Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

Abstract

Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the naïve state. Although naïve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether naïve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with b-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these naïve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these naïve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human naïve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:8-19 SIGNIFICANCEIn the present study, transgene-free naïve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with b-thalassemia in a defined culture system were generated. These naïve iPSCs, which show ground-state pluripotency, exhibited significantly improved singlecell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as b-thalassemia.

Clinical implementation of genome editing for correction of human diseases

“…A recent study reported efficiency of CRISPR/Cas9 system for correction of HBB in iPSCs|: up to 57% of the clones showed modification of at least one HBB allele in experimental series, as well as minimal off-target activity [28].…”

Achievements, sensations and problemsof molecular prenatal diagnostics

“…Этот подход уже был использован в экспериментах по коррекции мутаций, вызы-вающих тяжелые наследственные болезни (му-ковисцидоз [15], миодистрофия Дюшенна [16], дегенерация сетчатки глаза [17]), для лечения инфекционных заболеваний (вИЧ) [18] и в ис-следованиях тонкой структурно-функциональ-ной организации генома человека. Недавно с помощью этой технологии были проведены эксперименты на мышах, а затем на зиготах че-ловека для коррекции мутаций гена β-глобина, вызывающих тяжелое и частое наследственное заболевание -β-талассемию [19]. Результаты этих экспериментов вызвали большой науч-ный резонанс.…”

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

“…Precisely engineered iPSCs are needed for stem cell-based modelling of genetic disease, and for providing an unlimited source of gene-edited lines for potential use in regenerative medicine [228,229]. Recent studies have reported successful gene correction of beta thalassaemia mutations using Cas9WT in iPSCs [35][36][37]138]. Cooperative strategies, (i.e.…”

“…While the generation of iPSCs from the fibroblasts of beta thalassaemia individual [26][27][28][35][36][37] and amniotic fluids cells [38] has previously been reported [28,187] he production of footprint-free iPSCs from fibroblasts derived from patients with the Mediterranean mutation IVSII-1(G>A) of beta thalassaemia has not been reported to date (Table1-1). IVSII-1(G>A) is a splice junction mutation that affects the 5′ splice site leading to a severe form of beta thalassaemia in the homozygous state.…”

“…The Sendai virus [35] excisable integrating vector (STEMCCA/Cre/loxP) [36] and episomes [37,38] were used for the derivation of integration-free iPSCs from patients with inherited haemoglobinopathies (Table1-1). Although transgene-free iPSCs can be derived with the STEMCCA vector, the excision process leaves a short DNA sequence in the host genome [39] that may have consequences for the functionality of the gene [40].…”