Nature Neuroscience

AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset

Plasmids

The first-round viral DNA library was generated by amplification of a section of the AAV-PHP.eB capsid genome between AA450 and AA599 using NNK degenerate primers (Integrated DNA Technologies) to substitute AA452–AA458 with all possible variations. The resulting library inserts were then introduced into the rAAV-ΔCap-in-cis-Lox plasmid via Gibson assembly as previously described26. The resulting capsid DNA library, rAAV-Cap-in-cis-Lox, contained a diversity of ~1.28 billion variants at the AA level. The second-round viral DNA library was generated similarly to the first round, but, instead of NNK degenerate primers at the AA452–AA458 location, a synthesized oligo pool (Twist Bioscience) was used to generate only selected variants. This second-round DNA library contained a diversity of ~82,000 variants at the AA level.

The AAV2/9 REP-AAP-ΔCap plasmid transfected into HEK293T cells for library viral production was modified from the AAV2/9 REP-AAP plasmid previously used26 by deletion of the AAs between 450 and 592. This modification prevents production of a WT AAV9 capsid during viral library production after a plausible recombination event between this plasmid co-transfected with rAAV-ΔCap-in-cis-Lox containing the library inserts.

Three rAAV genomes were used in this study. The first, pAAV-CAG-mNeonGreen (Addgene, 99134), uses a single-stranded (ss) rAAV genome containing the fluorescent protein mNeonGreen under control of the ubiquitous CMV-β-actin-intron-α-globin hybrid promoter (CAG). The second, pAAV-CAG-NLS-GFP (Addgene, 104061), uses an ssAAV genome containing the fluorescent protein EGFP flanked by two nuclear localization sites, PKKKRKV, under control of the CAG promoter. The third, pAAV-CAG-FXN-HA, uses an ssAAV genome containing an HA-tagged FXN protein under control of the CAG promoter and harboring a unique 12-bp sequence in the 3′ untranslated region to differentiate different capsids packaging the same construct.

Viral production

rAAVs were generated according to established protocols59. In brief, HEK293T cells (American Type Culture Collection) were triple transfected using polyethylenimine; virus was collected after 120 h from both cell lysates and media and purified over iodixanol (OptiPrep, Sigma-Aldrich). The isolated variants investigated in vivo (AAV.CAP-B1, AAV.CAP-B2, AAV.CAP-B8, AAV.CAP-B10, AAV.CAP-B18 and AAV.CAP-B22) have similar production titer to AAV9, with normal titers around 1 ± 0.7 × 1012 viral genomes per 15-cm dish.

A modified protocol was used for transfection and purification of viral libraries. First, to prevent mosaic capsid formation, only 10 ng of rAAV-Cap-in-cis-Lox library DNA was transfected (per 150-mm plate) to decrease the likelihood of multiple library DNAs entering the same cell. Second, virus was collected after 60 h, instead of 120 h, to limit secondary transduction of producer cells. Finally, instead of polyethylene glycol precipitation of the viral particles from the media, as performed in the standard protocol, media were concentrated more than 60-fold for loading onto iodixanol.

Animals

All rodent procedures were approved by the Institutional Animal Use and Care Committee of the California Institute of Technology. Transgenic animals, expressing Cre under the control of various cell-type-specific promoters, and C57Bl/6J WT mice (000664) were purchased from Jackson Laboratory. Transgenic mice included Syn1-Cre (3966), GFAP-Cre (012886), Tek-Cre (8863) and TH-Cre (008601). Mice were housed under standard conditions between 71 °F and 75 °F, in 30–70% humidity and on a light cycle of 13 h on and 11 h off. For round 1 and round 2 selections from the viral library, we used one male and one female mouse from each transgenic line (aged 8–12 weeks), as well as a single male C57Bl/6J mouse. For validation of individual viral variants, male C57Bl/6J mice aged 6–8 weeks were used. Intravenous administration of rAAV vectors was performed via injection into the retro-orbital sinus.

Marmoset (C. jacchus) procedures for MPV1 and MPV2 and for marmoset single variant 1–4 (MSV1–4) and MPV11–13 were approved by the Animal Care and Use Committee of the National Institutes of Mental Health (NIMH). MPV1, MPV2, MSV1–4 and MSV11–13 were born and raised in NIMH colonies and housed in family groups under standard conditions of 27 °C and 50% humidity. They were fed ad libitum and received enrichment as part of the primate enrichment program for NHPs at the National Institutes of Health (NIH). For AAV infusions, animals were screened for endogenous neutralizing antibodies. None of the animals that were screened showed any detectible blocking reaction at 1:5 dilution of serum (Penn Vector Core, University of Pennsylvania). They were then housed individually for several days and acclimated to a new room before injections. Two animals were used for the pooled injection study, both males, aged 7.6 (MPV1) and 11.5 (MPV2) years (Extended Data Table 1). Five animals were injected with single variants for characterization, but only four were usable (Extended Data Table 2), as one animal (AAV.CAP-B22 injected, 6.9 years, female, 0.475 kg) was found dead (27 d after injection), and, at necropsy, the pathology report indicated chronic nephritis unrelated to the virus. The day before infusion, the animals’ food was removed. Animals were anesthetized with isoflurane in oxygen; the skin over the femoral vein was shaved and sanitized with an isopropanol scrub; and the virus (Extended Data Tables 1 and 2) was infused over several minutes. Anesthesia was withdrawn, and the animals were monitored until they became active, upon which they were returned to their cages. Activity and behavior were closely monitored over the next 3 d, with daily observations thereafter.

Marmoset procedures for MSV8–10 were approved by the Committee on Animal Care of the Massachusetts Institute of Technology (MIT), and all experiments were performed in accordance with the relevant guidelines and regulations. Marmosets were born and raised in an MIT facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Marmosets were housed in social groups under standard conditions of 23.3 ± 1.1 °C, 50% ± 20% humidity and a 12-h light/dark cycle. They were fed ad libitum with standard diet as well as fruits, vegetables and various protein sources. Periodic neutralizing antibody testing of animals in the facility did not reveal significant levels of neutralizing antibodies against AAV9. Each of the animals was injected with single variants for characterization. The day before infusion, the animals’ food was removed. Animals were sedated by alfaxalone; the skin over the cephalic vein was shaved and sanitized with an isopropanol scrub; and the virus (Extended Data Table 2) was infused through a 24-gauge catheter over several minutes. After the viral infusion was completed, animals were recovered on a warm water blanket (38 °C) until they regained normal motor functions. Then, animals were returned to their cages and monitored closely for normal behavior over the next 4 d, followed by daily observations thereafter.

Marmoset procedures for MSV5–7 were approved by the Institutional Animal Care and Use Committee of Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences. Marmosets were born and raised in SIAT colonies and housed in family groups under standard conditions of 22 ± 1 °C and 40–70% relative humidity. The marmoset breeding and housing facilities are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Although animals were not screened for endogenous neutralizing antibodies, the animals were born and raised in the animal facility, and the housing environment for each animal was clean and isolated to prevent bacterial and viral infection. Therefore, the possibility of the animal carrying neutralizing antibodies for AAV virus is low. Before injection, marmosets were separated from family groups, housed two animals per each room for several days and acclimated to a new room before injections. Each of the animals was injected with single variants for characterization, but one animal (AAV.CAP-B22 injected, 2 years, male, 0.364 kg) was found dead (29 d after injection); at necropsy, the pathology report indicated that the death was unrelated to the virus. The day before infusion, the animals’ food was removed. Animals were anesthetized with isoflurane in air; the skin over the saphenous vein was shaved and sanitized with an ethanol scrub; and the virus (Extended Data Table 2) was infused over several minutes. Anesthesia was withdrawn, and the animals were monitored until they became active, upon which they were returned to their cages. Activity and behavior were closely monitored over the next 3 d, followed by daily observations thereafter.

DNA/RNA recovery and sequencing

Round 1 and round 2 viral libraries were injected into C57Bl/6J and Cre transgenic animals (Syn1-Cre, GFAP-Cre, Tek-Cre and TH-Cre) at a dose of 8 × 1010 viral genomes per animal, and rAAV genomes were recovered 2 weeks after injection, as described in the M-CREATE protocol33. To determine the number of variants included in round 2, 0.01 times the enrichment of the top variant in each tissue was set as a threshold, and variants above that threshold were included. Mice were euthanized, and most major organs were recovered, snap-frozen on dry ice and placed into long-term storage at −80oC. Tissues collected included brain, spinal cord, DRG, liver, lungs, heart, stomach, intestines, kidneys, spleen, pancreas, testes, skeletal muscle and adipose tissue. Then, 100 mg of each tissue (~250 mg for brain hemispheres and <100 mg for DRG) was homogenized in TRIzol (Life Technologies, 15596) using a BeadBug (Benchmark Scientific, D1036), and viral DNA was isolated according to the manufacturer’s recommended protocol. Recovered viral DNA was treated with RNase, underwent restriction digestion with SmaI (found within the inverted terminal repeats) to improve later rAAV genome recovery by polymerase chain reaction (PCR) and purified with a Zymo DNA Clean and Concentrator kit (D4033). Viral genomes flipped by Cre recombinase in select transgenic lines (or pre-flipped in WT animals) were selectively recovered using the following primers: 5′-CTTCCAGTTCAGCTACGAGTTTGAGAAC-3′ and 5′-CAAGTAAAACCTCTACAAATGTGGTAAAATCG-3′, after 25 cycles of 98 °C for 10 s, 60 °C for 15 s and 72 °C for 40 s, using Q5 DNA polymerase in five 25-µl reactions with 50% of the total extracted viral DNA as a template.

After Zymo DNA purification, samples from the WT C57Bl/6J animals were serially diluted from 1:10 to 1:10,000, and each dilution was further amplified around the library variable region. This amplification was done using the following primers: 5′-ACGCTCTTCCGATCTAATACTTGTACTATCTCTCTAGAACTATT-3′ and 5′-TGTGCTCTTCCGATCTCACACTGAATTTTAGCGTTTG-3′, after ten cycles of 98 °C for 10 s, 61 °C for 15 s and 72 °C for 20 s, to recover 73 bp of viral genome around and including the 21-bp variable region and add adapters for Illumina NGS. After PCR cleanup, these products were further amplified using NEBNext Dual Index Primers for Illumina sequencing (New England Biolabs, E7600), after ten cycles of 98 °C for 10 s, 60 °C for 15 s and 72 °C for 20 s. The amplification products were run on a 2% low-melting-point agarose gel (Thermo Fisher Scientific, 16520050) for better separation and recovery of the 210-bp band. The dilution series was analyzed for each WT tissue and the highest concentration that resulted in no product from WT tissue on the gel was chosen for the amplification of the viral DNA from the transgenic animal tissues. This process was performed to differentiate between viral genomes flipped before packaging or due to Cre in the animal. Pre-flipped viral genomes should be avoided to minimize false positives in the NGS results.

All Cre-flipped viral genomes from transgenic animal tissues were similarly amplified (using the dilutions that do not produce pre-flipped viral genomes) to add Illumina sequencing adapters and subsequently for index labeling. The amplified products now containing unique indices for each tissue from each animal were run on a low-melting-point agarose gel, and the correct bands were extracted and purified with a Zymoclean Gel DNA Recovery kit.

Packaged viral library DNA was isolated from the injected viral library by digestion of the viral capsid and purification of the contained ssDNA. These viral genomes were amplified by two PCR amplification steps, like the viral DNA extracted from tissue, to add Illumina adapters and then indices. Correct bands were extracted and purified after gel electrophoresis. This viral library DNA, along with the viral DNA extracted from tissue, was sent for deep sequencing using an Illumina HiSeq 2500 System (Millard and Muriel Jacobs Genetics and Genomics Laboratory, California Institute of Technology).

A pool of eight viruses (AAV9, AAV-PHP.eB, AAV.CAP-B1, AAV.CAP-B2, AAV.CAP-B8, AAV.CAP-B10, AAV.CAP-B18 and AAV.CAP-B22) packaging CAG-FXN-HA with unique 12-bp barcodes were injected into two adult marmosets (Extended Data Table 1). After 6 weeks, animals were euthanized, and brain and liver were recovered and snap-frozen. Then, 1-mm coronal sections from each tissue (4 mm for the brain) were homogenized in TRIzol (Life Technologies, 15596) using a BeadBug (Benchmark Scientific, D1036), and total RNA was recovered according to the manufacturer’s recommended protocol. Recovered RNA was treated with DNase, and cDNA was generated from the mRNA using SuperScript III (Thermo Fisher Scientific, 18080093) and oligo(dT) primers according to the manufacturer’s recommended protocol. Barcoded FXN transcripts were recovered from the resulting cDNA library, as well as the injected pool, using the following primers: 5′-TGGACCTAAGCGTTATGACTGGAC-3′ and 5′-GGAGCAACATAGTTAAGAATACCAGTCAATC-3′, after 25 cycles of 98 °C for 10 s, 63 °C for 15 s and 72 °C for 20 s, using Q5 DNA polymerase in five reactions using 50 ng of cDNA or viral DNA each as a template. After Zymo DNA purification, samples were diluted 1:100 and further amplified around the barcode region using the following primers: 5′-ACGCTCTTCCGATCTTGTTCCAGATTACGCTTGAG-3′ and 5′-TGTGCTCTTCCGATCTTGTAATCCAGAGGTTGATTATCG-3′, after ten cycles of 98 °C for 10 s, 55 °C for 15 s and 72 °C for 20 s. After PCR cleanup, these products were further amplified using NEBNext Dual Index Primers for Illumina sequencing (New England Biolabs, E7600), after ten cycles of 98 °C for 10 s, 60 °C for 15 s and 72 °C for 20 s. The amplification products were run on a 2% low-melting-point agarose gel (Thermo Fisher Scientific, 16520050) for better separation and recovery of the 210-bp band. All indexed samples were sent for deep sequencing as before.

Individual variants (AAV9, AAV-PHP.eB, AAV.CAP-B10 and AAV.CAP-B22) packaging CAG-FXN-HA with unique 12-bp barcodes were injected into 13 individual adult marmosets (Extended Data Table 2). After between 5 and 6 weeks, animals were euthanized, and brain and liver were recovered and snap-frozen. For DNA extraction from these tissues, 25-mg sections from the cortex and the liver were processed using a QIAamp DNA Mini Kit (Qiagen, 51304) to obtain purified viral and genomic DNA from the samples. For RNA transcript extration, 100-mg sections from the cortex and the liver (taken from consistent sections of tissue across animals) were homogenized in TRIzol (Life Technologies, 15596) using a BeadBug (Benchmark Scientific, D1036), and total RNA was recovered according to the manufacturer’s recommended protocol. From purified RNA, cDNA was generated with SuperScript IV VILO MasterMix (Thermo Fisher Scientific, 11766050) and oligo (dT) primers according to the manufacturer’s recommended protocol (including the DNase step).

Viral genome and RNA transcript copy numbers were determined through qPCR as described59 using FXN-HA-specific primers: 5′-GACCTAAGCGTTATGACTGG-3′ and 5′-AATCTGGAACATCGTATGGG-3′. Within each sample, the viral genome or transcript copy number was normalized on a per-cell basis by quantifying GAPDH transcripts in each sample using the GAPDH-specific primers: 5′-TGTTCCAGTATGATTCCACC-3′ and 5′-GATGACCCTTTTGGCTCC-3′. DNA and RNA in tissues from animals MSV5, MSV6 and MSV7 were analyzed at the SIAT, whereas MPV1–2, MSV1–4 and MSV8–13 were analyzed at the California Institute of Technology.

NGS data alignment and processing

Raw FASTQ files from NGS runs were processed with M-CREATE data analysis code (available on GitHub at https://github.com/GradinaruLab/mCREATE) that align the data to an AAV9 template DNA fragment containing the 21-bp diversified region between AA452 and AA458, for the two rounds of AAV evolution/selection, or to an FXN-HA template containing the 12-bp unique barcode, for the marmoset virus pool. The pipeline to process these datasets involved filtering to remove low-quality reads, using a quality score for each sequence, and eliminating bias from PCR-induced mutations or high GC content. The filtered dataset was then aligned by a perfect string match algorithm and trimmed to improve the alignment quality. For the AAV engineering, read counts for each sequence were pulled out and displayed along with their enrichment score, defined as the relative abundance of the sequence found within the specific tissue over the relative abundance of that sequence within the injected viral library. For the pooled barcodes, read counts for each sequence were pulled out and normalized to the respective contribution of that barcode to the initial, injected pooled virus to account for small inequalities in the amount of each member of the pool that was injected into the marmosets.

Tissue preparation, immunohistochemistry and immunofluorescence

Mice were euthanized with Euthasol and transcardially perfused with ice-cold 1× PBS and then freshly prepared, ice-cold 4% paraformaldehyde (PFA) in 1× PBS. All organs were excised and post-fixed in 4% PFA at 4 °C for 48 h and then sectioned at 50 µm with a vibratome. Immunofluorescence was performed on floating sections with primary and secondary antibodies in PBS containing 10% donkey serum and 0.1% Triton X-100. Primary antibodies used were rabbit anti-NeuN (1:200, Abcam, 177487), rabbit anti-S100 (1:200, Abcam, 868), rabbit anti-Olig2 (1:200, Abcam, 109186) and rabbit anti-Calbindin (1:200, Abcam, 25085). Primary antibody incubations were performed for 16–20 h at room temperature. The sections were then washed and incubated with secondary Alexa Fluor 647-conjugated anti-rabbit FAB fragment antibody (1:200, Jackson ImmunoResearch, 711-607-003) for 6–8 h at room temperature. For nuclear staining, floating sections were incubated in PBS containing 0.2% Triton X-100 and DAPI (1:1,000, Sigma-Aldrich, 10236276001) for 6–8 h and then washed. Stained sections were then mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, P36970).

Marmosets were euthanized (Euthanasia, VetOne) and perfused with 1× PBS. One hemisphere of the brain (cut into coronal blocks) and the liver were flash-frozen in 2-methylbutane (Sigma-Aldrich, M32631) chilled with dry ice. The other hemisphere and organs were removed and post-fixed with 4% PFA at 4 °C for 48 h. These organs were then cryoprotected using 10% glycerol followed by 20% glycerol and flash-frozen in 2-methylbutane chilled with dry ice. The blocks of tissue were sectioned on an AO sliding microtome, except for spinal cord and DRG, which were cryosectioned. Then, 50-µm slices (20-µm for spinal cord and DRG) were collected in PBS, and immunohistochemistry or immunofluorescence was performed on floating sections.

To visualize cells expressing the HA-tagged FXN from the variant pool, slices were incubated overnight at room temperature with a rabbit anti-HA primary antibody (1:200, Cell Signaling Technologies, C29F4). After primary incubation, sections were washed in PBS and then incubated with a biotinylated goat anti-rabbit secondary antibody (1:200, Vector Laboratories, BA1000) for 1 h at room temperature. Sections were again washed in PBS and incubated for 2 h in ABC Elite (Vector Laboratories, PK6100) as outlined by the supplier. The ABC peroxidase complex was visualized using 3,3′-diaminobenzidine tetrahydrochloride hydrate (Sigma-Aldrich, D5637) for 5 min at room temperature. Sections were then mounted for visualization.

To visualize cells expressing the HA-tagged FXN for individual variant injections, immunofluorescence staining was performed on floating sections with primary and secondary antibodies in PBS containing 10% donkey serum and 0.1% Triton X-100. Primary antibodies used were rat anti-HA (1:200, Roche, 3F10), rabbit anti-NeuN (1:200, Abcam, 177487) and rabbit anti-S100 beta (1:200, Abcam, 52642). Primary antibody incubations were performed for 16–20 h at room temperature. The sections were then washed and incubated with secondary anti-rat Alexa Fluor 488 (1:200, Thermo Fisher Scientific, A-21208) and anti-rabbit Alexa Fluor 647 (1:200, Thermo Fisher Scientific, A32795). Stained sections were then washed with PBS and mounted with ProLong Diamond Antifade Mountant.

Imaging and quantification

All CAG-mNeonGreen-expressing tissues were imaged on a Zeiss LSM 880 confocal microscope using a Fluar ×5 0.25 M27 objective, with matched laser powers, gains and gamma across all samples of the same tissue. The acquired images were processed in Zen Black 2.3 SP1 (Zeiss).

All CAG-NLS-GFP-expressing tissues were imaged on a Keyence BZ-X all-in-one fluorescence microscope at 48-bit resolution with the following objectives: PlanApo-λ ×20/0.75 (1 mm working distance) or PlanApo-λ ×10/0.45 (4 mm working distance). For co-localization of GFP expression to antibody staining, in some cases the exposure time for the green (GFP) channel was adjusted to facilitate imaging of high- and low-expressing cells while avoiding oversaturation. In all cases in which fluorescence intensity was compared between samples, exposure settings and changes to gamma or contrast were maintained across images. To minimize bias, multiple fields of view per brain region and peripheral organ were acquired for each sample. For brain regions, the fields of view were matched between samples and chosen based on the antibody staining rather than GFP signal. For peripheral tissues, fields of view were chosen based on the DAPI or antibody staining to preclude observer bias.

Marmoset tissue sections transduced with the variant pool were examined and imaged on a Zeiss AxioImager Z1 with an Axiocam 506 color camera. Acquired images were processed in Zen Blue 2 (Zeiss).

Marmoset tissues transduced with individual variants were imaged on a Zeiss LSM 880 confocal microscope. Tissues from animals MSV5, MSV6 and MSV7 were imaged at the SIAT, whereas all other marmosets were imaged at the California Institute of Technology. Whole tissue sections were imaged using a Fluor ×5 0.25 M27 objective with a gallium arsenide phosphide photomultiplier tube detector at a pixel size of 1.25 µm × 1.25 µm. Images for cortex quantification were imaged with a LD-LCI Plan-Apochromat ×10/0.45 M27 objective with a photomultiplier tube detector at a pixel size of 0.42 µm × 0.42 µm. Images for liver quantification were taken with an LD-LCI Plan-Apochromat ×10/0.45 M27 objective with a photomultiplier tube detector at a pixel size of 0.42 µm × 0.42 µm or an LD-LCI Plan-Apochromat ×20/0.8 M27 with a gallium arsenide phosphide photomultiplier tube detector at a pixel size of 0.156 µm × 0.156 µm (MSV5, MSV6 and MSV7). Imaging at each magnification was performed with matched laser powers, gains and gamma across all samples of the same tissue imaged in the same location, whereas images taken in different locations were matched by maximizing the range indicator. The acquired images were processed in Zen Black 2.3 SP1 (Zeiss). Quantification of transgene expression in marmoset cortex was performed through manual cell counting of co-localized HA+ cells with NeuN+ staining in maximum intensity projection images. Quantification of transgene expression in the marmoset liver was performed through manual cell counting of co-localized HA+ cells with DAPI staining in maximum intensity projection images.

All image processing was performed with the Keyence BZ-X Analyzer (version 1.4.0.1). Co-localization between the GFP signal and antibody or DAPI staining was performed using the Keyence BZ-X Analyzer with the hybrid cell count automated plugin. Automated counts were validated and routinely monitored by comparison with manual hand counts and found to be below the margin of error for manual counts.

To compare total cell counts and fluorescence intensity throughout the brain between samples, an entire sagittal section located 1,200 µM from the midline was imaged using matched exposure conditions with the Keyence BZ-X automated XY stitching module. Stitched images were then deconstructed in the Keyence BZ-X Analyzer suite and run through the hybrid cell count automated plugin to count the total number of cells in the entire sagittal section. Average fluorescence intensity was calculated by creating a mask of all GFP-positive cells throughout the sagittal section and measuring the integrated pixel intensity of that mask. The total integrated pixel intensity was divided by the total cell count to obtain the fluorescence intensity per cell. In all cases where direct comparisons of fluorescence intensity were made, exposure settings and post-processing contrast adjustments were matched between samples.

Statistics and reproducibility

For the initial characterization of brain and liver expression in mice (Fig. 1d), the experiment was repeated with n = 3. For the statistical analysis in mice and related graphs (Fig. 2 and Extended Data Figs. 24), a single data point was defined as two tissue sections per animal, with multiple technical replicates per section when possible. Technical replicates were defined as multiple fields of view per section, with the following numbers for each region or tissue of interest: cerebellum = 3, cortex = 4, hippocampus = 3, midbrain = 1, striatum = 3, thalamus = 4, liver = 4, spleen = 2, testis = 2, kidney = 2, lung = 2, spine = 1, DRG = 1 and whole sagittal = 1. Unless otherwise noted, all experimental groups were n = 6, determined using preliminary data and experimental power analysis. Normality was tested to ensure that the data matched the assumptions of the statistical tests used.

For the initial pooled experiments in marmosets (Fig. 3), the experiment was repeated in two separate animals (Extended Data Table 1). For the statistical analysis in marmoset and related graphs (Fig. 4 and Extended Data Figs. 5 and 6), a single data point was defined as a single tissue section from a single animal. The data were collected across four separate cohorts in three separate locations (Extended Data Table 2), and the trend was recapitulated in each. For representative brain images and quantification (Fig. 4a,b and Extended Data Fig. 6b), the experiment was repeated n times, where n = 2 (AAV9), n = 3 (AAV.PHP.eB), n = 4 (AAV.CAP-B10) and n = 4 (AAV.CAP-B22), except for astrocyte staining (Extended Data Fig. 5), which was repeated n = 2 (AAV9), n = 3 (AAV.CAP-B10) and n = 2 (AAV.CAP-B22). For representative liver images and quantification (Fig. 4c,d and Extended Data Fig. 6a), the experiment was repeated n times, where n = 1 (AAV9), n = 3 (AAV.PHP.eB), n = 2 (AAV.CAP-B10) and n = 3 (AAV.CAP-B22). Global brain analysis of AAV.CAP-B10 nervous system expression (Fig. 4e,f) was performed with n = 2. No statistical methods were used to predetermine sample sizes, but our sample sizes were similar to those reported in our previous publications34,54. Data distribution was assumed to be normal, but this was not formally tested.

No data were excluded from analysis. Allocation of organisms and samples to separate groups was random, and animals were allocated to experimental conditions based on availability in each cohort. The investigators were not blinded to allocation during experiments and outcome assessment.

Microsoft Excel for Microsoft 365 (version 2107) and GraphPad Prism 8 for Windows (version 8.4.3 (686)) were used for statistical analysis and data representation. For all statistical analyses, significance is represented as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001; not significant, P ≥ 0.05.

Reporting Summary

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


Source link

Related Articles

Leave a Reply

Back to top button