Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells

Abstract

Induced pluripotent stem cells (iPSCs) generated from patient fibroblasts could potentially be used as a source of autologous cells for transplantation in retinal disease. Patient-derived iPSCs, however, would still harbor disease-causing mutations. To generate healthy patient-derived cells, mutations might be repaired with new gene-editing technology based on the bacterial system of clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9, thereby yielding grafts that require no patient immunosuppression. We tested whether CRISPR/Cas9 could be used in patient-specific iPSCs to precisely repair an RPGR point mutation that causes X-linked retinitis pigmentosa (XLRP). Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce iPSCs carrying the patient's c.3070G > T mutation. The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene's repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele. This is the first report using CRISPR to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This important proof-of-concept finding supports the development of personalized iPSC-based transplantation therapies for retinal disease.

Figure 1. Clinical findings and iPSCs from an adult male XLRP patient.

Autofluorescence imaging of the patient’s [A] right and [B] left eyes shows hyperautofluorescent bull’s-eye lesions in the macula corresponding to [C,D] loss of photoreceptor layers (red arrows) on optical coherence tomography. [E] A skin punch biopsy sample (star) was taken from the patient and used to culture fibroblasts (arrowheads) that were then transfected with transcription factors to yield efficient formation of [F] iPSC colonies (asterisk) that stain positively for alkaline phosphatase. With immunocytochemistry and fluorescence microscopy, expression of pluripotency markers [G] SSEA4, Sox2, and [H] TRA-1-60 and OCT4 is also detected. iPSCs injected into a severe combined immunodeficiency (SCID) mouse formed teratomas that were found on histology to contain cell types derived from all three germ layers: [I,J] neuronal rosettes and pigmented cells from the ectoderm (EC), [K] smooth muscle cells from the mesoderm (ME), and [L] gland cells from the endoderm (EN). The expression of pluripotency markers in the patient-derived cells and the ability to generate differentiated cells from all three primordial germ layers confirms that patient iPSCs are pluripotent.

Figure 2. Validating gRNAs for gene targeting.

[A] Two candidate gRNAs, g58 and g59, were selected based on their precise specificity for the mutation site within the ORF15 region of RPGR. [B] gRNAs were inserted into a combined expression vector alongside Cas9. The gRNA/Cas9 construct was transfected into HEK293 cells and the RPGR gene was amplified by PCR using both AccuPrime high fidelity taq polymerase as well as Herculase II polymerase (due to the GC-rich content of ORF15). [C] An in vitro SURVEYOR assay (Transgenomic, Omaha, NE) of the PCR products shows that g58 is more effective at directing cleavage by Cas9 (bands for the expected cleavage products are indicated by red arrowheads).

Figure 3. Correction of RPGR mutation in patient iPSC line.

[A] Dideoxy sequencing of patient DNA (top panel) shows the “T” point mutation (black arrow). After gRNA/Cas9 and antisense single-stranded donor oligonucleotide (ssODN) were transfected into the patient-derived iPSC line (bottom panel), sequencing shows a minor “G” peak at the same position (red arrowhead), demonstrating correction of the G > T mutation in a fraction of IPSC. [B,C] Deep sequencing revealed 13% of sequences in treated cells had the correct “GAG” codon at the position of the patient’s original “TAG” stop codon point mutation. This correction was not observed when untransfected control cells were sequenced.