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In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases

From Stem Cells
By Stuart P. Atkinson

Induced pluripotent stem cell (iPSC) technology not only has the capability to produce patient-specific but also disease-specific pluripotent cells. In monogenic diseases, there lies the potential for gene modification allowing differentiation of therapeutically relevant “corrected” cells, as has been demonstrated in mouse iPSCs where a sickle cell anemia mutation was targeted and corrected through the use of homologous recombination and an exogenous DNA donor template (Hanna et al). However, moving this technology into human cells is technically difficult due to the reported low efficiency of gene targeting and the requirement for large donor templates. However, the use of engineered zinc finger nucleases (ZFNs), customizable restriction enzymes which can be targeted to a desired DNA element (Kim et al) may allow higher gene targeting efficiency. The groups of Marius Wernig and J. Keith Joung from the Harvard Medical School, Boston, Massachusetts, USA have investigated the ability to efficiently correct a genetic mutation in human iPSCs (Sebastiano et al) and have shown the applicability of ZFN technology to human iPSCs containing a sickle cell anemia mutation, demonstrating a promising means of genetic correction.

An integrating lentivirus containing a polycistronic cassette encoding the four reprogramming factors (OSKM) flanked by loxP sites was used to reprogram fibroblasts sampled from two sickle cell anemia patients. Patient 1 harbours an E6V mutation in one b-globin allele and a splice donor site mutation at the end of exon 1 in the other allele, while patient 2 is homozygous for the E6V mutation. Colonies appeared after 4–6 weeks and from these four lines (I and II from patient 1 and III and IV from patient 2) were selected for continued expansion which, after a brief period of culture on feeders, were moved to feeder-free conditions. All lines were deemed pluripotent, however cells from Clone IV were unable to form teratomas. Southern blot analysis revealed the presence of three proviral integrations in clone I, two in clone II, one in clone III and one in clone IV, although the viral transgenes were effectively silenced. Upon transfection with a Cre recombinase plasmid into Clones I and II, vector sequences were removed and 2 subclones from each cell line retained a fully undifferentiated state as judged by the expression of a battery of pluripotency markers and a normal karyotype.

ZFNs were then specifically engineered to two sites within 25 base pairs (bp) of the sickle cell anemia mutation in patient-derived iPSC clones, one at the mutation site and one around 24bp away. These showed effective activity in human HEK293 cells and so ZFN pairs (A, B and C) were taken forward for testing in hiPSCs. This strategy also requires a donor template to introduce the correction and so a donor template was constructed that harboured 1.6 kb of b-globin gene sequence centred at the approximate location of the E6V mutation but which codes for the wild-type E6 codon. The donor template also contained a puromycin or neomycin-resistance gene cassette to allow for drug-selection to identify desired recombinants, and was flanked by loxP sites to enable its subsequent excision using Cre recombinase. In order to correct the mutation, ZFN pairs A, B or C were transfected alongside the donor template into Clone I iPSCs and each gave puromycin-resistant colonies. PCR screening for the puromycin-resistance gene in the endogenous b-globin demonstrated targeting efficiency as high as 37.9% (mean of 9.8%) and DNA sequencing of 13 positive lines revealed the presence of a successfully targeted allele in all of these cells. Of these 13 lines, seven were clonal populations with all but one having undergone gene targeting of the sickle cell allele while six of the 13 were mixed populations containing both targeted and non-targeted cells. Following further expansion in culture, the six mixed populations resolved into clonal lines: two consisting of cells that had successfully been targeted in the sickle E6V allele and four consisting of non-targeted cells. iPSC lines II and IV could also be corrected and, importantly, analysis revealed the absence of ZFN-induced indel (insertion or deletion) mutations suggesting that all three ZFN pairs can efficiently induce stable gene correction of the sickle cell mutation in hiPSCs without inducing additional mutagenesis of the other b-globin allele in the same cell. Although ZFNs did not cause mutagenic effects at the targeted locus, unwanted ‘‘off-target’’ mutagenic events are a concern, such as the potential targeting of γ-globin and δ-globin which are highly related paralogous genes each possessing DNA sequences that are very similar to the target sites for the ZFN pairs. Reassuringly, analysis of the 9 corrected clones had no evidence of alterations at these sites and nor did other related sites (differing by a single nucleotide) show any alterations.

Following this, a Cre-recombinase-GFP fusion protein was expressed in the iPSCs to remove the puromycin resistance gene and the randomly integrated reprogramming factors through adenoviral transduction. Following infection, selection for high-GFP and replating, single colonies were picked after two to three weeks and expanded. Analysis confirmed excision of the puromycin-resistance cassette and the reprogramming factor cassette in iPSCs, and cells were judged to be fully pluripotent by expression of multiple pluripotency markers and in vitro differentiation towards derivatives of the three germ layers. All cell lines tested were karyotypically normal with no evidence of translocations caused by transient ZFN and Cre recombinase expression.

Overall, this work provides an important proof-of-principle and lays down a framework for future studies, using widely available ZFN technology with specific and efficient results. However, further more detailed analysis to define the full extent of genetic alterations introduced into iPSCs during generation and repair may be required. Recent studies have been forging ahead in this area (Reviewed in Collin and Lako) and ZFNs and associated technologies should ultimately prove to be a very useful tool.



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