Correction of sickle cell disease by homologous recombination in embryonic stem cells

Abstract

Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing antisickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (beta(S)) with a normal copy of the gene (beta(A)). We recently derived embryonic stem (ES) cells from a novel knock-in mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. In this paper, we demonstrate the replacement of the human beta(S)-globin gene with a human beta(A)-globin gene and the derivation of mice from these cells. The animals produce high levels of normal human hemoglobin (HbA) and the pathology associated with SCD is corrected. Hematologic values are restored to normal levels and organ pathology is ameliorated. These experiments provide a foundation for similar studies in human ES cells derived from sickle cell patients. Although efficient methods for production of human ES cells by somatic nuclear transfer must be developed, the data in this paper demonstrate that sickle cell disease can be corrected without the risk of insertional mutagenesis.

Figure 1.

Replacement of the βS-globin gene with a βA-globin gene in knock-in sickle ES cells. (A) Schematic representation of the human β-globin locus, mouse β-globin locus, and the human –1400 ^Aγ-β^S knock-in locus in ES cells derived for this study. Arrows indicate DNase I hypersensitive sites (HSs) that mark the locus control region (LCR). Red and yellow boxes represent functional human and mouse genes, respectively. White boxes represent pseudogenes. Black circles indicate loxP sites. (B) Schematic representation of gene replacement in knock-in sickle ES cells. The 24-kb replacement vector contains a 2.1-kb PGK/TK marker gene, 1.7 kb of mouse 5′ flanking sequence, a –383 γ-β^A fragment (8.7 kb), a 1.8-kb floxed PGK/Hygro gene, and 7 kb of mouse 3′ flanking sequence. Homologous recombinants were identified by PCR with primers 1 and 2 to identify correct 5′ sequences (primer 1 is outside of the vector homology region) and with primers 5 and 6 to identify correct 3′ sequences (primer 6 is outside of the vector homology region). PCR with primers 3 and 4 followed by Bsu36I digestion was used to distinguish β^S and β^A alleles. (C) 5′ PCR (primers 1 and 2) and 3′ PCR (primers 5 and 6) from 2 positive homologous recombinant ES cell lines (clones 1 and 2). In clone 1, recombination at the 5′ end occurred downstream of the –383 γ sequence; therefore, the replacement allele maintained the –1400 γ promoter (2.8-kb PCR product). In clone 2, recombination at the 5′ end occurred upstream of the –383 γ sequence; therefore, the replacement allele contained the –383 ^Aγ promoter (1.8-kb PCR product). Bsu36I digestion of PCR fragments derived with primers 3 and 4 is presented in the second panel of panel C. β^A fragments are digested, but β^S fragments are resistant to digestion.

Figure 2.

Genomic DNA and hemoglobin analysis of mice derived from targeted ES cell lines 1 and 2. (A) After removal of the PGK/Hygro marker from corrected ES cell clone 1 (–1400 γ-β^A/–1400 γ-β^S) and clone 2 (–383 γ-β^A/–1400 γ-β^S), cells were injected into C57BL/6 blastocysts. Chimeric males obtained from these blastocysts were mated with hα/hα, –1400 γ-β^S/mβ females and offspring were screened for the corrected genotypes (–1400 γ-β^A/–1400 γ-β^S and –383 γ-β^A/–1400 γ-β^S) by PCR of tail DNA and Bsu36I digestion. (B) IEF gel of hemolysates from mice identified as sickle and corrected animals in panel A. The last 3 lanes are human control hemolysates from a sickle patient, an individual with sickle trait, and an unaffected individual. Of interest, the ratio of HbA to HbS in corrected animals mimics the ratio in humans with sickle trait.

Figure 3.

Correction of abnormal RBC morphology in –383 γ-β^A–corrected mice. (A) Blood smear of an hβ^A/hβ^A control. (B) Blood smear of an hβ^S/hβ^S sickle animal with characteristic sickled erythrocytes and a pronounced reticulocytosis. (C) Blood smear of an hβ^A/hβ^S-corrected mouse. No sickled cells were observed in any field examined. Blood smears were stained with Wright-Giemsa and the magnification is 100×/1.40 NA oil objective (Nikon Eclipse E800 inverted microscope [Nikon, Tokyo, Japan], Hamamatsu C5810 Color Chilled 3CCD camera [Hamamatsu City, Japan]; Adobe Photoshop CS version 8.0 imaging software [San Jose, CA]). Data from –1400 γ-β^A–corrected mice are identical to –383 γ-β^A–corrected animals.

Figure 4.

Amelioration of spleen, liver, and kidney pathology in –383 γ-β^A– corrected mice. (A) Spleen, liver, and kidney sections were analyzed at low (10×/0.45 NA objective for spleen and kidney, 40×/0.75 NA objective for liver) and high (100×/1.40 NA oil objective) magnification. In –383 γ-β^A–corrected mice, normal splenic red and white pulp is observed, and virtually no pools of sickle erythrocytes or infarcts are evident. In livers of –383 γ-β^A animals, focal areas of necrosis and aggregation of sickled erythrocytes are not observed; also, extramedullary hematopoiesis and hemosiderin deposition are absent. Kidneys of –383 γ-β^A mice appear normal and free of the disruptive vascular RBC pooling. All sections were stained with hematoxylin-eosin. (B) Correction of splenomegaly in –383 γ-β^A–corrected mice. Data from –1400 γ-β^A–corrected mice are identical to –383 γ-β^A–corrected animals.