TITLE: Individual AAV production and purification
AUTHORS: Miguel Chuapoco

[DESCRIPTION]
We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

[GUIDELINES]
Introduction

Recombinant AAVs (rAAVs) are commonly used vehicles for in vivo gene transfer and promising vectors for therapeutic applications1. However, AAVs that enable efficient and noninvasive gene delivery to defined cell populations are needed. Current gene delivery methods (e.g., intraparenchymal surgical injections) are invasive, and alternatives such as intravenous administration require high viral doses and provide relatively inefficient transduction of target cells. We previously developed Cre recombination-based AAV targeted evolution (CREATE) to engineer and screen for AAV capsids that are capable of more efficient gene transfer to specific cell types via the vasculature2–4. Compared to naturally occurring capsids (e.g., AAV9), the novel AAV-PHP capsids identified by CREATE exhibit markedly improved tropism for cells in the adult mouse central nervous system (CNS), peripheral nervous system (PNS), and visceral organs. In this protocol, we describe how to package genetic cargo into AAV-PHP capsids and intravenously administer AAVs for efficient, noninvasive, and targeted gene delivery at sites throughout the body (Fig. 1).
 

Among our new capsid variants2–4, AAV-PHP.B and the further evolved AAV-PHP.eB efficiently transduce neurons and glia throughout the CNS (Fig. 2); another variant, AAV-PHP.S, displays improved tropism for neurons within the PNS (Fig. 3) and organs, including the gut2 and heart (Fig. 4). Importantly, these capsids target cell populations that are normally difficult to access because of their location (e.g., sympathetic, nodose, dorsal root, and cardiac ganglia) (Figs. 3a–c and 4d) or broad distribution (e.g., throughout the brain or enteric nervous system) (Figs. 2 and 3d) and can be utilized in several mouse and rat strains (Fig. 5). Together with the capsid, the genetic cargo (or rAAV genome) can be customized to control transgene expression (Fig. 6 and Table 1). The rAAV genome contains the components required for gene expression, including promoters, transgenes, proteintrafficking signals, and recombinase-dependent expression schemes. Hence, different capsid–cargo combinations create a versatile AAV toolbox for genetic manipulation of diverse cell populations in wild-type and transgenic animals. Here, we provide researchers, especially those new to working with AAVs or systemic delivery, with resources that will help them utilize AAV-PHP viruses in their own research.

 
 
 
  
     
Table 1 | pAAV plasmids
 
        Vector name pAAV-  Expression class  Addgene no.    Tunable expression  TREa-mTurquoise2  tTA-dependent  99113      TRE-eYFP    104056      TRE-mRuby2  99114      TRE-DIOb-mTurquoise2  Cre- and tTAdependent  99115      TRE-DIO-eYFP  117383      TRE-DIO-tdTomato  99116      TRE-DIO-mRuby2  99117      CAGc-tTAd Inducer  99118      hSyn1e-tTA  99119      ihSyn1f-tTA  99120      ihSyn1-DIO-tTA  99121    Tissue-wide expression  CAG-mTurquoise2  Constitutive  99122      CAG-eYFP    104055      CAG-mRuby2  99123      CAG-NLSg-GFP  104061      CAG-DIO-mTurquoise2  Cre-dependent  104059      CAG-DIO-eYFP  104052      CAG-DIO-mRuby2  104058    Cell type–specific expression  hSyn1-mTurquoise2  Cell type–specific  99125      hSyn1-eYFP  117382      hSyn1-mRuby2  99126      GFAPh-2xNLS-mTurquoise2  104053      hSyn1-2xNLS-mTurquoise2  118025      MBPi-2xNLS-tdTomato  104054      mTHj-GFP  99128      hSyn1-tdTomato-fk  104060      GFAP-mKate2.5-f  99129      mDlxl-NLS-mRuby2  99130      CAG-eYFP-3x-miR204-5p-TSm  117380      CAG-eYFP-3x-miR708-5p-TSn  117381      CAG-GCaMP6f-3x-miR204-5p-3xmiR122-TSo  117384  
 
Overview of the protocol  

We provide an instruction manual for users of AAV-PHP variants. The procedure includes three main stages (Fig. 1): AAV production (Steps 1–42), intravenous delivery (Steps 43–49), and evaluation of transgene expression (Step 50). 

The AAV production protocol is adapted from established methods. First, HEK293T cells are transfected with three plasmids5–7 (Steps 1–3, Figs. 1 and 7): (i) pAAV, which contains the rAAV genome of interest (Fig. 6 and Table 1); (ii) pUCmini-iCAP-PHP, which encodes the viral replication and capsid proteins (Table 2); and (iii) pHelper, which encodes adenoviral proteins necessary for replication. Using this triple-transfection approach, a single-stranded rAAV genome is packaged into an AAV-PHP capsid in HEK293T cells. AAV-PHP viruses are then harvested8 (Steps 4–14), purified9,10 (Steps 15–31), and titered by quantitative PCR (qPCR)11 (Steps 32–42) (Fig. 7). Purified viruses are intravenously delivered to mice via retro-orbital injection12 (Steps 43–49), and gene expression is later assessed using molecular, histological, or functional methods relevant to the experimental aims (Step 50).

This protocol is optimized to produce AAVs at high titer (≥1 × 1013 vector genomes (vg)/ml and ≥1 × 1012 vg/dish) and with high transduction efficiency in vivo2,3.
  
Applications of the method

We anticipate that AAV-PHP capsids (Table 2) can be used to package rAAV genomes (contained in pAAV plasmids that are available through Addgene and elsewhere) (Fig. 6 and Table 1) to enable a wide range of biomedical applications. Below, we highlight current and potential applications of this method.

Table 2 | AAV-PHP capsid plasmids
 
      AAV-PHP capsid  Plasmid name  In vivo characteristics  Production efficiency  Addgene no.    AAV-PHP.B  pUCmini-iCAP-PHP.B  Broad CNS transduction  Good  103002    AAV-PHP.B2  pUCmini-iCAP-PHP.B2  Broad CNS transduction  Good  103003    AAV-PHP.B3  pUCmini-iCAP-PHP.B3  Broad CNS transduction  Good  103004    AAV-PHP.eB  pUCmini-iCAP-PHP.eB  Broad CNS transduction  Good  103005    AAV-PHP.S  pUCmini-iCAP-PHP.S  Broad transduction in PNS and visceral organs  Good  103006    AAV-PHP.Aa  piCAP-PHP.A  Broad astrocyte transduction in CNS  Poor  CLOVER  
 

Anatomical mapping

Fluorescent reporters are commonly used for cell type–specific mapping and phenotyping2,13,14 (Figs. 2–5). AAV-mediated multicolor labeling (e.g., Brainbow15) is especially advantageous for anatomical mapping approaches that require individual cells in the same population to be distinguished from one another. We and others have demonstrated the feasibility of this approach in the brain2,15, retina15, heart (Fig. 4b,c), and gut2, as well as the peripheral ganglia (Fig. 3c). Spectrally distinct labeling is well-suited for studying the organization of cells (e.g., cardiomyocytes (Fig. 4b)) in healthy and diseased tissues and long-range tract tracing of individual fibers through extensive neural networks (e.g., the enteric2 or cardiac nervous systems (Fig. 4c)).

Functional mapping

AAV-PHP capsids are also relevant for probing cell function. AAV-PHP.B was previously used to target distinct neural circuits throughout the brain for chemogenetic16,17 and optical imaging applications18,19. We predict that AAV-PHP viruses will be beneficial for manipulating neural networks that are typically difficult to access, such as peripheral circuits controlling the heart (Fig. 4d), lungs20, or gut21. AAV-PHP variants could also be utilized to interrogate the function of non-neuronal cell types, including cardiomyocytes22, pancreatic beta cells23,24, and hepatocytes25. Harnessing AAV-PHP viruses to modulate cell physiology may reveal novel roles for different cells in regulating organ function and/or animal behavior26.

Gene expression, silencing, and editing

AAV-PHP viruses can be used to test potential therapeutic strategies that would benefit from organwide or systemic transgene expression27. Recently, AAV-PHP.B was used to treat16 and model28 neurodegenerative diseases with widespread pathology. Other potential applications include gene editing (e.g., via CRISPR29–32) or silencing (e.g., via shRNA33); importantly, these approaches could be utilized to broadly and noninvasively manipulate cells in both healthy and diseased states for either basic research or therapeutically motivated studies.

AAV capsid engineering

AAV-PHP capsids can be further evolved for more efficient transduction of specific organs and cell types throughout the body. This protocol can be used for AAV engineering applications (e.g., our in vivo capsid selection method CREATE2,3). Using a modified transfection protocol (Steps 1–3 and online methods in ref. 3), DNA libraries (generated by diversification of the AAV cap gene) are packaged to produce AAV capsid libraries, which are then harvested (Steps 4–14 and online methods in ref. 3), purified (Steps 15–31), and titered (Steps 32–42). Libraries are systemically administered to Cre transgenic animals (Steps 43–49) or wild-type animals in which Cre is introduced (e.g., by AAV delivery), and Cre-dependent cap recovery from tissues of interest facilitates further rounds of selection to isolate enriched variants. This protocol can also be used to characterize novel serotypes identified with CREATE or other engineering methods34.

Limitations of the method

A major limitation of AAV capsids, including AAV-PHP variants, is their relatively small packaging capacity (<5 kb). Some elements of the rAAV genome, such as the woodchuck hepatitis posttranscriptional regulatory element (WPRE), can be truncated35 or removed36,37 to accommodate larger genetic components. The development of smaller promoters38,39 and dual expression systems40, in which genetic elements are split between two or more viruses (requiring efficient cotransduction), has also enabled the delivery of larger genomes. Continued development of these approaches will help bypass restrictions on rAAV genome size.

Intravenous administration of AAVs also presents unique challenges. For example, systemic transduction may be undesirable for applications in which highly restricted gene expression is vital to the experimental outcome. Possible off-target transduction, due to the broad tropism of AAV-PHP variants and/or lack of compatible cell type–specific promoters, can be reduced by miRNA-mediated gene silencing. Sequences complementary to miRNAs expressed in off-target cell populations can be introduced into the 3ʹ UTR of the rAAV genome (Fig. 6); this has been shown to reduce off-target transgene expression and better restrict expression to cell types of interest41,42 (Fig. 2d,e).

Another challenge of systemic delivery is that it requires a high viral load, which can elicit an immune response against the capsid and/or transgene and reduce transduction efficiency in vivo43. Immunogenicity of AAVs may be exacerbated by empty capsid contamination in viral preparations44,45. The viral purification protocol (Steps 15–31) reduces, but does not eliminate, empty capsids (Fig. 7b). If this poses a concern for specific applications, viruses can be purified using an alternative approach8,9,46.

Last, generation of viruses for systemic administration may impose a financial burden on laboratories due to the doses of virus required. Nevertheless, viral-mediated gene delivery is inexpensive compared to creating and maintaining transgenic animals. Moreover, intravenous injection is faster, less invasive, and less technically demanding than other routes of AAV administration, such as stereotaxic injection, thereby eliminating the need for specialized equipment and survival surgery training.

Experimental design

Before proceeding with the protocol, a number of factors should be considered, namely the expertise and resources available in the lab; the animal model, capsid, and rAAV genome to be used; the dose for intravenous administration; and the method(s) available for assessing transgene expression. Each of these topics is discussed below to guide users in designing their experiments.

Required expertise and resources

This protocol requires that the scientists have basic molecular biology, cell culture, and animal work experience. Users should be approved to handle laboratory animals, human cell lines, and AAVs. A background in molecular cloning is advantageous, although not necessary if relying on available plasmids.

In addition to having the above expertise, the labs must be equipped for the molecular and cell culture work relevant to the procedure; we suggest that users read through the entire ‘Materials’ and ‘Procedure’ sections beforehand to ensure that the required reagents and equipment are available and appropriate safety practices and institutional approvals are in place.

Animal model

This protocol describes the production of AAVs for intravenous delivery to 6- to 8-week-old male and female mice. AAV-PHP viruses have been validated in C57BL/6J mice2,3,16,47 (Figs. 2–5) and numerous Cre driver lines2,16–18, including, but not limited to, TH-IRES-Cre (Fig. 3), TRPV1-IRESCre (Figs. 3 and 4), and ChAT-IRES-Cre mice2 (Fig. 4). Intriguingly, AAV-PHP.B demonstrates low transduction throughout the brain when systemically administered to BALB/cJ mice48 (Fig. 5a). However, the neurotropic properties of AAV-PHP.B are not limited to the C57BL/6J strain in which they were selected. AAV-PHP.B transduces the brain more efficiently than AAV9 in both FVB/NCrl and 129S1/SvImJ mice (Fig. 5a). We also show examples of AAV-PHP.eB transducing neurons in C57BL/6NCrl (Fig. 2a–c) and 129T2/SvEmsJ mice (Fig. 5b), as well as Fischer (Fig. 5c) and Long- Evans rats (Fig. 5d). Compared to AAV9 and AAV-PHP.B, AAV-PHP.eB results in more efficient neuronal transduction in Sprague–Dawley rats after either intravenous administration or intraparenchymal stereotaxic injections28,49. We predict that AAV-PHP capsids can be used in multiple species and strains for diverse applications, such as those requiring fluorescent labeling (Fig. 5a–c) and calcium imaging (Fig. 5d,e). We have not compared the transduction efficiencies of AAV9 and AAV-PHP capsids across all rodent strains and species or determined the optimal dose for transducing specific organs and cell types in different animal models. Users should test these parameters to determine the utility of AAV-PHP variants in their research. See ‘Reagents’ for mouse and rat catalog numbers.

Selecting an AAV-PHP capsid

We recommend choosing an AAV-PHP capsid (Table 2) based on its tropism and production efficiency. Capsid properties are listed in Supplementary Table 1; we include species, organs, and cell populations examined to date and note typical viral yields. We anticipate that most researchers will use AAV-PHP.eB (Addgene, plasmid no. 103005) or AAV-PHP.S (Addgene, plasmid no. 103006) in their experiments. AAV-PHP.eB and AAV-PHP.S produce viral yields similar to those of other high-producing naturally occurring serotypes (e.g., AAV9) and enable efficient, noninvasive gene transfer to the CNS or PNS and visceral organs, respectively2 (Figs. 2–5).

The earlier capsid variants, which provide widespread CNS transduction, either produce suboptimal yields (AAV-PHP.A)3 or have since been further evolved for enhanced transduction efficiency in vivo (AAV-PHP.B (Addgene, plasmid no. 103002))2. We therefore recommend using AAV-PHP.eB for CNS applications, especially when targeting neurons. Note, however, that the chosen capsid will ultimately depend on the experimental circumstances; multiple factors, including species50, strain48 (Fig. 5), age51, gender52, and health53, can influence AAV tropism. Testing the AAV-PHP variants in a variety of experimental paradigms will continue to reveal the unique attributes of each capsid and identify those most suitable for different applications.

Selecting an rAAV genome

Users must select an rAAV genome, contained in a pAAV plasmid, to package into the capsid (Figs. 1 and 6; Table 1). In Table 1, we list the pAAVs used here (Figs. 2–4) and in our previous work2,3; we direct users to Addgene’s plasmid repository for additional pAAVs developed for various applications.

Depending on the experimental aims, users can elect to design their own genomes54 and clone from existing pAAVs. When customizing plasmids, it is imperative that the rAAV genome, the sequence between and including the two inverted terminal repeats (ITRs), does not exceed 4.7–5 kb (Fig. 6); larger genomes will not be fully packaged into AAV capsids, resulting in truncated genomes and low titers. The ITRs are 145-bp sequences that flank the expression cassette and are required for replication and encapsidation of the viral genome. ITRs are typically derived from the AAV2 genome and must match the serotype of the rep gene contained in the capsid plasmid; pUCmini-iCAP-PHP plasmids contain the AAV2 rep gene and are therefore capable of packaging genomes with AAV2 ITRs (i.e., almost any pAAV available from Addgene). Other genetic components (e.g., promoters, transgenes, localization signals, and recombination schemes) are interchangeable and can be customized for specific applications (Fig. 6).

Dosage for intravenous administration

The optimal dose for intravenous administration to target cell populations must be determined empirically. We encourage users to refer to Figs. 2–5 and related work for suggested AAV-PHP viral doses. AAV-PHP variants have been successfully administered to adult mice2,3,16,47 (Figs. 2–5), neonatal mice16, and neonatal and adult rats28,49 (Fig. 5c,d) for fluorescent labeling; they have also been used for calcium imaging18,19 and optogenetic (Fig. 4d), chemogenetic16,17, and therapeutic applications16,28.

We typically administer between 1 × 1011 and 5 × 1011 vg of AAV-PHP.eB or between 3 × 1011 and 1 × 1012 vg of AAV-PHP.S to adult mice (6–8 weeks old). However, dosage will vary depending on the target cell population, desired fraction of transduced cells, and expression level per cell. AAVs independently and stochastically transduce cells, typically resulting in multiple genome copies per cell2. Therefore, higher doses generally result in strong expression (i.e., high copy number) in a large fraction of cells, whereas lower doses result in weaker expression (i.e., low copy number) in a smaller fraction of cells. To achieve high expression in a sparse subset of cells, users can employ a two-component system in which transgene expression is dependent on co-transduction of an inducer (e.g., a vector expressing Cre55, Flp2, or the tetracycline-controlled transactivator (tTA)2); inducers are injected at a lower dose (typically 1 × 109 to 1 × 1011 vg) to limit the fraction of cells with transgene expression. Note that transgenes and gene regulatory elements (e.g., enhancers, promoters, and miRNA target sequences (Fig. 2d,e)) can influence gene expression levels. Therefore, users should assess transgene expression from a series of doses and at several time points after intravenous delivery to determine the optimal experimental conditions.

Evaluation of transgene expression

Following in vivo delivery, AAV transduction and transgene expression increase over the course of several weeks. Although expression is evident within days after transduction, it does not reach a steady-state level until at least 3–4 weeks after transduction. Therefore, we suggest waiting for a minimum of 2 weeks before evaluating fluorescent labeling2,3,16,28 (Figs. 2–5) and at least 3–4 weeks before beginning optogenetic (Fig. 4d), chemogenetic16,17, and calcium imaging18,19 experiments. Note that, like other AAVs, AAV-PHP variants are capable of providing long-term transgene expression. AAV-PHP.B-mediated cortical expression of a genetically encoded calcium indicator, GCaMP6s, was reported to last at least 10 weeks post-injection without toxic side effects19 (i.e., nuclear filling56), and we have observed GFP expression throughout the brain >1 year after viral administration (see Supplementary Fig. 4 in ref. 3). However, the time points suggested here are only meant to serve as guidelines; gene expression is contingent on multiple factors, including the animal model, capsid, genome, and dose.

The appropriate method(s) for evaluating transgene expression will vary among users and may include functional (e.g., optical imaging56), histological57 (e.g., using endogenous fluorescence, antibodies, or molecular probes), or molecular (e.g., Western blot58 or qPCR3) approaches59. To assess transduction efficiency across different organs, users can perform a qPCR-based vector biodistribution assay, in which vector genomes are quantified and normalized to the mouse genome (e.g., a housekeeping gene)3. Other approaches typically involve examining fluorescent protein expression in thin or thick (≥100 μm) tissue samples. The CLARITY-based methods such as passive CLARITY technique (PACT) and perfusion-assisted agent release in situ (PARS)60 render thick tissues optically transparent while preserving their three-dimensional molecular and cellular architecture, and facilitate deep imaging of large volumes (e.g., using confocal or light-sheet microscopy)61–63. Cleared tissues are compatible with endogenous fluorophores, including commonly used markers such as GFP3,57,60, eYFP60, and tdTomato57. However, some fluorescent signals, such as those from mTurquoise2, mNeonGreen, and mRuby2, can deteriorate in chemical clearing reagents. To visualize these reporters, we suggest using optical clearing reagents such as refractive index-matching solution (RIMS)57 or ScaleSQ64 (Figs. 3a,c,d, 4b–d, and 5e) or commercially available mounting media (Step 50) (Fig. 5a,c). Some fluorescent proteins are sensitive to photobleaching. For example, mRuby2 may bleach over long imaging sessions or at high magnification; tdTomato exhibits similar spectral properties and may be a more suitable alternative, given its photostability65. Also, note that autofluorescent lipofuscin accumulates in aging postmitotic tissues (e.g., the brain and heart)66 and may interfere with examination of transduced cells; in this case, either reduce autofluorescence using histological methods57,67 or, if possible, inject younger adults (≤8 weeks old) and determine the minimum time required for transgene expression.

Troubleshooting
Troubleshooting advice can be found in Table 3.

Table 3 | Troubleshooting table
 
            Step  Problem  Possible reason  Solution    2 (Transfection)  Transfection solution is not cloudy  DNA–PEI complexes have not formed  Thoroughly vortex the transfection solution for 10 s and incubate at RT   for 2–10 min before use; always use PEI at RT        Transfection miscalculation  Carefully follow the instructions in the Reagent setup and Supplementary   Table 2 (‘Transfection calculator’ sheet) to prepare the PEI + DPBS master   mix and DNA + DPBS solutions  
 
Timing

Refer to Fig. 7a for a time line of the Procedure.
Steps 23–25, triple transient transfection of HEK293T cells: 1–2 h
Steps 26–36, AAV harvest: 5 d
Steps 37–53, AAV purification: 1 d
Steps 54–64, AAV titration: 1 d
Steps 65–71, intravenous (retro-orbital) injection: <5 min per mouse, excluding setup and cleanup time
Step 72, evaluation of transgene expression: variable


Acknowledgements

We thank M. Fabiszak (W. Freiwald lab, Rockefeller University) and N.C. Flytzanis (V. Gradinaru lab) for the images in Fig. 5d and e, respectively. We also thank M.S. Ladinsky at the Biological and Cryogenic Transmission Electron Microscopy Center (California Institute of Technology (Caltech)) for preparing transmission electron microscopy samples and for acquiring the image shown in Fig. 7b. We are grateful to Y. Lei for help with cloning and K. Lencioni for performing tail-vein injections in rats. The images in Fig. 5a,b were acquired in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. AAV-PHP capsids are dedicated to the memory of Paul H. Patterson (P.H.P.), who passed away during the preparation of the manuscript describing AAV-PHP.B[3]. This work was primarily supported by the National Institutes of Health (NIH) through grants to V.G.: Director's New Innovator grant DP2NS087949 and PECASE; National Institute on Aging grant R01AG047664; BRAIN grant U01NS090577; SPARC grant OT2OD023848-01 (to V.G. and K.S.); and the Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO). Additional funding included the NSF NeuroNex Technology Hub grant 1707316, and funds from the Curci Foundation, the Beckman Institute, and the Rosen Center at Caltech. V.G. is a Heritage Principal Investigator supported by the Heritage Medical Research Institute. R.C.C. was supported by an American Heart Association Postdoctoral Fellowship (17POST33410404). C.C. was funded by the National Institute on Aging (F32AG054101), and P.S.R. was funded by the National Heart, Lung, and Blood Institute (F31HL127974).


 


[STEPS]
SECTION: Reagent setup: Plasmid DNA
1. 
 
Grow bacterial stocks in LB or Plasmid+ media containing the appropriate selective antibiotic; refer
to the Addgene catalog for suggested growth conditions. Use a large-scale endotoxin-free plasmid purification kit to isolate plasmids; elute plasmid DNA with the supplied Tris-EDTA (TE) buffer. Measure the DNA purity and concentration using a spectrophotometer and freeze at -20 °C or
-80 °C for up to several years. 
 

SECTION: Reagent setup: Cell culture media
2. Add 
 
Invert to mix and store at 4 °C for up to several months; warm to 37 °C before use. The resulting cell culture media should have a final concentration of 5% (vol/vol) FBS, 1× NEAA, and 50 1946 pen–strep.
 

SECTION: Reagent setup: Cell culture
3. Thaw HEK293T cells according to the manufacturer’s recommendations. Passage cells using either
TrypLE Express enzyme or a standard trypsinization protocol for adherent cultures69. Seed cells in 150-mm tissue culture dishes with a final volume of 20 mL of media per dish. Maintain in a cell culture incubator at 37 °C with 5% CO2. 
 

SECTION: Reagent setup: PEI stock solution
4. Pipette 50 mL of WFI water into a 50-ml conical centrifuge tube for later use. Add 323 mg of PEI to the remaining 950 mL bottle of WFI water and adjust the pH to 2–3 by adding 1 1833 HCl suitable for cell culture, keeping track of the volume of HCl added. Heat in a 37 °C water bath for several hours (or 1440 min) and occasionally shake to mix. Once dissolved, add reserved WFI water to a total volume of 1 L. Filter-sterilize, make aliquots in 50-ml conical centrifuge tubes, and store at -20 °C for up to 1 year. We routinely freeze–thaw our PEI aliquots. 
 

SECTION: Reagent setup: PEI + DPBS master mix
5. Thaw PEI in a 37 °C water bath. Bring the PEI to Room temperature (23 °C) and vortex to mix. Add PEI and DPBS to a 50-ml conical centrifuge tube and vortex again to mix. Use Supplementary Table 2 (‘Transfection calculator’ sheet) to calculate the volumes of PEI (cell I9) and DPBS (cell J9) needed. 
 

SECTION: Reagent setup: DNA + DPBS
6. Bring plasmid DNA to Room temperature and briefly vortex to mix. For each viral prep, add DNA and DPBS to a 50-ml conical centrifuge tube; the solution is vortexed in Step 24. Use Supplementary Table 2 (‘Transfection calculator’ sheet) to calculate the quantities of DNA (e.g., cells E9, E11, and E13) and DPBS (e.g., cell F9) needed. 
 

SECTION: Reagent setup: SAN digestion buffer
7. Add 
 
and shake to mix. Filter-sterilize and store at Room temperature for up to several months. The resulting SAN digestion buffer should have a final pH of ~pH 10.0 and a final concentration of 500 millimolar (mM) NaCl, 40 millimolar (mM) Tris base, and 10 millimolar (mM) MgCl2.
SECTION: Reagent setup: SAN + SAN digestion buffer
8. Add 100 1834 of SAN (4 µL of 25 1923 SAN) per milliliter of SAN digestion buffer; pipette to mix.
 

SECTION: Reagent setup: 40% (wt/vol) PEG stock solution
9. Decant ~500 mL of WFI water into a 500-ml sterile bottle for later use. Add 146.1 g of NaCl to the remaining 500 mL (in the 1-liter bottle of WFI water) and shake/heat until dissolved. 
Once completely dissolved, add 400 g of PEG and heat at 37 °C 1440 min for up to 2 nights. Add reserved WFI water to a total volume of 1 L. Filter-sterilize and store at Room temperature for up to several months. The resulting stock solution should have a final concentration of 2.5 Molarity (M) NaCl and 40% (wt/vol) PEG. 
 

SECTION: Reagent setup: DPBS + high salt
10. Add
 
and shake to mix. Filter-sterilize and store at Room temperature for up to several months. The resulting buffer should have a final concentration of 1 Molarity (M) NaCl, 2.5 millimolar (mM) KCl, and 1 millimolar (mM) MgCl2 (in addition to the salts in the DPBS).

SECTION: Reagent setup: DPBS + low salt
11. Add
 
and shake to mix. Filter-sterilize and store at Room temperature for up to several months. The resulting buffer should have a final concentration of 100 millimolar (mM) NaCl, 2.5 millimolar (mM) KCl, and 1 millimolar (mM) MgCl2 (in addition to the salts in the DPBS).
SECTION: Reagent setup: DNase digestion buffer
13. Use a 50-ml serological pipette to measure 247.5 mL of UltraPure water into a 250-ml sterile bottle. Add
 
and shake to mix. Filter sterilize and store at Room temperature for up to several months. The resulting buffer should have a final concentration of 2 Molarity (m) CaCl2, 10 Molarity (m) Tris-HCl, and 10 Molarity (m) MgCl2.
SECTION: Reagent setup: Iodixanol density gradient solutions (15%, 25%, 40%, and 60% (wt/vol) iodixanol)
12. For each layer, add iodixanol (OptiPrep), DPBS + high salt or DPBS + low salt, and phenol red (if
applicable) to a 50-ml conical centrifuge tube. Invert or briefly vortex to mix. Use Supplementary Table 3 to determine the volumes of each reagent needed. The 25% and 60% layers contain phenol red, which turns the solutions red and yellow, respectively, and facilitates clear demarcation of the gradient boundaries (Fig. 8). 
 
  
SECTION: Reagent setup: Proteinase K solution
15. Use a 50-ml serological pipette to measure 250 mL of UltraPure water into a 250-ml sterile bottle. Add 14.61 g of NaCl and shake to mix. Add 2.5 g of N-lauroylsarcosine sodium salt to the mixture and gently swirl to mix; N-lauroylsarcosine sodium salt is a surfactant and will generate bubbles during vigorous mixing. Filter-sterilize and store at Room temperature for up to several months. The resulting solution should have a final concentration of 1 Molarity (M) NaCl and 1% (wt/vol) N-lauroylsarcosine sodium salt.

SECTION: Reagent setup: DNase I + DNase digestion buffer
14. Add 50 1834 of DNase I per milliliter of digestion buffer (a 1:200 dilution of 10 1923 DNase); pipette to mix. 
 


SECTION: Reagent setup: DNA standard stock
17. Set up a single 50-μl restriction digest reaction; use 60-80 1834 (3–4 μl) of ScaI (or another suitable enzyme) to linearize 20 µg of the plasmid DNA containing the target sequence. Run a small amount of the reaction on an agarose gel to ensure complete digestion. Purify the reaction using two DNA clean-up columns. Measure the DNA concentration (ng/μl) using a spectrophotometer. Dilute to ~5–10 × 109 single-stranded (ss) DNA molecules/μl and use the Qubit assay to verify the concentration (ng/μl). Divide into 20 µL aliquots in DNA/RNA LoBind microcentrifuge tubes and freeze at -20 °C for up to 1 year. 
 


SECTION: Reagent setup: Proteinase K + proteinase K solution
16. Add 100 µg of proteinase K per milliliter of solution (a 1:200 dilution of 50 1808 (2.5 U/mg) proteinase K); pipette to mix. 
 


SECTION: Reagent setup: DNA standard dilutions
18. Prepare three sets of eight (1:10) serial dilutions of the DNA standard stock. For each set, begin by pipetting 5 µL of the standard into 45 µL of UltraPure water (standard no. 8). Mix by vortexing for 3 s and proceed with the seven remaining dilutions (standard no. 7 to standard no. 1). The final concentrations of the standard dilutions should range from 5–10 × 108 (standard no. 8) to 5–10 × 101 (standard no. 1) ssDNA molecules/μl. 
 

SECTION: Equipment setup: Clamp setup for AAV purification
20. Attach the rod to the support stand. Secure the clamp 25–30 cm above the stand (Fig. 8f).
   
SECTION: Reagent setup: qPCR master mix
19. Prepare a qPCR master mix for the total number of reactions (i.e., wells) needed. One reaction requires 
 
Pipette or vortex for 1–2 s to mix.
 

SECTION: Procedure
21. 
 

 

SECTION: Procedure: Triple transient transfection of HEK293T cells ● Timing 1–2 h
22. 
 

24 h before transfection, seed HEK293T cells in 150-mm dishes to attain 80–90% confluency the next day69. Incubate the cells in a cell culture incubator at 37 °C with 5% CO2.
SECTION: Procedure: Triple transient transfection of HEK293T cells ● Timing 1–2 h
23. At the time of transfection, make the PEI + DPBS master mix and the DNA + DPBS solution for each viral prep (Reagent setup and Supplementary Table 2, ‘Transfection calculator’ sheet). Using a 5- or 10-ml serological pipette, add the required volume of the PEI + DPBS master mix (e.g., ‘Transfection calculator’ cell G9) dropwise to the DNA + DPBS solution (e.g., ‘Transfection calculator’ cells E9 + E11 + E13 + F9) while gently vortexing to mix. Cap the tube and thoroughly vortex for 10 s to mix. Allow the mixture to sit at Room temperature for 2–10 min. Add 2 mL of the transfection solution dropwise to each dish and swirl to mix before returning the dishes to the cell culture incubator. 
 

SECTION: Procedure: Triple transient transfection of HEK293T cells ● Timing 1–2 h
24. Change the media 12–24 h post transfection by aspirating the old media in 10% (vol/vol) bleach and replacing it with 20 mL of fresh, warmed media (Reagent setup).
 

SECTION: Procedure: AAV harvest ● Timing 5 d
25. 
 

 

Harvest the cell culture media 72 h (3 d) post transfection. Tilt each dish at a 30° angle and use a 25-ml serological pipette to collect the media. Store in an empty, sterile media bottle or sterile 500-ml bottle at 4 °C until Step 28. Replace the media with 20 mL of fresh, warmed media (Reagent setup). 
 

SECTION: Procedure: AAV harvest ● Timing 5 d
26. Harvest the media and cells 120 h (5 d) post-transfection. Use a cell scraper to gently scrape the cells in the media. After scraping the first dish, prop it at a 30° angle, using an empty 1.5-ml microcentrifuge tube rack for support. Scrape down the residual cells and media such that they are pooled together. Return the dish lid and scrape the next plate; prop dishes up against one another along the length of the biosafety cabinet until scraping is complete. Use a 25-ml serological pipette to collect the media and cells from each dish; transfer to a 250-ml conical centrifuge tube. Pool the media and cells from up to 10 dishes in a single tube. 
 

SECTION: Procedure: AAV harvest ● Timing 5 d
27. Combine the media collected at 72 h post transfection (Step 26) with the media and cells collected at 120 h post transfection (Step 27). For smaller viral preps (1–5 dishes), use option step 28.1. For larger preps (6–10 dishes), use option step 28.2. 
SECTION: Procedure: AAV harvest ● Timing 5 d
27.1. Harvest from 1–5 dishes
Pour the media collected in Step 26 into the corresponding 250-ml tube of media and cells
collected in Step 27.
 

SECTION: Procedure: AAV harvest ● Timing 5 d
27.2. Harvest from 6–10 dishes
Pour the media collected in Step 26 into a new 250-ml tube.
 

SECTION: Procedure: AAV harvest ● Timing 5 d
28. Centrifuge the media and cells at 2000 x g, 15 min. Ensure that the tube caps are tightly secured. Centrifugation will result in the formation of a cell pellet (Fig. 7b).
  
SECTION: Procedure: AAV harvest ● Timing 5 d
30. Cell pellet resuspension. Prepare 5 mL of SAN + SAN digestion buffer (Reagent setup) per viral prep. For smaller viral preps (1–5 dishes), use option 31.1. For larger preps (6–10 dishes), use option 31.2.
SECTION: Procedure: AAV harvest ● Timing 5 d
29. Pour off the supernatant (i.e., the clarified media) into the corresponding bottle from Step 26. Allow excess media to drip back down onto the beveled edge of the 250-ml tube; remove using a P1000 pipette and add to the supernatant. Store the supernatant at 4 °C until Step 32. 
 

SECTION: Procedure: AAV harvest ● Timing 5 d
30.1. Harvest from 1–5 dishes
Use a 5-ml serological pipette to gently resuspend the cell pellet in 5 mL of SAN + SAN digestion buffer; pipette into a 50-ml tube to finish resuspending the pellet (Fig. 7b).
Incubate in a 37 °C water bath for 60 min and store at 4 °C until Step 36 (up to 1 d).
 

SECTION: Procedure: AAV harvest ● Timing 5 d
30.2. Harvest from 6–10 dishes
Use a 10-ml serological pipette to partially resuspend the smaller cell pellet in 5 mL of SAN + SAN digestion buffer. Pipette into the second 250-ml tube containing the larger pellet and resuspend together; pipette into a 50-ml tube to finish resuspending the pellet (Fig. 7b).
Incubate in a 37 °C water bath for 60 minand store at 4 °C until Step 36 (up to 1 d).
 

SECTION: Procedure: AAV harvest ● Timing 5 d
31. Retrieve the supernatant collected in Step 30. For smaller viral preps (1–5 dishes), use option 32.1. For larger preps (6–10 dishes), use option 32.2.
SECTION: Procedure: AAV harvest ● Timing 5 d
31.1. Harvest from 1–5 dishes
Pour the supernatant from Step 30 into the corresponding 250-ml tube from Step 31. 
 

SECTION: Procedure: AAV harvest ● Timing 5 d
31.2. Harvest from 6–10 dishes
Equally divide the supernatant from Step 30 between the two corresponding 250-ml tubes from Step 31. 
 

SECTION: Procedure: AAV harvest ● Timing 5 d
34. Pour off the supernatant (i.e., the PEG-clarified media) into a used media collection bottle for bleaching. Allow excess media to drip back down onto the beveled edge of the 250-ml tube; aspirate or pipette to remove.
SECTION: Procedure: AAV harvest ● Timing 5 d
33. Centrifuge the PEG–media mixture at 4000 x g, 30 min, 4 °C. Centrifugation will result in the formation of a PEG pellet (Fig. 7b).

SECTION: Procedure: AAV purification ● Timing 1 d
36. 
 
Determine the number of gradients needed and prepare the iodixanol density gradient solutions (Reagent setup and Supplementary Table 3). Set the OptiSeal tubes in the rack provided in the OptiSeal tube kit; alternatively, use the long edge of a 50-ml tube Styrofoam rack. 
 

SECTION: Procedure: AAV harvest ● Timing 5 d
35. PEG pellet resuspension. Prepare 1 mL of SAN + SAN digestion buffer (Reagent setup) per viral prep. For smaller viral preps (1–5 dishes), use option 36.1. For larger preps (6–10 dishes), use option 36.2. 
SECTION: Procedure: AAV harvest ● Timing 5 d
35.1. Harvest from 1–5 dishes
Use a P1000 pipette to carefully resuspend the PEG pellet in 1 mL of SAN + SAN digestion buffer; pipette into the corresponding lysate from Step 31 (Fig. 7b).
Incubate in a 37 °C water bath for an additional 30 min. 
 

SECTION: Procedure: AAV harvest ● Timing 5 d
35.2. Harvest from 6–10 dishes
Use a P1000 pipette to partially resuspend one of the PEG pellets in 1 mL of SAN + SAN digestion buffer. Pipette into the second 250-ml tube containing the second pellet and carefully resuspend together; pipette into the corresponding lysate from Step 31 (Fig. 7b).
Incubate in a 37 °C water bath for an additional 30 min.
 

SECTION: Procedure: AAV purification ● Timing 1 d
40. Using a 2-ml serological pipette, fill each tube up to the neck with SAN digestion buffer. Firmly insert a black cap (Fig. 8d) and place a spacer on top (Fig. 8e). Caps and spacers are provided with the OptiSeal tubes and in the OptiSeal tube kit, respectively. 
 

SECTION: Procedure: AAV purification ● Timing 1 d
37. Pour the density gradients (Fig. 8a,b and Supplementary Video 1, 0:00–1:45, or Supplementary Video 2, 0:00–1:13). Each gradient is composed of the following layers: 6 mL of 15% (wt/vol) iodixanol, 6 mL of 25% (wt/vol) iodixanol, 5 mL of 40% (wt/vol) iodixanol, and 5 mL of 60% (wt/vol) iodixanol (Supplementary Table 3). Pour the layers with a 2- or 5-ml serological pipette. We typically use a 2-ml pipette; using a 5-ml pipette is faster but requires the use of PTFE and Tygon tubing and extra reagents. To load the layers with a 2-ml pipette, choose option 38.1. To load the layers with a 5-ml pipette, choose option 38.2.
SECTION: Procedure: AAV purification ● Timing 1 d
37.1. Loading with a 2-ml pipette
Begin by pipetting 6 mL (measure to the 3 ml mark twice) of 15% (wt/vol) iodixanol to each tube. Next, add 6 mL of 25% (wt/vol) iodixanol under the 15% layer by lightly touching the pipette tip to the bottom of the tube and slowly releasing the solution (Fig. 8a and Supplementary Video 1, 0:13–1:29). Continue adding layers of increasing density under the previous layer. The gradients should have a sharp delineation between layers (Fig. 8b).
 

SECTION: Procedure: AAV purification ● Timing 1 d
37.2. Loading with a 5-ml pipette
Attach a piece of tubing (see Equipment) to a 5-ml pipette. Begin by pipetting 6 mL of 15% (wt/vol) iodixanol into each tube. Next, add 6 mL of 25% (wt/vol) iodixanol under the 15% layer by lightly touching the tubing to the bottom of the tube and slowly releasing the solution (Supplementary Video 2, 0:17–1:13). Continue adding layers of increasing density under the previous layer. The gradients should have a sharp delineation between layers (Fig. 8b). 
 

SECTION: Procedure: AAV purification ● Timing 1 d
38. Centrifuge the lysate from Step 36 at 2000 x g, 10 min. Centrifugation will result in the formation of a pellet (Fig. 7b).

SECTION: Procedure: AAV purification ● Timing 1 d
39. Use a 2-ml serological pipette to load the supernatant (i.e., the clarified lysate) (~6–7 ml total) from Step 39 above the 15% (wt/vol) iodixanol layer (Fig. 8c and Supplementary Video 1, 1:46–2:22 or Supplementary Video 2, 1:14–1:55). Touch the pipette tip to the surface of the 15% layer and slowly release the lysate such that a layer forms on top.
 

SECTION: Procedure: AAV purification ● Timing 1 d
41. Weigh the tubes with the caps and spacers on. Balance the tubes to within 5-10 mg of each other using SAN digestion buffer. Be sure to adjust the tube weight in the biosafety cabinet; use the tube removal tool provided with the OptiSeal tube kit to remove the black cap and add the appropriate volume of SAN digestion buffer with a P20 or P200 pipette. 
 

SECTION: Procedure: AAV purification ● Timing 1 d
42. Place the ultracentrifuge rotor in the biosafety cabinet. Load the tubes and fasten the lid. 
 

SECTION: Procedure: AAV purification ● Timing 1 d
43. Carefully transfer the rotor to the ultracentrifuge. Spin the Type 70 Ti rotor at 350000 x g, 145 min, 18 °C (58,400 r.p.m.)  with slow acceleration (no. 3; the instrument will take 3 min to accelerate to 500 r.p.m., followed by maximum acceleration) and deceleration (no. 9; the instrument will deccelerate at maximum speed until it reaches 500 r.p.m., then take 6 min to stop) profiles. Alternatively, a Type 60 Ti rotor can be used at 358000 x g (59,000 r.p.m.). 
 

SECTION: Procedure: AAV purification ● Timing 1 d
44. During ultracentrifugation, gather the supplies and equipment for Steps 46–49. Assemble the clamp setup (Equipment setup) and collect one of each of the following per gradient: PES Ultra-15 centrifugal filter device, 5-ml syringe, 10-ml syringe, 0.22-μm syringe filter unit, and a 16-gauge needle.
SECTION: Procedure: AAV purification ● Timing 1 d
45. After ultracentrifugation, bring the rotor inside the biosafety cabinet and remove the lid. Use the spacer removal tool provided with the OptiSeal tube kit to remove the spacer from the first tube. Next, use the tube removal tool to grip the tube neck. Slowly remove the tube from the rotor and secure it into the clamp setup above a 500-ml or 1-liter beaker containing fresh 10% (vol/vol) bleach (Fig. 8f). Clean the side of the tube with a paper towel or a Kimwipe sprayed with 70% (vol/vol) ethanol. 
 

SECTION: Procedure: AAV purification ● Timing 1 d
46. Prepare the supplies for Steps 48 and 49. First, remove and save the plunger from a 10-ml syringe. Attach a 0.22-μm syringe filter unit to the syringe barrel and place it on top of an PES filter device. Next, add 10 mL of DPBS supplemented with 0.001% of Pluronic F-68 (DPBS + 0.001% Pluornic F-68) to the barrel and allow the solution to begin dripping through the syringe filter unit and into the filter device (Fig. 8f). Last, attach a 16-gauge needle to a 5-ml syringe. 
 

SECTION: Procedure: AAV purification ● Timing 1 d
47. From the tube clamped in Step 46, collect the virus from the 40/60% interface and 40% layer9,10 (Fig. 8g and Supplementary Video 3, 0:00–1:30). Hold the top of the OptiSeal tube with your nondominant hand; use your dominant hand to hold the needle/syringe. Use a forward-twisting motion to insert the needle ~4 mm below the 40/60% interface (i.e., where the tube just starts to curve). Use the tube removal tool in your non-dominant hand to remove the black cap from the tube to provide a hole for air entry. With the needle bevel up, use the needle/syringe to collect 4.0–4.5 ml of virus/ iodixanol from the 40/60% interface and 40% layer. Do not collect from the white protein layer at the 25/40% interface; as this interface is approached, rotate the needle bevel down and continue collecting from the 40% layer. Firmly replace the black cap before removing the needle from the tube. 
 

 

SECTION: Procedure: AAV purification ● Timing 1 d
48. Add the 4.0-4.5 mL of virus/iodixanol to the syringe barrel containing 10 mL of DPBS (prepared in Step 47) (Fig. 8h and Supplementary Video 3, 1:31–2:06). Layer the virus below the DPBS by placing the needle near the bottom of the barrel and pressing on the plunger. Insert the 10-ml syringe plunger into the barrel and push the virus/DPBS mixture through the syringe filter unit and into the PES filter device (Supplementary Video 3, 2:07–2:32). Mix well using a P1000 pipette. 
 

SECTION: Procedure: AAV purification ● Timing 1 d
49. Centrifuge the virus/DPBS mixture at 3000 x g for 5–8 min, or until the volume of the solution remaining in the top chamber of the PES filter device is 500-1500 µL (>10× concentrated). 
 

SECTION: Procedure: AAV purification ● Timing 1 d
50. Discard the flow-through for bleaching. Add 13 mL of DPBS + 0.001% Pluornic F-68 to the virus in the top chamber and use a P1000 pipette to mix. 
 

SECTION: Procedure: AAV purification ● Timing 1 d
51. Centrifuge the virus/DPBS mixture as in Step 50. Wash the virus two more times for a total of four buffer exchanges. During the last spin, retain 300-500 µL of solution in the top chamber. 
 


SECTION: Procedure: AAV purification ● Timing 1 d
52. Use a P200 pipette to transfer the virus from the top chamber of the PES filter device directly to a 1.6-ml screw-cap vial; store at 4 °C. 
 

SECTION: Procedure: AAV titration ● Timing 1 d
53. 
 
Prepare a plan for the PCR plate setup. Allocate the first 24 wells (A1–B12) for the DNA standards such that standard no. 1 occupies wells A1–A3, standard no. 2 occupies wells A4–A6, and so on. Use the remaining wells for the virus samples such that the first virus sample occupies wells C1–C3, the second sample occupies wells C4–C6, and so on. 
 

SECTION: Procedure: AAV titration ● Timing 1 d
54. Use DNase I to digest DNA that was not packaged into the viral capsid. Prepare DNase I + DNase digestion buffer (Reagent setup) and add 100 µL to each 1.5-ml tube. Vortex each virus for 1–2 s to mix; alternatively, use a P200 pipette to mix. Add 2 µL of the virus to each of three tubes. Vortex for 1–2 s to mix and spin down (2000 x g, 10 s); incubate in a 37 °C water bath for 60 min. 
 

SECTION: Procedure: AAV titration ● Timing 1 d
55. Inactivate the DNase. Add 5 µL of EDTA to each tube; vortex for 1–2 s to mix, spin down (2000 x g, 10 s), and incubate in a 70 °C dry bath for 10 min. 
 

SECTION: Procedure: AAV titration ● Timing 1 d
56. Use proteinase K to digest the viral capsid and release the viral genome. Prepare proteinase K + proteinase K solution (Reagent setup) and add 120 µL to each tube. Vortex for 1–2 s to mix and spin down (2000 x g, 10 s); incubate in a 50 °C dry bath for 120 min. 
 

SECTION: Procedure: AAV titration ● Timing 1 d
57. During the last 20 min of the proteinase K digestion, prepare the DNA standard dilutions (Reagent setup) and use the Qubit assay to measure the concentration (ng/μl) of the DNA standard stock. 
 

SECTION: Procedure: AAV titration ● Timing 1 d
58. Inactivate the proteinase K. Incubate the tubes in a 95 °C dry bath for 10 min. 
 

 

SECTION: Procedure: AAV titration ● Timing 1 d
59. Allow the tubes to cool for 5 min. Vortex each sample for 1–2 s to mix and add 3 µL to a new tube containing 897 µL of UltraPure water (a 1:300 dilution). Vortex the diluted samples for 3 s to mix.

SECTION: Procedure: AAV titration ● Timing 1 d
60. Prepare the qPCR master mix (Reagent setup).
SECTION: Procedure: AAV titration ● Timing 1 d
61. Load the PCR plate based on the experimental plan from Step 32. First, pipette 23 µL of qPCR master mix into each designated well. Next, pipette 2 µL of each standard into wells A1–B12. Last, pipette 2 µL of each diluted sample from Step 38 into wells C1 and onward. Seal the plate with sealing film and briefly spin down (500 x g, 10 s) in a plate spinner.
SECTION: Procedure: AAV titration ● Timing 1 d
63. When the qPCR run is complete, export the cycle threshold (Ct) values to an Excel file. Copy and paste the Ct values into Supplementary Table 4 (‘AAV titration calculator’ sheet) to generate a standard curve and calculate the titer (vg/ml) (cell G27) of each virus; calculate per-plate production (vg/dish) (cell K27) to assess production efficiency. Be sure to enter the appropriate values in cells B7–10 and B18; see ‘Example’ sheet. 
 

SECTION: Procedure: AAV titration ● Timing 1 d
62. Place the PCR plate into the qPCR machine. Use the following cycling parameters:
    Step 63.1: 95 °C, 10 min
    Step 63.2: 95 °C, 15 s
    Step 63.3: 60 °C, 20 s
    Step 63.4: 60 °C, 40 s
    Repeat steps 63.2–63.4 40×.
SECTION: Procedure: AAV harvest ● Timing 5 d
32. Use a 25-ml or 50-ml serological pipette to add a 1/5 final volume of 40% (wt/vol) PEG stock solution to the supernatant (i.e., the supernatant should contain a final concentration of 8% (wt/vol) PEG solution). Tighten the cap and thoroughly invert ten times to mix. Incubate on ice for 120 min.