In vivo base editing rescues ADPKD in a humanized mouse model

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: major_revision

Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is a genetic kidney disease, caused by mutations of the PKD1 and PKD2 genes, characterized by the development of renal cysts and extrarenal complications, such as cardiac hypertrophy. Recently, a revolutionary approach, adeno-associated virus (AAV) delivered CRISPR-Cas9 gene editing, has been developed to treat inherited diseases. However, the use of this technology in kidney diseases in vivo is challenged. In this study, we adapt one of the gene editing systems, adenine base editor (ABE9), to develop a broadly expressed and a kidney-specific promoter mediated base editors, and test the effects of these two systems delivered by AAV9 on preventing disease in humanized Pkd1RC/RC mice carrying an arginine (R) to cystine (C) mutation that mimics a mutation in ADPKD patients. We show that one dose of the broadly expressed dual ABE9-AAV9 treatment corrects the pathogenic variant in kidneys, hearts and livers, and result in delaying cyst growth, decrease heart hypertrophy and improve liver function. To confirm the specificity of the base editor system in kidneys, we show that one dose of the kidney specific promoter mediated dual-ABE9-AAV9 treatment corrects the Pkd1 gene mutation in the kidney, and not in the heart, resulting in delaying cyst growth in Pkd1RC/RC kidneys, supporting a promising strategy of using base editor to target specific organs. Treatment with ABE9 base editors mediated by either the broadly expressed or kidney specific promoter increased the survival rate of Pkd1RC/null mice. These preclinical studies support a potential that single-dose genetic therapies may be through the correction of pathogenic variants to prevent ADPKD development in the clinic.

Introduction
Autosomal dominant polycystic kidney disease (ADPKD) is a genetic kidney disease, caused by mutations of the PKD1 and PKD2 genes, encoding polycystin 1 (PC1) and polycystin 2 (PC2), respectively1,2. PKD gene mutations drive disease progression in both renal (kidney) and extrarenal (outside the kidney) organs such as heart3,4. Point mutations represent the largest class of known human ADPKD variants5. ADPKD is a slowly progressive disease, beginning with the formation of renal cysts in utero. Cyst formation and enlargement continue over decades with diverse mechanisms6,7. ultimately leading to kidney failure. As such, it would be advantageous to prevent the initiation of cyst formation by correcting disease-causing point mutations to stop cyst formation altogether in ADPKD kidneys.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based gene editing is a powerful therapeutic tool to correct gene mutations and thus to ameliorate genetic diseases8. Adeno-associated virus-mediated (AAV-mediated) CRISPR editing, in particular the AAV delivered base editors (BEs), is a revolutionary approach for treating inherited diseases. There are two kinds of BEs including adenine base editors (ABEs) and cytidine base editors (CBEs), and both of them are comprised of a single guide RNA (sgRNA) and a nuclease-inactivated Cas9 (or nickase form) fused to a deaminase enzyme, which enables precise installation of A•T to G•C (in the case of ABEs) or C•G to T•A (in the case of CBEs) substitutions9,10. The architecture of ABEs ensures that only the targeted adenine within a specific window (usually around 4-7 nucleotides) near the protospacer adjacent motif (PAM) sequence is deaminated. Although CRISPR-Cas9 base editing has been applied in animal models of various gene mutation diseases11,12, its application in ADPKD remains untested.

In humans, the hypomorphic p.Arg3277Cys (RC) variant in exon 29 of the PKD1 gene has been linked with typical ADPKD when found in homozygosity13. Pkd1RC/RC mice have been generated to mimic human ADPKD, and include phenotypes in the kidneys and heart. In this study, we modify the ABE9 system to generate a broadly expressed and a kidney specific promoter mediated ABE9 system to genetically correct the RC disease-causing mutation in Pkd1RC/RC mice. In particularly, the kidney specific promoter mediated ABE9 base editor has better editing efficiency in the kidneys than the broadly expressed ABE9 base editor, pointing a promising strategy for using this technology to target specific organs in vivo.

Results
Develop a dual-AAV-encoded ABE9 base editor and protospacer
For the R3277C transition, the “AGA” codon for arginine was changed to a “TGC” cystine codon by changing two nucleotides, A>T and A>C (Fig. 1a). Both AGA and CGC encode Arg (R). Therefore, if we can convert the “T” in TGC to “C”, it should generate the “CGC” codon, also resulting in conversion of the amino acid Cys (C) to Arg (R) in Pkd1RC/RC mice (Fig. 1a). To achieve this goal, we use an adenine base editor (ABE), which can target A•T base pairs to produce G•C8. We generated a chicken-beta actin hybrid (Cbh)-AAV-ABE9 N-terminal vector by the replacement of the TadA with a TadA deaminase domain (ABE9) into the AAV-ABE7max vector (Fig. 1b), in which the ABE9 has N108Q and L145T variants to reduce both adenine and cytosine bystander editing, eliminate cytosine editing, and decrease off-target effects as described14,15. We then designed and cloned a single guide RNA (sgRNA) (5’-ACGCAAGTAAAGCGGCTACG-3’) into Cbh-AAV-ABE9 N-terminal and Cbh-AAV-ABE9 C-terminal vectors (Fig. 1a, b). This sgRNA uses a nearby ‘NGG’ PAM, which can be recognized efficiently by a Streptococcus pyogenes Cas9 variant SpCas916,17.

To evaluate the delivery efficiency of AAV9-Cbh vectors, we generated AAV9-Cbh-EGFP particles, and evaluated their distribution in organs 2 weeks after intravenous (IV) injection by an in vivo imaging system (IVIS). AAV9-Cbh-EGFP particles entered (Supplementary Fig. 1a) and GFP was expressed (Supplementary Fig. 1b) in heart, kidney, and liver in AAV9-Cbh-EGFP injected mice, in which the liver was the organ with the strongest GFP expression as examined with IVIS. By performing immunofluorescence (IF) staining in kidneys with GFP antibody and co-stained with different markers, including dolichos biflorus agglutinin (DBA, a collecting ducts marker), lotus Tetragonolobus Lectin (LTL, a proximal tube marker), Na+–K+–Cl− cotransporter 2 (NKCC2, a loop of Henle marker), and Na+-Cl− cotransporter (NCC, a distal convoluted tubule marker), we found that the GFP positive cells could be detected in collecting duct cells (about 26% in total DBA positive cells), proximal tubule cells (about 24% in total LTL positive cells), and distal convoluted tubules cells (about 10% in total NCC positive cells), but not in loop of Henle (Supplementary Fig. 1c–f). The average percentage of GFP-positive cells was about 19% among those cells in AAV9-Cbh-EGFP treated kidneys.

Treatment with Cbh-ABE9-dual-AAV9 delays cyst growth in Pkd1RC/RC mice
We evaluate the effect of the broadly expressed Cbh promoter-directed Cbh-ABE9-dual-AAV9 in Pkd1RC/RC mice, in which renal cysts could be detected at about one month of age in the C57BL/6J background (Supplementary Fig. 1g)13, by intravenous (IV) injection at either postnatal day 14 (PN14) or PN28. The total dose of the Cbh-ABE9-dual-AAV9 base editor was 1×1011 (low dosage) or 1×1012 vg (high dosage) per mouse, and the mice were euthanized at three month. We found that one dose of Cbh-ABE9-dual-AAV9 particles at either dose delayed cyst growth, as seen by the decrease of cyst index and kidney weight/body weight (KW/BW) ratios as well as blood urea nitrogen (BUN) and serum creatinine levels in Pkd1RC/RC mice (n=10) compared to vehicle treated mice (n=10) (Fig. 1c–g, Supplementary Fig. 2a). Importantly, injection at PN14 was more effective at delaying cyst growth than injection at PN28 (Supplementary Fig. 2b–d). It is consistent with the finding that PN14 is a critical time point for renal cyst development in Pkd1 mutant mouse models18. Treatment with high dose of Cbh-ABE9-dual-AAV9 through IV injection at PN14 did not induce renal cyst (Supplementary Fig. 2e) and had no effect on KW/BW ratio, BUN and serum creatinine levels in 3-month-old wild type mice (n=10) (Fig. 1d–g) and Pkd1RC/+ mice (n=10) (Supplementary Fig. 2f,g) compared to vehicle treated age matched wild type and Pkd1RC/+ mice (n=10). Treatment with high dose of Cbh-ABE9-dual-AAV9 also did not induce renal cysts and heart hypertrophy in Pkd1RC/+ mice (Supplementary Fig. 2e). In addition, the editing efficiency was around 9.8% in kidneys in Pkd1RC/+ mice treated with Cbh-ABE9-dual-AAV9 (Supplementary Fig. 2h).

It has been reported that a PC1 triplet can be detected in wild type kidneys by Western blot analysis, in which the top PC1 band of the triplet represents the full length PC1, and the middle band, the N-terminal (NT) endoglycosidase H (EndoH) resistant product (NTR), which is the mature and likely surface localized PC1, and the lower band, EndoH sensitive product (NTS, 440kDa), which may be ER localized PC1. In Pkd1RC/RC kidneys, the PC1-NTS product can be detected as that in Pkd1 wild type kidneys, whereas the PC1-NTR product is decreased in Pkd1RC/RC kidneys, resulting in more robust cyst development19. We found that treatment with Cbh-ABE9-dual-AAV9 particles has no effect on the level of PC1 in wild type kidneys (Fig. 1h, Supplementary Fig. 2i), but could partially normalize the expression of PC1, representing by an increase of PC1-NTR product in Pkd1RC/+ and Pkd1RC/RC kidneys compared to that seen in vehicle treated age matched control kidneys (Fig. 1h, Supplementary Fig. 2j, and Supplementary Fig. 3a, b). The Pkd1 mRNA was also increased in Pkd1RC/RC kidneys treated with Cbh-ABE9-dual-AAV9 compared to vehicle-treated controls as examined by qRT-PCR analysis (Supplementary Fig. 3c).

To determine the editing efficiency of the Cbh-ABE9-dual-AAV9 base editor in vivo, we found that DNA base-editing was 7.7±1.6% and 11.6±1.5% in kidneys, 15.2±2.2% and 21.3±3.2% in hearts, and 31.9±4.2% to 40.1±8.7% in livers in low dosage vs. high dosage PN14-treated mice harvested at 3-month Pkd1RC/RC mice (Supplementary Fig. 3d), suggesting that high dosage injected at PN14 have the higher editing efficiency in kidneys from 3-month Pkd1RC/RC mice. In addition, we found that high dose PN14 treatment also achieved higher DNA base-editing efficiency than the same dose treated at PN28 and analyzed in 3-month Pkd1RC/RC tissues; DNA base-editing efficiency was 11.6±1.5% (PN14) vs. 7.1±3.0% (PN28) in kidneys, 21.3±3.2% (PN14) vs. 17.4±4.7% (PN28) in hearts, and 40.1±8.7% (PN14) vs. 30±8.2% (PN28) in livers (Fig. 1i). Treatment with non-targeting sgRNA did not convert the “T” in TGC to “C” in Pkd1RC/RC kidneys and treatment with AAV-ABE9 also did not convert the codon around the editing window in wide type kidneys (Supplementary Fig. 4a,b). These results suggest that a single dose injection of a broadly expressed ABE9-encoding AAV delays cyst growth through the partial correction of the RC variant to restore the expression of Pkd1 gene and the mature protein form in the kidneys.

Treatment with Cbh-ABE9-dual-AAV9 decreases heart hypertrophy in Pkd1RC/RC mice
ADPKD gene mutations not only drive cyst progression in the kidney but also result in extrarenal complications, including left ventricular hypertrophy (LVH)20, which has also been shown in Pkd1RC/RC mice21. We found that one injection of Cbh-ABE9-dual-AAV9 particles, total 1×1011 or 1×1012 vg also decreased cardiac hypertrophy as seen by a decrease of heart weight/body weight (HW/BW) ratios in three-month-old Pkd1RC/RC mice compared to vehicle treated age matched control mice. Again, early injection at PN14 with a high dosage of 1×1012 vg had a stronger effect on decreasing heart hypertrophy compared to injection at PN28 with the low dosage in those mice (Fig. 2a, b, Supplementary Fig. 5a). We detected a doublet of PC1 around 470kDa in three-month-old wild type hearts, in which the PC1-NTR was decreased in vehicle treated age matched Pkd1RC/RC hearts compared to WT hearts and was partially increased in Cbh-ABE9-dual-AAV9 treated age matched Pkd1RC/RC hearts as examined by Western blot analysis (Fig. 2c, Supplementary Fig. 5b). In addition, the expression of Pkd1 mRNA was also increased in Cbh-ABE9-dual-AAV9 treated Pkd1RC/RC hearts compared to vehicle treated controls as examined by qRT-PCR analysis (Fig. 2d).

A long-term duration effect of Cbh-ABE9-dual-AAV9 treatment in Pkd1RC/RC mice
To evaluate the long-term effectiveness of injected Cbh-ABE9-dual-AAV9 particles in vivo, we found that one-time injection of Cbh-ABE9-dual-AAV9 particles of 1×1011 vg (n=10) or 1×1012 vg (n=10, equal numbers of male and female mice) delayed cyst growth in six-month-old Pkd1RC/RC mice as seen by a decrease of cystic index and KW/BW ratio as well as BUN and serum creatinine levels (Fig. 3a–d, Supplementary Fig. 5c, 5d), and treatment with high dose of Cbh-ABE9-dual-AAV9 particles decreased HW/BW ratios in Pkd1RC/RC mice compared to vehicle treated controls (n=10) (Fig. 3e, f, Supplementary Fig. 5c). An increase of PC1-NTR product was detected in Cbh-ABE9-dual-AAV9 particle treated Pkd1RC/RC kidneys and hearts compared to that in vehicle treated controls (Fig. 3g, h, Supplementary Fig. 5e, 5f). These results suggest that there is a long-term or lasting effect of Cbh-ABE9-dual-AAV9 particles in vivo. Furthermore, DNA base-editing outcomes were still high in organs from 6-month Pkd1RC/RC mice when treated with the high dose of Cbh-ABE9-dual-AAV9 at PN14; 12.1±0.5% in kidneys, 22.4±1.4% in hearts and 38.2±3.9% in livers, but these were not significant differences compared to 3-month Pkd1RC/RC mice when treated with the high dose of Cbh-ABE9-dual-AAV9 at PN14 (Fig. 3i). These results suggest that DNA base-editing on the correction of the RC variant may continue from 3 months to 6 months in vivo. Also, there is a possibility that edited cells may have a survival advantage over uncorrected cells in those organs, increasing the prevalence of edited alleles over time, which is consistent with the known persistence of AAV-ABE in mammals11.

Assessment of bystander editing in Cbh-ABE9-dual-AAV9 treated Pkd1RC/RC mice
As amino acid off-target substitutions could be deleterious, we assessed the frequency of the editing of bystander mutations of adenosine residues at protospacer positions 1 and 6 and the introduction of indels in base-edited Pkd1RC/RC mice using genomic DNA (gDNA) and RT–PCR-amplified Pkd1 mRNAs from kidneys in three-month Pkd1RC/RC mice treated with Cbh-ABE9-dual-AAV9 particles (1×1012 vg) at PN14 (Supplementary Fig. 6a–e). The result showed levels of Pkd1 cDNA editing efficiency in kidney is 28.7±3.6% (Supplementary Fig. 6c), in heart is 40.89±4.7% (Supplementary Fig. 6d), and in liver is 56.96±2.8% (Supplementary Fig. 6e). We did not detect the bystander edit around the potential editing site in gDNA and RNA. In addition, no significant differences were observed in the frequency of A-to-I RNA nucleotide changes in AAV-ABE9 treated mice (Supplementary Fig. 6f). Furthermore, to evaluate the Cas9-dependent off-target effect, we predict potential off-target sites using CRISPOR, a tool ranks the sgRNA with off-target effects and on-target activity, and then conducted targeted amplicon deep sequencing (Amplicon-seq). There were no detectable off-target editing above background levels at the top candidate sites analyzed (Supplementary Fig. 6g), supporting the high specificity and safety of the Cbh-ABE9-dual-AVV9 base editing system in vivo.

Establish kidney specific promoter mediated ABE9-AAV delivery system
To test whether base editing can be used to specifically target the genome in kidneys and to convert the RC mutation in the Pkd1 gene in kidney only, we generated a kidney specific promoter mediated dual AAV base editor by replacing the broadly expressed Cbh promoter as well as the cytomegalovirus enhancer and hybrid intron in the established Cbh-ABE9-dual-AAV9 vectors with a kidney specific promoter mediated cadherin-16 promoter (Ksp), Ksp-ABE9-dual-AAV9 (Fig. 4a). Before testing the effect of Ksp-ABE9-dual-AAV9 in vivo, we first determined the distribution and expression of AAV-Ksp-EGFP particles. We found that the AAV-Ksp-EGFP particles entered and the GFP auto-fluorescent signal was observed only in kidney and liver but not in other organs, such as heart (Supplementary Fig. 7a,b). The expression of GFP was also detected by immunofluorescence staining with GFP antibody, which co-localized with DBA (about 39% in total DBA positive cells), LTL (about 36% in total LTL positive cells), and NCC (about 6% in total NCC positive cells) in kidneys treated with AAV-Ksp-EGFP (Supplementary Fig. 7c). The average percentage of GFP positive cells was 27% among those cells in AAV9-Ksp-EGFP treated kidneys. These results indicate that the transduction of AAV-Ksp-EGFP particles is expressed in specific renal tubular cells in the kidney, which is consistent with prior reports showing that AAV9 can efficiently target renal tubules, even though those reports did not quantify the percentages of GFP-positive cells in kidneys22–25. In addition, we found that GFP is expressed in kidneys injected with AAV9-Cbh-EGFP and AAV9-Ksp-EGFP but not in kidneys injected with empty AAV vectors as examined with immunohistochemistry (IHC) staining (Supplementary Fig. 7d, 7e), confirming the specificity of AAV-mediated GFP expression in kidneys.

Treatment with Ksp-ABE9-dual-AAV9 particles delays cyst growth in Pkd1RC/RC mice
To test the effect of Ksp-ABE9-dual-AAV9 particles in vivo, we found that treatment with Ksp-ABE9-dual-AAV9 particles (1×1012 vg) at PN14 delayed cyst growth as seen by a decrease of cystic index and KW/BW ratio as well as BUN and serum creatinine levels in 3-month Pkd1RC/RC mice compared to vehicle treated age matched mice (n=10) (Fig. 4b–f, Supplementary Fig. 8a). In contrast, this treatment did not normalize HW/BW ratio and cardiac hypertrophy in those mice (n=10) (Fig. 4g, h, Supplementary Fig. 8b). Treatment with high dosage of 1×1012 vg Ksp-ABE9-dual-AAV9 particles had no significant effect on the KW/BW ratio, BUN and serum creatinine levels as well as HW/BW ratio in WT mice (n=10) (Fig. 4d–f and Fig. 4h).

We further found that DNA base-editing was 23.4±0.4% in 3-month Ksp-ABE9-dual-AAV9 treated kidneys, which was higher than the DNA base-editing efficiency in kidneys in age matched Pkd1RC/RC mice treated with the broadly expressed Cbh-ABE9-dual-AAV9 particles (Fig. 4i). The on-target DNA base-editing was very low in heart tissues in 3-month Pkd1RC/RC mice treated with Ksp-ABE9-dual-AAV9 particles (1×1012 vg) at PN14, consistent with the Ksp promoter’s kidney-specific expression (Fig. 4i). Treatment with Ksp-ABE9-dual-AAV9 could partially rescue the PC1-NTR product in kidneys (Fig. 4j, Supplementary Fig. 8c) and livers (Fig. 4k, Supplementary Fig. 8d) but not that in hearts compared to that in vehicle treated controls (Fig. 4l, Supplementary Fig. 8e).

Treatment with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles corrects mutations in primary renal tubular cells isolated from Pkd1RC/RC mice
To determine the editing efficiency in primary renal tubular cells, we isolated the renal tubules and then tubular cells from Pkd1RC/RC kidneys injected with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles. We found that the editing efficiency in renal tubular cells isolated from, 1) Cbh-ABE9-dual-AAV9 particle injected Pkd1RC/RC kidneys is about 7.21% ± 0.3, similar to that in the whole kidneys (Supplementary Fig. 9a), and 2) Ksp-ABE9-dual-AAV9 injected Pkd1RC/RC kidneys was 34.2% ± 8.8, which is higher than that in the whole kidneys (Supplementary Fig. 9a), possibly due to the fact that the Ksp promoter is expressed exclusively in tubular epithelial cells in the kidney but not in other cells26.

It has been reported that chromatin accessibility differs substantially between C57BL/6J and 129S1/SvImJ mice, contributing to distinct regulatory landscapes and potentially affecting gene editing outcomes27. By treating the C57BL/6 (B6) background Pkd1RC/RC mice with Cbh- and Ksp-ABE9-dual-AAV9 particles, we found that the editing efficiency was approximately 4.9% and 20.4% (Supplementary Fig. 9b) in the kidneys of the B6 background Pkd1RC/RC mice treated with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-AAV particles, respectively. These values are lower than those observed in 129 Pkd1RC/RC mice (7.7% for Cbh-ABE9-AAV and 23.4% for Ksp-ABE9-AAV), supporting the notion that mouse genetic background can affect base editing outcomes.

Treatment with Cbh-ABE9-dual-AAV9 rescues the liver function and did not induce liver toxicity and immune infiltration in Pkd1RC/RC mice
In ADPKD, liver cysts are not a primary or early feature of the disease, and liver function is generally not significantly affected despite the presence of liver cysts28. To evaluate the effect of base editors in the liver, we first found that treatment with Cbh-ABE9-dual-AAV9 led to an increase of Pkd1 mRNA in 3- and 6-month-old livers compared to vehicle treated age matched controls, even though it was still lower than that in wild type livers (Fig. 5a, b), and high dose treatment achieved a better effect on the expression of Pkd1 mRNA in livers (Fig. 5a, b). We also found that the level of PC1-NTR was decreased in 3- and 6-month-old vehicle treated Pkd1RC/RC livers, which could be restored in Cbh-ABE9-dual-AAV9 treated age matched Pkd1RC/RC livers (Fig. 5c, d, Supplementary Fig. 9c, Supplementary Fig. 9d).

With H&E staining, we found that there was no massive hepatocyte necrosis in Pkd1RC/RC livers, and only mild structural disorganization and liver cyst were observed in 3- and 6-month-old Pkd1RC/RC livers (Fig. 5e, f), and treatment with Cbh-ABE9-dual-AAV particles ameliorated liver cyst in 3- and 6-month-old Pkd1RC/RC livers, suggesting a protective effect of Cbh-ABE9-AAV on hepatocellular injury in Pkd1RC/RC mice (Fig. 5e, f). In liver function tests, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) are key enzymes that can indicate liver health, and the elevated levels of ALT, AST, and ALP often suggest liver cell damage29. We found that the levels of ALT, AST and ALP were increased in 3-month old Pkd1RC/RC livers compared to age-matched wild type livers, while treatment with Cbh-ABE9-dual-AAV9 could decrease the level of ALT, AST, and ALP in Pkd1RC/RC livers comparable to wild type livers compared to Ksp-ABE9-AAV treated Pkd1RC/RC (Supplementary Fig. 10a–c), suggesting that treatment with Pkd1 base editors could rescue liver function in Pkd1RC/RC mice.

To evaluate the liver toxicity of base editors, we detected the levels of white blood cell (WBC), red blood cell (RBC), platelet in blood of wild type and Pkd1RC/Null mice at PN20 as well as in blood of 3-month-old wild-type and Pkd1RC/RC mice treated with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles. We found that treatment with high dose Cbh-ABE9-dual-AAV9 or Ksp-ABE9-dual-AAV9 has no significant effect on the level of WBC, RBC, platelets in the blood from wild-type and age-matched Pkd1RC/null and Pkd1RC/RC mice. These results suggested that the used doses of Ksp-ABE9-dual-AAV9 and Cbh-ABE9-dual-AAV9 did not cause an obvious immune response in mice (Supplementary Fig. 10d–i). C-reactive protein (CRP) is primarily produced by the liver in response to inflammation, and as an acute-phase reactant, its levels rise in the blood when there is inflammation in the body30. We found that treatment with high dose Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles decreased the level of CRP in blood, and IL-6 and TNF-α in livers in Pkd1RC/RC mice compared to age matched control mice treated with vehicle, whereas those treatments had no obvious effect on these parameters in wild type mice compared to age matched WT mice treated with vehicle (Supplementary Fig. 10j–l).

To further evaluate the hepatic immune infiltration in Pkd1RC/RC livers with the treatment of Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles, we performed the immunofluorescence staining with CD45 (a pan-leukocyte marker), CD3 (T cell marker), CD4 (helper T cell marker) as well as F4/80 antibodies. We found that the numbers of CD45, CD3+, CD4+ T cells and macrophage were increased in Pkd1RC/RC livers compared to age-matched wild-type controls, indicating enhanced immune infiltration, whereas treatment with either Cbh-ABE9-dual-AAV9 or Ksp-ABE9-dual-AAV9 decreased the levels of CD45, CD3+, CD4+ T cells and macrophage cells in 3-month-old Pkd1RC/RC livers, and had no significant effect on those in livers of wild-type controls (Supplementary Figs. 11 and 12). Because the doses of AAV that we used in this study is within the safety range as reported in previous studies11,31,32, these results further indicate that either Cbh-ABE9-AAV or Ksp-ABE9-AAV treatment leads to the decrease of immune infiltration and early signs of hepatocyte stress in Pkd1RC/RC livers.

Treatment with ABE9-dual-AAV9 particles extends lifespan of Pkd1RC/null mice
By crossing Pkd1RC/RC mice with Pkd1null/+ mice, we generated more rapidly progressing Pkd1RC/null mice, which only survive up to about 25 days13. We evaluated the effect of the ABE9-dual-AAV9 treatment on the lifespan of these Pkd1RC/null mice by injection of 1×1012 vg broadly expressed Cbh-ABE9-dual-AAV9 and kidney specific promoter mediated Ksp-ABE9-dual-AAV9 particles at PN7 (see details in Methods). We found that treatment with ABE9-dual-AAV9 particles did not affect the body weight of Pkd1RC/null mice (n=10) compared to vehicle treated controls (n=10) (Fig. 6a). We further found that the broadly expressed Cbh-ABE9-dual-AAV9 treated Pkd1RC/null mice (n=10) lived to a mean age of 33.0 days, while vehicle treated mice (n=10) died of PKD at a mean age of 23.0 days (p<0.0001) (Fig. 6b). Importantly, we found that treatment with Ksp-ABE9-dual-AAV9 further increased the lifespan of Pkd1RC/null mice (n=10) to a mean age of 37 days (p<0.0001) (Fig. 6b). These PN7-injected Pkd1RC/null mice exhibited normal behavior and vitality at ages well beyond the lifespan of vehicle treated Pkd1RC/null mice. We also saw a significant decrease of cystic index, KW/BW ratio and BUN level, as well as serum creatinine levels (Supplementary Fig. 13a and 13b) in Pkd1RC/null mice collected at 20 days, after treatment with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 (Fig. 6c–f). In addition, treatment with Ksp-ABE9-dual-AAV9 increased editing efficiency in kidneys compared to that in Cbh-ABE9-dual-AAV9 treated kidneys (Fig. 6g). We further found that treatment with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles increased 1) the PC1-NTR level in Pkd1RC/Null kidneys (Fig. 6h, Supplementary Fig. 13c) and 2) Pkd1 mRNA and protein in PN20 Pkd1RC/+ and Pkd1RC/null kidneys compared to that in vehicle treated controls (Supplementary Fig. 13d).

Treatment with AAV9-ABE9 reduces the recruitment/accumulation of macrophages at interstitial and pericystic regions and decreases cyst cell proliferation
It has been reported that an abundance of M2-like macrophages can be recruited to interstitial and pericystic regions in ADPKD mouse kidneys to promote cystic renal cell proliferation and cyst growth33–35. We found that one time injection of broadly expressed Cbh-ABE9-dual-AAV9 particles of 1×1011 and 1×1012 vg at either PN14 or PN28 decreased the accumulation/recruitment of macrophages to pericystic and interstitial sites (Supplementary Fig. 14a) and cyst lining epithelial cell proliferation (Supplementary Fig. 14b) in three-month-old Pkd1RC/RC kidneys compared to vehicle treated age matched Pkd1RC/RC mice. In addition, treatment with broadly expressed Cbh-ABE9-dual-AAV9 particles of 1×1011 and 1×1012 vg at PN14 also decreased the accumulation/recruitment of macrophages and cyst lining epithelial cell proliferation in 6-month Pkd1RC/RC kidneys compared to age-matched vehicle-treated Pkd1RC/RC kidneys (Supplementary Fig. 15a,b). Furthermore, one-dose injection of Ksp-ABE9-dual-AAV9 particles of 1×1012 vg at PN14 also decreased the accumulation/recruitment of macrophages and cyst lining epithelial cell proliferation in three-month-old Pkd1RC/RC kidneys compared to vehicle treated age matched Pkd1RC/RC mice (Supplementary Fig. 16a,b).

Treatment with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 decreases fibrosis in Pkd1RC/RC mice
Renal fibrosis is a hallmark of ADPKD36. As the progression of disease, renal fibrosis increases and kidney function declines37. Treatment with high dose of either Cbh-ABE9-dual-AAV9 or Ksp-ABE9-dual-AAV9 particles decreased renal fibrosis in 3- and 6-month-old Pkd1RC/RC kidneys compared to that in age matched vehicle treated Pkd1RC/RC kidneys (Supplementary Fig. 17a and 17b). Myocardial fibrosis is also prominently increased in mouse models of ADPKD38,39. We found that treatment with Cbh-ABE9-dual-AAV9 particles significantly decreased fibrosis in 3- and 6-month-old Pkd1RC/RC hearts compared to that in hearts from vehicle treated Pkd1RC/RC mice (Supplementary Fig. 17c and 17d). We also found that treatment with Cbh-ABE9-dual-AAV9 decreased heart and renal fibrosis in Pkd1RC/Null mice compared to that in vehicle treated control mice (Supplementary Fig. 17e), whereas treatment with Ksp-ABE9-dual-AAV9 decreased renal fibrosis only but had no effect on heart fibrosis compared to vehicle treated and Cbh-ABE9-dual-AAV9 treated kidneys and hearts (Supplementary Fig. 17f). Furthermore, we found that treatment with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 significantly decreased fibrosis in 3-month and 6-month-old Pkd1RC/RC and PN20 Pkd1RC/null livers compared to that in vehicle treated age matched livers (Supplementary Fig. 18a–18c). Together, these results indicate that Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 treatments decrease organ fibrosis in Pkd1RC/RC mice.

Methods
Ethics statement
All animal protocols were approved by and conducted in accordance with Laboratory Animal Resources of the Mayo Clinic and Institutional Animal Care and Use Committee regulations.

AAV vector cloning
The ABE9 was cloned into dual-vector split-intein base editor AAV genome plasmids (Addgene plasmids 137176 and 137177) as described in ref. 8. In brief, the ABE9 fragments were amplified by PCR, and then cloned into the N-AAV vector using isothermal assembly in NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs (NEB), E2621S). The protospacer sequence targeting the pathogenic variant R3277C was cloned into the sgRNA cassette in AAV genome vectors as previously described in ref. 11. In brief, AAV genome vectors were digested with BsmBI and ligated with pre-annealed oligos encoding the spacer sequence. SgRNA Forward oligo: aaaccgtagccgctttactTGCgtC. Reverse oligo: caccGacGCAagtaaagcggctacg. Non-target sgRNA Oligo-scramble-F: caccGTGTAGTTCGACCATTCGTG, scramble-R: aaacCACGAATGGTCGAACTACAC. For the vehicle AAV, we used the Cas9-ABE9 vectors with the non-targeted sgRNA and injection with 1×1012 vg/mice.

To generate the Ksp-AAV vectors, we replaced the ubiquitously expressing synthetic Cbh promoter with the AAV9 vectors with the kidney-specific cadherin 16 promoter (Ksp) promoter (1.3 kb), which was amplified from mouse genomic DNA with the following primers, including forward oligo: ATGGTACCagcttgctctgccatg and reverse oligo: CGACCGGTgcaaatttggcttagg.

AAV production
AAV production was performed as previous reported8. Briefly, HEK293T/17 cells (ATCC, cat: CRL-11268) were plate in 10 cm dishes with DMEM/10% FBS without antibiotic (Thermo Fisher Scientific 157150). Each plate was transfected with 5 μg AAV genome, 5 μg pAd-DeltaF6 and 5 μg pAAV2/9n plasmid. PEI 40k transfect regent mix with plasmids and incubate in 37°C for 15 min. Three days after transfection, cells were scraped and used the AAVpro purification kit (All serotypes, Takara) to purify the AAV particles. Then the concentration of AAV particles was detected by AAVpro® Titration Kit (for Real Time PCR) Ver.2 (Takara).

Mouse strains and treatments
All mice were housed and maintained under pathogen-free conditions. The housing conditions were: 12 h dark/light cycle (lights on at 8:00 AM), 22°C temperature, and 30%–70% humidity. 129S/6 Pkd1RC/RC mice were treated with AAV particles by intravenous injection at postnatal day 14 and day 28, and then those mice were euthanized at three-month-old and six-month-old for further analysis. To evaluate the effect of gene editing on the lifespan of ADPKD mice, we generated Pkd1null/RC mice by crossing Pkd1null/+ mice with Pkd1RC/RC. Pkd1null/RC mice and Pkd1null/RC mice were treated with AAV particles by intravenous injection at postnatal day 7 (retro-orbital injection).

High-throughput sequencing of in vivo samples
Fresh tissues (5-10 mg) were digested with 600 lysis buffers (10 mM Tris-HCl, pH 7.5, 0.05% SDS, 25 μg/ml proteinase K (NEB)). Lysing tissues were incubated at 55°C overnight. Isolated DNA was amplified as described for the genomic DNA samples using mouse Pkd1 specific primers (F: 5-CAGTTCCAGCGGCTCCTC-3, R: 5-GTCCAGCAAACCTCAGGA-3). The sgRNA library readout was performed using two steps of PCR, where the first PCR includes enough genomic DNA to preserve full library complexity, and the second PCR adds appropriate sequencing adapters to the products from the first PCR The sgRNA library for each sample (plasmid, genomic DNA from cells and tissues) was amplified and prepared for Illumina sequencing using the following two-step PCR procedure. All PCR was performed using Phusion Flash High Fidelity Master Mix (Thermo). For PCR#1, the thermocycling parameters were: 98oC for 30s, 18–24 cycles of (98oC for 1s, 62oC for 5s, 72oC for 35s), and 72oC for 1 minute. In each PCR#1 reaction, we used 3 μg of gDNA. Then the PCR products were harvested and send to GENEWIZ (Amplicon EZ; GENEWIZ Germany GmbH) for a second PCR and deep sequencing. As for the editing assay at the RNA level, cDNA was synthesized using 1 μg of RNA, and reverse transcription was performed11. As for the editing assay at the RNA level, cDNA was synthesized using 1 μg of RNA, and reverse transcription was performed. PCR amplification was performed using 50 ng of cDNA, and the PCR program was performed at 95°C for 5 min, followed by 39 cycles of 94°C for 30s and 60°C for 30s at a slope of 2°C/s. Raw sequencing data are available at the NCBI Sequence Read Archive (SRA) under the Bioproject accession no PRJNA1157189. Amplicon sequencing results were analyzed using CRISPResso2.

AAV copy number detection
DNA was extracted from kidneys and livers tissues using the QIAGEN DNeasy Blood and Tissue kit and resuspended in AE buffer. The AAV copy number was detected by using AAVpro® Titration Kit (for Real Time PCR) Ver.2 (Takara, Cat 6233). Brifely, qPCR reactions were prepared in 10 μL reaction volume with AAV9 primers, TB Green Premix, positive control. Thermocycling parameters were as follows: initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 5s and elongation at 60°C for 30s. The standard curve was created with positive AAV9 vector and diluted to an appropriate range for primer efficiency to estimate vector copies per reaction.

RNA-sequencing and analysis of transcriptome-wide off-target RNA base editing
Mouse kidneys were collected and homogenized in Trizol Reagent (Life Technologies, USA) with TissueLyzer (QIAGEN, Inc., Valencia, CA) for the RNA extraction. The construction and sequencing of the RNA-seq library were performed by CD Genomics (USA) on an Illumina PE150 platform. Analysis of transcriptome-wide off-target RNA base editing was performed as previously described63. Data were processed as previously described and normalized to the total number of reads per million exon kilobases (RPKM). Reads were aligned to the GRCm38 mouse genome using Spliced Transcripts Alignment to a Reference (STAR). RNA edits in ABE9 compared with the negative controls were filtered to include only loci with ten or more reads and greater than 0% reads containing an alternate allele. In brief, REDItools version 1.312 was used to quantify the average percentage of A-to-I editing among all sequenced adenosines in each sample. Any adenosines with a read depth less than 10 or a read quality score below 30 were removed from the analysis. The transcriptome-wide A-to-I editing frequency was calculated as: (number of reads in which an adenosine was called as a guanosine) divided by (total number of reads covering all analyzed adenosines).

Tissue histology and Immunohistochemistry
The sections of the kidney, liver, and heart were fixed in 4% paraformaldehyde (pH 7.4), and stained with hematoxylin and eosin (H&E) and Masson trichrome staining methods. For the Immunohistochemistry (IHC) staining, a monoclonal mouse anti-GFP antibody (Santa Cruz, Dallas, TX, USA, sc-9996, 1:500), biotinylated secondary antibody (Santa Cruz Biotechnology Inc., 1:500), and DAB substrate system were used. Kidney sections were counterstained by hematoxylin. For the Masson trichrome staining, collagen fibers in kidney sections were stained with Masson trichrome. Images were analyzed using a NIKON ECLIPSE 80i microscope.

Primary renal tubular isolation
Fresh renal tissue (approximately 10 mg) was placed in a sterile Petri dish and soaked with PBS to remove blood. The outer cortex was cut from the medulla to form pieces about 1 mm3 and then washed three times. The tissue was then digested in 1 mg/ml collagenase type IV with 0.1% DNase I in fetal bovine serum (FBS) at 37°C for 30 min in a shaking water bath. The digestion was then stopped by adding an equal volume of ice-cold PBS/10% FBS. The digestion was stopped by adding an equal volume of ice-cold PBS containing 10% FBS. The cell suspension was first passed through a 100 μm sterile cell strainer to remove undigested tissue chunks and large structures such as glomeruli. The filtrate was collected and centrifuged at 4°C for 200 g for 5 min. The cell pellet was gently resuspended in cold PBS and layered onto a discontinuous Percoll gradient (25%–50%), followed by centrifugation at 3000×g for 30 min at 4°C. The layer of renal tubular epithelial cells was harvested, then diluted in ice-cold PBS and centrifuged at 200×g at 4°C for 5 min to remove any contaminated Percoll. After centrifugation, the supernatant was removed carefully, and the pellets were resuspended in culture medium for further analyses.

Immunofluorescence (IF) staining
The sections of the kidney and liver were fixed in 4% paraformaldehyde (pH 7.4) and stained with IF staining with a pan-macrophage marker, F4/80. After antigen retrieval, tissue sections were incubated with a rat anti-mouse F4/80 antibody (14-4801-82; eBioscience Inc.; 1:100 dilution) overnight, and then were incubated with Fluro-555 anti-rat IgG secondary antibody and mounted in Prolong Gold Anti-fade reagent with DAPI (Invitrogen). For Ki67 staining, a rabbit anti-Ki67 antibody (ab15580; Abcam) and Alexa Fluor 488 anti-rabbit IgG secondary antibody were used. For DBA, LTL, NCC and NKCC2 staining, the fixed kidney sections was stained with DBA Rhodamine (RL-1032-2, Vector Laboratories, 1:200), LTL Texas Red (21761195-1, bioPLUS™, 1:200), NCC (GeneTex, GTX41969, 1:1000), NKCC2 (Proteintech, 18970-1-AP, 1:1000) and mouse monoclonal GFP antibody (Santa cruz, sc-9996, 1:1000). For liver immune infiltration staining, the mouse FITC-CD45 antibody (Biolegend, Cat: 103107), PE-CD3 antibody (Biolegend, Cat: 100205) and PE-CD4 antibody (Biolegend, Cat: 100407) were used. Images were analyzed using a Nikon TI2E microscope.

Quantitative reverse-transcription PCR
The RNA extraction and reverse transcription (RT) were performed as previously described in ref. 64. Total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN). Total RNA (1 μg) was used for RT reactions in a 20 μl reaction to synthesize cDNA using a iScript cDNA Synthesis Kit (Bio-Rad). RNA expression profiles were analyzed by real-time PCR using iTaq SYBR Green Supermix with ROX (Bio-Rad) in an iCycler iQ Real-Time PCR Detection System. The complete reactions were subjected to the following program of thermal cycling: 40 cycles of 10 seconds at 95°C and 20 seconds at 60°C. A melting curve was run after the PCR cycles, followed by a cooling step. Each sample was run in triplicate in each experiment, and each experiment was repeated 3 times. Expression levels of target genes were normalized to the expression level of actin. Mouse Pkd1 primer used as: Forward 5’-TCAATGGGTCTGTGTCAAGG-3’, Reward: 5’-CCAGGGAGCATAGGAACATC-3’. Cas9 primer used as Forward 5’: TCTGAGACACCTGGCA CAAG-3’, Reward: 5’-: CTGGGCACCTTGTACTCGTC-3’. Mouse TNF-α Forward 5′-GCCTCTTCTCATTCCTGCTTG-3′, Reward 5′-CTGATGAGGGAGGCCATT-3′, Mouse IL-6 Forward 5′-ACGGCCTTCCCTACTTCACA-3′, Reward 5′-CATTTCCACGATTTCCCAGA-3′.

Western blot
Western blotting analysis was performed on 3%–8% TA gels (Invitrogen) for 4.5 hours at 150 V at 4°C. Gels were transferred to 0.45 μm nitrocellulose membranes at 40 V overnight at 4°C. The blots were probed with anti-PC1 antibody (7e12), Cas9 Antibody (Santa Cruz Biotechnology Inc, sc-517386), and then with HRP-conjugated secondary antibodies (Southern Biotech, 1:1000).

Measurement of cyst area
The cyst volume was quantified in the whole kidney after H&E staining using Image-Pro Plus v5 software (Media Cybernetics). The cyst area was calculated as (cyst area/total area) × 100. Three sections from both kidneys were analyzed for each mouse.

Quantitative BUN determination
Serum samples were diluted 5-fold in distilled water prior to assay. Next, 5 μl water (blank), 5 μl standard (50 mg/dl) and 5 μl sample were transferred in triplicate into wells of a clear-bottom 96-well plate. Approximately 200 μl of working reagent was added and tapped lightly to mix, and the samples were incubated 20 min at room temperature. Optical density was read at 520 nm.

Analysis of serum creatinine and C-reactive protein (CRP)
Creatinine levels were measured with the QuantiChrom Creatinine Assay Kit from Bio Assay Systems (Hayward, CA). The CRP levels were analyzed with the Mouse C-Reactive Protein/CRP ELISA Kit (Protentech, Cat No. KE10085). Serum was obtained by centrifuging the blood samples at 4000×g/min for 15 min following 2 h resting period. The collected serum samples were subjected to ELISA, and serum levels of creatinine and CRP were quantified in accordance with the manufacturer’s instructions.

Analysis of liver aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP)
Liver tissues were cut into about 1 mm3 blocks with a sterile knife and washed twice with PBS and lysate to grind tissue homogenates on ice. After centrifuging for 5 minutes at 5000×g then collect the supernatant and detect total protein concentration by bicinchoninic acid assay (BCA assay). The levels of AST, ALT, and ALP in the liver supernatant were measured by AST (ELK Biotechnology, Cat: ELK1778), ALT (Biocompare, Cat: EM0829), and ALP (ebiohippo, Cat: BHE10810355). ELISA kits according to the manufacturer’s instructions. The absorbance (450 nm) of each sample was detected on a standard automatic microplate reader.

Analysis of complete blood count (CBC) parameters
The whole blood was collected within an hour before the analysis of CBC, including white blood cell (WBC), red blood cell (RBC), platelets. The Piccolo XpressTM Chemistry Analyzer was utilized according to manufacturer instructions65 along with the Abaxis VetScan HM5 Analyzer for veterinary hematology in Mayo Clinic.

Biodistribution of injected EGFP-AAV
To assess the biodistribution of cystic cell-derived AAV-Ksp-EGFP and AAV-Cbh-EGFP particle, 1.5-month-old wide type and Pkd1RC/RC mice were treated with a dose of 1×1012 vg AAV-Ksp-EGFP and AAV-Cbh-EGFP by intravenous injection. The AAV-Ksp-EGFP and AAV-Cbh-EGFP treated wide type and Pkd1RC/RC mice were sacrificed 2 weeks post-administration, and then the organs (kidney, heart, liver, lung, and spleen) were harvested and analyzed with vivo imaging system (IVIS Perklin Elmer, MA, USA). In addition, fresh frozen sections of whole kidney, liver, and heart were examined under fluorescence microscopy, where GFP auto-fluorescence confirmed the presence of AAV-derived signals in these organs.

Junction-spanning RT-qPCR analysis
To analyze the expression of GFP transcripts in kidneys, the primers were designed to target the spliced 5′-UTR–GFP junction in the mature mRNA: Forward 5′-ATCACTCTCGGCATGGACGA-3’, and Reward 5′-AAGCAGCGTATCCACATAGC-3’. The RNA extraction and reverse transcription (RT) were performed as previous described in ref. 64. RT-qPCR was conducted using the following program: an initial denaturation at 95°C for 30s, followed by 40 cycles of 95°C for 10s and annealing/extension at 60°C for 20s. A melting curve analysis was performed at the end of amplification, followed by a cooling step. The experiment was repeated three times in AAV-Ksp-EGFP and AAV-Cbh-EGFP groups vs. vehicle controls. GFP transcript abundance was normalized to the expression of actin.

Statistics & reproducibility
All statistical data are presented as mean ± SEM. All statistical analyses were performed using SPSS Statistics 22 software. P-values were calculated by two-tailed unpaired Student’s t-test and one-way ANOVA; and a P-value less than 0.05 was considered significant. The Investigators were blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Cheng et al. utilized an adenine base editor (ABE) split system delivered via dual AAV9 vectors to correct a pathogenic point mutation in a humanized Pkd1RC/RC mouse model of autosomal dominant polycystic kidney disease (ADPKD). They demonstrated that both a broadly expressed ABE9 and a kidney-specific ABE9 system led to an increase in PKD1 mRNA levels and a slight increase in PKD protein, resulting in a reduction in renal cyst progression. This was also accompanied by an improvement in cardiac defects.\nThis is the first study to employ gene editing to correct a point mutation in a mouse model of PKD, making the findings novel and of interest to the scientific community. However, several concerns limit the overall impact and significance of this study.\nMajor comments:\n1- AAV9 Transduction Efficiency\nMany studies, including those published in Nature Communications, have reported limited efficiency of AAV9 transduction in the kidney via intravenous (IV) injection (PubMed IDs: 39737896, 24784447, 29976586). The near-100% AAV9 transduction efficiency shown in Supplementary Figure 1 is highly unusual and suggests non-specific staining. Additional evidence should be provided to confirm transduction efficiency, including staining with multiple GFP antibodies via both immunofluorescence and immunohistochemistry on tissue sections. Moreover, proper negative controls are missing from the figures and should be included.\nEven in the study cited by the authors, AAV9 transduction was largely restricted to mesangial cells within the glomeruli, with only minimal transduction in proximal tubule cells (~9% efficiency) and higher expression in podocytes.\nThe investigator utilized the split-base editor system to express the base editor (due to size limitation). This requires the same cell to be transduced simultaneously by two different AAV9 viruses for the system to work, which will further reduce the editing. To validate transduction efficiency, vector genome and vector genome transcript levels should be quantified in various organs. Additionally, measuring AAV copy number in the liver and kidney is crucial to assess how long the base editor remains active and whether immune clearance affects AAV persistence over time. The stability or decline of AAV expression over time should also be addressed. If AAV expression levels are disproportionately high, the observed effects may not be due to successful base editing alone but could instead result from AAV overload, potentially affecting cellular functions in unintended ways. The authors should clarify whether their findings are influenced by non-specific AAV effects rather than precise gene editing.\n2- Base Editing Efficiency in Specific Kidney Cell Populations\nThe assessment of base editing efficiency is not entirely convincing, as the analysis appears to consider the whole kidney rather than specific cell types. It is essential to determine whether editing occurs in epithelial cells, vasculature, or glomeruli. To confirm that editing is occurring in renal tubules, efficiency should be assessed in isolated tubular cells rather than the entire kidney.\n3- Safety Assessments Following AAV Administration\nThe study does not adequately address safety concerns related to AAV administration, apart from measuring renal BUN levels. Liver toxicity is a primary concern with AAV-based therapies; therefore, assessments of liver function markers (ALT, AST, ALP, bilirubin) should be included. Additionally, complete blood count (CBC) parameters (WBC, RBC, platelet count) should be evaluated to monitor immune responses. Systemic inflammatory markers such as CRP and cytokines (IL-6, TNF-α) should be measured to assess immune activation. Histological analyses of hepatocyte damage, immune cell infiltration, and fibrosis should also be performed. Given that high AAV vector doses can induce hepatotoxicity due to excessive transgene expression, measuring AAV copy number in the liver is critical.\n4- Influence of Genetic Background on Gene Editing Efficiency\nGene editing efficiency and outcomes can be significantly influenced by the genetic background of the mouse strain. Differences in DNA repair mechanisms, chromatin accessibility, and immune responses can contribute to variability in editing efficiency. The authors should consider using an additional mouse model, such as C57BL/6 (B6) background, to validate their findings in correcting the mutation.\n5- Liver Phenotype:\nGiven that gene editing via AAV9 is most efficient in the liver, one would expect the most pronounced phenotypic improvement to occur in this organ. However, in this study, the liver is barely discussed, and the only provided histological evidence consists of a single low-resolution H&E image. Why is that?\nTo better assess the extent of cystogenesis rescue, could you provide full liver section images from Figure 4g? This would offer a more comprehensive view of the overall effect of gene editing on hepatic pathology. Additionally, biliary abnormalities is more frequently reported than cyst area in the liver in similar models. It would be valuable for the investigators to analyze and report this aspect as well.\nOther Comments:\n1. H&E Staining Quality: The resolution of H&E staining across all organs is relatively low and not very convincing. Please provide higher-quality images with both low and high magnification to better visualize the overall tissue structure and finer details.\n2. Segment-Specific Rescue Analysis: To determine whether gene editing affects specific nephron segments, could you perform IF staining using segment-specific nephron markers? Suggested markers include LTL (proximal tubule), NKCC2 (loop of Henle), NCC (distal convoluted tubule), and cytokeratin (collecting duct).\n3. Serum Creatinine Measurement: Have serum creatinine levels been measured? If so, do they change in a manner similar to blood urea nitrogen (BUN) levels in rescued animals?\n4. Western Blot Data Presentation: Since Pkd1 expression in gene-edited animals appears weak, Western blot data should include all individual data points (three independent samples) run on the same gel, rather than presenting only a single representative sample.\n5. Antibody Validation for PKD1 Detection: Can you confirm the observed Pkd1 upregulation with an alternative antibody? Several validated antibodies are available from the PKDRRC, which could help strengthen the findings.\n6. GFP Expression and Specificity in Extended Fig. 6: The GFP signal driven by the Ksp construct appears excessively high, suggesting potential nonspecific expression. Please provide additional controls, including more antibody staining, negative controls, and high-resolution images at higher magnification. Additionally, co-staining with renal tubule markers is needed to demonstrate colocalization, particularly given previous reports of inefficient AAV9 transduction using the Ksp promoter.\n7. Western Blot for PN28 in Fig. 1g: Please include a Western blot for PN28 in Figure 1g.\n8. Methodology Details: More details are needed in the Methods section to enhance reproducibility and clarity.\n9. Text Clarity and Grammar: The clarity of the text should be improved throughout. For example, in the abstract, \"causing by mutations\" should be corrected to \"caused by mutations.\"", "reviewer_2": "In this manuscript, the authors have demonstrated the use of adenine base editing, delivered by adeno-associated viral (AAV) vectors, to correct a pathogenic variant in the Pkd1 gene that causes kidney and heart phenotypes (renal cyst growth, cardiac hypertrophy) in mice consistent with human autosomal dominant polycystic kidney disease (ADPKD). They achieve sufficient correction to partially revert the kidney and heart phenotypes. In a mouse model with exaggerated disease (early mortality), the base editing is even able to modestly improve lifespan. To my knowledge, this is the first in vivo demonstration of the use of corrective editing to improve ADPKD and, more generally, any genetic kidney disease in a mouse model.\nThe work is technically sound, and I do not have substantive criticisms in that respect. I have the following suggestions to improve the manuscript.\n(1) The authors give themselves more credit than is warranted. \"In this study, we developed two adenine base editors (ABE9) delivered by AAV9\" makes it seem like they invented ABE9 for this study, when in fact it was simply adapted from a publication from another group. \"We first developed a broadly expressed ABE9 base editor system\"--they simply swapped out a deaminase domain from ABE8 to ABE9 in existing AAV vectors from another group. \"We developed a kidney specific ABE9 base editor system\"--they simply swapped out a promoter from a generally expressed promoter to an established kidney promoter. From a technical perspective, these are incremental changes. The mouse data are novel enough on their own to warrant publication--the authors do not need to represent themselves as having developed whole new technologies. I would advise toning down the language and focusing on what is truly novel about this work.\n(2) \"We developed a kidney specific ABE9 base editor system\"--their data plainly demonstrate that it is not kidney specific, as it produces editing in the liver as well. They did not perform editing biodistribution studies to assess many organs in the mouse. The major difference they show is that the Ksp promoter compared to the Cbh promoter reduces expression in the heart. Is this actually a desirable effect, given that ADPKD causes cardiac hypertrophy, and an optimal ADPKD therapy would target the heart as well as the kidneys? Are there any meaningful downsides of having corrective editing of Pkd1 to wild-type in organs other than the kidneys and heart? If not, why is it important to have a kidney specific system? The authors should better justify this.\n(3) Can the authors better explain the rationale for using ABE9? In the Discussion section, the authors imply that ABE8e is ineffective in the kidney (whereas ABE9 is effective), but this is based on studies that did not focus on the kidney or on kidney-specific genes and so are not that relevant. Furthermore the major advantage of ABE9 over ABE8e is that it has less bystander editing without losing much efficiency (there is no claim of increased efficiency)--or, to put it another way, it has a narrower editing window. But it is not clear that ABE9 is a better choice than a well-established narrow-window editor like ABE8.8. The authors do not report any initial screening of ABE9 or other ABEs in cells--this seems odd (and unlikely). Did the authors really just guess that ABE9 would work the best and go straight to experiments in mice without any testing in vitro?\n(4) As a proof-of-concept study, the work in this manuscript has considerable merit. However, it seems unlikely that the dual AAV approach used in this work would advance to the clinic. Indeed, no AAV-based gene editing therapy has made it very far in the clinic (especially compared to non-viral approaches, namely lipid nanoparticles), due to the serious downsides of AAV: prolonged expression of the editor causing a substantial immune response (not typically evident in mice, but quite apparent in large animal models), along with increased off-target editing; inability to redose the therapy, which is important because the authors have shown only partial correction of disease in this study, and full correction of the disease would likely need higher levels of editing that could only be achieved with repeat dosing; potentially dangerous and even fatal toxicities from having to use high AAV doses in order to (1) account for the need to use dual AAV vectors to deliver a base editor and (2) get enough vector into the kidneys, given the broad distribution of AAV9 to many organs throughout the body. The authors should have a limitations paragraph in the Discussion acknowledging these barriers to translation.\n(5) Line 139: \"Both AGA and CGC encode Arg (A)\"--this should be \"Both AGA and CGC encode Arg (R)\".", "reviewer_3": "The present manuscript describes a base editing strategy delivered via AAV to correct a mutation in the PKD1 gene, as a potential therapeutic approach for autosomal dominant polycystic kidney disease (ADPKD). The authors demonstrate in vivo editing in a mouse model using ABE9 and also engineer a kidney-specific promoter for targeted AAV expression.\nOverall, the manuscript is well-written and supported by comprehensive datasets. The clarity of the language could be improved by streamlining several sentences that are currently difficult to follow. The manuscript's structure would also benefit from reorganization for improved data flow (see detailed comments below). I would recommend the manuscript for publication if authors could sufficiently address the following concerns:\nMajor comments:\n1. While the authors present robust characterization of editing efficiency and physiological outcomes, the manuscript lacks any assessment of Cas9-dependent off-target effects. A comprehensive nomination of potential off-target sites, followed by experimental validation in edited cells or tissues, is critical to support the therapeutic relevance and safety of the proposed strategy.\n2. The current structure presents data from AAV injections at PN14 and PN28 (Figures 1 and 2) early in the Results, while the corresponding editing efficiency data appear later (Figure 4). For improved clarity and narrative flow, it would be helpful to present the editing outcomes alongside the relevant experimental context (e.g., incorporating Figure 4 data into Figures 1–3).\n3. In the Discussion section (Ling 438-444), authors should discuss the limitation of using Ksp promoter for kidney-specific expression. Since ADPKD also affects other organs, including the heart, the therapeutic strategy’s limitation in targeting extra-renal tissues should be discussed.\nMinor comment:\n4. Line 118-120: original ABE paper [PMID: 29160308] should be cited in this sentence.\n5. Please specify the administration route used for IV injection (e.g., retro-orbital vs. tail vein) in the Methods section for reproducibility.\n6. Figure 1d and other similar plots have both pink and blue dots. What is the difference between the two colored dots? Authors should specify in the figure legend."}, "author_response": "Response to Reviewer’s Comments\nReviewer #1\nIn this study, Cheng et al. utilized an adenine base editor (ABE) split system delivered via dual AAV9\nvectors to correct a pathogenic point mutation in a humanized Pkd1RC/RC mouse model of autosomal\ndominant polycystic kidney disease (ADPKD). They demonstrated that both a broadly expressed ABE9\nand a kidney-specific ABE9 system led to an increase in PKD1 mRNA levels and a slight increase in PKD\nprotein, resulting in a reduction in renal cyst progression. This was also accompanied by an improvement\nin cardiac defects.\nThis is the first study to employ gene editing to correct a point mutation in a mouse model of PKD,\nmaking the findings novel and of interest to the scientific community. However, several concerns limit\nthe overall impact and significance of this study.\nMajor comments:\n1- AAV9 Transduction Efficiency\nMany studies, including those published in Nature Communications, have reported limited efficiency of\nAAV9 transduction in the kidney via intravenous (IV) injection (PubMed IDs: 39737896, 24784447,\n29976586). The near-100% AAV9 transduction efficiency shown in Supplementary Figure 1 is highly\nunusual and suggests non-specific staining. Additional evidence should be provided to confirm\ntransduction efficiency, including staining with multiple GFP antibodies via both\nimmunofluorescence and immunohistochemistry on tissue sections. Moreover, proper negative controls are missing from the\nfigures and should be included.\nResponse: Thanks for your comments and suggestions. As suggested, we performed the\nimmunofluorescence staining in kidneys treated with AAV9-Cbh-EGFP and AAV9-Ksp-EGFP as\nwell as blank AAV (no GFP expression cassette) with i dolichos biflorus agglutinin (DBA, a\ncollecting ducts marker), lotus Tetragonolobus Lectin (LTL, a proximal tube marker), Na+–K+–Cl−\ncotransporter 2 (NKCC2, a loop of Henle marker), and Na+-Cl− cotransporter (NCC, a distal\nconvoluted tubule marker), and found that GFP signal co-localize with collecting ducts/tubules\nand proximal tubules as well as distal convoluted tubule (new Extended Data Fig. 1, new\nExtended Data Fig. 7c). In addition, we performed immunohistochemistry (IHC) staining with GFP\nantibody, and found that GFP is expressed in kidneys injected with AAV9-Cbh-EGFP and AAV9-\nKsp-EGFP but not that in kidneys injected with blank AAV (new Extended Data Fig. 8), confirming\nthe specificity of AAV-mediated GFP expression in kidneys.\nEven in the study cited by the authors, AAV9 transduction was largely restricted to mesangial cells within\nthe glomeruli, with only minimal transduction in proximal tubule cells (~9% efficiency) and higher\nexpression in podocytes. The investigator utilized the split-base editor system to express the base editor\n(due to size limitation). This requires the same cell to be transduced simultaneously by two different\nAAV9 viruses for the system to work, which will further reduce the editing. To validate transduction\nefficiency, vector genome and vector genome transcript levels should be quantified in various organs.\nAdditionally, measuring AAV copy number in the liver and kidney is crucial to assess how long the base\neditor remains active and whether immune clearance affects AAV persistence over time. The stability or\ndecline of AAV expression over time should also be addressed. If AAV expression levels are\ndisproportionately high, the observed effects may not be due to successful base editing alone but could\ninstead result from AAV overload, potentially affecting cellular functions in unintended ways. The\nauthors should clarify whether their findings are influenced by non-specific AAV effects rather than\nprecise gene editing.\nResponse: Thanks for your comments and suggestions. We address these concerns point-by-\npoint below:\n1) To validate transduction efficiency, vector genome and vector genome transcript levels should be\nquantified in various organs.\nResponse: We have evaluated the transduction efficiency of Cbh-ABE9-dual-AAV9 and Ksp-\nABE9-dual-AAV9 in the kidney, liver, and heart by examining the levels of Cas9 protein in those\norgans. In addition, we also detected the Cas9 mRNA level in the kidneys, livers, and hearts from\n3- and 6-month-old Pkd1RC/RC mice after injected with Cbh-ABE9-dual-AAV9. The new results\nshowed that the expression of Cas9 protein level could be detected in kidneys, livers and hearts\nin Cbh-ABE9-dual-AAV9 injected Pkd1RC/RC mice, and the expression of Cas9 could mainly be\ndetected in kidneys and livers in Pkd1RC/RC mice treated with Ksp-ABE9-dual-AAV9 (new\nExtended Data Fig. 21a-21d). Furthermore, both protein and mRNA level of Cas9 are decreased\nin the kidneys, livers, and hearts from 6-month-old Pkd1RC/RC mice.\n2) measuring AAV copy number in the liver and kidney is crucial to assess how long the base editor remains\nactive and whether immune clearance affects AAV persistence over time.\nResponse: Thanks for your comments and suggestions. We have cited the papers which showed\nthe duration of AAV in the liver and kidney (PMID: 29599079). In addition, to validate transduction\nefficiency of Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles in vivo, we found that\nCas9 protein and mRNA could be detected in kidneys, livers and hearts in Cbh-ABE9-dual-AAV9\ninjected Pkd1RC/RC mice, and Cas9 protein could mainly be detected in kidneys and livers in Ksp-\nABE9-dual-AAV9 injected Pkd1RC/RC mice (Extended Data Fig. 21a-21d). In addition, we found\nthat there was a significant reduction of the AAV copy number and Cas9 expression over time in\n6-month Pkd1RC/RC mice kidneys and livers after post-injection with Cbh-ABE9-dual-AAV9\n(Extended Data Fig. 21e and 21f). The transient expression of Cas9 following AAV delivery\nsupports a “hit-and-go” gene editing model, in which Cas9 is briefly expressed to introduce the\ndesired genomic changes and then cleared gradually, thereby minimizing long-term Cas9\nexposure and associated risks. Depending on the route of administration, choice of serotype and\ndosage used, the general AAVs (including AAV9) exhibits a relatively tolerable safety profile when\ncompared to other gene transfer devices like adenovirus (Ad) and lentivirus (PMID: 33837614).\nWe have also revised the discussion section as that “AAVs are favored for their safety, low\nimmunogenicity, and ability to provide long-lasting gene expressions, which are particularly useful\nfor targeting non-dividing cells, such as those in the heart, kidney and liver (PMCID: PMC9418752,\nPMCID: PMC12272985, PMCID: PMC3799231). A number of theories have been proposed to\nexplain the relatively low immunogenicity of AAV. First, most AAV serotypes may be poor\ntransducers of professional antigen-presenting cells (APCs), such as dendritic cells, and may only\nresult in minimal upregulation of major histocompatibility complex (MHC) proteins, key molecular\nplayers in the presentation of antigens to the immune system, in target cell types. Second, there\nis a balance between immune tolerance and the clearance of AAV-delivered components (e.g.,\nCas9) after AAVs treatment. High dosage of AAV is easy to activate T cells to induce the immune\nclearance of AAV, resulting in the loss of efficacy, but low dose is more likely to be neutralized by\nanti-AAV antibodies (PMID: 36635967). However, in some contexts, moderate doses may\npromote immune tolerance, rather than immune activation, particularly in immune-modulatory\norgans, such as the liver. Certain degree of base editing vector uptake/accumulation and the\nexpression of base editor is inevitable in livers, even with tissue-enriched promoters. This is a\nknown limitation of AAV9 delivery and does not necessarily reflect promoter leakiness but rather\na vector biodistribution.”\nIn our study, we observed that although the copy number of AAV vector in the kidney and liver\ndecreased over time, the base editing efficiency remained stable. This suggests that the AAV\nlevels of base editor during the early post-injection window was sufficient to induce durable and\nirreversible genomic editing. Such a phenomenon is consistent with the mechanism of base\nediting, where even short-term expression of the editor can result in permanent genetic correction\n(PMCID: PMC12284509). Therefore, while immune clearance may reduce AAV vector\npersistency, it does not necessarily compromise therapeutic efficacy, providing that sufficient\nediting occurs before vector elimination.\n3) The stability or decline of AAV expression over time should also be addressed.\nResponse: Thanks for your suggestion. We have revised the discussion section as that “The\nstability of AAV mediated base editor expression can be influenced by several factors, including\nthe specific serotype, the target tissue, specific promoters, and the route of administration (PMID:\n9156805; PMID: 18414476). It has been reported that AAV vector-mediated the expression of the\ntransgene is generally maintained in vivo for at least 6 months (PMCID: PMC9702554). In\nessence, the initial burst of AAV gene delivery, possibly resulting in a higher copy number, and\nthe stability of episomal genomes in non-dividing cells and the potential for low-level integration,\ncan collectively contribute to the observed long-term maintenance of transgene expression\ndespite a decline in the overall AAV copy number over time (PMCID: PMC9077371). As such,\ncopy number and transgene expression are not always directly proportional. In our study, we\nobserved a decline in AAV vector copy number and Cas9 protein expression over time in the heat,\nkidney and liver, whereas the base editing efficiency remained stable, which is consistent with the\nmechanism of base editing that even short-term expression of the editor can result in permanent\ngenetic correction (PMID: 40704025). These results suggest that the subsequent declines in AAV\ncopy number and Cas9 expression do not negate those already established genomic changes,\nwhich supports a “hit-and-go” editing model that minimizes long-term exposure to the editing\nmachinery. This concept is crucial for the development of safe and effective long-term gene\ntherapies using CRISPR-Cas9 delivered via AAV vectors (PMCID: PMC9077371, PMCID:\nPMC110396, PMID: 12946324).”\n4) The authors should clarify whether their findings are influenced by non-specific AAV effects rather than\nprecise gene editing.\nResponse: Thanks for your comments. To clarify whether the observed phenotypic changes were\ncaused by precise gene editing rather than non-specific effects of AAV delivery or transgene\nexpression, we treated Pkd1RC/RC and Pkd1RC/null mice with AAVs carrying a non-targeting sgRNA\nthat does not recognize any genomic locus. This non-targeting sgRNA control allowed us to\nevaluate the effects arising from viral infection, vector expression, or dCas9 activity independent\nof gene-specific targeting. As shown in figures (Figure 1- 6, Extended Data Fig. 4), the non-\ntargeting control sgRNA did not show the phenotypic or base changes observed in the\nexperimental group, supporting that the observed effects are due to specific gene editing rather\nthan off-target or AAV-related artifacts. In addition, we also evaluated the bystander editing\nefficiency include the non-targeting sgRNA AAV and the off-target effect between non-targeting\nsgRNA AAV and targeting sgRNA AAV and we did not observe the bystander editing and off-target\neffect caused by non-specific AAV effects (Extended Data Fig. 4).\n2. Base Editing Efficiency in Specific Kidney Cell Populations.\nThe assessment of base editing efficiency is not entirely convincing, as the analysis appears to consider\nthe whole kidney rather than specific cell types. It is essential to determine whether editing occurs in\nepithelial cells, vasculature, or glomeruli. To confirm that editing is occurring in renal tubules, efficiency\nshould be assessed in isolated tubular cells rather than the entire kidney.\nResponse: Thanks for your comments and suggestions. As suggested, to determine the editing\nefficiency in primary renal tubular cells, we isolated the renal tubules and then tubular cells from\nPkd1RC/RC kidneys injected with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles. We\nfound that the editing efficiency in renal tubular cells isolated from, 1) Cbh-ABE9-dual-AAV9\nparticle injected Pkd1RC/RC kidneys is about 7.21% ± 0.3, similar to that in the whole kidneys (new\nExtended Data Fig. 10a), and 2) Ksp-ABE9-dual-AAV9 injected Pkd1RC/RC kidneys was 34.2% ±\n8.8 (new Extended Data Fig. 10a), possibly due to the fact that the Ksp promoter is expressed\nexclusively in tubular epithelial cells in the kidney but not in other cells (PMID: 10516285).\n3. Safety Assessments Following AAV Administration\nThe study does not adequately address safety concerns related to AAV administration, apart from\nmeasuring renal BUN levels. Liver toxicity is a primary concern with AAV-based therapies; therefore,\nassessments of liver function markers (ALT, AST, ALP, bilirubin) should be included. Additionally, complete\nblood count (CBC) parameters (WBC, RBC, platelet count) should be evaluated to monitor immune\nresponses. Systemic inflammatory markers such as CRP and cytokines (IL-6, TNF-α) should be measured\nto assess immune activation. Histological analyses of hepatocyte damage, immune cell infiltration, and\nfibrosis should also be performed. Given that high AAV vector doses can induce hepatotoxicity due to\nexcessive transgene expression, measuring AAV copy number in the liver is critical.\nResponse: Thanks for your comments and suggestions. We address these concerns point-by-\npoint below:\n1) Liver toxicity is a primary concern with AAV-based therapies; therefore, assessments of liver function\nmarkers (ALT, AST, ALP, bilirubin) should be included.\nResponse: Thanks for your comments and suggestions. We evaluated the liver ALT, AST and\nALP after treatment with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9. We found that the\nlevels of ALT, AST and ALP were increased in 3-month old Pkd1RC/RC livers compared to age-\nmatched wild type livers, while treatment with Cbh-ABE9-dual-AAV9 could decrease the level of\nALT, AST, and ALP to the normal value as in wild type livers(new Extended Data Fig. 11a-11c),\nsuggesting that treatment with Pkd1 base editors could rescue liver function in Pkd1RC/RC mice.\n2) Complete blood count (CBC) parameters (WBC, RBC, platelet count) should be evaluated to monitor\nimmune responses\nResponse: Thanks for your comments and suggestions. To evaluate the liver toxicity of base\neditors, we detected the levels of white blood cell (WBC), red blood cell (RBC), platelet in blood\nof wild type and Pkd1RC/Null mice at PN20 as well as in blood of 3-month-old wild type and\nPkd1RC/RC mice treated with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particles. We found\nthat treatment with high dose Cbh-ABE9-dual-AAV9 or Ksp-ABE9-dual-AAV9 has no significant\neffect on the level of WBC, RBC, platelet in the blood from wild type and aged matched Pkd1RC/null\nand Pkd1RC/RC mice (new Extended Data Fig. 11d-11i). These results suggested that the doses of\nKsp-ABE9-dual-AAV9 and Cbh-ABE9-dual-AAV9 used might not cause immune response in\nmice.\n3) Systemic inflammatory markers such as CRP and cytokines (IL-6, TNF-α) should be measured to assess\nimmune activation.\nResponse: Thanks for your comments and suggestions. We evaluated the serum CRP, and\ncytokine levels of IL-6, and TNF-α in livers. We found that treatment with high dose Cbh-ABE9-\ndual-AAV9 and Ksp-ABE9-dual-AAV9 particles decreased the level of CRP in blood, and IL-6 and\nTNF-α in livers in Pkd1RC/RC mice compared to age matched control mice treated with vehicle,\nwhereas those treatments had no obvious effect on these parameters in wild type mice compared\nto age matched wild type mice treated with vehicle (new Extended Data Fig. 11j-11l).\n4) Histological analyses of hepatocyte damage, immune cell infiltration, and fibrosis should also be\nperformed.\nResponse: Thanks for your comments and suggestions. We performed additional histological\nanalyses focusing on hepatocyte damage, immune cell infiltration, and liver fibrosis after AAV\ntreatment in both 3-month and 6-month-old Pkd1RC/RC and WT mice. In brief,\ni) for hepatocyte damage, we conducted H&E staining and assessed hepatocyte damage. We\nfound that there was no massive hepatocyte necrosis in Pkd1RC/RC livers, and only mild structural\ndisorganization and liver cyst were observed in 3- and 6-month-old Pkd1RC/RC livers (new Figure\n5e and 5f), and treatment with Cbh-ABE9-dual-AAV particles ameliorated liver cyst in 3- and 6-\nmonth-old Pkd1RC/RC livers, suggesting a protective effect of Cbh-ABE9-dual-AAV on\nhepatocellular injury in Pkd1RC/RC mice (new Figure 5e and 5f).\nii) for immune cell infiltration, we performed co-immunofluorescence staining for CD45 (pan-\nleukocyte marker), CD3 (T cell marker), and CD4 (helper T cell marker) as well as F4/80 in liver\ntissues. We found that the numbers of CD3+, CD4+ T and macrophage cells were increased in\nPkd1RC/RC livers compared to age-matched wild-type controls, indicating enhanced immune\ninfiltration, whereas treatment with either Cbh-ABE9-dual-AAV9 or Ksp-ABE9-dual-AAV9\ndecreased the levels of CD3+, CD4+ T cells as well as F4/80 in 3-month-old Pkd1RC/RC livers, and\nhad no significant effect on those in livers of wild-type controls (new Extended Data Fig. 12 and\nnew Extended Data Fig. 13). Because the doses of AAV that we used in this study is within the\nsafety range as reported in previous studies (PMID: 33408413, PMID: 36797478, PMID: 38796705),\nthese results further indicate that either Cbh-ABE9-dual-AAV9 or Ksp-ABE9-dual-AAV9 treatment leads to the decrease of immune infiltration and early signs of hepatocyte stress\nin Pkd1RC/RC livers.\niii) for the evaluation of fibrosis, we performed Masson staining in the livers from 3- and 6-month-\nold Pkd1RC/RC and age-matched WT mice treated with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-\nAAV9. We found that treatment with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 significantly\ndecreased fibrosis in 3-month and 6-month-old Pkd1RC/RC and PN20 Pkd1RC/null livers compared\nto that in vehicle treated age matched livers (new Extended Data Fig. 19). Together, these results\nindicate that Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 treatments leads to the decrease\nof immune infiltration, early signs of hepatocyte stress and fibrosis in Pkd1RC/RC livers.\nIn summary, we did not observe histological signs of hepatotoxicity in H&E-stained liver sections,\nfurther supporting the safety of our AAV treatment regimen. In addition, we have carefully\nconsidered the concern regarding potential hepatotoxicity from high AAV vector doses. In our\nstudy, we used an AAV dosage that is well known within the safety range as reported in previous\nstudies (PMID: 33408413; PMID: 36797478; PMCID: PMC11286820), none of which reported\nliver toxicity at comparable doses.\n5) Given that high AAV vector doses can induce hepatotoxicity due to excessive transgene expression,\nmeasuring AAV copy number in the liver is critical.\nResponse: To assess the safety and persistence of AAV in vivo, we quantified AAV vector\ngenome copy number and Cas9 protein expression in the liver and kidney at different time points\npost-injection. As shown in new Extended Data Fig. 21, the AAV copy number is decreased\nkidneys, livers and Cas9 expression were also decreased in kidneys, livers, and hearts of 6-\nmonth-old Pkd1RC/RC mice treated with Cbh-ABE9-dual-AAV9 at PN14. The repaid decrease of\nAAV copy number and Cas9 expression in 6-month liver supports a “hit-and-go” gene editing\nmodel, in which Cas9 is briefly expressed to introduce the desired genomic changes and then\ncleared gradually, thereby minimizing long-term Cas9 exposure and associated risks. Depending\non the route of administration, choice of serotype and dosage used, the general AAVs (including\nAAV9) exhibits a relatively tolerable safety profile when compared to other gene transfer devices\nlike adenovirus (Ad) and lentivirus (PMID: 33837614). These results indicate that “AAV vector\nmay not induce hepatotoxicity due to the clearance of AAV and the decreased expression of Cas9,\nmeasuring AAV copy number in the liver is critical.”\n4- Influence of Genetic Background on Gene Editing Efficiency\nGene editing efficiency and outcomes can be significantly influenced by the genetic background of the\nmouse strain. Differences in DNA repair mechanisms, chromatin accessibility, and immune responses can\ncontribute to variability in editing efficiency. The authors should consider using an additional mouse\nmodel, such as C57BL/6 (B6) background, to validate their findings in correcting the mutation.\nResponse: Thanks for your comments. We fully agree that genetic background can influence\ngene editing efficiency. The adenine base editors (ABE), which do not rely on double-strand break\nformation or the associated NHEJ/HDR DNA repair pathways (PMCID: PMC10586758). The\ndifferences in DNA repair mechanisms are less likely to account for the observed variability in\nediting efficiency across mouse strains. However, other strain-dependent factors—such as\nchromatin accessibility, promoter activity, AAV transduction efficiency, and immune responses—\ncan influence editing efficiency.\nTo determine whether the genetic background of mice affects base editing efficiency, we treated\nthe C57BL/6 (B6) background Pkd1RC/RC mice with Cbh- and Ksp-ABE9-dual-AAV9 particles, and\nfound that the editing efficiency was approximately 4.9% and 20.4% (new Extended Data Fig.\n10b) in kidneys of the B6 background Pkd1RC/RC mice treated with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 particle, respectively. These values are lower than those observed in 129\nPkd1RC/RC mice (7.7% for Cbh-ABE9-dual-AAV9 and 23.4% for Ksp-ABE9-dual-AAV9), supporting\nthe notion that mouse genetic background can affect base editing outcomes.\nIt has been reported that C57BL/6J miceexisting neutralizing antibodies and increased immune\ncell infiltration, which may reduce the effectiveness of AAV-mediated gene therapy (PMCID:\nPMC6749937; PMCID: PMC8584150). Furthermore, recent research has demonstrated that\nchromatin accessibility differs substantially between C57BL/6J and 129S1/SvImJ mice,\ncontributing to distinct regulatory landscapes and potentially affecting gene editing outcomes\n(PMID: 38153160). These findings reinforce the importance of considering genetic background in\ntherapeutic gene editing studies and provide validation of our approach in an independent mouse\nmodel.\n5- Liver Phenotype:\nGiven that gene editing via AAV9 is most efficient in the liver, one would expect the most pronounced\nphenotypic improvement to occur in this organ. However, in this study, the liver is barely discussed, and\nthe only provided histological evidence consists of a single low-resolution H&E image. Why is that?\nResponse: Thanks for your comments. As a proof-of-concept study, we mainly focus on the base\nediting in kidneys. We agree that AAV9 is known to transduce the liver with high efficiency, and\nwe appreciate your comment regarding the expectation of a phenotypic response in the liver. To\naddress your comment, we have now included additional high-resolution histological analysis of\nthe liver (new Figure 5e and 5f). Specifically, we performed H&E staining, immune cell infiltration\n(CD3, CD4, CD45, and F4/80), and fibrosis analysis in livers in 3-month-old Pkd1RC/RC mice. The\nresults showed treatment with either Cbh-ABE9-dual-AAV9 or Ksp-ABE9-dual-AAV9 decreases\nliver pathology or fibrosis and immune cell infiltration in Pkd1RC/RC mice (new Extended Data Fig.\n12 and new Extended Data Fig. 13, new Extended Data Fig. 19).\nTo better assess the extent of cystogenesis rescue, could you provide full liver section images from Figure\n4g? This would offer a more comprehensive view of the overall effect of gene editing on hepatic pathology.\nAdditionally, biliary abnormalities is more frequently reported than cyst area in the liver in similar models.\nIt would be valuable for the investigators to analyze and report this aspect as well.\nResponse: Thank you very much for your comments. We have provided the full liver section\nimages for new Figure 5e and 5f. To address biliary abnormalities, we performed the masson\nstaining and analyzed the liver sections with a focus on biliary morphology and found that the\nlivers from 6-month old Pkd1RC/RC have more massive biliary abnormalities compared to the livers\nfrom 3-month old Pkd1RC/RC and PN20 Pkd1RC/null mice and treatment with either Cbh-ABE9-dual-\nAAV9 or Ksp-ABE9-dual-AAV9 particles can decreased fibrosis and biliary abnormalities in\nPkd1RC/RC livers compared to aged matched vehicle group (new Extended Data Fig. 19).\nOther comments:\n1. H&E Staining Quality: The resolution of H&E staining across all organs is relatively low and not very\nconvincing. Please provide higher-quality images with both low and high magnification to better visualize\nthe overall tissue structure and finer details.\nResponse: Thanks for your comments. We have provided higher-quality images with low\nmagnification in figure 1-6 and high magnification figures of H&E staining in the new Extended\nData Fig. 2a, new Extended Data Fig. 5a and 5c, new Extended Data Fig. 9a and 9c and new\nExtended Data Fig. 14a.\n2. Segment-Specific Rescue Analysis: To determine whether gene editing affects specific nephron\nsegments, could you perform IF staining using segment-specific nephron markers? Suggested markers\ninclude LTL (proximal tubule), NKCC2 (loop of Henle), NCC (distal convoluted tubule), and cytokeratin\n(collecting duct).\nResponse: Thanks for your comments. We have performed the immunofluorescence (IF)\nstaining\nwith markers of DBA, LTL, NKCC2, as well as NCC to determine whether gene editing affects the\nspecific nephron segments in bothAAV9-Cbh-EGFP and AAV9-Ksp-EGFP treated kidneys.\nWe\nfound that the GFP signal is mainly co-localized with DBA, LTL, NCC but not with NKCC2,\nsuggesting that the AAV mediated gene editing mainly affects proximal tubule cells, collecting\nducts cells, distal convoluted tubule but not in loop of Henle (new Extended Data Fig. 1 and new\nExtended Data Fig. 7).\n3. Serum Creatinine Measurement: Have serum creatinine levels been measured? If so, do they change in\na manner similar to blood urea nitrogen (BUN) levels in rescued animals?\nResponse: Thanks for your comments. We evaluated the serum creatinine levels in 3- and 6-\nmonth-old Pkd1RC/RC and WT as well as PN20 Pkd1RC/null mice and age-matched WT mice treated\nwith the Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 and vehicle control. The new results\nshowed that treatment with Cbh-ABE9-dual-AAV9 and Ksp-ABE9-dual-AAV9 could decrease\ncreatinine level in Pkd1RC/RC and PN20 Pkd1RC/null mice compared to aged-match WT (new\nExtended Data Fig. 2b, new Extended Data Fig. 5d, new Extended Data Fig. 9b, new Extended\nData Fig. 14b).\n4. Western Blot Data Presentation: Since Pkd1 expression in gene-edited animals appears weak, Western\nblot data should include all individual data points (three independent samples) run on the same gel, rather\nthan presenting only a single representative sample.\nResponse: Thanks for your comments. We have repeated the Western blot data with all individual\ndata points (three independent samples) run on the same gel and updated the figures in the new\nExtended Data Fig. 2j and 2k, new Extended Data Fig. 3a and 3b, new Extended Data Fig. 5b,\n5e and 5f, new Extended Data Fig. 9d-9f, new Extended Data Fig. 10c and 10d, and new\nExtended Data Fig. 14c.\n5. Antibody Validation for PKD1 Detection: Can you confirm the observed Pkd1 upregulation with an\nalternative antibody? Several validated antibodies are available from the PKDRRC, which could help\nstrengthen the findings.\nResponse: Thanks for your comments. In our study, we used the well-characterized 7e-12\nmonoclonal antibody developed by Peter Harris’ lab, which has been extensively validated and\nwidely used in the PKD research field to detect endogenous Polycystin-1 protein (PMID:\n25574838). This antibody has demonstrated high specificity and sensitivity in both immunoblotting\nwith the full length, NTR and NTS of PC1. Given its excellent performance and broad acceptance\nin the PKD community, we did not use additional antibody. However, we appreciate the suggestion\nand will consider including further validation in future studies to expand reproducibility.\n6. GFP Expression and Specificity in Extended Fig. 6: The GFP signal driven by the Ksp construct appears\nexcessively high, suggesting potential nonspecific expression. Please provide additional controls, including\nmore antibody staining, negative controls, and high-resolution images at higher magnification.\nAdditionally, co-staining with renal tubule markers is needed to demonstrate colocalization, particularly\ngiven previous reports of inefficient AAV9 transduction using the Ksp promoter.\nResponse: Thanks for your comments. We have performed the immunofluorescence (IF)\nstaining with markers of DBA, LTL, NKCC2, as well as NCC to determine whether gene editing\naffects the specific nephron segments in both AAV9-Cbh-EGFP and AAV9-Ksp-EGFP treated\nkidneys. We found that the GFP signal is mainly co-localized with DBA, LTL, NCC but not with\nNKCC2, suggesting that the AAV mediated gene editing mainly affects proximal tubule cells,\ncollecting ducts cells, distal convoluted tubule but not in loop of Henle (new Extended Data Fig.\n1 and new Extended Data Fig. 7). In addition, we also performed immunohistochemistry staining\nand found that GFP is expressed kidneys injected with AAV9-Cbh-EGFP and AAV9-Ksp-EGFP\nbut not that in kidneys injected with blank AAV (new Extended Data Fig. 8), confirming the\nspecificity of AAV-mediated GFP expression.\n7. Western Blot for PN28 in Fig. 1g: Please include a Western blot for PN28 in Figure 1g.\nResponse: Thanks for your comments. We have added the Western blot for PN28 in new\nExtended Data Fig. 3b.\n8. Methodology Details: More details are needed in the Methods section to enhance reproducibility and\nclarity.\nResponse: Thanks for your comments. We have added the details in the Methods section and\nhighlighted them.\n9. Text Clarity and Grammar: The clarity of the text should be improved throughout. For example, in the\nabstract, \"causing by mutations\" should be corrected to \"caused by mutations.\"\nResponse: Thanks for your comments. We have checked and corrected the grammar errors in\nthe revised manuscript.\nReviewer #2\nIn this manuscript, the authors have demonstrated the use of adenine base editing, delivered by adeno-\nassociated viral (AAV) vectors, to correct a pathogenic variant in the Pkd1 gene that causes kidney and\nheart phenotypes (renal cyst growth, cardiac hypertrophy) in mice consistent with human autosomal\ndominant polycystic kidney disease (ADPKD). They achieve sufficient correction to partially revert the\nkidney and heart phenotypes. In a mouse model with exaggerated disease (early mortality), the base\nediting is even able to modestly improve lifespan. To my knowledge, this is the first in vivo\ndemonstration of the use of corrective editing to improve ADPKD and, more generally, any genetic\nkidney disease in a mouse model.\nThe work is technically sound, and I do not have substantive criticisms in that respect. I have the\nfollowing suggestions to improve the manuscript.\n(1) The authors give themselves more credit than is warranted. \"In this study, we developed two adenine\nbase editors (ABE9) delivered by AAV9\" makes it seem like they invented ABE9 for this study, when in\nfact it was simply adapted from a publication from another group. \"We first developed a broadly\nexpressed ABE9 base editor system\"--they simply swapped out a deaminase domain from ABE8 to ABE9\nin existing AAV vectors from another group. \"We developed a kidney specific ABE9 base editor system\"--\nthey simply swapped out a promoter from a generally expressed promoter to an established kidney\npromoter. From a technical perspective, these are incremental changes. The mouse data are novel\nenough on their own to warrant publication--the authors do not need to represent themselves as having\ndeveloped whole new technologies. I would advise toning down the language and focusing on what is\ntruly novel about this work.\nResponse: Thanks for these comments and suggestions. We appreciate the reviewer’s comment\nthat “the mouse data are novel enough on their own to warrant publication”. We have revised the\nmanuscript to tone down the language in multiple sections, including the Abstract, Results, and\nDiscussion, to more accurately reflect the nature of our contribution. For example, in the revised\nabstract, we modified as: “In this study, we adapted one of the gene editing systems, adenine\nbase editor (ABE9), to develop a broadly and a kidney-specific base editors, and test the effects\nof these two systems delivered by AAV9 on preventing disease in humanized Pkd1RC/RC mice\ncarrying an arginine (R) to cystine (C) mutation that mimics a mutation in ADPKD patients”.\n(2) \"We developed a kidney specific ABE9 base editor system\"--their data plainly demonstrate that it is not\nkidney specific, as it produces editing in the liver as well. They did not perform editing biodistribution\nstudies to assess many organs in the mouse. The major difference they show is that the Ksp promoter\ncompared to the Cbh promoter reduces expression in the heart. Is this actually a desirable effect, given\nthat ADPKD causes cardiac hypertrophy, and an optimal ADPKD therapy would target the heart as well as\nthe kidneys? Are there any meaningful downsides of having corrective editing of Pkd1 to wild-type in\norgans other than the kidneys and heart? If not, why is it important to have a kidney specific system? The\nauthors should better justify this.\nResponse: Thanks for this thoughtful comment. The rationale for generating both an universal\nand a kidney specific base editor is to, 1) determine whether base editor can work in specific\norgan, such as kidney, 2) minimize off-target editing and potential toxicity in non-renal tissues,\nand 3) explore whether restricting base editing activity to renal epithelial cells can enhance\ntherapeutic precision compared to universal base editor. Importantly, our results support that\nkidney specific base editor works better than the universal base editor in correcting point mutation\nin kidney. In addition, our results that kidney specific base editor does not expressed in the heart\nsupport the potential to target specific organs with organ specific base editor in the clinic. Although\nkidney specific base editor may avoid off-target effects in non-renal tissues, for that ADPKD is a\nsystemic disease and cardiac manifestations, such as cardiac hypertrophy, are clinically relevant\n(PMCID: PMC7136326), the base editing in other organs—particularly the heart—may not\nnecessarily be undesirable. The application of universal base editor which can correct mutation\nin organs other than kidney may be beneficial in the context of systemic manifestations of ADPKD.\nTh liver is a major metabolic and clearance organ that inevitably receives a large proportion of\nsystemically administered AAV9 vectors. As a result, both universal and kidney specific base\neditors can be eventually accumulated in the liver. The certain degree of vector uptake and the\nexpression of base editor is inevitable, even with tissue-enriched promoters (PMID: 24959122).\nThis is a known limitation of AAV9 delivery and does not necessarily reflect promoter leakiness\nbut rather a vector biodistribution. In the revised Discussion, we elaborate on this point.\nFor the words limitation, we have revised the discussion section as that “With kidney specific base\neditor, we intend to, 1) determine whether base editor can work in specific organ, such as kidney,\n2) minimize off-target editing and potential toxicity in non-renal tissues, and 3) explore whether\nrestricting base editing activity to renal epithelial cells can enhance therapeutic precision\ncompared to universal base editor. Importantly, our results support that kidney specific base editor\nworks better than the universal base editor in correcting point mutation in kidney. In addition, our\nresults that kidney specific base editor does not expressed in the heart support the potential to\ntarget specific organs with organ specific base editor in the clinic. We should point out that ADPKD\nis a systemic disease with extrarenal manifestations, such as cardiac hypertrophy (PMCID:\nPMC7136326) and liver cysts (PMCID: PMC5576944), thus, the application of universal base\neditor which can correct mutation in organs other than kidney may be beneficial in the context of\nsystemic manifestations of ADPKD.”\n(3) Can the authors better explain the rationale for using ABE9? In the Discussion section, the authors\nimply that ABE8e is ineffective in the kidney (whereas ABE9 is effective), but this is based on studies that\ndid not focus on the kidney or on kidney-specific genes and so are not that relevant. Furthermore, the\nmajor advantage of ABE9 over ABE8e is that it has less bystander editing without losing much efficiency\n(there is no claim of increased efficiency)--or, to put it another way, it has a narrower editing window. But\nit is not clear that ABE9 is a better choice than a well-established narrow-window editor like ABE8.8. The\nauthors do not report any initial screening of ABE9 or other ABEs in cells--this seems odd (and unlikely).\nDid the authors really just guess that ABE9 would work the best and go straight to experiments in mice\nwithout any testing in vitro?\nResponse: We appreciate the reviewer’s insightful comment. The rationale for choosing ABE9\nover ABE8e or other variants is based on the following reasons. First, the target mutation in\nPkd1RC/RC mice lies precisely at position A5 within the protospacer. According to the literature\n(PMID: 36229683), ABE9 was specifically engineered to achieve high precision editing at A5 with\nminimizing bystander editing at adjacent adenines and cytosines. In fact, ABE9 demonstrates a\nhighly constrained 1–2 nucleotide editing window centered on A5, with >340-fold higher precision\nat this site compared to ABE8e (PMID: 36229683). Second, the broad editing window of ABE8e\n(positions A3–A9) and its known off-target cytosine editing make it unsuitable for our disease\nmodel, where specificity is critical. As it has been demonstrated that ABE8e induces substantial\noff-target DNA and RNA edits, whereas ABE9 shows background-level off-target activity (PMID:\n36229683). Thus, the choose of ABE9 in this study is guided by the editing window required by\nour target and by evidence that ABE9 is the most precise editor to date for single-nucleotide A-to-\nG conversions, particularly at A5.\nFor the words limitation, we have revised the discussion section as that “Recently, the ABE7 and\nABE8e systems have been developed and tested in Hutchinson-Gilford progeria syndrome and\ncardiac disease (PMID: 33408413, PMID: 36797483, PMID: 36634166) However, it has been\nreported that ABE7 and ABE8e base editing achieves no obvious editing efficiency in kidneys\n(PMID: 33408413). In addition, ABE8e has a broad editing window (positions A3–A9) and a known\noff-target cytosine editing (PMCID: PMC7136326). For that the target mutation in Pkd1RC/RC mice\nlies precisely at position A5 within the protospacer (Extended Data Fig. 6a), and ABE9 has been\nspecifically engineered to achieve high precision editing at A5 with minimized bystander editing\nat adjacent adenines and cytosines (PMID: 36229683), thus, we adapted the ABE9 system to\ngenerate broad and kidney specific base editors.”\n(4) As a proof-of-concept study, the work in this manuscript has considerable merit. However, it seems\nunlikely that the dual AAV approach used in this work would advance to the clinic. Indeed, no AAV-based\ngene editing therapy has made it very far in the clinic (especially compared to non-viral approaches,\nnamely lipid nanoparticles), due to the serious downsides of AAV: prolonged expression of the editor\ncausing a substantial immune response (not typically evident in mice, but quite apparent in large animal\nmodels), along with increased off-target editing; inability to redose the therapy, which is important\nbecause the authors have shown only partial correction of disease in this study, and full correction of the\ndisease would likely need higher levels of editing that could only be achieved with repeat dosing;\npotentially dangerous and even fatal toxicities from having to use high AAV doses in order to (1) account\nfor the need to use dual AAV vectors to deliver a base editor and (2) get enough vector into the kidneys,\ngiven the broad distribution of AAV9 to many organs throughout the body. The authors should have a\nlimitations paragraph in the Discussion acknowledging these barriers to translation.\nResponse: Thanks for this important and thoughtful comment. Actually, the reviewer has already\nprovided an excellent limitation paragraph. Thank you. We agree that AAV-based delivery\nsystems—especially dual AAV systems—face significant translational hurdles. While AAV9 was\nselected here for its well-characterized in vivo performance and ability to transduce the kidney,\nthere are alternative delivery platforms—such as lipid nanoparticles (LNPs)—are emerging as\nmore clinically viable options due to their transient expression profile and potential for repeat\ndosing (PMID: 34880218, PMCID: PMC10616378). Indeed, the first FDA-approved\ninvestigational new drug (IND) application for in vivo base editing therapy (BEAM-101, targeting\nsickle cell disease) employs a non-viral, LNP-based approach, underscoring the clinical\nmomentum behind such systems (PMCID: PMC9868311). We revised the manuscript to reflect\nthese limitations as that “While our study demonstrates the feasibility of in vivo adenine base\nediting to partially correct Pkd1 mutations in a mouse model of ADPKD, several limitations must\nbe clarified. First, prolonged expression of Cas9 and the deaminase may increase the risk of off-\ntarget editing and elicit immune responses, particularly in larger animals and humans59, 60.\nSecond, the use of high AAV doses required for dual-vector delivery to efficiently target kidney\nraises safety concerns in both preclinical and clinical settings61. Future efforts are necessary to\ndevelop more compact base editor systems that can be packaged into a single AAV, and non-viral\ndelivery methods, such as lipid nanoparticles (LNPs) that are testing in sickle cell disease for\nhuman gene editing applications62. In sum, our current study demonstrates therapeutic potential\nand kidney accessibility of base editor, laying the groundwork for further optimization of the\ndelivery technologies/methods for its clinic potential.”\n(5) Line 139: \"Both AGA and CGC encode Arg (A)\"--this should be \"Both AGA and CGC encode Arg (R)\".\nResponse: Thanks for your comments. We have corrected this error in the revised manuscript.\nReviewer #3:\nThe present manuscript describes a base editing strategy delivered via AAV to correct a mutation in the\nPKD1 gene, as a potential therapeutic approach for autosomal dominant polycystic kidney disease\n(ADPKD). The authors demonstrate in vivo editing in a mouse model using ABE9 and also engineer a\nkidney-specific promoter for targeted AAV expression.\nOverall, the manuscript is well-written and supported by comprehensive datasets. The clarity of the\nlanguage could be improved by streamlining several sentences that are currently difficult to follow. The\nmanuscript's structure would also benefit from reorganization for improved data flow (see detailed\ncomments below). I would recommend the manuscript for publication if authors could sufficiently\naddress the following concerns:\nMajor comments:\n1. While the authors present robust characterization of editing efficiency and physiological outcomes,\nthe manuscript lacks any assessment of Cas9-dependent off-target effects. A comprehensive nomination\nof potential off-target sites, followed by experimental validation in edited cells or tissues, is critical to\nsupport the therapeutic relevance and safety of the proposed strategy.\nResponse: Thanks for your comments and suggestions. To address this concern, we predict\npotential off-target sites using CRISPOR, a tool ranks the sgRNA with off-target effects and on-\ntarget activity (PMCID: PMC6037314), and then conducted targeted amplicon deep sequencing\n(Amplicon-seq). There were no detectable off-target editing above background levels at the top\ncandidate sites analyzed (new Extended Data Fig. 6g), supporting the high specificity and safety\nof the base editing system in vivo.\n2. The current structure presents data from AAV injections at PN14 and PN28 (Figures 1 and 2) early in the\nResults, while the corresponding editing efficiency data appear later (Figure 4). For improved clarity and\nnarrative flow, it would be helpful to present the editing outcomes alongside the relevant experimental\ncontext (e.g., incorporating Figure 4 data into Figures 1–3).\nResponse: Thanks for your comments. We have modified the figures based on this comment by\nincorporating Figure 4 data into Figures 1–3.\n3. In the Discussion section (Ling 438-444), authors should discuss the limitation of using Ksp promoter\nfor kidney-specific expression. Since ADPKD also affects other organs, including the heart, the\ntherapeutic strategy’s limitation in targeting extra-renal tissues should be discussed.\nResponse: Thanks for the comments and suggestions. We revised the manuscript based on this\ncomment as: “With kidney specific base editor, we intend to, 1) determine whether base editor\ncan work in specific organ, such as kidney, 2) minimize off-target editing and potential toxicity in\nnon-renal tissues, and 3) explore whether restricting base editing activity to renal epithelial cells\ncan enhance therapeutic precision compared to universal base editor. Importantly, our results\nsupport that kidney specific base editor works better than the universal base editor in correcting\npoint mutation in kidney. In addition, our results that kidney specific base editor does not\nexpressed in the heart support the potential to target specific organs with organ specific base\neditor in the clinic. We should point out that ADPKD is a systemic disease with extrarenal\nmanifestations, such as cardiac hypertrophy (PMCID: PMC7136326) and liver cysts (PMCID:\nPMC5576944), thus, the application of universal base editor which can correct mutation in organs\nother than kidney may be beneficial in the context of systemic manifestations of ADPKD.”\nMinor comment:\n4. Line 118-120: original ABE paper [PMID: 29160308] should be cited in this sentence.\nResponse: Thanks for your comments. We have cited this paper in the revised manuscript.\n5. Please specify the administration route used for IV injection (e.g., retro-orbital vs. tail vein) in the\nMethods section for reproducibility.\nResponse: Thanks for your comments. We have revised the methods section to clarify this point\nas that the administration route used is retro-orbital injection.\n6. Figure 1d and other similar plots have both pink and blue dots. What is the difference between the two-\ncolored dots? Authors should specify in the figure legend.\nResponse: Thanks for your comments. The two colors represent female (pink) and male mice\n(blue), respectively. We clarified this in the revised figure legends."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I still have concerns with the staining data. The IF staining shows markedly more GFP signal in the kidney than in the liver, which is unexpected. Some figures are also unclear—for example, in Fig. 7b, it is not obvious whether the panel represents whole-mount autofluorescence or antibody staining. Additional details are needed for proper interpretation.\nFurthermore, the IHC and IF results for GFP are strikingly different. The IF staining suggests transduction efficiencies approaching 90%, which is extraordinarily high for intravenous AAV delivery, whereas the IHC staining shows much lower efficiency (~20%) with an unusual nuclear localization pattern. This discrepancy should be addressed directly. The authors should discuss why their observed kidney transduction efficiency appears unusually high compared with other published studies, and provide possible explanations (e.g., vector dose, construct design, detection sensitivity, or protein uptake artifacts). Additional IHC images of full kidney sections shown side by side with IF images would help clarify this issue.\nAt present, I remain unconvinced that the GFP signal represents true viral transduction of kidney cells rather than GFP protein synthesized in the liver, released into the bloodstream, and subsequently taken up by renal cells.\nRegarding Fig. 21e–f, the vector genome quantification does not convincingly demonstrate cellular transduction. Measuring total AAV DNA in bulk tissue does not prove that the virus actually entered the GFP-positive cells—it could reflect background signal or a minority of infected cells while the GFP protein itself disseminated systemically.\nTo resolve this, I recommend a straightforward assay such as RNAscope (or intron-spanning RT-qPCR) to directly detect GFP/Cas9 transcripts in kidney, liver, and heart sections. Demonstrating the presence of mRNA within the relevant cells would provide definitive evidence of transcriptional activity, distinguishing genuine transduction from simple protein uptake.", "reviewer_2": "The authors have satisfactorily addressed my concerns.", "reviewer_3": "The authors have sufficiently addressed my previous concerns, and the revision has greatly strengthened the manuscript. I have only a few additional minor comments:\n• Abstract: The statement that the authors developed “a broadly and a kidney-specific base editors” is inaccurate and potentially misleading. The base editors themselves are not “kidney-specific”; rather, it is the promoters that confer tissue-specific expression. Please revise such phrasing for accuracy for the remainder of the manuscript as well.\n• Line 144: Correct “Tada” to “TadA.”\n• Lines 161–162: Please provide the percentage of transduction in each of the cell types that the authors describe as being “mainly expressed.”"}, "author_response": "Response to Reviewer’s Comments\nReviewer #1\nI still have concerns with the staining data. The IF staining shows markedly more GFP signal in the kidney than in the liver, which is unexpected. Some figures are also unclear—for example, in Fig. 7b, it is not obvious whether the panel represents whole-mount autofluorescence or antibody staining. Additional details are needed for proper interpretation.\nResponse: Thanks for the comments. We thought the reviewer’s concern was the images in\nthe Extended Data Fig. 7b instead of Fig. 7b. In Extended Data Fig. 7b, the images showed the\nwhole-mount GFP auto-fluorescence but not the immunofluorescence (IF) staining of GFP with\nantibody in the kidney and liver of mice injected with a Ksp-GFP-AAV vector. The GFP signal in\nthese images was captured by the microscope with the setting of auto-exposure. Thus, the\nmore or less density of the green signals captured in the whole kidney and liver sections could\nnot be compared which only represented that the entry of AAV-Ksp-GFP particles and the\nexpression of GFP in those organs (Extended Data Fig. 7b). We clarified this point in the revised\nfigure legend of this panel.\nFurthermore, the IHC and IF results for GFP are strikingly different. The IF staining suggests\ntransduction efficiencies approaching 90%, which is extraordinarily high for intravenous AAV\ndelivery, whereas the IHC staining shows much lower efficiency (~20%) with an unusual nuclear\nlocalization pattern. This discrepancy should be addressed directly. The authors should discuss\nwhy their observed kidney transduction efficiency appears unusually high compared with other\npublished studies, and provide possible explanations (e.g., vector dose, construct design,\ndetection sensitivity, or protein uptake artifacts). Additional IHC images of full kidney sections\nshown side by side with IF images would help clarify this issue.\nResponse: Thanks for the comments and suggestions. As we response to the concern above,\nthe GFP auto-fluorescence images but not the IF staining of GFP in Extended Data Fig. 7b were\ncaptured from the whole kidney by the microscope with the setting of auto-exposure, which looked\nlike the transduction efficiencies were extraordinarily high (about 90% as the reviewer stated).\nThe appearance of GFP auto-fluorescence in the whole kidney only indicated the entry of Ksp-\nGFP-AAV9 particles and the expression of GFP in the kidney but did not represent the\ntransduction efficiency in kidneys with the injection of Ksp-GFP-AAV9 particles. When we\nperformed the IF staining with GFP antibody as shown in Extended Data Fig. 7c, we found a\nmosaic distribution of GFP epithelial cells within specific nephron segments co-stained with\ndiverse markers, including DBA, LTL, NCC, NKCC2. With the quantification of the GFP-positive\ntubular cells, we found that o⁺nly about 27 % of GFP-positive tubular cells could be detected in the\nkidney treated with Ksp-GFP-AAV9 particles. These results indicate that the transduction of AAV-\nKsp-EGFP particles are expressed in specific renal tubular cells in the kidney, which is consistent\nwith prior reports showing that AAV9 can efficiently target renal tubules, even though those reports\ndid not quantify the percentages of GFP-positive cells in kidneys (PMID: 37556557, PMCID:\nPMC11685967,PMID: 39225099, PMID: 40774951). As suggested, we showed GFP IHC images\nside by side with the GFP IF staining in new Extended Data Fig. 7d. The IHC images of the whole\nkidney sections were also showed in new Extended Data Fig. 7e.\nThe nuclear GFP IHC staining might be a technical artifact of the chromogenic IHC procedure,\nincluding chromogen overdevelopment, weak or uneven hematoxylin counterstaining, and the\nfixation/antigen retrieval conditions, etc. We repeated the IHC staining with optimized conditions,\nincluding a shorter diaminobenzidine (DAB) development time and an improved nuclear\ncounterstaining. The new images showed cytoplasmic and membranous GFP staining in tubular\nepithelial cells, consistent with the IF staining results (Extended Data Fig. 7d and 7e).\nAt present, I remain unconvinced that the GFP signal represents true viral transduction of kidney\ncells rather than GFP protein synthesized in the liver, released into the bloodstream, and\nsubsequently taken up by renal cells.\nResponse: Thanks for the comments. To address this concern, first, we observed that the\ndistribution pattern of GFP in the images of IF and IHC staining was restricted to renal tubular\nepithelial cells (Extended Data Fig. 7c and 7d), rather than a diffuse or vascular pattern that was\nusually observed for protein uptake from the circulation. Second, by performing the junction-\nspanning RT-qPCR analysis, we confirmed a robust expression of GFP transcript in kidneys\n(new Extended Data Fig. 20a-20b), indicating an active transcription of the true viral\ntransduction of GFP transgene. Together, these results support that the GFP signal reflects true\nrenal cell transduction and expression, rather than nonspecific uptake of GFP synthesized\nelsewhere.\nRegarding Fig. 21e–f, the vector genome quantification does not convincingly demonstrate cellular\ntransduction. Measuring total AAV DNA in bulk tissue does not prove that the virus actually entered\nthe GFP-positive cells—it could reflect background signal or a minority of infected cells while the\nGFP protein itself disseminated systemically. To resolve this, I recommend a straightforward assay\nsuch as RNA scope (or intron-spanning RT-qPCR) to directly detect GFP/Cas9 transcripts in kidney,\nliver, and heart sections. Demonstrating the presence of mRNA within the relevant cells would\nprovide definitive evidence of transcriptional activity, distinguishing genuine transduction from\nsimple protein uptake.\nResponse: Thanks for the comments and suggestions. Because our AAV expression cassette\ndoes not contain introns within the GFP transcript, the classical intron-spanning RT-qPCR\nacross GFP exons is not applicable. Instead, we use junction-spanning RT-qPCR targeting the\nspliced 5′-UTR–GFP junction present in the mature mRNA. This strategy provides spatial\nevidence of GFP mRNA-level to distinguish genuine transduction from protein uptake. With this\napproach, we detected robust GFP transcripts in kidney, liver, and heart, as shown in the new\nExtended Data Fig. 20a–20b. These results provide direct evidence of the active transcription of\nGFP transgene in the kidney, liver and heart treated with Cbh- and Ksp-GFP-AAV9 particles,\nrespectively.\nReviewer #3\nThe authors have sufficiently addressed my previous concerns, and the revision has greatly\nstrengthened the manuscript. I have only a few additional minor comments:\n• Abstract: The statement that the authors developed “a broadly and a kidney-specific base editors”\nis inaccurate and potentially misleading. The base editors themselves are not “kidney-specific”;\nrather, it is the promoters that confer tissue-specific expression. Please revise such phrasing for\naccuracy for the remainder of the manuscript as well.\nResponse: Thanks for your comments. We have modified the sentences in the revised\nmanuscript.\n• Line 144: Correct “Tada” to “TadA.”\nResponse: Thanks for your comments. We have corrected it in manuscript.\n• Lines 161–162: Please provide the percentage of transduction in each of the cell types that the\nauthors describe as being “mainly expressed.”\nResponse: Thanks for your comments. We have added the percentage of transduction in each\nof the cell types in the revised manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed all major concerns."}, "author_response": ""}]

Decision letter