Disease modeling with iPSCs can be challenging due

Disease modeling with iPSCs can be challenging due to inherent patient variability and line-to-line differences (Hu et al., 2010; Boulting et al., 2011). Importantly, very few differences were observed among the three independent control lines. The SNCA (3×) iPSC line was not different from controls in the parameters tested, but one SNCA (3×) line is not sufficient to conclude this line does not exhibit other PD-related phenotypes. The two homozygous LRRK2 G2019S iPSC lines used here showed slight variation in the extent of the dysfunction, with LRRK2 G2019S 1 generally being more affected. However, both lines were consistently impaired relative to control mct2 pathway and the SNCA (3×) line. The heterozygous LRRK2 G2019S iPSC-derived sensory neurons exhibited a less-severe phenotype than sensory neurons derived from either homozygous LRRK2 G2019S iPSC line. This could be due to the fact that the heterozygous LRRK2 G2019S iPSC line was generated from an asymptomatic patient. Alternatively, it could be due to a dosage effect of mutant LRRK2. Nevertheless, the heterozygous mutant LRRK2 iPSC-derived neurons did exhibit significant structural and functional abnormalities consistent with the dominant nature of LRRK2 mutations. Despite minor variations among the three LRRK2 G2019S iPSC lines, our data report a robust aggregate and calcium phenotype in LRRK2 G2019S iPSC-derived sensory neurons that provides the foundation for additional optimization and evaluation across a larger cohort of LRRK2 patient samples. The iPSC-based model of PD offers a valuable tool to study the pathophysiology of LRRK2-related defects in multiple cell types affected in PD and may provide a mechanistic link between cytoskeletal changes, neuron dysfunction, and the appearance of motor and non-motor symptoms in PD.
Experimental Procedures
Acknowledgments
Introduction Pyruvate kinase deficiency (PKD; OMIM: 266200) is a rare metabolic erythroid disease caused by mutations in the PKLR gene, which codes the R-type pyruvate kinase (RPK) in erythrocytes and L-type pyruvate kinase (LPK) in hepatocytes. Pyruvate kinase (PK) catalyzes the last step of glycolysis, the main source of ATP in mature erythrocytes (Zanella et al., 2007). PKD is an autosomal-recessive disease and the most common cause of chronic non-spherocytic hemolytic anemia. The disease becomes clinically relevant when RPK activity decreases below 25% of the normal activity in erythrocytes. PKD treatment is based on supportive measures, such as periodic blood transfusions and splenectomy. The only definitive cure for PKD is allogeneic bone marrow transplantation (Suvatte et al., 1998; Tanphaichitr et al., 2000). However, the low availability of compatible donors and the risks associated with allogeneic bone marrow transplantation limit its clinical application. Transplantation of gene-corrected autologous hematopoietic progenitors might solve these problems. We have developed different gamma-retroviral and lentiviral vectors to correct a mouse PKD model (Meza et al., 2009), and their efficacy is currently being tested in hematopoietic progenitors from PKD patients (M. Garcia-Gomez et al., personal communication). However, the main drawback of current gene therapy approaches based on retro-/lentiviral vectors is the random integration of transgenes, which can promote insertional mutagenesis by disrupting tumor suppressor genes or cis-activating proto-oncogenes (Cavazza et al., 2013). Over the last few years, gene editing by homologous recombination (HR) has been widely used in human cells to avoid undesirable transgene insertion. HR efficacy is very limited in human cells, estimated at one HR event per 106 cells; however, the potential application of HR in human cells has been enhanced considerably by sequence-specific DNA nucleases (Carroll, 2011; Porteus and Carroll, 2005). Three different gene-editing strategies can be applied: gene correction, where a mutation is exchanged directly by the wild-type sequence; knockin, where a partial cDNA is inserted in the target locus to express a chimeric mRNA formed by endogenous first exons and partial cDNA under the endogenous promoter control; and safe harbor, in which the transgene is inserted by HR in a safe place in the genome, such as AAVS1 or CCR5 loci (Garate et al., 2013).