Enhancing gene transfer to renal tubules and podocytes by context-dependent selection of AAV capsids

AAV vectors and plasmids

AAV-CAG-VBC library, AAV9-CAG-tdTomato, AAV-KP1-CAG-tdTomato, and AAV-KP1-CAG-nlsGFP were produced in HEK293 cells (RRID: CVCL-6871, Agilent) by an adenovirus-free plasmid transfection method and purified by two rounds of cesium chloride density-gradient ultracentrifugation followed by dialysis as described previously⁶³. AAV9 and AAV-KP1 helper plasmids were provided by J. M. Wilson and M. A. Kay, respectively. pAAV-CAG-tdTomato and pAAV-CAG-nlsGFP were gifts from E. Boyden and V. Gradinaru (59462 and 104061, Addgene, Watertown, MA), respectively. For AAV barcode library production, we used pdsAAV-CAG-VBCx plasmids (where VBC is the viral barcode and x is an integer identification number indicating each different viral barcode contained in each plasmid). pdsAAV-CAG-VBCx plasmids are double-stranded (ds) AAV vector plasmids and same as pdsAAV-U6-VBCx described previously⁶³ except that the human U6 small nuclear RNA gene promoter has been replaced by the CAG promoter and a SV40 polyadenylation signal has been incorporated. For 47 AAV capsids in the library (Supplementary Table 1), we used a total of 114 barcodes. Two barcoded capsid clones were allocated to each capsid except for AAV9 and AAV2R585E, for which 15 and 9 barcoded clones were allocated, respectively. Each AAV capsid clone was produced in separate culture vessels. Subsequently, all the produced clones were mixed into a pool after titer adjustment and purified to create an AAV library stock as described elsewhere¹⁶.

Mouse experiments

All the mouse experiments were performed according to the guidelines for animal care at Oregon Health & Science University (OHSU). Mice were kept with free access to food (5L0D, LabDiet, St. Louis, MO) and water in a controlled environment (12-h light-dark cycle with an average humidity range of 30–70% and temperatures of 20–23 °C). C57BL/6 J mice and B6.Cg-Col4a5^tm1Yseg/J mice were purchased from the Jackson Laboratory (Strain IDs are 664 and 6183, respectively). For the AAV Barcode-Seq analysis, 8-week-old C57BL/6 J male mice were injected with the AAV barcode library via the tail vein at a dose of 2.0 × 10¹³ vg/kg (n = 3), and via a renal vein (n = 4) and renal pelvis (n = 3) at a dose of 3.0 × 10¹¹ vg/mouse. Six weeks post-injection, mice were euthanized, and kidneys were harvested. For the AAV9 and AAV-KP1 individual capsid validation study, 8-week-old C57BL/6 J male mice were injected with AAV9-CAG-tdTomato or AAV-KP1-CAG-tdTomato via the tail vein (i.e., IV injection), renal vein (RV), and renal pelvis (RP) at a dose of 3.0 × 10¹¹ vg/mouse (n = 4 per group). Two weeks post-injection, mice were euthanized, and kidneys and livers were harvested for vector genome quantification and histological assessment by immunofluorescence microscopy. For a pharmacokinetic study, 8-week-old C57BL/6 J male mice were injected with AAV9-CAG-tdTomato or AAV-KP1-CAG-tdTomato via IV, RV, or RP injection at a dose of 1.0 × 10¹³ vg/kg (n = 4 per group). Subsequently, whole blood samples were collected from the retro-orbital plexus at 6-time points (0 min for RV and RP when the 15-min dwelling was completed or 1 min for IV, followed by 10 min, 30 min, 1 h, 4 h, and 8 h) following vector injection, and vector genome copy numbers in the blood samples were determined as detailed in the AAV vector genome quantification section. To quantify vector genome copy numbers in the kidney after local vector injection, 8-week-old C57BL/6 J male mice were injected with AAV9-CAG-tdTomato or AAV-KP1-CAG-tdTomato via RV or RP injection at a dose of 1.0 × 10¹³ vg/kg (n = 4 per group). Subsequently, injected kidneys were harvested 10 min after the completion of 15-min dwelling time following the RV and RP injections, and vector genome copy numbers in the kidney were determined as detailed in the AAV vector genome quantification section. To investigate the renal transduction in CKD, 25 to 30-week-old B6.Cg-Col4a5^tm1Yseg/J hemizygous male mice and age-matched wild-type controls from the colony were randomly allocated to two groups and intravenously injected with AAV9-CAG-tdTomato or AAV-KP1-CAG-tdTomato at a dose of 1.0 × 10¹³ vg/kg (n = 3–5 per group). After injection, mice were put in the metabolic cage (MMC100, Hatteras Instruments, Grantsboro, NC) for 5 h to collect the urine samples. Two weeks post-injection, mice were euthanized, and the kidneys and hearts were harvested for downstream analysis. For active immunization with AAV-KP1, 20-week-old C57BL/6 J male mice were injected via IV with AAV-KP1-CAG-nlsGFP at a dose of 2.3 × 10¹¹ vg/mouse and incubated for 3 weeks for anti-AAV-KP1 antibody production. For passive immunization with AAV-KP1, six 11-week-old C57BL/6 J male mice were injected via IV with AAV-KP1-CAG-tdTomato at a dose of 1.0 × 10¹¹ vg/mouse. Four weeks post-injection, sera were collected from these mice, pooled, and kept frozen until use. Passive immunization of mice was then established by infusing 100 μL of the pooled sera immediately before the experimental treatment. As a control, one mouse received 100 μL of naive mouse sera instead of the anti-sera and was subjected to the same experimental treatment. To address sex as a biological variable, we injected the AAV-KP1-CAG-tdTomato vector into C57BL/6 J female mice via RV (n = 2) and RP (n = 3).

Mouse surgical procedures

For RV injection, modified from the previous protocol¹⁰, mice were anesthetized by isoflurane inhalation and placed on a heated surgical pad (8002062012, Stryker Medical, Portage, MI) to maintain a constant body temperature. A medial abdominal incision was made, and intestines were removed from the abdominal cavity to expose the left renal vasculature and the kidney. Removed intestines were kept moist throughout the surgery. A non-traumatic micro-serrefine clamp was placed on the renal artery and vein, and 50 μL of AAV vector solution was injected using a 31-gauge needle (328468, BD Medical, Franklin Lakes, NJ) by hand as quickly as possible. Following the 15-minute ischemic time, the clamp was removed to observe the restoration of the blood flow (verified by color change), and the incision was closed. For RP injection, modified from the previous protocol⁶⁴, a flank incision was made to expose the left kidney to place a non-traumatic micro-serrefine clamp on the renal pedicle and the ureter under general anesthesia. Fifty μL of AAV solution was injected into the pelvic cavity over 1 min using a syringe pump (70-4507, Harvard Apparatus, Holliston, MA) to prevent parenchymal damage and leakage. Fourteen minutes post-injection (total ischemic time, 15 min), the clamp was removed. After ensuring the restoration of the blood flow, the incision was closed. To assess the effect of ischemia on renal transduction, a flank incision was made and a clamp was placed on the renal artery and vein for 15 min. Following the 15-minute ischemic time, the clamp was removed, and the vector solution was injected through the tail vein. RV and RP injections were also performed with varying durations of ischemia: < 1 min (RV injection) or 0 min (RP injection), 5, 10, and 15 min. In the < 1 min group, RV injection was performed with a minimal length of ischemia required for the injection. The 0 min condition was not possible for RV injection because the renal blood flow needs to be stopped when the agent is injected via RV into the kidney. RP injection was performed without the blockade of the blood flow of the renal artery and vein for the 0-min group and a clamp was placed on the ureter for 15 min regardless of the ischemic time.

Retrograde transureteral renal pelvis (RP) injection into NHPs

NHPs were managed according to the Oregon National Primate Research Center (ONPRC) program for animal care, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International, and based on guidelines, laws, regulations and principles stated in the Animal Welfare Act (United States Department of Agriculture), Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research), and the Public Health Service Policy on Humane Care and Use of Laboratory Animals (National Institutes of Health). Experimental procedures were reviewed and approved by the OHSU Institutional Biosafety Committee and the ONPRC Institutional Animal Care and Use Committee. Study animals were housed individually on a 12-h light-dark cycle in a climate-controlled, Animal Biosafety Level 2 (ABSL-2) facility that allowed protected interaction with other rhesus macaques in the same room. Seven female rhesus macaques were used for the study: one (NHP1) received the vehicle only, four (NHP2, 3, 5, 6) received AAV-KP1-CAG-tdTomoato vector, two (NHP4, 7) received AAV9-CAG-tdTomoato vector. We used female animals because the shorter urethra in females than in males would make the insertion of the fibrotic scope easier. Anti-AAV-KP1 or anti-AAV9 NAb titers were determined in the AAV vector-injected animals before the injection as described previously with minor modifications⁶⁵. Serum samples were diluted by serial two-fold dilutions ranging from 1:5 to 1:5120. NAb titer was defined as the 50% inhibition of virus transduction calculated based on the global fitting of the data obtained from each of the serum dilutions. For the first set of the three animals (NHP2, NHP3, NHP4), we selected those that were negative (< 1:5, NHP3, and NHP4) or marginally positive (1:7.5, NHP2) for NAbs. For the second set of three animals (NHP5, NHP6, and NHP7), we selected those that were unambiguously positive for NAb on purpose. Renal transduction and off-target liver transduction were assessed three weeks after injection. Serial blood and urine samples were collected to determine vector pharmacokinetics and assess the safety of the procedure. The procedure comprised the following steps: (1) Under general anesthesia, a fiber bronchoscope (11282BN1, Karl Storz) was inserted into the bladder through the urethra and the right and left ureteral orifices were identified; (2) A 0.025-inch Glidewire (Terumo) was advanced via the ureter to the right or left renal pelvis depending on the choice of the target kidney; (3) The scope was removed and a 4 French 40 cm Fogarty balloon catheter (Edwards Medical) was advanced over the wire; (4) The balloon of the Fogarty catheter was inflated at or just before the ureteropelvic junction; (5) The volume of the pelvic cavity was estimated by injecting a contrast agent under the fluoroscope; (6) The estimated pelvic volume of AAV vector solution was injected over 1 min into the pelvic cavity, followed by injection of air (~ 0.5 mL) to increase the pelvic pressure; (7) After 5 min dwelling time, the animal position was changed to the ipsilateral decubitus position so that the injected air into the renal pelvis lied over the injected AAV vector solution, and the animal was maintained in this position for an additional 5 min; (8) After a total of 10 min dwelling time, the animal position was returned to the supine position and the balloon was deflated; and (9) The balloon catheter was removed after confirming the appropriate placement of the catheter by injecting a small volume of the contrast agent.

AAV Barcode-seq analysis

Total DNA was extracted from tissues using the QIAamp MinElute Virus Spin Kit (57704, Qiagen, Venlo, Netherlands) or the KingFisher Cell and Tissue DNA Kit (97030196, Fisher Scientific, Hampton, NH) following Proteinase K (25530049, Invitrogen, Waltham, MA) treatment. Total RNA was extracted from tissues using TRIzol (15596018, Invitrogen, Waltham, MA) followed by DNase treatment using the TURBO DNA-free Kit (AM1907, Invitrogen, Waltham, MA). Point eight μg of DNase-treated RNA was reverse-transcribed with reverse transcription (RT)-specific primer (5’-GGCGGCGGTCACGAA-3’) using the High-Capacity cDNA Reverse Transcription Kit (4368813, Applied Biosystems, Waltham, MA) or SuperScript IV Reverse Transcriptase (18090200, Invitrogen, Waltham, MA) in a total volume of 20 μL. One μg DNA or 4 μL cDNA was used to PCR-amplify virus barcode (VBC) using Platinum SuperFi II DNA Polymerase (12361010, Invitrogen, Waltham, MA). Following primers were used to amplify left and right VBCs: left VBC forward (5’- FSN-SBC-ACCTACGTACTTCCGCTCAT-3’), left VBC reverse (5’-FSN-SBC-TCCCGACATCGTATTTCCGT-3’), right VBC forward (5’-FSN-SBC-ACGGAAATACGATGTCGGGA-3’) and right VBC reverse (5’-FSN-SBC-CTTCTCGTTGGGGTCTTTGC-3’). Each primer has an 8 nucleotide-long sample-specific Barcode (SBC) and a 0-4 nucleotide-long frame-shifting nucleotide (FSN) as previously described^16,63. PCR products were mixed at an approximately equimolar ratio and sequenced at the OHSU Massively Parallel Sequencing Shared Resource (MPSSR) or Novogene (Sacramento, CA). The sequencing was performed using the following configurations on an Illumina NextSeq 500 or NovaSeq 6000 instrument: 75-cycle single-end, 150-cycle single-end, 180-cycle single-end, or 300-cycle paired-end. The quality of Illumina raw sequence reads was assessed by FastQC, in which the following four quality measures, per base sequence quality, per sequence quality scores, per base N content, and sequence length, were all met in all the data sets we used in this study. The Illumina sequencing data were then analyzed at the Pittsburgh Supercomputing Center or the Advanced Computing Center at OHSU to determine Phenotypic Difference (PD) values of each AAV capsid^16,63. PD values indicate the ‘fold change’ of a phenotype compared with that of the reference control, which was AAV9 in this study. The yields of reverse-transcription (RT)-PCR products for RNA Barcode-Seq relative to the quantity of vector genomes could vary depending on the VBC sequences. This is because, as opposed to the DNA Barcode-Seq, the RNA Barcode-Seq involves additional steps, i.e., in vivo mRNA transcription and in vitro reverse transcription, in which the VBC-dependent differences cannot be canceled out by determining the ratios between input vector genome DNA read counts and vector genome transcript RNA read counts. Thus, in our RNA Barcode-Seq analysis, we introduced correction factors to normalize RNA Barcode-Seq PD values and make the PD values independent of VBC sequences. This approach helps minimize undesired variations in VBC sequence-dependent RNA Barcode-Seq readouts. The correction factor for each VBC pair in the RNA Barcode-Seq analysis was experimentally determined as follows and summarized in Supplementary Data 1. In brief, we made two AAV9-CAG-VBCx libraries that contain all the AAV-CAG-VBCx genomes packaged with the same AAV9 capsid. These two libraries were produced independently from two independent pools of all the pdsAAV-CAG-VBCx plasmids mixed at an equimolar ratio. Each of the two AAV9-CAG-VBCx virus libraries was intravenously injected into 8-week-old C57BL/6 J male mice (n = 3 each) to obtain an independent, duplicated set of data, each of which was obtained from 3 mice. The livers were harvested from the library-injected mice 6 weeks post-injection. Liver DNA and RNA were extracted and then subjected to the DNA and RNA Barcode-Seq analysis, which provides RNA and DNA barcode reads, respectively. The correction factors obtained by the ratio of RNA and DNA barcode reads were used to cancel out the barcode sequence-dependent differences in the in vivo mRNA transcription and the RT-PCR amplification efficiencies between VBCs. Data variations of the benchmark AAV9 capsids between biological replicates were quantified by calculating the proportion of each barcode relative to the total barcode count in the tissue of interest. While our data were normalized by the composition of the initial library, we did not perform additional adjustments by multiplying the data with vector genome per diploid genomic equivalent (vg/dge), the approach employed by Weinmann et al.¹⁸. Our approach is justified because our analysis focused exclusively on the relative transduction efficiencies within the kidney treated with the same procedure and did not compare transduction profiles across different organs or different procedures, making the data independent of renal transduction levels. Nonetheless, for completeness, we conducted additional adjustments on the Supplementary Fig. 1 data by vg/dge of each sample as reported by Weinmann et al.¹⁸ and presented the results in Supplementary Fig. 11.

AAV vector genome quantification

Total DNA was extracted from tissues as described in the AAV Barcode-Seq analysis section. Blood DNA sample was prepared using the Extract-N-Amp Blood PCR Kit (XNAB2R, Sigma, St. Louis, MO) and diluted 100 times. Total DNA was extracted from 100 μL urine samples using the Wako DNA Extraction Kit (29550201, Wako Chemicals, Richmond, VA) following Proteinase K treatment. The urine DNA pellet was dissolved in 15 μL Tris-HCl buffer. AAV vector genome copy numbers were quantified by quantitative PCR (qPCR). In brief, 10–100 ng of tissue DNA or 1 μL of blood or urine DNA sample was mixed with Power SYBR Green PCR Master Mix (43-676-59, Fisher Scientific, Hampton, NH) and 25 pmol primers in a 25 μL reaction volume and subjected to qPCR using Rotor-Gene Q (Qiagen, Venlo, Netherlands). We amplified the tdTomato gene sequence and the barcode sequence for vector genome quantification using the following primers: tdTomato forward (5’-ATGGACCTGTGATGCAGAAG-3’), tdTomato reverse (5’-TTCAGCTTCAGAGCCTGGTG-3’), barcode forward (5’-ACCTACGTACTTCCGCTCAT-3’) and barcode reverse (5’-CTTCTCGTTGGGGTCTTTGC-3’). Information on copy number standards and normalization for mouse study was described previously¹⁶. For the NHP study, the following primers were used: ACTB forward (5’-AGCTGCGCCCTTTCTCACTG-3’) and ACTB reverse (5’-CAGAGTTCCAAAGGAGACTC-3’). Vector genome copy numbers were expressed as double-stranded vector genome copy numbers per diploid genomic equivalent (ds-vg/dge) except in blood and urine samples, where single-stranded vector genomes per unit sample volume (vg/μL) were used for quantification and in Fig. 4a, where single-stranded vector genome copy numbers per diploid genomic equivalent (vg/dge) were presented due to the likelihood that most vector genomes in the kidneys are in single-stranded form and protected by AAV capsids 10 min post-injection. For the NHP kidneys, vector genome copy numbers were assessed in three samples obtained from the upper, middle, and lower thirds of the kidney and averaged. For the NHP livers, vector genome copy numbers of the right and left lobes were averaged.

Histological processing and analysis

Organs were harvested from mice following PBS perfusion. Harvested organs were fixed in 4% paraformaldehyde (PFA) (158127, Sigma, St. Louis, MO) and subsequently equilibrated in 30% sucrose (S5, Fisher Scientific, Hampton, NH) for cryoprotection. The fixed tissues were cryo-embedded in Tissue-Tek O.C.T. Compound (4583, Sakura Finetek, St. Torrance, CA), cut into 5 μm-thick sections using a cryostat, analyzed by immunofluorescence and light microscopy. Immunostaining was performed as described previously⁶⁶ using rabbit anti-WT1 (1:100, ab89901, Abcam, Cambridge, UK), rat anti-CD31 (1:100, 550274, BD Biosciences, Franklin Lakes, NJ), Lotus tetragonolobus lectin (LTL)-fluorescein (1:400, FL-1321-2, Vector Laboratories, Newark, CA), rabbit anti-HNF4α (1:200, 3113, Cell Signaling Technology, Danvers, MA), rabbit anti-NCAM1 (1:200, 99746, Cell Signaling Technology, Danvers, MA), rabbit anti-NKCC2 (1:100, SPC-401, StressMarq, Victoria, BC, Canada), rat anti-PDGFRβ (1:100, 14-1402-81, ThermoFisher Scientific, Waltham, MA), rabbit anti-AQP2 (1:100 ab199975, Abcam, Cambridge, UK), goat anti-rabbit IgG Alexa Fluor 647 (1:500, 111-605-144, Jackson ImmunoResearch, West Grove, PA), goat anti-rat IgG Alexa Fluor 647 (1:500, 112-605-167, Jackson ImmunoResearch, West Grove, PA) and Hoechst (1:10000, H3570, ThermoFisher Scientific, Waltham, MA). Immunofluorescence images were obtained using a BZ-X700 Fluorescence Microscope (Keyence, Osaka, Japan) or an LSM900 Confocal Microscope with Airyscan2 (Carl Zeiss, Oberkochen, Germany). For the quantification of tdTomato-positive proximal tubule cells, we used HNF4α instead of LTL as a proximal tubule marker because nuclear labeling by anti-HNF4α antibody enables separation of individual proximal tubule cells in contrast to the brush border labeling by LTL, which labels proximal tubule as a segment. As shown in Supplementary Fig. 12a, anti-HNF4α antibody and LTL label the same population of cells in the kidney. We manually counted the tdTomato-positive and -negative proximal tubule cells. At least 4 images per kidney were analyzed. To determine the percentage of tdTomato-positive podocytes, five glomeruli per kidney were selected from sections that were cut in close proximity to the middle of the glomeruli. Z-stacked images were taken, and tdTomato-positive and -negative podocytes were manually counted. Representative high-magnification images used for quantification were presented in Supplementary Fig. 12 and Supplementary Movies 1–4. For the quantitative analysis of heart transduction, the heart area was determined through thresholding, and the mean fluorescent intensities (MFIs) of both the heart and the background were measured by ImageJ. The net intensity obtained by subtracting the background MFI from the heart MFI was used for the quantification of transduction. Hematoxylin and eosin (H&E) staining was also performed on NHP kidney sections to assess tissue damage.

Nanoparticle distribution in the kidney following injection

Eight-week-old C57BL/6 J male mice were injected with fluorescent polystyrene beads (G25, ThermoFisher Scientific, Waltham, MA)⁶⁷ via IV (300 μL), RV (50 μL) or RP (50 μL). The beads are fluorescent microspheres with a diameter of 25 nm, which is comparable in size to AAV. Therefore, they can be used as surrogates to investigate the distribution of AAV particles following injection. We used 4 conditions IV, RV, RP with blockade of the blood flow in the renal artery and vein, and RP without the blockade (n = 3 per condition). For IV injection, the distribution was assessed 30 min post-injection. For RV and RP injections, the distribution was assessed immediately after the completion of each injection procedure. Organs were harvested after PBS perfusion to remove beads from the blood vessels. Staining was performed as described above.

Assessment of albuminuria

Spot urine was collected for the quantification of albuminuria. Urine albumin concentration was measured using Albuwell M (1011, Ethos Biosciences, Logan Township, NJ) and normalized by creatinine concentration measured by Creatinine Reagent Assay (C75391250, Pointe Scientific, Canton, MI). The data was expressed as gram-per-gram creatinine (g/gCre).

Blinding

The following procedures were conducted in a blinded fashion in which the investigator remained unaware of the specifics of the vectors and samples: AAV Barcode-Seq analyses, urine albumin measurement, and a part of fluorescence microscopic studies in healthy and CKD kidneys. Although other experiments were conducted in an unblinded fashion, efforts were made to minimize bias and ensure objective observations.

Statistics

Statistical analyses were performed using GraphPad Prism 9 (version 9.5.0). Data are presented as mean ± standard error of the mean. Due to the small sample sizes in our study, normality tests such as the Shapiro-Wilk test may have limited power in detecting departures from normality. Therefore, we assumed approximate normality based on the visual inspection of the data and conducted parametric tests, the results of which are presented in this paper. In this approach, comparisons between two groups were performed using a two-tailed Welch’s t test. For data sets with more than two groups, significance was determined by a one-way ANOVA or two-way ANOVA followed by Tukey’s post hoc test or a two-tailed Welch’s t test with Bonferroni correction. P < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54475-9