Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.
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This work was supported by grants from the NIH 2R01HL06427418A1 (M.A.K.), the Falk Medical Research Trust (M.A.K.), NIH DK098132 (C.J.S.) and the National Hemophilia Foundation (C.J.S.). A.F.M. was supported by intramural funds. We wish to acknowledge the Stanford Cell Sciences Imaging Facility for use of imaging equipment, the Stanford Genomics Facility for performing next-generation sequencing and the Stanford Department of Comparative Medicine’s Animal Histology Services and Stanford Veterinary Service Center for animal health examinations. We thank M.J. Finegold and J.G. Vilches-Moure for histology analysis. We also thank the Genome Engineering and iPSC Center at Washington University in St. Louis for performing next-generation sequencing and analysis. The microscope was funded by the Stanford Beckman Center. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the various funding bodies or universities involved.
S.T. and M.A.K. are named on patent applications related to this article. M.A.K. has commercial affiliations and stock and/or equity in companies with technology broadly related to this article. S.T. is a current employee of Daiichi-Sankyo Co., Ltd. G.A. is a current employee of Sangamo Therapeutics. The remaining authors declare no competing interests.
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Extended Data Fig. 1 rAAV-mediated gene targeting efficiency in vitro with various compounds and the assessment of toxicity from fludarabine or hydroxyurea administration in mice.
a, The effect of each compound on rAAV-mediated gene targeting efficiency was tested in Huh7 cells by treatment with each drug followed by transduction with an AAVDJ gene targeting vector, GAPDH-P2A-GFP. Flow cytometry analysis of GFP positive cells 2 days after treatment is shown. Data is representative of two independent experiments, each with three biological replicates. Data is displayed as the group mean with error bars representing s.d.; n = 3 biological replicate wells. Significance testing was performed by a one-way ANOVA with Dunnett’s multiple comparison test and two-tailed t test. P-value for Torin-1 was .0046, MG132 was .076, TSA was .301, and .0003 for Teniposide. b, Mice were treated with hydroxyurea (HU) (300 mg/kg/day) or fludarabine (Flu) (375 mg/kg/day) for three days. Body weight was monitored over 6 weeks after drug administration. Data is displayed as the group mean with error bars representing s.d.; n = 5 individual mice per group. Significance testing was performed by a two-way ANOVA. c, Serum alanine transaminase (ALT) levels were evaluated 3 days after the last drug administration. Data is displayed as the group mean with error bars representing s.d.; n = 5 mice per group. Significance testing was performed by a one-way ANOVA with Dunnett’s multiple comparison test. P-value for HU-treatment was <0.0001 and .4343 for Flu-treatment. d, Mice were treated with fludarabine (Flu) (375 mg/kg/day) or PBS for three days. Groups of two PBS mice or three fludarabine-treated mice were submitted 12 hours after the final drug injection (hpi). A second cohort of mice was submitted 30 days after injections (dpi). Livers were collected for H&E staining and subsequent blinded analysis by a trained veterinary pathologist. Representative images of H&E-stained liver sections from fludarabine treated mice are shown, at 12 hpi (left column) and 28 dpi (right column). Top panel is images with a 5x objective and bottom panel with a 20x objective. e, Livers were weighed at the time of collection at either 12 hpi or 30 dpi. Hematological analysis of these mice is available in Supplementary Table 2. Each point represents data from one animal with n = 2 or 3 per group from one independent experiment. Error bars represent s.d., where applicable. f, Total RNA was extracted from liver tissue from the animals in Fig. 2. qPCR using primers (Fw1 and Rv1) (Fig. 2d) determined the levels of endogenous albumin mRNA following treatment with PBS, HU, or Flu. Data is shown as the mean and error bars represent s.d; n = 5 animals per group. Statistical testing was performed with a one-way ANOVA analysis followed by Dunnett’s t-test from one independent experiment.
a, Detection of hF9 mRNA (red) in liver sections using RNAScope in situ hybridization. Liver sections of mice from non-injected, PBS-treated, and Flu-treated groups at the end of experiment in Fig. 2 (60 days after rAAV transduction, ~12 weeks of age) were used for hybridization and counterstained with hematoxylin. Representative images from each injected group are shown. b. Representative images of RNAScope in situ hybridization are provided. In the first two columns, mAlb mRNA was stained in a non-injected normal mouse to determine the albumin locus expression characteristics. Specificity for RNA was confirmed by digestion of tissue with RNAseA. c, Additional images of hF9 mRNA staining in mice injected with Alb-P2A-hF9 vector, with or without fludarabine treatment are shown at various levels of magnification.
a, Female neonatal mice were injected with i.p. with PBS or Fludarabine (375 mg/kg) and four hours later with rAAV8-Alb-P2A-hF9 (2.5 × 1013 vg/kg) at one week of age. Fludarabine was administered once more, one day after vector injection. Four weeks later, plasma was drawn and hF9 levels measured at various time points by ELISA. Data shown is from n = 9 mice per group and displayed as the group mean with error bars representing s.d. Significance testing was performed by two-way ANOVA analysis. b, Genomic DNA was extracted from liver tissues of male neonates (from Fig. 2h) 84 days after rAAV8-Alb-P2A-hF9 vector injection and qPCR was performed to quantify the amount of total AAV genomes. Actb primers were used for quantification of the number of diploid genomes. Data is displayed as the group mean with error bars representing s.d.; n = 6 PBS-treated and n = 8 fludarabine-treated mice. Significance in all qPCR data in this figure was determined using two-tailed Student’s T tests, after testing for normal distribution with Shapiro-Wilk test and F tests for variance. For all qPCR data Actb mRNA was used for normalization and data is shown as relative expression to the PBS-treated group. c, Male and female mouse weights were monitored following treatment with PBS or Fludarabine and rAAV8-Alb-P2A-hF9. Data is displayed as the group mean with error bars representing s.d.; n = 17 PBS-treated and n = 18 fludarabine-treated mice. Significance testing was performed by 2-way ANOVA analysis. d, On-target fusion hF9 mRNA was quantified from liver tissues of the male mice at the end of the time course, using primers Fw1 and Rv2 (from Fig. 2e). Data is displayed as the group mean with error bars representing s.d.; n = 6 PBS-treated and n = 8 fludarabine-treated mice. A Shapiro-Wilk test was used to test for normal distribution, and F test determined variation between groups, and two-tailed Student’s t-test was used to test for significance in d-g. p-value was .008. e, Total hF9 mRNA was also quantified from the male mice using primers Fw2 and Rv3 (from Fig. 2f). Data is displayed as the group mean with error bars representing s.d.; n = 6 PBS-treated and n = 8 fludarabine-treated mice. p-value was .0035. f, The fraction of hF9 fusion mRNA derived from on-target HR out of the total amount of hF9 mRNA is shown. Data is displayed as the group mean with error bars representing s.d.; n = 6 PBS-treated and n = 8 fludarabine-treated mice. g, mAlb mRNA was quantified from the male mice using primers Fw1 and Rv1 (from Fig. 2). Data is displayed as the group mean with error bars representing s.d.; n = 6 PBS-treated and n = 8 fludarabine-treated mice.
a, Four-week-old mice were treated with various Flu doses, differing in the length of administration, as described in Fig. 2i, as well as injected i.v. with the gene targeting Alb-P2A-hF9 vector (1 × 1011 vg/mouse) on Day 1 of Flu treatment. Plasma hF9 protein levels from the same mice in Fig. 2i were determined via ELISA for a 90-day time course. Data is displayed as the group mean with error bars representing s.d.; n = 4 mice per group. Significance testing was performed by a two-way ANOVA with Dunnett’s multiple comparison test. P-value of the 3-day treatment was .0004 and < .0001 for the 5-day treatment. b, Body weight of mice in each treated group were monitored throughout the time course. Data is displayed as the group mean with error bars representing s.d.; n = 4 mice per group. Statistics were performed by two-way ANOVA. P-value for the 1-day treatment was .001.
Extended Data Fig. 5 The effect of DEN administration on the efficiency of gene targeting in mice liver.
a, DEN (10 or 30 mg/kg) was administered through a single i.p. injection per day for three days. Mice were also injected i.v. with rAAV8 packaged Alb-P2A-hF9 gene targeting vector (1.0 × 1011 vg/mouse) on Day 1. Body weight was measured at the indicated time points. Data is displayed as the group mean with error bars representing s.d.; n = 4 mice per group. Significance testing was performed by two-way ANOVA with Dunnett’s multiple comparison test. P-value for DEN-10 treatment was .1589 and .0065 for DEN-30 treatment. b, Plasma hF9 protein levels in each treatment group was determined by ELISA. Data is displayed as the group mean with error bars representing s.d.; n = 4 mice per group. Significance testing was performed by one-way ANOVA with Dunnett’s multiple comparison test. P-value for DEN-10 treatment was .1301 and < .0001 for DEN-30 treatment. c-f, Total RNA was extracted from liver tissues and qPCR was performed to quantify the expression levels of (c) total hF9 mRNA, (d) endogenous albumin mRNA and (e) on-target integration derived Alb-P2A-hF9 fusion mRNA. (f) The fraction of hF9 fusion mRNA derived from on-target HR out of the total amount of hF9 mRNA is given. Actb mRNA was used for normalization and each data is shown as relative expression to the vehicle (saline)-injected control group. Data is displayed as the group mean with error bars representing s.d.; n = 4 mice per group. Significance was determined using two-tailed unpaired t tests for normally distributed data with equal variance or Welch’s correction for data with significantly different variance. Testing of non-normally distributed data was performed with a non-parametric Mann-Whitney U test.
Extended Data Fig. 6 Fludarabine does not increase transduction of CRISPR/Cas9 encoded rAAV vectors or saCas9 mRNA.
a-b, At the conclusion of the two-week experiment in Fig. 5, DNA was extracted from livers of rAAV-injected animals. Viral genomes were quantified by qPCR and normalized to cellular genomic DNA. The donor rAAV repair template AAV-GFP-HDR is presented on the top panel (a), while AAV-Cas9 is displayed in the middle panel (b). Each point represents data from one animal, with n = 3 to 5 mice per group. Error bars represent s.d. Significance testing was performed using two-tailed t tests or Mann-Whitney U test if data was non-normally distributed. c, Mice were injected with SaCas9 (6.0E12 vg/kg) following fludarabine or PBS treatment as in Fig. 5f–g. Two weeks later, total RNA was extracted and saCas9 mRNA levels quantified by qPCR. n = 3 mice per group. Error bars represent s.d. Significance was determined using two-tailed t test.
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Tsuji, S., Stephens, C.J., Bortolussi, G. et al. Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice. Nat Biotechnol 40, 1285–1294 (2022). https://doi.org/10.1038/s41587-022-01240-2