The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.

SUMMARY Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell-replacement therapies. However one crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age-onset disorders where in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc-finger nuclease (ZFN)-mediated genome editing and iPSC technology we provide a generally applicable solution to this problem by generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson’s disease by modifying the underlying point mutations (A53T/E46K) in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents not only a significant progress for basic biomedical research but also a major advancement towards hiPSC-based cell-replacement therapies.

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNA mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin.

The accession numbers for sequencing data are SRP111763 and SRP111797.

The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and ΔF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was P o = 0.047 ± 0.019. Addition of genistein activated Cl currents ∼10-fold, and the P o of G551D-CFTR increased to 0.49 ± 0.12, which is a P o similar to wild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous for the trafficking-impaired ΔF508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These data suggested that genistein activated CFTR mutants that were present in the cell membrane. Therefore, we tested the effects of genistein in CF patients with the G551D mutation in nasal potential difference (PD) measurements in vivo. The perfusion of the nasal mucosa of G551D CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, −2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to β-adrenergic stimulation) found in healthy subjects.