Intralysosomal Cystine Accumulation in Mice Lacking Cystinosin, the Protein Defective in Cystinosis

Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-βgal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(−/−) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(−/−) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(−/−) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(−/−) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis

Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient

Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC–derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model

Visual phenotype of the <i>Fatp1</i>^−/− mouse.

<p>A. Histology of the retina. Ch: choroid. RPE: retinal pigment epithelium. POS: PR outer segments. PIS: PR inner segments. ONL: outer nuclear layer. OPL: outer plexiform layer. INL: inner nuclear layer. IPL: inner plexiform layer. GCL: ganglion cell layer. WT: <i>Fatp1</i>^+/+. KO: <i>Fatp1^−/−</i>. B. TEM of WT and KO retinas. BM: Bruch membrane. C. Electroretinography of <i>Fatp1</i>^+/+ (<i>n</i> = 29) and <i>Fatp1^−/−</i> (<i>n</i> = 23) mice. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. The statistical analyses were performed initially for the whole curve (line above the graph) and subsequently for each point. D. b-wave on a-wave ratio of the ERG experiments. Error bars represent SEM. E. Quantification of the retinal coupled-rhodopsin (<i>n</i> = 4 <i>Fatp1</i>^+/+ and <i>n</i> = 4 <i>Fatp1^−/−</i>) in mice aged 5 to 8 months. Error bars represent SEM. The retinal histology is unchanged in the <i>Fatp1^−/−</i> mice. <i>Fatp1^−/−</i> PRs present a lower response to light flashes and this decrease is not due to a decrease in the level of retinal coupled-rhodopsin in these animals.</p

Aging of the <i>Fatp1</i><i>^−/−</i> retina.

<p>A. Electroretinography of <i>Fatp1</i>^+/+ (<i>n</i> = 5) and <i>Fatp1^−/−</i> (<i>n</i> = 11) mice, * <i>p</i> = 0.021 for the whole curve. B. Quantification of autofluorescence in young (<i>n</i> = 3 <i>Fatp1</i>^+/+ and <i>n</i> = 4 <i>Fatp1^−/−</i>) and old (<i>n</i> = 6 <i>Fatp1</i>^+/+ and <i>n</i> = 5 <i>Fatp1^−/−</i>) mice. The values are expressed in arbitrary fluorescent units from the machine. For each mouse, the result is obtained by summing up all values between 520 and 640 nm taken every 10 nm. Error bars represent SEM. <i>Fatp1^−/−</i> PRs remains less responsive to light with age. <i>Fatp1^−/−</i> mice do not accumulate more autofluorescence, thus presumably more lipofuscin, than <i>Fatp1</i>^+/+ with age. C. Upper panel: neutral lipid labeling of <i>Fatp1</i>^+/+ (WT) and <i>Fatp1^−/−</i> (KO) retinas. Ch: choroid. RPE: retinal pigment epithelium. POS: PR outer segments. PIS: PR inner segments. ONL: outer nuclear layer. OPL: outer plexiform layer. INL: inner nuclear layer. IPL: inner plexiform layer. GCL: ganglion cell layer. Arrows indicate the BM. Lower panel: positive control at the same magnification (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050231#s2" target="_blank">Material and methods</a>). Arrows indicate the BM as in the upper panel. The asterisk indicates positive ORO labeling. <i>Fatp1^−/−</i> or <i>Fatp1</i>^+/+ mice showed no Oil red O labeling in the BM.</p

Fatty acid composition in the NR and RPE in 8 mo-old <i>Fatp1</i>^+/+ and <i>Fatp1^−/−</i> mice.

<p><i>Fatp1</i>^+/+ (n = 5) and <i>Fatp1^−/−</i> (n = 7). 16:0: palmitate. 22:6n-3: DHA. The functional visual difference between <i>Fatp1^−/−</i> and <i>Fatp1</i>^+/+ mice is not due to a different fatty acid composition.</p