1
Widespread and targeted gene expression
by systemic AAV vectors:
Production, purification, and administration
Rosemary C Challis
1
, Sripriya Ravindra Kumar
1
, Ken Y Chan
1
, Collin Challis
1
, Min J Jang
1
,
Pradeep S Rajendran
2
, John D Tompkins
2
, Kalyanam
Shivkumar
2
, Benjamin E Deverman
1
,
and Viviana Gradinaru
1
*
1
Division of Biology and Biological Engineering,
California Institute of Technology
Pasadena, CA
2
Cardiac Arrhythmia Center and Neurocardiology Research Center of Excellence, University of
Calif
ornia, Los Angeles
Los Angeles, CA
*To whom correspondence should be addressed:
Viviana Gradinaru, Ph.D.
Division of Biology and Biological Engineering
California Institute of Technology
1200 East California Blvd. MC 156-29
Pasadena, CA 91125
Phone: (626) 395-6813
viviana@caltech.edu
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not
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2
ABSTRACT
We recently developed novel AAV capsids for efficient and noninvasive gene transfer across the
central and peripheral nervous systems. In this
protocol, we describe how to produce and
systemically
administer AAV-PHP viruses to label
and/or genetically
manipulate cells in the
mouse 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
eight days, excluding the time required to assess gene expression, and can
be readily adopted by laboratories with standard molecular and cell culture capabilities. We
provide guidelines for experimental design
and choosing 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.
INTRODUCTION
Recombinant adeno-associated viruses (AAVs) are commonly used
vehicles for
in vivo
gene
transfer
and promising vectors for therapeutic applications
1
. However, AAVs that enable efficient
and noninvasive gene delivery across 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 still provide relatively inefficient
transduction of target cells. We previously developed CREATE (Cre REcombination-based AAV
Targeted Evolution)
to
engineer and screen for
AAV capsids capable of more efficient
gene
transfer to specific cell types
via the vasculature
2,3
. Compared
to naturally occurring capsids, the
novel AAV
-PHP capsids exhibit markedl
y 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 and noninvasive
gene delivery
throughout the body (
Fig. 1
).
We recently identified several new capsid variants with distinct tropisms
2,3
. AAV-PHP.B
and the further evolved
AAV-PHP.eB
efficiently transduce
neurons and glia throughout the CNS
(
Fig. 2
); a second
variant, AAV
-PHP.S,
display
s improved tropism for neurons within the PNS
(
Fig. 3
)
and
organs
including the gut
2
and heart (
Fig. 4
).
Importantly, these capsids
target cell
populations
that are
normally
difficult to access due to their location
(e.g., sympathetic, nodose,
dorsal root, and cardiac ganglia) (
Fig. 3a-c
and
Fig. 4d
)
or broad distribution
(e.g.,
throughout
the brain or
enteric nervous system) (
Figs. 2
and
3d
).
Together with the capsid, the genetic
cargo (or AAV genome) can be customized to control transgene expression (
Fig. 5
and
Table
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3
1
). The
recombinant AAV (rAAV)
genome contains the components required for gene
expression including promoters, transgenes, protein trafficking
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
res
ources to utilize AAV
-PHP viruse
s in their own research.
Overview of the protocol
We provide an instruct
ion 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 plasmids
4-6
(Steps 1-3) (
Figs
. 1
and
6
): (1)
pAAV, which contains the
rAAV genome of interest (
Fig. 5
and
Table 1
);
(2) AAV-PHP Rep
-Cap, which encodes the viral
replication and capsid proteins; and (3) pHelper, which encodes adenoviral
proteins
necessary
for
replication.
Using this triple transfection approach, the
rAAV genome is packaged into an
AAV-PHP capsid
in HEK293T cells. AAV-PHP viruses
are then harv
ested
7
(Steps
4-14),
purified
8,9
(Steps
15-31), and titered
10
(Steps
32-42) (
Fig. 6
). Purified
viruses are
intravenously
delivered
to mice
via retro-orbital injection
11
(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 (over 10
13
vector genomes/ml)
and with high transduction efficiency
in vivo
2,3
.
Experimental design
Before proceeding with
the protocol, a number of factors should be considered, namely the
expertise and resources available in the lab; the 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 and intended to guide users in designing their
experiments.
Required expertise
and resources.
This
protocol requires
that scientists have basic molecular
biology, cell culture, and animal work experience.
Users should be approved to handle
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laboratory animals, human cell lines, and AAVs.
A background in molecular cloning is
advantageous
though not necessary if relying on available plasmids.
In addition to having the above expertise, 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.
Selecting an AAV
-PHP capsid.
We recommend choosing an AAV
-PHP capsid
based on its
tropism and viral 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 ID 103005) or AAV
-PHP.S
(Addgene ID 103006) in their
experiments.
AAV-PHP.
eB and AAV-PHP.S
produce viral yields
similar to other high producing naturally occurring serotypes (e.g., AAV9) and enable efficient,
noninvasive gene transfer to the CNS or PNS and visceral organs, respectively
2
(
Figs. 2-4
).
The earlier
capsid
variants, which provide broad 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 ID
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 species
12
, age
13
, gender
14
, and health
15
influence
AAV trop
ism. 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 a rAAV genome.
Users must select a rAAV genome, contained in a pAAV plasmid,
to package into the capsid (
Figs
. 1
and
5
and
Table 1
). In
Table 1
, we list pAAVs used here
(
Figs. 2-4
) and in our previous work
2,3
; we direct users to Addgene’s plasmid repository for
additional pAAVs developed for various applications.
Depending on the experimental aims, users may elect to design their own genomes
16
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. 5
); larger genomes will not be fully packaged into AAV capsids,
resulting in truncated genomes and low titers. The ITRs are 145 base pair sequences that flank
the expression cassette and are required for replication and encapsidation of the viral genome.
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ITRs are typically derived from the AAV2 genome and must match the serotype of the
rep
gene
contained in the AAV Rep-C
ap plasmid; AAV
-PHP Rep
-Cap plasmids contain the AAV2
rep
gene and are therefore capable of packaging genomes with AAV2 ITRs.
Other genetic
components (e.g., promoters, transgenes, localization signals, and recombination scheme
s) are
interchangeable and can be customized for specific
applications
(
Fig. 5
).
Dos
age for intravenous administration.
The optimal dose for intravenous
administration to
target cell populations must be determined empirically. We encourage users to consult
Figures
2-4
and related work for suggested AAV
-PHP viral doses. The variants have been successfully
employed for
fluorescent labeling
in adult mice
2,3,17
(
Figs. 2-4
), neonatal mice
17
, and neonatal
and adult rats
18
; they have also been administered for calcium imaging
19,20
and optogenetic (
Fig.
4d
),
chemogenetic
17
, and therapeutic
applications
17,18
.
For applications using AAV
-PHP.eB and AAV-PHP.S, we typically administer between 1
x 10
11
and 3 x 10
12
vector genomes (vg) of virus to adult mice (≥6 weeks of age)
. 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 cell
2
. Therefore, higher doses generally result in
strong
expression (i.e., high copy number) in a large fraction of cells, whereas low
er
doses result in
weaker
expression (i.e., low copy number) in a small
er
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
the tetracycline-controlled transactivator (tTA))
2
; inducers are injected at a lower dose (typically
1 x 10
9
to 1 x 10
11
vg) to limit t
he fraction of cells with transgene expression.
Note that gene
regulatory elements
(e.g., enhancers and promoters)
also 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. While expression is evident
within days after transduction, it does not reach a steady state level until at least 3-
4 weeks.
Therefore, we suggest waiting a minimum of 2 weeks before evaluating
fluorescent
labeling
2,3,17,18
(
Figs. 2-4
) and at least 3-4 weeks before beginning optogenetic (
Fig. 4d
),
chemogenetic
17
, and calcium imaging
19,20
experiments.
Note that
like other AAVs, AAV-PHP
variants are capable of providing long-term transgene expression. AAV-PHP.B-
mediated
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cortical
expression of a genetically encoded calcium indicator,
GCaMP6s, was reported to last
at least 10 weeks post-injection without toxic side effects
20
(i.e., nucl
ear filling
21
), and we have
observed
GFP expression
throughout the brain
mo
re than one year after viral administration
(see Supplementary Figure 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.
Th
e appropriate method(s) for evaluating transgene expression will vary among users
.
Fluorescent protein
expression
can be assessed in thin or
thick
(≥
100 μm
) tissue samples
. The
CLARITY
-based
methods PACT (passive CLARITY technique) and PARS (perfusion-assisted
agent release
in situ
)
22
render thick tissues optically transparent while preserving their
three-
dimensional
molecular and cellular architecture and facilitate deep imaging of large volumes
(e.g., via confocal or light-sheet microscopy)
23
. Cleared tissues are compatible with endogenous
fluorophores including
commonly used markers like GFP
3,22,24
, eYFP
22
, and tdTomato
24
.
However, some fluorescent signals, such as those from mTur
quoise2, mNeonGreen, and
mRuby2
, can deteriorate
in chemical clearing reagents. To visualize these reporters, we
suggest using optical clearing methods
like
RIMS (refractive index matching solution)
24
or
Sca
l
eSQ
25
(
Figs. 3a
and
c
, and
4b-c
), or commercially available mounting media like Prolong
Diamond Antifade (Thermo Fisher Scientific, cat. no.
P36965)
2
(
Fig. 2
).
See the Anticipated
Results section for
details on expected outcomes when using fluorescent reporters.
We recognize that fluorescent labelling is not desired or feasible for every application
. In
such cases, users must identify the appropriate method(s) for examining transduced cells,
which may include molecular (e.g., qPCR or Western blot), histological
(e.g. with antibodies,
small molecule dyes, or molecular probes) or functional (e.g., optical
imaging)
approaches.
Limitations of the method
A major limitation of AAV capsids, including AAV-PHP variants, is their relatively small
packaging capa
city
(<5 kb).
Some elements of the rAAV genome
, such as the WPRE (see
legend in
Fig
ure
5
), can be truncated
26
or
removed
27,28
to
accommodate larger genetic
components. T
he development
of smaller promoters
29,30
and
dual expression systems
31
, in
which genetic elements are split between two or more viruses (requiring efficient
cotransduction),
hav
e 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
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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 microRNA (miRNA)
-mediated gene silencing. Sequences complementary to
miRNAs expressed in off
-target cell populations can be introduced in
to
the 3’ UTR
of the rAAV
genome (
Fig. 5
); this has been shown to
reduce off-target
transgene expression
and restrict
expression to cell types
of interest
32,33
.
Another challenge of systemic delivery is that it requires a high viral load, which can
illicit
an
immune response against the capsid
and/or transgene and reduce transduction efficiency
in
vivo
34
. Immunogenicity of
AAVs may be exacerbated by empty capsid contamination in viral
preparations
35,36
. The
viral purification protocol (Steps 15-31)
provided here reduces, but does
not eliminate, empty capsids (
Fig. 6b
). If
this poses a concern for specific applications, viruses
can be purified using an alternative approach
7,8,37
.
Lastly, generating 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.
Applications of the method
We anticipate that AAV-PHP
capsids can be used with the growing pAAV plasmid repository
available through Addgene and elsewhere to enable
a wide range of biomedical
applications
(
Fig. 5
and
Table 1
).
Below, we highlight a few current and potential
applications
of this method.
Anatomical mapping.
F
luorescent reporters are commonly used for cell type-specific mapping
and phenotyping
2,38,39
(
Figs. 2-4
). AAV-mediated
multicolor labeling (e.g., Brainbow
40
) 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 brain
2,40
, retina
40
, heart (
Figs
. 4b
and
c
), and gut
2
, as well as
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 enteric
2
or
cardiac nervous systems (
Fig. 4c
)).
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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
chemogenetic
17
and optical imaging
applications
19,20
. 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
), lungs
41
, or gut
42
. AAV-PHP variants could also be utilized to
interrogate the function of non-neuronal cell types including cardiomyocytes
43
, pancreatic beta
cells
44,45
, and hepatocytes
46
. Harnessing AAV-PHP viruses
to
modulate cell physiology may
reveal novel roles for different cells in regulating
organ function and/or animal behavior.
Gene
expression, silencing,
and editing.
AAV-PHP viruses are well
-suited for assessing
potential therapeutic strategies that would benefit from organ-wide or systemic transgene
expression. Recently, AAV-PHP.B was used to treat
17
and model
18
neurodegenerative diseases
with widespread pathology
. Other potential applications include gene editing
(e.g., via
CRISPR
47,48
) or silencing (e.g., via short hairpin RNA (shRNA)
49,50
); 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.
Summary
Systemically delivered AAV-PHP v
iruses
provide
efficient, noninvasive, and long-term
gene
transfer
to cell types throughout the adult CNS, PNS, and visceral organs. Together with the use
of custom rAAV genomes (
Fig. 5
and
Table 1
), researchers can target genetic elements to
defined c
ell populations for diverse applications.
MATERIALS
REAGENTS
Plasmid DNA preparation
●
Plasmids, supplied as bacterial stabs (Addgene; see
Table 1
for plasmids used in this and
related work)
CRITICAL
Three plasmids (pAAV, capsid, and
pH
elper) are required for
transfection (
Fig. 1
).
●
Agarose (Amresco, cat. no. N605-250G)
●
Antibiotics (e.g., carbenicillin disodium salt, Alfa Aesar, cat. no. J61949-
06; all plasmids
used in this work carry antibiotic resistance genes to ampicillin/carbenicillin)
●
DNA ladder, 2-log, 0.1-10.0 kb (New England Biolabs, cat. no. N0550S)
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●
Lysogeny broth (LB) (Amresco, cat. no. J106-1KG); for large-scale plasmid preparations,
such
as maxi
- and giga-preps, we typically use Plasmid+ media (Thomson Instrument
Company, cat. no. 446300), an enriched media formulated to support higher cell densities
and plasmid yields compared to LB
●
Lysogeny broth (LB) with agar (Sigma-Aldrich, cat. no. L3147-1KG)
●
NucleoBond Xtra Maxi Endotoxin-Free (EF) plasmid purification kit (Macherey
-Nagel, cat.
no. 740424.50) CRITICAL
Triple transient transfection requires large amounts of capsid
(22.8 μg/dish) and pHelper plasmid DNA (11.4 μg/dish) (
Supplementar
y Table 2
, sheet
‘Detailed calculations’
); isolating these plasmids may be more convenient with a giga
-
scale purification kit (NucleoBond PC 10000 EF, Macherey
-Nagel, cat. no. 740548). All
plasmids should be purified under endotoxin-free conditions. Endotoxin contamination in
plasmid preparations can reduce transfection efficiency, and contaminating endotoxins in
viral preparations could elicit immune reactions in mammals
in vivo
.
●
Res
triction enzymes
, including SmaI
(New England Biolabs, cat. no. R0141S); used for
verifying plasmid and ITR integrity
●
Sequencing primers (Integrated DNA Technologies); used for verifying plasmid integrity
●
SYBR Safe DNA gel stain (Invitrogen, cat. no. S33102)
●
Tris-acetate
-EDTA (TAE) buffer, 50X (Invitrogen, cat. no. B49)
Cell culture
●
Human embryonic kidney (HEK) cells, 293 or 293T (ATCC, cat. no.
CRL 1573 or CRL
3216)
CAUTION
HEK cells pose a moderate risk to laboratory workers and the
surrounding environment and must be handled according to governmental and
institutional regulations. Experiments involving HEK cells are performed using Biosafety
Level 2 practices as required by
the California Institute of Technology and the U.S. Centers
for Disease Control and Prevention. The cell line identity has not been validated, nor do
we routinely test for mycoplasma.
CRITICAL
HEK293
and HEK293T cells constitutively
express two
adenoviral
genes, E1a
and E1b, which are required for AAV production
in
these cells
6
; we
do not
recommend using an alternative
producer
cell line
with this
protocol
.
●
Dulbecco’s Modified Eagle Medium (DMEM), high glucose, GlutaMAX supplement,
pyruvate (Gibco, cat. no. 10569-044)
●
Ethanol, 70% (v/v); prepare from absolute ethanol (J.T. Baker, cat. no. 8025) CAUTION
Ethanol is flammable.
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●
Fetal bovine serum (FBS) (GE Healthcare, cat. no. SH30070.03)
CRITICAL
Aliquot and
store at -
20 ̊C for up to 1 year. Avoid freeze/thaw
cycles.
●
MEM Non
-Essential Amino Acids (NEAA) solution, 100X (Gibco, cat. no. 11140-050)
●
Penicillin
-Streptomycin (Pen-Strep), 5000 U/ml (Gibco, cat. no. 15070-063)
CRITICAL
Aliquot and store at -
20 ̊C for up to 1 year. Avoid freeze/thaw cycles.
●
TrypLE Express enzyme, 1X, phenol red (Gibco, cat. no. 12605-036)
Transfection
●
Plasmid DNA CRITICAL
We use a pAAV:capsid:pHelper plasmid ratio of 1:4:2 based on
μg of DNA. We use 40 μg of total DNA per 150 mm dish (5.7 μg of pAAV, 22.8 μg of
capsid, and 11.4 μg of pHelper) (
Supplementary Table 2
, sheet
‘Detailed calculations’
).
●
Polyethylenimine (PEI), linear, MW 25000 (Polysciences, Inc., cat. no. 23966-2)
●
Water for Injection (WFI) for cell culture (Gibco, cat. no. A1287304)
●
1X Dulbecco’s PBS (DPBS), no calcium, no magnesium (Gibco, cat. no. 14190-250)
●
1 N HCl solution, suitable for cell culture (Sigma
-Aldrich, cat. no. H9892)
CAUTION
HCl
is corrosive. Use personal protective equipment.
AAV production
●
Bleach, 10% (v/v); prepare fresh from concentrated liquid bleach (e.g., Clorox)
CRITICAL
AAV-contaminated materials must be disinfected with bleach prior to disposal; ethanol is
not an effective disinfectant. Experiments involving AAVs follow a standard operating
procedure in which contaminated equipment, surfaces, and labware are disinfected for 10
min with 10% bleach. AAV waste disposal should be conducted according to federal, state,
and local regulations.
●
Dry ice; optional
●
KCl (any)
●
MgCl
2
(any)
●
NaCl (any)
●
OptiPrep (60% (w/v) iodixanol) density gradient media (Cosmo Bio USA, cat. no. AXS
-
1114542-5)
●
Phenol red solution (Millipore, cat. no. 1072420100)
●
Pluronic F-68 non-ionic surfactant, 100X (Gibco, cat. no. 24040-032); optional
●
Polyethylene glycol (PEG), MW 8000 (Sigma-Aldrich, 89510
-1KG-F)
●
Salt-active nuclease (SAN) (ArcticZymes, cat. no. 70910-202)
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●
Tris base (any)
●
Ultrapure DNAse/RNAse-free distilled water (Invitrogen, cat. no. 10977-023)
●
Water for Injection (WFI) for cell culture (Gibco, cat. no. A1287304)
●
1X Dulbecco’s PBS (DPBS), no calcium, no magnesium
(Gibco, cat. no. 14190-250)
AAV titration
●
AAVs
●
CaCl
2
(any)
●
DNAse I recombinant, RNAse-free (Sigma-Aldrich, cat. no. 4716728001)
●
HCl, 37% (wt/wt) (Sigma
-Aldrich, cat. no. 320331
-500ML)
●
MgCl
2
(any)
●
NaCl (any)
●
N
-lauroylsarcosine
sodium salt (Sigma-Aldrich, cat. no. L9150
-50G)
●
Plasmid DNA containing the target sequence (e.g., pAAV
-CAG-eYFP, Addgene ID
104055); used for preparing the DNA standard stock
CRITICAL
The plasmid used to
make
the DNA standard must contain the same target sequence as the pAAV plasmid used to
generate virus. The target sequence must be within the rAAV genome; we typically amplify
a portion of the WPRE in the 3’ UTR of pAAVs (see legend in
Fig. 5
).
●
Primers corresponding to the target sequence (Integrated DNA Technologies)
WPRE
-forward:
GGCTGTTGGGCACTGACAAT
WPRE
-reverse:
CCGAAGGGACGTAGCAGAAG
CRITICAL
The proximity of the primer binding sites to the ITRs can affect titering
results;
therefore, titers measured with different primers or across laboratories may not be directly
comparable.
●
Proteinase K, recombinant, PCR grade (Sigma-
Aldrich, cat. no. 03115828001)
●
Qubit dsDNA HS assay kit (Invitrogen, cat. no. Q32854)
●
ScaI-HF restriction enzyme (New England Biolabs, cat. no. R3122S) or other enzyme that
cuts outside of the rAAV genome and within the pAAV backbone
●
SYBR green master mix (Roche Diagnostics, cat. no. 04913850001)
●
Tris base (any)
●
Ultrapure DNAse/RNAse-free distilled water (Invitrogen, cat. no. 10977-
023)
●
Ultrapure EDTA, 0.5 M, pH 8.0 (Invitrogen, cat. no. 15575-020)
Intravenous
(retro
-orbital) injection
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●
AAVs
●
Animals to be injected. This protocol describes the production of AAVs for intravenous
delivery to
6-8 week old
wild-type (C57BL/6J), ChAT-IRES
-Cre (Jackson Laboratory,
stock no. 028861, heterozygous), TH
-IRES
-Cre (European Mutant Mouse Archive, stock
no. EM00254, heterozygous), and TRPV1-IRES-Cre mice (Jackson Laboratory, stock no.
017769, homozygous). CAUTION
Experiments on vertebrates must conform to all
relevant governmental and institutional regulations. Animal husbandry and experimental
procedures involving mice were approved by the Institutional Animal Care and Use
Committee (IACUC) and the Office of Laboratory Animal Resources at the California
Institute of Technology.
●
Bleach, 10% (v/v) prepared fresh, or equivalent disinfectant (e.g., Accel TB surface
cleaner, Health Care Logistics, cat. no. 18692)
●
Isoflurane, USP (Piramal Healthcare, 66794-017-25)
CAUTION
Isoflurane is a
halogenated anesthetic gas associated with adverse health outcomes in humans and
must be handled according to governmental and institutional regulations. To reduce the
risk of occupational exposure during rodent anesthesia, waste gas is collected in a
biosafety cabinet using a charcoal scavenging system as approved by the California
Institute of Technology.
●
Proparacaine hydrochloride ophthalmic solution, USP, 0.5% (Akorn Pharmaceuticals, cat.
no. 17478-263-12)
●
1X Dulbecco’s PBS (DPBS), no calcium, no magnesium (Gibco, cat. no. 14190-250)
-------------------------------------------------------------------------------------------------------------------------------
EQUIPMENT
Plasmid DNA preparation equipment
●
Autoclave (any, as available)
●
Bunsen burner and lighter (Fisher Scientific, cat. nos. 03-917Q and S41878A)
●
Centrifuge (see requirements for chosen plasmid purification kit)
●
Gel electrophoresis system (Bio-Rad horizontal electrophoresis system)
●
Gel imaging system (Bio-Rad Gel Doc EZ model)
●
Incubating shaker (Eppendorf I24 model)
●
Incubator (Thermo Fisher Scientific Heratherm model) or 37 ̊C warm room
●
Sequence editing and annotation software (e.g., Lasergene by DNASTAR, SnapGene by
GSL Biotech, or VectorNTI by Thermo Fisher Scientific)
●
Spectrophotometer (Thermo Fisher Scientific NanoDrop model)
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13
Plasmid DNA preparation supplies
●
Petri dishes, 100 mm x 15 mm (Corning, cat. no. 351029)
●
Test tubes, 14 ml (Corning, cat. no. 352059)
●
Ultra Yield flasks and AirOtop seals, 250 ml (Thomson Instrument Company, cat. nos.
931144 and 899423); use with Plasmid+ media. Alternatively, use LB and standard
Erlenmeyer flasks.
AAV production equipment
●
Biological safety cabinet
CAUTION
HEK293T cells and AAVs are biohazardous materials
and must be handled according to governmental and institutional regulations. All
experiments involving the aforementioned materials are performed in a Class II biosafety
cabinet with annual certification as required by the California Institute of Technology and
the U.S. Centers for Disease Control and Prevention.
●
Centrifuge (any, provided the instrument can reach speeds up to 4,000 x
g
, refrigerate to
4 ̊C, and accommodate 250 ml conical centrifuge tubes; we
use the Beckman Coulter
Allegra X
-15R model)
●
Fluorescence microscope for cell culture (Zeiss Axio Vert.A1 model)
●
Incubator for cell culture, humidified at 37 ̊C with 5% CO
2
(Thermo Fisher Scientific
Heracell 240i model)
●
Laboratory balance (any, with a readability of 5-
10 mg)
●
Support stand with rod and clamp (VWR International, cat. nos. 12985-
070, 60079-
534,
and
89202-624) (
Fig. 7f
)
●
Ultracentrifuge (any preparative ultracentrifuge for
in vitro
diagnostic use; we use the
Beckman Coulter Optima XE-9 model with the Type 70Ti rotor) CAUTION
During
ultracentrifugation, rotors are subjected to enormous forces (up to 350,000 x
g
in this
protocol). Rotor failure can have catastrophic consequences including irreparable damage
to the centrifuge and laboratory and fatal injuries to personnel. Inspect rotors for signs of
damage or weakness prior to each use, and always follow the manufacturer’s instructions
while operating an ultracentrifuge.
●
Water bath (Fisher Scientific Isotemp model)
AAV production supplies
●
Amicon Ultra-15 centrifugal filter devices, 100 KDa molecular weight cutoff (Millipore, cat.
no. UFC910024)
●
Barrier pipet tips, 1000 μl (Genesee Scientific, cat. no. 23-430)
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14
●
Cell scrapers, 25 cm handle x 3 cm blade (Corning, cat. no. 353089)
●
Conical centrifuge tubes, 50 ml and 250 ml (Corning, cat. nos. 352098 and 430776)
●
Empty, sterile media bottles
●
OptiSeal tubes (Beckman Coulter, cat. no. 361625); includes black caps
●
OptiSeal tube kit (Beckman Coulter, cat. no. 361662); includes a tube rack, spacers, and
spacer and tube removal tools
●
Pipet Aid XL portable pipetting device (Drummond, cat. no.
4-000-105)
CRITICAL
Use a
pipetting device with precise control, which is essential for pouring the density gradients
in Step 16.
●
pH indicator strips (Millipore, cat. nos. 109532 and 109584)
●
Screw-cap vials, 1.6 ml (National Scientific Supply Co., cat. no. BC16NA-PS)
●
Serolo
gical pipets, 2 ml, 5 ml, 10 ml, 25 ml, and 50 ml (Corning, cat. no. 356507 and
Genesee Scientific, cat. nos. 12-102, 12-104, 12-106, and 12-107)
CRITICAL
Only
Corning brand 2 ml serological pipets consistently fit into OptiSeal tubes while pouring the
density gradients in Step 16. Alternatively, attach a small piece of clear tubing
(6 mm)
(e.g., Tygon Tubing) to a
5 ml pipet to pour the gradients.
●
Stericup
sterile vacuum filtration system, 0.22 μm, 500 ml and 1 L (Millipore, cat. nos.
SCGPU05RE and SCGPU11RE)
●
Sterile bottles, 500 ml (VWR International, cat. no. 89166-106)
●
Syringes, 5 ml and 10 ml (BD, cat. nos. 309646 and 309604)
●
Syringe filter unit, 0.22 μm (Millipore, cat. no. SLGP033RS)
●
Tissue culture dishes, 150 mm x 25 mm (Corning, cat. no. 430599)
●
16 G x 1 ½ in needles (BD, cat. no. 305198)
AAV titration equipment
●
Centrifuge (Eppendorf, 5418 model)
●
Dry bath and heating blocks (Fisher Scientific Isotemp models)
●
PCR plate spinner (VWR International, cat. no. 89184) or centrifuge equipped with plate
adapters
●
Quantitative PCR machine (any)
●
Qubit 3.0 fluorometer (Invitrogen, cat. no. Q33216)
AAV titration supplies
●
Barrier pipet tips, 10 μl, 20 μl, 200
μl, and 1000 μl (Genesee Scientific, cat. nos. 23-
401,
23-404, 23-412, and 23
-430)
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15
●
DNA clean
-up kit, for purification of up to 25
μg of DNA standard (Zymo Research, cat.
no. D4033)
●
Microcentrifuge tubes, 1.5 ml DNA/RNA LoBind (Eppendorf, cat. no. 86-923)
●
Qubit assay tubes (Invitrogen, cat. no. Q32856)
●
Sealing film for 96-well PCR plates (Genesee Scientific, cat. no. 12-529)
●
Stericup sterile vacuum filtration system, 0.22 μm, 250 ml (Millipore, cat. no.
SCGPU02RE)
●
Sterile bottles, 250 ml (VWR International, cat. no. 89166-104)
●
96-well PCR plates (Genesee Scientific, cat. no. 24-310W)
Intravenous (retro-orbital) injection equipment
●
Animal anesthesia system (VetEquip, cat. nos. 901806, 901807, or 901810) CRITICAL
Most animal facilities provide anesthesia systems equipped with an induction chamber,
isoflurane vaporizer, nose cone, and waste gas scavenging system. In our experience, a
mobile anesthesia system is most convenient for administering AAVs in a biosafety
cabinet.
Intravenous
(retro
-orbital) injection supplies
●
Activated charcoal adsorption filters (VetEquip, cat. no. 931401)
●
Insulin syringes with permanently attached needles, 31 G x 5/16 in (BD, cat. no. 328438)
●
Oxygen gas supply (any)
●
Screw-cap vials, 1.6 ml (National Scientific Supply Co., cat. no. BC16NA
-PS)
---------------------------------------------------------------------------------------------------------------------
REAGENT SETUP
CRITICAL All solutions should be prepared in a biosafety cabinet using sterile technique and
endotoxin-free reagents and supplies. Glassware, stir bars, and pH meters are not endotoxin-
free; autoclaving does not eliminate endotoxins. To prepare solutions, use pH indicator strips,
dissolve reagents by heating and/or inverting to mix, and use demarcations on bottles to bring
solutions up to the final volume. Reagents can be weighed outside of a biosafety cabinet since
all solutions are filter sterilized before use.
Plasmid DNA
Grow bacterial stocks in LB or Plasmid+ media containing the appropriate
selective antibiotic. Use a large-scale endotoxin-free plasmid purification kit to isolate plasmids;
elute plasmid DNA with the supplied Tris
-EDTA (TE) buffer. Measure DNA purity and
concentration using a spectrophotometer and freeze at -
20 ̊C or
-
80 ̊C for up to several years.
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CRITICAL Always verify the integrity of purified plasmids by sequencing and restriction digest
before proceeding with downstream applications. pAAV plasmids contain inverted terminal
repeats (ITRs) (
Fig. 5
) th
at are prone to recombination in
E. coli
. pAAVs should be propagated
in recombination deficient strains such as NEB Stable (New England Biolabs, cat. no. C3040H),
Stbl3 (Invitrogen, cat. no. C737303),
or SURE 2 competent cells (Agilent, cat. no. 200152) to
prevent unwanted recombination. After purification, pAAVs should be digested with SmaI to
confirm the presence of ITRs, which are required for replication and encapsidation of the viral
genome; use sequence editing and annotation software to determine expected band sizes
. Note
that it is difficult to sequence through
the secondary structure of ITRs
51
; avoid ITRs when
designing sequencing primers.
DMEM + 5% (v/v) FBS
Add 25 ml of FBS, 5 ml of NEAA, and 5 ml of Pen-Strep to a 500 ml
bottle of DMEM. Invert to mix and store at 4 ̊C for up to several months. The resulting cell
culture media should have a final concentration of 5% (v/v) FBS, 1X NEAA, and 50 U/ml Pen-
Strep.
Cell culture
Thaw HEK293T cells according to the manufacturer’s recommendations. Passage
cells using either TrypLE Express enzyme or a standard trypsinization protocol for adherent
cultures
52
. Seed cells in 150 mm tissue culture dishes with a final volume of 20 ml of DMEM +
5% FBS per di
sh. Maintain in a cell culture incubator at 37 ̊C with 5% CO
2
. CRITICAL
We
suggest a passage ratio of 1:3 (i.e., divide one dish of cells into three new dishes of cells every
other day) when expanding cells for viral production; split cells at 1:2 (or 6 x 10
4
cells/cm
2
) 24 hr
before transfection. Always use sterile technique.
PEI stock solution
Pipet 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 N HCl suitable for cell culture, keeping track of the volume of HCl added. Heat in a
37 ̊C water bath for several hours (or overnight) and occasionally shake to mix. Once dissolved,
add reserved WFI water to a total volume of 1 L. Filter sterilize, aliquot to 50 ml conical
centrifuge tubes, and store at -
20 ̊C for up to 1 year. We routinely freeze/thaw our PEI aliquots.
CRITICAL
Both our PEI stock solution recipe and PEI calculations (
Supplementary Table 2
,
sheet ‘Detailed calculations’) are based on ref.
4
.
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PEI + DPBS master mix
Thaw PEI in a 37 ̊C water bath. Bring PEI to RT and vortex to mix.
Add PEI and DPBS to a 50 ml conical centrifuge tube and vortex again to mix. Use
Supplementary Table 2
(sheet ‘Transfection
calculator’)
to calculate the volumes of PEI (cell
I9) and DPBS (cell J9)
needed.
CRITICAL
Prepare fresh the day of transfection.
DNA + DPBS
Bring plasmid DNA to RT and briefly vortex to mix.
For each viral prep, add DNA
and DPBS to a 50 ml conical centrifuge tube and use a P1000 pipet to mix. Use
Supplementary Table 2
(sheet ‘Transfection
calculator’)
to calculate the quantities of DNA
(e.g., cells E9+E11+E13) and DPBS (e.g., cell F9
) needed.
CRITICAL Prepare fresh the day of
transfection. Re-measure plasmid DNA concentrations
immediately prior to
use; multiple
freeze/thaw cycles may cause DNA degradation.
SAN digestion buffer
Add 29.22 g of NaCl, 4.85 g of Tris base, and 952 mg of MgCl
2
to a 1 L
bottle of WFI water and shake to mix. Filter sterilize and store at RT for up to several months.
The resulting SAN digestion buffer should have a final pH of 9.5-10.0 and a final concentration
of 500 mM NaCl, 40 mM Tris base, and 10 mM MgCl
2
.
SAN + SAN digestion buffer
Add 100 U of SAN per ml of SAN
digestion buffer; pipet to mix.
CRITICAL
Prepare fresh prior to use.
40% (w/v) PEG stock solution
Decant approximately 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 bottle of WFI water and
shake/heat until dissolved. Once completely dissolved, add 400 g of PEG and heat at 37 ̊C for
several hours to overnight. Add reserved WFI water to a total volume of 1 L. Filter sterilize and
store at RT for up to several months. The resulting stock solution should have a final
concentration of 2.5 M NaCl and 40% (w/v) PEG. CRITICAL
Prepare in advance. To expedite
the procedure, heat the solution at 65 ̊C until the PEG is dissolved. The solution will appear
tur
bid but no flecks of PEG should remain; the mixture will become clear upon cooling.
CRITICAL
Pre-wet the entire filter surface with a minimal volume of water prior to adding the
solution. This solution is extremely viscous and will take 1-2 h to filter.
DPBS +
high salt
Add 29.22 g of NaCl, 93.2 mg of KCl, and 47.6 mg of MgCl
2
to a 500 ml
bottle of DPBS and shake to mix. Filter sterilize and store at RT for up to several months. The
resulting buffer should have a final concentration of 1 M NaCl (in addition to the salt in the
DPBS), 2.5 mM KCl, and 1 mM MgCl
2
.
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DPBS + low salt
Add 2.92 g of NaCl, 93.2 mg of KCl, and 47.6 mg of MgCl
2
to a 500 ml bottle
of DPBS and shake to mix. Filter sterilize and store at RT for up to several months. The
resulting buffer should have a final concentration of 100 mM NaCl (in addition to the salt in the
DPBS), 2.5 mM KCl, and 1 mM MgCl
2
.
Iodixanol density step solutions (15%, 25%, 40%, and 60% (w/v) iodixanol)
For each step,
add iodixanol, DPBS + high salt or DPBS + low
salt, and phenol red (if applicable) to a 50 ml
conical centrifuge tube. Briefly invert or vortex to mix. Use
Supplementary Table 3
to
determine the volumes of each reagent needed. The 25% and 60% steps contain phenol red,
which turns the solutions red and yellow, respectively, and facilitates clear demarcation of the
gradient boundaries (
Fig. 7
).
CRITICAL
Prepare fresh the day of AAV purification. Alternatively,
prepare up to 1 d in advance; store under sterile conditions at RT and protect from light. Do not
pour the density gradients until Step 16.
1 M Tris-Cl stock solution
Pipet 80 ml of Ultrapure or WFI water into a 250 ml sterile bottle.
Add 12.11 g of Tris base and 7 ml of concentrated HCl and shake to mix; fine adjust the pH to
7.5 by adding more concentrated HCl. Add water to a total volume of 100 ml. Filter sterilize and
store at RT for up to several months.
DNAse digestion buffer
Decant 250 ml of Ultrapure water into a 250 ml sterile bottle. Add 55.5
mg of CaCl
2
, 2.5 ml of 1 M Tris-Cl stock sol
ution, and 238 mg of MgCl
2
and shake to mix. Filter
sterilize and store at RT for up to several months. The resulting buffer should have a final
concentration of 2 mM CaCl
2
, 10 mM Tris
-Cl, and 10 mM MgCl
2
.
DNAse I + DNAse digestion buffer
Add 50 U of DNAse I per ml of digestion buffer; pipet to
mix.
CRITICAL Prepare fresh prior to use.
Proteinase K solution
Decant 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 RT for up to several months. The resulting
solution should have a final concentration of 1 M NaCl and 1% (w/v)
N
-lauroylsarcosine sodium
salt.
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Proteinase K + proteinase K solution
Add 100 μg of proteinase K per ml of solution; pipet to
mix.
CRITICAL Prepare fresh prior to use.
DNA standard stock
Set up a single 50 μl restriction digest reaction; use 60-80 U (3-4 μl) of
ScaI (or other 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 approximately 5-10 x 10
9
single-
stranded (ss) DNA
molecules/μl and use the Qubit assay to verify the concentration (ng/μl). Divide into 20 μl
aliquots and freeze at -
20 ̊C for up to 1 year.
CRITICAL
Prior to preparing the standard, use
sequence editing and annotation software to confirm that the plasmid contains a single ScaI site
in the ampicillin resistance gene. Refer to ref.
10
and use
Supplementary Table 4
(cell B13)
to
calculate the number of ssDNA molecules in a given plasmid. We typically use pAAV-CAG-
mNeonGreen to prepare the standard; following restriction digest we dilute the linearized
plasmid to 10 ng/μl, which corresponds to 6.6 x 10
9
ssDNA molecules/μl.
DNA standard dilutions
Prepare three sets of 8 (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 #8). Mix by pipetting and proceed with the 7 remaining dilutions (standard #7 to
standard #1). The final concentrations of the standard dilutions should range from 5-10 x 10
8
(standard #8) to 5-10 x 10
1
(standard #1) ssDNA molecules/μl. CRITICAL
Prepare fresh in
DNA/RNA LoBind microcentrifuge tubes immediately prior to use; at low concentrations, the
linearized DNA is prone to
degradation and/or sticking to the walls of the tube
10
. One 20 μl
aliquot of the DNA standard stock will provide enough DNA for preparing the dilutions and
verifying the concentration via the Qubit assay prior to qPCR.
qPCR master mix
Prepare a qPCR master mix for the total number of reactions (i.e., wells)
needed. One reaction requires 12.5 μl of SYBR green master mix, 9.5 μl of Ultrapure water, and
0.5 μl of each primer (from a 2.5 μM stock concentration), for a total of 23 μl/well. Briefly vortex
to m
ix.
CRITICAL
Prepare fresh prior to use.
-------------------------------------------------------------------------------------------------------------------------------
EQUIPMENT SETUP
Clamp setup for AAV purification
Attach the rod to the support stand. Secure the clamp
approximately 25-30 cm above the stand (
Fig. 7f
).
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