Gene correction by homologous recombination with zinc finger nucleases in primary cells from a mouse model of a generic recessive genetic disease

Abstract

Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

Figure 1

Construction of targeting vector and screening of ES clones. (a) Schematic of targeting vector and screening strategy. Correct knock-in of the GFP* reporter cassette to the ROSA26 locus causes the addition of an upstream EcoRV site resulting in a 2.2 kb fragment upon digestion. (b) Southern blot analysis of correct knock-in ES clones. Genomic DNA was purified from ES clones, digested with EcoRV and used for Southern analysis. ES, embryonic stem; kb, kilobase; PGK-DTA, diphtheria toxin cassette; PGK-Neo, neomycin resistance cassette; SA, splice acceptor.

Figure 2

Gene targeting in ES cells. (a) Titration of donor plasmid and ZFNs in ES cells. Cells were transfected using Lipofectamine 2000 with different amounts of donor and ZFN plasmids. From left to right, amounts of donor plasmid increased, whereas ZFN amounts decreased. Transfection was performed with Lipofectamine 2000, and the amounts of donor plasmid and ZFNs in each lane are indicated as donor (ng), ZFN1 (ng)/ZFN2 (ng). (1) 100, 350/350; (2) 400, 200/200; (3) 600, 100/100; (4) 700, 50/50; (5) 750, 25/25; (6) 775, 13/13. Fifteen hours after transfection, media were changed to normal ESLX. Gene-targeting events were analyzed 4 days after transfection using flow cytometry. (b) Gene targeting in ES cells using two sets of GFP-ZFNs, with and without vinblastine treatment. Cells were plated in ESLX with and without vinblastine (100 nmol/l). Transfection mix was added, and 15 hours later, removed and replated with ESLX. Gene-targeting events were analyzed as in a. In the upper left of each graph is a representative flow cytometry plot after targeting in which GFP fluorescence is measured in the y axis and background orange fluorescence along the x axis. The number in the left corner of the flow plot is the percentage of GFP⁺ cells. ES, embryonic stem; ZFN, zinc finger nuclease.

Figure 3

Teratoma formation assay. Two teratomas resulting from subcutaneous injection of targeted cells were harvested from nude mice. (a) Teratomas were initiated by gene-targeted ES cells as demonstrated by GFP fluorescence. Teratomas were also photographed under a Cy3 filter to show background fluorescence. (b) Sections were cut and stained with hematoxylin and eosin to identify tissue structures indicated in the figure. n, neuroepithelial cells; o, osteoid tissue; e, epithelium.

Figure 4

ZFN-mediated gene targeting in primary cells. (a) Gene targeting in homozygous and heterozygous ROSA-3T3s: transfections were performed using Lipofectamine 2000 with the indicated amounts of donor plasmid and ZFNs. The next day, media were changed, and on day 4, gene-targeting events were analyzed. (b,c) Gene targeting in MAF/MEFs: transfection of plasmids was performed by nucleofection using 2 µg of each ZFN and the indicated amounts of donor plasmid. Gene-targeting events were analyzed 4 days after transfection. (d) Gene targeting in astrocytes: targeting was performed in the same manner as MAFs/MEFs. In the upper left of each graph is a representative flow cytometry plot after targeting in which GFP fluorescence is measured in the y axis and background orange fluorescence along the x axis. The number in the left corner of the flow plot is the percentage of GFP⁺ cells. The corrected ROSA-3T3 cells show much higher GFP fluorescence than the primary cells demonstrating that although the ROSA26 locus is ubiquitously expressed, expression levels from the locus vary significantly depending on the cell type. Data are presented as mean ± SEM (*P < 0.05). ZFN, zinc finger nuclease.

Figure 5

Toxicity assay and transplantation of gene-targeted adult fibroblasts. (a) Toxicity of GFP-ZFNs in adult fibroblasts was measured using a fluorescence reporter assay. Cells were transfected with a tdTomato reporter along with an I-SceI, GFP-ZFN, or CCR5-ZFN expression plasmids. ZFN toxicity in cells is reported as fluorescence lost (due to cytotoxic effect) compared to cells transfected with the I-SceI expression plasmid. (b) For transplantation, adult fibroblasts underwent gene correction by nucleofection of 2 µg of each ZFN expression plasmid and 10 µg of donor plasmid. Gene targeting was measured by flow cytometry immediately before transplantation 6 days after nucleofection. Fibroblasts were then injected subcutaneously in a Matrigel matrix. Two weeks after transplantation, the Matrigel plug and surrounding skin were excised, cells dissociated, and plated to allow for fibroblast enrichment from other host-derived infiltrating cell types. On day 6, cells were harvested and analyzed using flow cytometry. ZFN, zinc finger nuclease.