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. 2016 Apr;24(4):697-706.
doi: 10.1038/mt.2016.35. Epub 2016 Feb 11.

In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA

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Free PMC article

In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA

Dustin J Landau et al. Mol Ther. .
Free PMC article

Abstract

Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P < 0.04). Furthermore, transgene integration has been confirmed by sequencing in the majority of the mice treated with both vectors. Targeted alleles were 4.6-fold more common in livers of mice with GSD Ia, as compared with normal littermates, at 8 months following vector administration (P < 0.02). This suggests a selective advantage for vector-transduced hepatocytes following ZFN-mediated integration of the G6Pase vector. A short-term experiment also showed that 3-month-old mice receiving the ZFN had significantly-improved biochemical correction, in comparison with mice that received the donor vector alone. These data suggest that the use of ZFNs to drive integration of G6Pase at a safe harbor locus might improve vector persistence and efficacy, and lower mortality in GSD Ia.

Figures

Figure 1
Figure 1
Gene editing at the ROSA26 locus occurs specifically in mice administered AAV-ZFN/8. (a) The Surveyor assay demonstrated that indels indicative of nonhomologous end-joining occurred only in mice that received AAV-ZFN/8. G6pc-/- knockout mice had significantly more ZFN target site-specific DNA repair events than wild-type mice. (b) Representative gel images illustrating the difference between knockout and wild-type mice. Surveyor nuclease-digested product bands are indicated by black arrows. “WT, UT”: untreated wild-type mouse control, representative of the control mice that did not receive AAV2/8-ZFN. “-n”: no-Surveyor nuclease control of wild-type mouse PCR product. Error bars: mean ± SD.
Figure 2
Figure 2
Vector transgenes are integrated into the ROSA26 locus. G6pc-/- knockout and wild-type mice were treated with both AAV2/8-ZFN and AAV2/8-RoG6P or only AAV2/8-RoG6P prior to analysis at 8 months of age. (a) Illustration of the predicted integration structure following homology-directed repair between the mouse genomic ROSA26 cleavage site and the AAV2/8-RoG6P viral genome donor homology arms. Primer locations are denoted P1-P4. (b) Representative gel of PCR products from all mice following the nested round of PCR. Predicted product size from an HDR event is 1,335 bp. Ladder is 1 kb Plus (Invitrogen, Waltham, MA). White arrow indicates the 1 kb position. “PBS”: PCR reaction was run on DNA from an affected mouse injected with PBS instead of vector and collected at 2 weeks of age. “NTC”: No template control PCR reaction, run using water instead of DNA as template.
Figure 3
Figure 3
Donor vector genomes persist better than ZFN genomes in hepatocytes. (a) AAV2/8-RoG6P and AAV2/8-ZFN genomes were quantified by qPCR of G6pc-/- mouse liver DNA at 8 months of age. The ZFN genome was found to exist at much lower levels than the RoG6P genome when both were delivered in equal amounts by AAV vectors (P < 0.014). (b) AAV2/9-RoG6P genomes were quantified by qPCR of knockout mouse liver DNA at 3 months of age. The G6Pase transgene levels did not differ between treatment groups. Error bars: mean ± SD.
Figure 4
Figure 4
Prolonged survival of G6pc-/- mice depends on administration of AAV2/8-ZFN. (a) All mice that received AAV-ZFN/8 in addition to AAV-RoG6P/8 survived for 8 months. In contrast, only 43% of mice that received AAV-RoG6P/8 alone survived for the same duration, showing a significant difference (P < 0.04, using the log-rank test). (b) Female mice showed the same requirement for ZFN as the population as a whole, with 100% survival among mice receiving both vectors and only 33% among those receiving the donor vector alone (P < 0.05 using the log-rank test). Only postweaning mice are shown, because sex cannot be determined prior to weaning.
Figure 5
Figure 5
Markers of therapeutic effect show no difference between treatment groups at 8 months of age. (a) Blood glucose at 5 months following treatment. Both treatment groups showed similar blood glucose. Both were significantly different than previously published values for untreated G6pc-/- knockout mice. Two-week-old untreated mice were used because knockout mice do not survive past 3 weeks. (b) Hepatic G6Pase activity at 8 months of age in G6pc-/- knockout mice. No difference was observed between the two treatment groups or between either group and untreated knockout mice. Six-month-old WT mice are shown for positive reference. (c) Hepatic glycogen accumulation in treated and untreated knockout mice, and wild-type mice. Both treatment groups had comparable glycogen accumulation in their livers, though both groups had significantly more glycogen than wild-type controls. Glycogen in treated mice was also reduced compared with historical data on hepatic glycogen in knockout mice. An untreated control used to verify day-to-day experimental consistency was consistent with historical results. (d) Relative human G6Pase expression (vector-specific) normalized to mouse β-actin, as determined by RT-qPCR, was equivalent whether or not mice received AAV2/8-ZFN. However, both groups showed highly elevated expression compared with the background signal for untreated mice. Error bars: mean ± SD.
Figure 6
Figure 6
Administration of AAV2/9-ZFN enhanced biochemical correction at 3 months of age. Groups of mice with GSD Ia were treated with AAV2/9-RoG6P+ AAV2/9-ZFN (n = 11), or AAV2/9-RoG6P alone (n = 7). (a) Blood glucose at 3 months of age following an 8-hour fast. No difference was observed between the two groups in this assay, but both were improved compared with untreated knockout mice. Groups of “no vector” G6pc-/- mice (n = 7); and untreated wild-type mice (WT; n = 4) were controls. (b) G6Pase activity in the liver was significantly higher in dual-vector-treated mice, in comparison with either “no vector” G6pc-/- mice or single-vector-treated mice. Groups of “no vector” G6pc-/- mice (n = 3); and untreated wild-type mice (WT; n = 6) were controls. (c) Hepatic glycogen accumulation was reduced in in dual-vector treated mice, in comparison with either “no vector” G6pc-/- mice or single-vector-treated mice. No significant difference was observed between double-vector mice and wild-type mice. Error bars: mean ± SD.
Figure 7
Figure 7
G6Pase histochemical staining in the livers of 3-month-old mice reveals an improvement in G6Pase-expressing cells in mice receiving dual-vector treatment. (a) Representative G6Pase staining of an untreated knockout mouse at 10 days of age. Note the absence of expression throughout the liver. (b) Representative staining section of a wild-type mouse liver. Note the uniform brown stain throughout all hepatocytes. (c,d) Representative sections from mice that received the AAV2/9-RoG6P donor vector only, or the donor vector as well as AAV2/9-ZFN, respectively. (e) G6Pase-positive cell counts from both treatment groups (n = 7 each), demonstrating a significant enhancement in positive cell counts when both vectors were administered (P < 0.03). Error bars: mean ± SD.
Figure 8
Figure 8
Transduction of mouse Liver with AAV2/8-RoGFP. (a) Liver section one month following administration of AAV2/8-RoGFP or PBS. (b) Three representative liver sections 6 months following AAV2/8-RoGFP administration. (c) Three representative liver sections 6 months following AAV2/8-RoGFP and AAV2/8-ZFN administration. (d) Quantification of GFP+ cells per high magnification field using a Zeiss LSM 510 inverted confocal microscope; GFP+ cells counted from two representative fields of view (10× objective) of four mouse livers per each group and average number of cells calculated. Magnification 100×; bar is 100 µm. Mice received 1E13 vp/g IP at 2 weeks of age. Liver sections are 30 µm. Error bars: mean ± SD.

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