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. 2019 Dec 24;17:234-245.
doi: 10.1016/j.omtm.2019.12.004. eCollection 2020 Jun 12.

AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria

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Free PMC article

AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria

Daelyn Y Richards et al. Mol Ther Methods Clin Dev. .
Free PMC article

Abstract

Phenylketonuria (PKU) due to recessively inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia, which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake. In this study, liver-tropic recombinant AAV2/8 vectors were used to deliver CRISPR/Cas9 machinery and facilitate correction of the Pah enu2 allele by homologous recombination. Additionally, a non-homologous end joining (NHEJ) inhibitor, vanillin, was co-administered with the viral drug to promote homology-directed repair (HDR) with the AAV-provided repair template. This combinatorial drug administration allowed for lifelong, permanent correction of the Pah enu2 allele in a portion of treated hepatocytes of mice with PKU, yielding partial restoration of liver PAH activity, substantial reduction of blood phenylalanine, and prevention of maternal PKU effects during breeding. This work reveals that CRISPR/Cas9 gene editing is a promising tool for permanent PKU gene editing.

Keywords: CRISPR/Cas9; gene correction; gene editing; gene therapy; homology directed repair; phenylalanine; phenylalanine hydroxylase; phenylketonuria.

Figures

Figure 1
Figure 1
Design of In Vivo CRISPR/Cas9 Gene-Editing Strategy in Pahenu2 Mice (A) Dual AAV constructs. The Cas9-expressing rAAV2/8 vector genome was 5.2 kb in length and contained the S. pyogenes Cas9 gene driven by a transthyretin-based liver-specific promoter (LSP). The second rAAV2/8 vector genome was 2.7 kb in length and contained a 2-kb repair template flanking 1 kb in either direction of the enu2 missense mutation, as well as guide 1 driven by a human U6 promoter. The repair template sequence pictured below the vector genomes contained the wild-type Pah exon 7 sequence with correction of the Pahenu2 mutation, as well as purposefully introduced synonymous and intronic mutations (denoted in red) to hinder Cas9 cutting of corrected alleles. Uppercase denotes exonic Pah sequence, and lowercase indicates intronic sequence. The wild-type protein sequence of the repair template demonstrates correction of the Ser263Phe and a synonymous mutation at Pro279. The Pahenu2 allele below reveals the c.835T > C mutation in green and the guide targeted sequence in blue. The symbol “//” indicates 46-bp DNA and 15-aa separation. (B) Diagram of expected Cas9-mediated DNA cutting and cellular repair. Guide 1, indicated in blue, directs Cas9 (yellow oval) to induce a double-strand break (DSB) (indicated by the lightning bolt) 46 bp downstream of the c.835T > C mutation (black arrow) in the Pahenu2 allele; double lines indicate double-stranded DNA (dsDNA), where thick lines are exons and thin lines are introns. Potential DSB intracellular pathways that the cell may utilize to repair the Cas9 DSB are depicted below. The repair template, indicated in red, may be incorporated into the genome by homology-directed repair (HDR), pictured on the left. Alternatively, non-homologous end joining (NHEJ) restores the mutant allele (not shown); or alternate end joining (Alt-EJ), pictured at the right, results in a small insertion or deletion (indel, indicated by the bold X). Either NHEJ or Alt-EJ fails to incorporate the repair template. Vanillin is a potent NHEJ inhibitor (indicated by the smaller arrow at the right) that directs DSB repair toward HDR (indicated by the larger arrow at the left). (C) Table of experimental animal cohorts. Three experimental animal cohorts were tested in this study. The cohort with dual AAV plus vanillin (dAAV+Van) received both viral vectors plus vanillin, with a total of 11 animals treated: 5 males and 6 females. The dual-AAV (dAAV) cohort received both viruses but no vanillin, with a total of 10 animals treated: 4 males and 6 females. The cohort with repair template vector plus vanillin (rtAAV+Van) received only the repair template virus with vanillin, with a total of 9 animals treated: 2 males and 7 females.
Figure 2
Figure 2
Efficacy of In Vivo CRISPR/Cas9 Gene Editing in Pahenu2 Mice (A) Experimental timeline. Pahenu2/enu2 mice were born, administered the initial dose of drug in the first week of life, weaned at 4 weeks, and placed on standard mouse chow. At week 5, mice were re-dosed with drug. Between 16 and 24 weeks, mice from each cohort were harvested, and one breeding pair per treatment group was placed on high-energy mouse chow and allowed to breed. By 28 weeks, one successful litter was produced by the dAAV+Van pair, while the dAAV and rtAAV+Van pairs did not successfully breed. At this point, the dAAV and rtAAV+Van breeding pairs were euthanized for tissue collection. The dAAV+Van breeder pair produced three more successful litters and were euthanized at 65 weeks. (B) Serum phenylalanine. The graph shows serum phenylalanine levels of dAAV+Van, dAAV, and rtAAV+Van animals at time of euthanasia. The left y axis indicates serum Phe in micromol/l (Mmicromol/l, while the right y axis indicates serum Phe in micrograms per deciliter. The red line indicates the mean serum phenylalanine concentration of an unrelated cohort of untreated male and female Pahenu2/enu2 animals combined consuming standard mouse chow. The black line indicates the upper limit of serum Phe levels in wild-type mice (180 μM or 3 mg/dL). The x axis is separated by treatment group, in the order of dAAV+Van, dAAV, and rtAAV+Van from left to right, whisker plots depict mean serum phenylalanine ± 2 SD, and each dot on the graph indicates an individual animal. Males are depicted with triangles and females with circles. Red indicates 65-week-old animals, and black indicates 16- to 24-week-old animals. The average Phe levels were 685 μM, 1,518 μM, and 1,314 μM, respectively; and ranges were 252–1,168 μM, 1,231–1,863 μM, and 827–1,850 μM, respectively. Intergroup comparisons (depicted as brackets above the treatment groups) revealed significant differences between dAAV+Van and dAAV (p < 0.0001, ∗∗∗∗) and between dAAV+Van and rtAAV+Van (p = 0.0001, ∗∗∗). (C) PAH enzyme activity. The graph shows the PAH enzyme activity levels of dAAV+Van, dAAV, and rtAAV+Van animals at time of euthanasia. The y axis indicates percent wild-type PAH activity; the x axis is separated by treatment group in the order of dAAV+Van, dAAV, and rtAAV+Van from left to right, whisker plots depict mean liver PAH activity ± 2 SD, and each dot on the graph indicates an individual animal. Males are depicted with triangles, and females are depicted with circles. Red indicates 65-week-old animals, and black indicates 16- to 24-week-old animals. The average activity was 9.5%, 1.2%, and 0.5%, respectively, and ranges were 3.3%–25%, 0%–5.7%, and 0%–2.3%, respectively. Intergroup comparison reveals a significant difference between dAAV+Van and dAAV (p = 0.0002, ∗∗∗) and between dAAV+Van and rtAAV+Van (p < 0.0001, ∗∗∗∗). (D) Coat color at euthanasia. Photograph of coat colors of mice from each treatment group at 16–24 weeks of age in comparison to wild-type or untreated Pahenu2/enu2 mice. Mice are lined up from left to right as wild-type, untreated Pahenu2/enu2, dAAV+Van, rtAAV+Van, and dAAV.
Figure 3
Figure 3
On- and Off-Target CRISPR/Cas9 DNA Analyses (A) Table of Top Four Cutting Sites for SpCas9+Guide 1 in the Mouse Genome. Two in silico tools, Cas-OFFinder and COSMID, were used to identify the top four SpCas9-Guide1 cutting sites in the mouse genome. Locations of cutting sites are described in the first four columns, followed by target sequence (black) and PAM (red) in the next column. The last column indicates the COSMID score, in which the lowest, 0, indicated the highest probability of cutting (the target site) and ranged to over 40, with the lowest probability of cutting. The top three off-target sites located on chr 10, chr 12, and chr X were scored as 0.17, 1.33, and 2.93, respectively, as indicated in the last column. (B) On-target repair. The graph shows percent reads of indels (blue) or “full” HDR (including exonic and intronic sequences) (black) in dAAV+Van, dAAV, and rtAAV+Van animals at time of euthanasia. The y axis indicates percent reads of overall NGS reads; the x axis is separated by treatment group in order of dAAV+Van, dAAV, and rtAAV+Van from left to right, whisker plots depict the mean percent reads ± 2 SD, and each dot on the graph indicates an individual animal. Males are depicted with triangles, and females are depicted with circles. Red indicates animals at 65 weeks, and black indicates animals at 16–24 weeks. The average percentages of indel reads were 21.27%, 12.92%, and 6.58%, respectively, with ranges of 12.25%–39.29%, 2.2%–39.54%, and 2.52%–19.34%, respectively. The average percentages of HDR reads were 13.06%, 0.96%, and 3.51%, respectively, and ranges were 4.13%–36.78%, 0%– 3.90%, and 0%–12.57%, respectively. Two-way ANOVA revealed a significant difference between treatment groups, F(2, 54) = 12.87, p < 0.0001; and DNA repair, F(1, 54) = 13.11, p = 0.0006. Brackets above the treatment groups depict post hoc intergroup statistical comparisons. - p < 0.05; ∗∗ - p < 0.01, ∗∗∗ - p < 0.001, ∗∗∗∗ - p < 0.0001 (C) Functional reads. The graph indicates functional reads of exonic-only NGS reads in dAAV+Van, dAAV, and rtAAV+Van animals at time of euthanasia. The y axis indicates the percentages of functional Pah-exon7 NGS reads. The x axis is separated by treatment group in order of dAAV+Van, dAAV, and rtAAV+Van from left to right, whisker plots depict the mean percent functional reads ± 2 SD, and each dot on the graph indicates an individual animal. Males are depicted with triangles, and females are depicted with circles. Red indicates 65-week-old animals, and black indicates 16- to 24-week-old animals. The averages of functional reads were 7.54%, 0.70%, and 2.06%, respectively; and ranges were 1.94%–23.97%, 0.11%–2.54%, and 0.08%–5.58%, respectively. One-way ANOVA revealed significant difference of treatment groups, F(2, 27) = 5.805, p = 0.0080; with a p value of 0.0093 between dAAV+Van and dAAV and a p value of 0.048 between dAAV+Van and rtAAV+Van. Brackets above the treatment groups depict post hoc intergroup statistical comparisons. - p < 0.05; ∗∗ - p < 0.01. (D) Sanger sequencing of dAAV+Van enu2 allele correction. The Sanger sequencing chromatogram shows low levels of correction in liver genomic DNA, as indicated with the large arrow pointing to the Pahenu2 c.835T > C (indicated with “C”) site that contains a small peak of corrected (indicated with “T”) DNA. (E) Percent off-target indels. The graph shows the percentage of overall NGS reads containing indels in the top three off-target regions identified in Figure 3A. The y axis shows the percentage of indels; the x axis is separated by treatment groups in the order of dAAV+Van, dAAV, and rtAAV+Van from left to right, with further nested separation of each chromosomal region in order of highest likelihood of cutting to lowest; chr 10, chr 12, and chr X, for each treatment group. Each animal is represented by a blue, a red, and a black dot, indicating chr 10, chr 12, and chr X, respectively. Whisker plots depict the mean percent off target indels ± 2 SD. The average percentages of indels for chr 10 were 0.29%, 0.35%, and 0.18%, respectively; for chr 12, they were 0.33%, 0.38%, and 0.89%, respectively; and for chr X, they were 0.05%, 0.09%, and 0.04%, respectively. The ranges of percent indels for chr 10 were 0%–0.94%, 0.07%–1.44%, and 0%–0.34%, respectively; for chr 12, they were 0%–0.91%, 0%–0.80%, and 0.19%–1.66%, respectively; and for chr X, they were 0%–0.17%, 0%–0.33%, and 0%–0.16%, respectively. One-way ANOVA of each chromosomal region between treatment groups revealed significant difference in only the chr 12 region, F(2, 27) = 5.709, p = 0.0086; with a p value of 0.012 between rtAAV+Van and dAAV+Van and a p value of 0.0248 between rtAAV+Van and dAAV. Brackets above the treatment groups depict post hoc intergroup statistical comparisons. - p < 0.05.
Figure 4
Figure 4
Efficacy of In Vivo CRISPR/Cas9 Gene Editing in dAAV+Van-Treated Animals (A) Longitudinal Phe in dAAV+Van animals. Plot of long-term (up to 65 weeks) serum phenylalanine concentration in the dAAV+Van cohort mice. The red line denotes the mean serum phenylalanine concentration of an unrelated cohort of untreated male and female combined Pahenu2/enu2 animals consuming standard mouse chow. The black line indicates the upper limit of serum Phe concentration in wild-type C57BL/6 mice (180 μM). For the time points for which Phe measurements in multiple mice were available, the data are presented as mean ± SEM. Data at 40–65 weeks are means of the two dAAV+Van animals, one male and one female, that remained a breeding pair until euthanasia. (B) Improved growth in dAAV+Van-treated animals. Three untreated age-matched males and three untreated age matched females were compared to dAAV+Van-treated animals. A three-way ANOVA comparing age, treatment group (control versus treated), and sex showed statistically significant differences between these animals, albeit low sample sizes, F(1, 10) = 10.44, p = 0.0090. Post hoc intergroup comparisons revealed consistent significant differences between each treated and untreated group of either sex (p < 0.0001) as depicted by the brackets with asterisks (∗∗∗∗). (C) Correlation between liver PAH activity and functional Pah exon 7 reads in dAAV+Van-treated mice. Plot of percent wild-type liver PAH enzyme activity on y axis versus the percent functional Pah exon 7 reads on the x axis with linear regression analysis. Each point is a single dAAV+Van mouse at euthanasia. Graph shows percent functional Pah exon 7 reads on the x axis and percent wild-type PAH enzyme activity on the y axis. Each point represents a dAAV+Van animal at time of euthanasia. (D) Correlation between serum Phe and percent functional Pah exon 7 reads in dAAV+Van-treated animals. Plot of serum Phe on the y axis versus percent functional Pah exon 7 reads on the x axis. Each point represents a dAAV+Van animal at time of euthanasia. The data were fit with a non-weighted, non-linear exponential one-phase decay using least-squares regression. (E) Correlation between serum Phe and liver PAH enzyme activity in dAAV+Van-treated animals. Plot of serum Phe on the y axis versus percent wild-type liver PAH enzyme activity on the x axis. Each point represents a dAAV+Van animal at time of euthanasia. The data were fit with a non-weighted, non-linear exponential one-phase decay using least-squares regression.

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