinosis
              • Thesis
              • Peer Reviewed
              UC San Diego Electronic Theses and Dissertations (2024)

              Mucopolysaccharidosis type IIIC (MPS IIIC) is a severe neurodegenerative lysosomal storage disease stemming from the loss of function of the lysosomal transmembrane protein Heparan glucosamine N-acetyltransferase (HGSNAT). This leads to the accumulation of the Glycosaminoglycan: Heparan Sulfate. Currently there are no available treatments for this disease. To address this, we developed a new mouse model which displays key disease features such as GAG buildup, Splenomegaly, neurological impairment, and the presence of disease specific non reducing end carbohydrates. In order to investigate a new therapeutic approach for this disease, we transplanted hematopoietic stem and progenitor cells (HSPCs) isolated from Wildtype mice into Hgsnat -/- mice and observed differences between the treated and untreated Hgsnat -/- mice. Transplanted HSPCs differentiated into macrophages in peripheral tissues and microglia-like cells in the brain. The exogenous supply of HSPCs resulted in partial restoration of HGSNAT expression and enzymatic activity which led to a notable reduction in MPS IIIC specific non reducing end carbohydrates within the Hgsnat -/- mice treated with WT HSPCs compared to the untreated Hgsnat -/-mice as well as Hgsnat -/-mice treated with HSPCs harvested from fellow Hgsnat -/-mice. Additionally, WT HSPC transplantation was able to improve neurological deficits, decrease splenomegaly and urine retention in the Hgsnat -/-mice. Histological analysis of the diseased mice kidneys revealed the presence of glomerular hyaline bodies with focal fibrosis and sclerosis which were mitigated in the mutant mice treated with WT HSPCs. The findings from this study provide evidence for the potential therapeutic benefits that WT HSPC transplantation offers MPS IIIC and could serve as an early step toward HSPC gene therapy becoming a clinical approach for rare hereditary diseases such as MPS IIIC.

                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2019)
                Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFNγ) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFNγ polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.
                  Cover page: Macrophage polarization impacts tunneling nanotube formation and intercellular organelle trafficking
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