Cre-dependent selection yields AAV variants for widespread
gene transfer to the adult brain
Benjamin E. Deverman
1
,
Piers L. Pravdo
1
,
Bryan P. Simpson
1
,
Sripriya Ravindra Kumar
1
,
Ken Y. Chan
1
,
Abhik Banerjee
1
,
Wei-Li Wu
1
,
Bin Yang
1
,
Nina Huber
2
,
Sergiu P. Pasca
2
, and
Viviana Gradinaru
1
1
Division of Biology and Biological Engineering, California Institute of Technology Pasadena, CA
2
Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine,
Stanford, CA
Abstract
Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for
in vivo
gene
transfer
1
-
6
. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a
search for novel AAV capsids with desired characteristics
7
-
13
. Here we describe a capsid selection
method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the
development of AAV capsids that more efficiently transduce defined Cre-expressing cell
populations
in vivo
. We use CREATE to generate AAV variants that efficiently and widely
transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant,
AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater
than that of the current standard, AAV9
14
-
17
, and transduces the majority of astrocytes and neurons
across multiple CNS regions.
In vitro
, it transduces human neurons and astrocytes more efficiently
than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for
biomedical applications.
rAAVs are the preferred vehicles for many
in vivo
gene transfer applications to non-dividing
cell populations and are proving safe in clinical trials
18
-
20
. However, therapeutic applications
have been limited by inefficient transduction of target cell populations. A prime example is
gene transfer to the CNS; the transduction efficiency of currently available vectors in the
mouse CNS after intravenous administration is at least an order of magnitude lower than in
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To whom correspondence should be addressed: Benjamin E. Deverman, Ph.D., Division of Biology and Biological Engineering,
California Institute of Technology, 1200 East California Blvd. MC 156-29, Pasadena, CA 91125, Phone: (626) 395-2776,
bd@caltech.edu, 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.
AUTHOR CONTRIBUTIONS
B.E.D designed and performed experiments, analyzed data, prepared figures and wrote the manuscript. P.L.P., B.P.S., S.K., A.B., and
K.C. performed experiments, virus production and characterization. W.L.W. performed tissue processing and IHC. B.Y. assisted with
tissue clearing and imaging. N.H. and S.P.P. performed the experiments with human cells, analyzed the data, and prepared the
associated figure and text. V.G. helped with study design and data analysis, manuscript and figure preparation and supervised the
project. All authors edited and approved the manuscript.
Competing Financial Interests
B.E.D. is listed as an inventor on a patent application relating to this work.
HHS Public Access
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Nat Biotechnol
. 2016 February ; 34(2): 204–209. doi:10.1038/nbt.3440.
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the liver
21
. Encouragingly, the transduction efficiency of rAAVs can be enhanced by creating
AAV capsid libraries and selecting for variants with more desirable characteristics
8
-
13
. This
approach, however, has not been successfully applied to the development of more effective
gene delivery vehicles for global CNS transduction. Because the highly selective blood brain
barrier (BBB) and cellular heterogeneity of the CNS present challenges for gene transfer to
the CNS through the vasculature, we reasoned that it would be beneficial to provide selective
pressure for capsids that cross the BBB and functionally transduce specific cell types. To
meet these needs, we devised CREATE—a Cre recombination-dependent approach to
selectively recover capsids that transduce predefined Cre expressing target cell populations
(
Fig. 1a
).
CREATE uses an rAAV capsid genome (rAAV-Cap-in-cis-lox) that couples a full-length
AAV
Cap
gene, controlled by regulatory elements from the AAV
Rep
gene (
Fig. 1b
and
Methods
), with a Cre-invertible switch (
Fig. 1b
). By building capsid libraries within the
rAAV-Cap-in-cis-lox backbone and delivering the virus libraries to animals with Cre
expression in a defined cell population, the system enables the selective amplification and
recovery of sequences that have transduced the target population (
Fig. 1c
). Because the
rAAV-Cap-in-cis-lox genome lacks a functional
Rep
gene,
Rep
must be provided in trans for
virus production. For this purpose, we modified an AAV2/9 rep-cap plasmid to eliminate
capsid protein expression by inserting in-frame stop codons within the reading frame for
each capsid protein, VP1-3 (
Fig. 1d
). These stop codons do not alter the amino acid
sequence of the assembly-activating protein (AAP), which is expressed from an alternative
reading frame within the
Cap
gene
22
. This split rAAV-Cap-in-cis-lox genome and Rep-AAP
AAV helper system efficiently generates rAAV (
Fig. 1e
) and is the foundation of the
CREATE selection platform, which enables capsid sequence recovery from genetically
defined Cre-expressing cell populations within heterogeneous tissue samples (see
Supplementary Fig. 1
).
Within the rAAV-Cap-in-cis-lox acceptor genome we generated a library of AAV variants by
inserting 7 amino acids (AA) of randomized sequence (7-mer) between AA588-589 (VP1
position) of the AAV9 capsid (
Fig. 1f, g
). To select for vectors that crossed the BBB and
transduced cells throughout the CNS, we administered the capsid library intravenously into
adult GFAP-Cre mice, which express Cre in astrocytes
23
. One week later, we isolated DNA
from brain and spinal cord tissue and recovered capsid sequences by PCR from viral
genomes that had undergone Cre-mediated recombination. We cloned the entire library of
recovered Cap sequences back into the rAAV-Cap-in-cis-lox acceptor genome to generate
the library GFAP1 and randomly chose 13 clones for sequencing. All tested sequences
recovered from the GFAP1 library were unique, and therefore we used the GFAP1 plasmid
library to generate a second virus library and performed an additional round of selection in
GFAP-Cre mice. After the second selection, several variants were enriched (
Supplementary
Table 2
) and showed enhanced CNS transduction (
Supplementary Fig. 2a
). We chose the
most enriched variant, AAV-PHP.B, which represented 25% of recovered library sequences
and encodes the 7-mer sequence TLAVPFK, for further tropism evaluation
in vivo
.
AAV-PHP.B and AAV9 capsids were used to package a single-stranded (ss) GFP reporter
vector driven by the ubiquitous CAG promoter (ssAAV-CAG-GFP). Both AAV-PHP.B and
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AAV9 produced virus with similar efficiencies (
Supplementary Fig. 2b
). We next
administered 1×10
12
vector genomes (vg) of either vector to six-week-old mice by
intravenous injection and assessed transduction by GFP expression three weeks later. Our
data show that AAV-PHP.B transduced the entire adult CNS with high efficiency as indicated
by imaging GFP immunohistochemistry (IHC) (
Fig. 2a
) or native eGFP fluorescence in
several brain regions (
Fig. 2b
), the spinal cord (
Fig. 2c
) and retina (
Fig. 2d
). Using PARS-
based CLARITY for whole body tissue clearing
24
, we imaged native eGFP fluorescence
through cleared sections of tissue from the spinal cord (
Fig. 2c
), cortex and striatum (
Fig.
2e
). These 3D renderings (also see
Supplementary Movies 1-3
) further demonstrate the
broad and efficient CNS transduction with the AAV-PHP.B vector. Outside the CNS, the
cellular level tropism of AAV-PHP.B and AAV9 appeared similar in several organs, with the
exception of the pancreas where the efficiency of transduction by AAV-PHP.B was reduced
as compared with AAV9 (
Fig. 2f
).
To quantify the efficiency of gene transfer to the CNS and peripheral organs by AAV-PHP.B
as compared with AAV9, we measured the number of viral genomes present in several brain
regions and organs 25 days post-injection (
Fig. 2g
). AAV-PHP.B provided significantly
greater gene transfer than AAV9 to each of the CNS regions examined: cortex (40-fold),
striatum (92-fold), thalamus (76-fold), cerebellum (41-fold) and spinal cord (75-fold).
Vector genome biodistribution outside the CNS showed that AAV-PHP.B transferred genes
to the pancreas and adrenal gland less efficiently than AAV9 (
Fig. 2g
). No significant
differences were found between the two vectors in the liver, heart, skeletal muscle and
kidneys. When considered together with the CNS biodistribution data, in all CNS areas
except the cerebellum, the number of viral genomes detected in mice treated with AAV-
PHP.B was similar to that measured in the liver, an organ efficiently transduced by
AAV9
21
,
25
, and greater than that observed in other organs. In contrast, AAV9-mediated gene
transfer to each of the examined CNS regions was at least 120-fold lower than in the liver.
Therefore, although the tropism of AAV-PHP.B is not CNS specific, the enhanced gene
transfer characteristics of this vector are CNS specific.
AAV9 preferentially transduces astrocytes when delivered intravenously to adult mice and
non-human primates, but it also transduces neurons in several regions
14
,
26
. To examine the
cell types transduced by AAV-PHP.B, we analyzed the co-localization of GFP with several
cell-type markers. Owing to the highly efficient transduction, individual GFP expressing
astrocytes were difficult to discern in mice that received 1×10
12
vg AAV-PHP.B (
Fig. 2a
),
but could be more easily identified morphologically by their compact, highly ramified
processes in animals that received 10-fold less virus (
Fig. 2a, b
) and by co-localization with
IHC for GFAP (
Fig. 3a
). In addition to astrocytes, AAV-PHP.B transduced CC1
+
oligodendrocytes (
Fig. 3b
) and all neuronal subtypes examined, including NeuN
+
throughout the brain (
Fig. 3c
) as well as midbrain tyrosine hydroxylase (TH)
+
dopaminergic
neurons (
Fig. 3d
), Calbindin
+
cerebellar Purkinje cells (
Fig. 3e
), and several interneuron
populations (
Supplementary Fig. 3a-d
). AAV-PHP.B also transduced CD31
+
endothelial
cells (
Supplementary Fig. 3e
) but did not appear to transduce Iba1
+
microglia
(
Supplementary Fig. 3f,g
). The paucity of GFP
+
microglia seen after intravenous AAV-
PHP.B delivery is consistent with previous reports of rare or nonexistent AAV-mediated gene
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expression in this cell population
14
,
27
-
30
. We have observed native GFP expression
throughout the brain over a year after administration of AAV-PHP.B, suggesting that AAV-
PHP.B can provide long-term, CNS-directed transgene expression (
Supplementary Fig. 4
).
We next quantified the fraction of several cell types transduced by AAV-PHP.B as compared
to AAV9. To facilitate reliable individual cell counting, we constructed a vector expressing a
nuclear-localized GFP (NLS-GFP) under the control of the CAG promoter, ssAAV-CAG-
NLS-GFP. A single injection of 2×10
12
vg/mouse of ssAAV-PHP.B:CAG-NLS-GFP
transduced the majority of Aldh1L1
+
astrocytes (
Fig. 3f
and
Supplementary Fig. 5a
) and
NeuN
+
neurons (
Fig. 3g
and
Supplementary Fig. 5b
), as well as a modest fraction of
Olig2
+
oligodendrocyte lineage cells (
Fig.3h
and
Supplementary Fig. 5c
) across all brain
regions examined. In all cases, AAV-PHP.B provided significantly enhanced transduction as
compared to the same dose of AAV9. Notably, AAV-PHP.B also transduced over 94% of
Chat
+
motor neurons throughout the spinal cord (
Fig. 3i
), 91.4±1.6% of TH
+
midbrain
dopaminergic neurons (
Supplemental Fig. 5d
) and 91.7±5.8% of Calbindin
+
Purkinje cells
(
n
=5). In sum, adult intravenous administration of AAV-PHP.B efficiently targets multiple
neuronal and glial cell types in the adult mouse.
The method used to identify AAV-PHP.B only selects for transduction of the target cell
population; it does not necessarily select for specificity. Nevertheless, in a separate trial in
GFAP-Cre mice, we identified (after two rounds of
in vivo
selection) another AAV capsid
variant, AAV-PHP.A, with the 7-mer sequence, YTLSQGW, that exhibits both more efficient
and selective CNS astrocyte transduction (
Fig. 4a-d
), as well as reduced tropism for the liver
(
Fig. 4e, f
) and other peripheral organs (
Fig. 4f
), as compared to AAV9. The increase in
specificity for gene transfer to the CNS over the liver provided by AAV-PHP.A versus AAV9
is 400- to 1200-fold, resulting from a combination of enhanced adult CNS gene transfer
(2.6- to 8-fold more depending on the specific region) and reduced liver gene transfer (152-
fold). Two other variants enriched in this trial (
Supplementary Fig. 2a
) did not show
enhanced GFP expression in CNS neurons or glia as compared with AAV9.
To determine whether AAV-PHP.A and AAV-PHP.B can also transduce human neural cells,
we tested them on cortical neurons and astrocytes derived from human induced pluripotent
stem cells (hiPSCs) using a 3D differentiation method
31
. HiPSC lines from two individuals
were differentiated into 3D cerebral cortex-like structures (cortical spheroids), and
maintained
in vitro
for up to 200 days. Aged cortical spheroids contain superficial and deep
layer cortical excitatory neurons and up to 20% astrocytes
31
. In dissociated cortical
spheroids that were exposed to the three viruses in monolayer, AAV-PHP.B more efficiently
transduced both GFAP-expressing astrocytes and MAP2-expresing neurons in comparison
with either AAV9 or AAV-PHP.A (Supplemental Fig. 5; two-way ANOVA p<0.01, n=3). In
addition, all three viruses were capable of transducing intact 3D cortical spheroids
(Supplementary Fig. 5c).
Using CREATE, we have developed new AAV variants that enable efficient widespread gene
transfer to the adult mouse CNS after intravenous administration. An important advantage of
this system is that it introduces selective pressure for capsids that mediate efficient
intracellular trafficking and conversion of the single-stranded viral genome to persistent
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double-stranded DNA (dsDNA) forms necessary for long-term transduction (only the
dsDNA genomes should serve as substrates for Cre). This additional selective pressure for
functional capsids may have contributed to the identification of AAV-PHP.A and the AAV-
PHP.B variants in independent trials after only two rounds of
in vivo
selection. In
comparison, many previous
in vivo
and
in vitro
AAV capsid selection methods have applied
3-10 rounds of selection to identify capsid variants with enhanced properties
9
-
13
,
32
.
In principle, CREATE could be applied to discover AAV capsid variants that target defined,
CRE-expressing cell types in any organ. Thus, it could be used not only in transgenic
animals, as shown here, but also to develop AAV variants that target (i) specific Cre
+
cell
types in spheroid cultures
31
or organoid cultures, (ii) cells made Cre
+
in non-transgenic
animals by, for example, viral injections that achieve population-, projection
33
-, or activity-
based Cre expression
34
,
35
or (iii) Cre
+
human cells in human/mouse chimeric animals.
Given the reported AAV tropism differences between animal models and humans
11
,
selection schemes that use human Cre
+
cells
in vivo,
cell-specific Cre expression in three-
dimensional hiPSC-derived cellular models, or future Cre transgenic marmosets
36
may be
desirable for developing improved vectors for clinical applications. In addition, the success
of AAV-based gene therapies, especially those requiring systemic delivery, can be hindered
by the presence of neutralizing AAV capsid antibodies in the human population
37
-
39
. By
using CREATE together with exposure of AAV libraries to pooled human sera, one could
envision simultaneously selecting for capsids with retained or enhanced transduction
characteristics that are also less susceptible to antibody-mediated neutralization.
A limitation of CREATE and other reported capsid selection methods is that it is difficult to
predict, beyond an increase in target cell transduction efficiency, what characteristics the
enriched variants will have before they are tested. In our two trials for astrocyte targeting, we
identified several variants, AAV-PHP.B, B2, and B3 (
Supplementary Fig. 2
) that provide
broad CNS transduction of both neurons and glia, and AAV-PHP.A that provides selectively
more efficient astrocyte transduction. Identification of capsids with distinct properties from
the same selection scheme was expected given that the recovery method we used selected for
astrocyte transduction rather than for any specific intermediate step(s), e.g., brain vascular
association, BBB transcytosis or astrocyte binding/internalization. Therefore, capsid variants
that are more efficient at any of these intermediate steps should be recovered in our selection
process. Indeed, by immunostaining for capsids we found that unlike AAV9, both AAV-PHP
capsids readily localized to the brain vasculature shortly after intravenous administration
(
Supplementary Fig. 7a, b
). In addition, by 24 hours post-administration, significantly
more GFP-expressing cells were observed along the brain vasculature of mice that received
AAV-PHP.B as compared with those that received AAV9 or AAV-PHP.A (
Supplementary
Fig. 7c, d
). Considered together with the transduction characteristics of AAV-PHP.B and
AAV-PHP.A
in vivo
(
Figs. 2-4
) and in human neural cultures (
Supplementary Fig. 6
), these
data suggest that while both AAV-PHP vectors more efficiently associate with the brain
vasculature, they may differ in subsequent cell type-specific entry or transport step(s).
In the future, CREATE could be used with next-generation sequencing to better predict the
characteristics of the recovered sequences prior to testing the variants individually.
Sequencing both the entire pool of variants recovered from the Cre-expressing target cells
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along with the unselected virus library should enable quantification of the relative extent of
enrichment of each recovered sequence. Furthermore, sequencing capsids recovered from
multiple Cre-expressing or Cre non-expressing populations could provide a means to
perform both positive and negative selection in multiple cell types in a single experiment.
Such
in vivo-in silico
selection approaches should increase the power of CREATE to
enhance gene transfer to the CNS and other difficult-to-target cell populations.
METHODS
Plasmids
The rAAV-Cap-in-cis-lox genome plasmid contains three main elements flanked by AAV2
ITRs: (i) an mCherry expression cassette, which is comprised of a 398bp fragment of the
human UBC gene upstream of the mCherry cDNA followed by a synthetic polyadenylation
sequence
40
; (ii) the AAV9 capsid gene and regulatory sequences, which are comprised of the
AAV5 p41 promoter sequence (1680-1974 of GenBank AF085716.1)
41
,
42
and splicing
sequences taken from the AAV2 rep gene; and (iii) a Cre-dependent switch, which is
comprised of the SV40 polyadenylation sequence (pA) flanked by inverted lox71 and lox66
sites
43
(Fig. 1b). The rAAV-Cap-in-cis-lox genome plasmid was further modified to
introduce two unique restriction sites, XbaI and AgeI, within the capsid sequence. These
sites flank the region (AA450-592) that is replaced by the randomized library fragment. The
introduction of the XbaI site introduces a K449R mutation, which does not have an overt
effect on vector production or transduction. The mutations required to insert the AgeI site
are silent. For the rAAV-ΔCap-in-cis acceptor plasmid used for the capsid library cloning,
the coding region between the XbaI and AgeI sites was removed to prevent virus production
from the acceptor plasmid lacking the library fragment.
As a template for the library fragment, we PCR amplified the region spanning the XbaI and
AgeI sites of the modified AAV9. This sequence was modified to remove a unique EarI
restriction site and insert a unique KpnI site (both silent mutations) to create the xE
fragment. The modified xE fragment was TA cloned into pCRII (Life Technologies) to
generate pCRII-9Cap-xE. Eliminating the EarI site provided a second method that could be
used, if necessary, to selectively digest contaminating (AAV9) capsid sequences recovered
by PCR, but not digest the library-derived sequences. We did not find it necessary to use this
digestion step. Using the rAAV-ΔCap-in-cis acceptor for cloning the libraries and taking
standard PCR precautions (e.g., UV treating reagents and pipettors) was sufficient to prevent
contamination.
The AAV2/9 REP-AAP helper plasmid was constructed by introducing five stop codons into
the coding sequence of the VP reading frame of the AAV9 gene at VP1 AAs: 6, 10, 142, 148
and 216. The stop codon at AA216 was designed not to disrupt the coding sequence of the
AAP protein, which is encoded within an alternative reading frame.
Several rAAV genomes were used in this study. Each is constructed within a single stranded
(ss) rAAV genome with a reporter driven by the ubiquitous CMV-
β
-Actin-intron-
β
-Globin
hybrid promoter (CAG). For simplicity, the vector descriptions have been abbreviated in the
text. ssAAV-CAG-GFP refers to ssAAV-CAG-eGFP-2A-Luc-WPRE-SV40 polyA. ssAAV-
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CAG-NLS-GFP refers to ssAAV-CAG-NLS-GFP-WPRE-SV40 polyA, which was
constructed by inserting the nuclear localization sequence PKKKRKV at both the N- and C-
termini of GFP. ssAAV-CAG-mNeGreen-f refers to ssAAV-CAG-mNeonGreen-f-WPRE
with a human growth hormone polyA signal. The mNeonGreen
44
was modified with the
membrane targeting (farnesylation and palmitoylation signals) sequence from c-Ha-Ras
45
.
Capsid library generation
The random 7-mer library fragment (inserted between amino acids 588 and 589) was
generated by PCR using Q5 Hot Start High-Fidelity DNA Polymerase (NEB; M0493),
primers XF and 7×MNN and pCRII-9Cap-xE as a template. A schematic showing the
approximate primer binding sites and a table of the primer sequences are given in
Supplementary Figure 1
and
Supplementary Table 1
, respectively. To generate the rAAV-
based library, the PCR products containing the library and the XbaI- and AgeI-digested
rAAV-ΔCap-in-cis acceptor plasmid were assembled using Gibson Assembly (NEB; E2611).
The reaction products were then treated with Plasmid Safe (PS) DNase (Epicentre; E3105K)
to digest any unassembled fragments and purified using a QIAquick PCR Purification Kit
(Qiagen). This reaction typically yielded over 100 ng of assembled plasmid (as defined by
the amount of DNA remaining after the PS DNase digestion step). 100 ng is sufficient to
transfect ten 150mm tissue culture dishes at 10 ng/dish. Note, the libraries can also be
constructed by ligation or Gibson Assembly and then amplified in
E. coli
, but bacterial
transformation reduces the library diversity. By directly transfecting the assembled reaction
products, the library diversity is limited instead by the number of successfully transfected
HEK293 producer cells.
Virus production and purification
Recombinant AAVs were generated by triple transfection of 293T cells (ATCC) using
polyethylenimine (PEI)
46
. Viral particles were harvested from the media at 72 hrs post
transfection and from the cells and media at 120 hrs. Cell pellets were resuspended in 10mM
Tris with 2mM MgCl
2
, pH 8, freeze-thawed three times, and treated with 100 U/mL
Benzonase (Epicentre) at 37°C for at least 1 hr. Viral media was concentrated by
precipitation with 8% polyethylene glycol 8000 (Sigma-Aldrich) with 500 mM sodium
chloride
47
, resuspended in Tris-MgCl
2
, and then added to the lysates. The combined stocks
were then adjusted to 500 mM NaCl, incubated at 37°C for 30 minutes, and clarified by
centrifugation at 2000 × g. The clarified stocks were then purified over iodixanol (Optiprep,
Sigma; D1556) step gradients (15%, 25%, 40% and 60%)
48
. Viruses were concentrated and
formulated in phosphate buffered saline (PBS). Virus titers were determined by measuring
the number of DNaseI-resistant vg using qPCR with linearized genome plasmid as a
standard
46
.
For capsid library virus generation, two modifications were made to the above virus
production protocol to reduce the production of mosaic capsids that could arise from the
presence of multiple capsid sequences in the same cell. First, only 10 ng of the rAAV-Cap-
in-cis library plasmid was transfected (per 150 mm dish) to increase the likelihood that most
transfected cells only received one capsid variant sequence. Second, the virus was collected
at 48 hrs (media) and 60 hrs (cells and media), rather than at 72 hrs and 120 hrs as described
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above, to minimize the secondary transduction of producer cells with rAAV library virus
released into the medium.
Animals
All procedures were approved by the California Institute of Technology Institutional Animal
Care and Use Committee (IACUC). GFAP-Cre mice expressing Cre under the control of the
mouse GFAP promoter (012886)
23
and C57Bl/6J mice (000664) were purchased from the
Jackson Laboratory (JAX). Intravenous administration of rAAV vectors was performed by
injecting the virus into the retro-orbital sinus. Mice were randomly assigned to groups of
predetermined sample size. No mice were excluded from these analyses. Experimenters
were not blinded to sample groups.
In vivo
selection
For the selections in GFAP-Cre mice, 1×10
11
vg of the capsid libraries were injected
intravenously into adult Cre
+
mice of either sex. Seven to eight days post-injection, mice
were euthanized and the brain and spinal cord were collected. Vector DNA was recovered
from one hemisphere of the brain and half of the spinal cord using 4-5 ml of Trizol (Life
Technologies; 15596). To purify viral DNA, the upper aqueous fraction was collected
according to the manufacturer's extraction protocol. We found that the aqueous fraction
contains a significant portion of the viral DNA genomes as well as RNA. RNA was then
digested by treatment with 1 uL of RNase A (Qiagen) at 37 °C overnight. Next, a two-step
PCR amplification strategy was used to selectively recover
Cap
sequences from Cre-
recombined genomes. The first amplification step preferentially amplifies Cre-recombined
rAAV-Cap-in-cis-lox sequences using the primers 9CapF and CDF (see
Supplementary
Fig. 1
). The PCR was performed for 20-26 cycles of 95°C for 20 sec, 60°C for 20 sec and
72°C for 30 sec using Q5 Hot Start High-fidelity DNA Polymerase. The PCR product was
then diluted 1:10 or 1:100 and then used as a template for a second Cre-independent PCR
reaction using primers XF and AR (
Supplementary Fig. 1C
). The second PCR generated
the fragment that was cloned back into the rAAV-ΔCap-in-cis acceptor plasmid as described
above. 1 μL of the Gibson Assembly reactions were then diluted 1:10 and transformed into
SURE2 competent cells (Agilent; 200152) to generate individual clones for sequencing.
Variants that showed evidence of enrichment were cloned into an AAV Rep-Cap plasmid
and transformed into DH5
α
competent cells (NEB). The novel AAV Rep-Cap variants, or
AAV2/9 Rep-Cap as a control, were then evaluated using one of the reporter genomes
described above.
Vector biodistribution
Six-week-old mice female C57Bl/6 mice were injected intravenously with 1×10
11
vg of the
ssAAV-CAG-GFP vector packaged into the indicated AAV capsid. Animals were randomly
assigned to groups. 25 days after injection, the mice were euthanized and tissues and
indicated brain regions were collected and frozen at −80°C. DNA was isolated from the
tissue samples using Qiagen DNeasy Blood and Tissue kit. Vector genomes were detected
using PCR primers that bind to the WPRE element and were normalized to mouse genomes
using primers specific to the mouse glucagon gene. Absolute quantification was performed
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using serial dilutions of linearized plasmid standards of known concentration
46
. One
randomly chosen animal injected with AAV-PHP.B was removed from the bio-distribution
study for histological analysis.
Tissue preparation, immunohistochemistry and imaging
Mice were anesthetized with Nembutal and transcardially perfused with 0.1 M phosphate
buffer (PB) at room temperature (RT) at pH 7.4 and then with freshly prepared, ice-cold 4%
paraformaldehyde (PFA) in PB. Brains were post-fixed in 4% PFA overnight and then
sectioned by vibratome or cryoprotected and sectioned by cryostat. IHC was performed on
floating sections with primary and secondary antibodies in PBS containing 10% goat or
donkey serum and 0.5% Triton X-100 (no detergent was used for GAD67 staining). Primary
antibodies used were mouse anti-AAV capsid (1:20; American Research Products,
03-65158, clone B1), rabbit anti-GFP (1:1000; Invitrogen, A11122), chicken anti-GFP
(1:1000; Abcam, ab13970), mouse anti-CC1 (1:200; Calbiochem, OP80), rabbit anti-GFAP
(1:1000; Dako, Z0334), mouse anti-NeuN (1:500; Millipore, MAB377), rabbit anti-IbaI
(1:500; Biocare Medical, CP290), mouse anti-Calbindin D28K (1:200; Sigma, CB-955),
rabbit anti-Calretinin (1:1000; Chemicon, AB5054), mouse anti-GAD67 (1:1000; Millipore,
MAB5406), guinea pig anti–MAP2 (1:1000; Synaptic Systems, 188004), mouse anti-
Parvalbumin (1:1000; Sigma), Tyrosine Hydroxlyase (1:1000, Aves) and rabbit anti-CD31
(1:50; Abcam, ab28364). Primary antibodies incubations were performed for 16-24 hrs at
RT. The sections were then washed and incubated with secondary Alexa-conjugated
antibodies (1:1000; Invitrogen) for 2-16 hrs. For capsid detection with the B1 antibody that
recognizes an internal epitope
49
, floating sections were treated with 2M HCl for 15 minutes
at 37°C and then washed extensively with PBS prior to incubation with the primary
antibody. For some images, the 16-bit green channel (GFP) gamma was adjusted to enable
visualization (without oversaturation) of both low and high GFP-expressing cells present
within the same field of view. In all cases, changes to gamma or contrast as well as
microscope and laser settings remained consistent across sets of images. Images were taken
with a Zeiss LSM 780 confocal microscope fitted with the following objectives: Fluar 5×/
0.25 M27 Plan-Apochromat 10×/0.45 M27 (working distance 2.0 mm), Plan-Apochromat
25×/0.8 Imm Corr DIC M27 multi-immersion and LD C-Apochromat 40×/1.1 W Korr and
Plan-Apochromat 100×/1.46 Oil DIC objectives. 3D MIP images and Supplementary
Movies were generated with Imaris (Bitplane).
Quantification of cell type-specific transduction
Six-week-old female mice were randomly assigned to groups and injected intravenously
with 2×10
12
vg of ssAAV-CAG-NLS-GFP packaged into AAV9, AAV-PHP.B or AAV-
PHP.A. Three weeks later, the mice were perfused and the brains were processed and
immunostained for the indicated antigen as described above. The number of animals per
group was pre-established; no animals were excluded from the analysis. Confocal single-
plane images of the cell type-specific immunostaining and native NLS-GFP fluorescence
were taken. To prevent bias, images were taken from the indicated matched regions
identified by viewing only the cell type-specific immunostaining channel, rather than from
GFP expression, prior to image acquisition. Likewise, cell counting was performed by first
counting and marking each cell stained by the cell-specific antigen by viewing the IHC
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channel. Next those marked IHC
+
cells that were positive for native GFP fluorescence were
counted. Due to the stark transduction efficiency differences between capsids, the counting
was not blinded by group.
Tissue clearing
Mice were perfused as above with 60-80 mL of ice-cold 4% PFA in PB at a flow rate of 14
mL per minute. The flow rate was then reduced to 2-3 mL/min and continued for 2 hrs at
RT. The mice were then placed in individual custom-built chambers
24
and perfused with 200
mL of recycling RT 4% acrylamide in PB at 2-3 mL/min overnight followed by a 2-hr
perfusion flush with PB to remove residual polymers/monomers from the vasculature. The
polymerization process was initiated by placing the chambers in a 42°C water bath and
delivering, by perfusion (2-3 mL/min), of 200 mL of recycling, degassed PB containing
0.25% VA-044 initiator for 2-4 hrs. The mice were then perfused with 8% SDS in PB, pH
7.5 for 7 days. The SDS containing solution was refreshed two times during the 7 days and
then flushed out by perfusion of 2 L of non-recirculating PB overnight. Cleared tissue
samples were mounted in RIMS solution (refractive index of 1.46)
24
for imaging.
Generation of cortical spheroids from human iPSC
Human cortical spheroids were generated from iPSC as previously described
31
. Briefly,
iPSC lines derived from two healthy control individuals were grown on inactivated mouse
embryonic fibroblast feeders in the following medium: DMEM/F12, Knockout Serum 20%,
1 mM non-essential amino acids (1:100), GlutaMax (1:200),
β
-mercaptoethanol (0.1 mM),
penicillin and streptomycin (1:100) (Life Technologies). Cultures were regularly tested and
maintained mycoplasma free. Colonies of iPSCs were detached intact with dispase (0.35
mg/ml, Invitrogen) and transferred into low-attachment plates in iPSC medium
supplemented with dorsomorphin (5 μM, Sigma) and SB-431542 (10 μM, Tocris), and the
medium was changed daily. On day six of
in vitro
differentiation, neural spheroids were
transferred to NPC medium (Neurobasal A, B27 without vitamin A, GlutaMax (1:100),
penicillin and streptomycin; Life Technologies), which was supplemented with EGF (20
ng/ml) and FGF2 (20 ng/ml) until day 24, and then supplemented with BDNF (20 ng/ml)
and NT3 (20 ng/ml) from day 25 to 42. From day 43 onwards, cortical spheroids were
maintained in NPC medium, which was changed every 4 days.
Dissociation and viral infection of cortical spheroids
For enzymatic dissociation and culture in monolayer, cortical spheroids at day 170-200 of
in
vitro
differentiation (two independent neural differentiations of one iPSC line from one
individual and one differentiation of an iPSC line from another individual) were incubated
with Accutase (Innovative Cell Technologies) for 25 min at 37°C, washed three times with
NPC media and gently triturated with a P-200 pipette. Cells were plated on poly-ornithine
and laminin coated glass coverslip (15 mm) at ~300,000 cells/well and maintained in NPC
media supplemented with BDNF (20 ng/ml) and NT3 (20 ng/ml) for the first 24 hrs, and
then maintained in NPC media without growth factors.
Cultures grown on coverslips were infected with each of the viruses at a titer of 1×10
9
vg/
well and fixed 5 days later with 4% paraformaldehyde (PFA) for 10 min. For
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immunocytochemistry, cells were permeabilized with 0.2% Triton X-100 for 10 min and
blocked with 10% goat serum in PBS for 1 hr. Coverslips were then incubated with
antibodies diluted in blocking solution for 2 hr. Nuclei were visualized with Hoechst 33258
(Life Technologies, 1:10,000).
Cells were imaged with a Zeiss M1 Axioscope using a 40× objective. The proportion of
GFP
+
cells co-labeled with either GFAP or MAP2 was quantified in images of 10 random
fields per coverslip for each experimental condition. Results presented are the average of
two separate dissociation and infection experiments.
To infect intact 3D cultures with AAVs, single human cortical spheroids at day 197 days of
in vitro
differentiation were transferred overnight into 1.5 ml Eppendorf tubes containing
6×10
9
vg/400 μl in NPC media, and were fixed 7 days later in 4% PFA overnight. Fixed
spheroids were then transferred into 30% sucrose for 24 hrs, embedded in O.C.T. (Fisher
Scientific) and cut at 14 μm sections. For immunohistochemistry, sections were blocked with
10% goat serum in PBS containing 0.3% Triton-X100 for 1 hr. Images were collected with a
Leica TCS SP8 confocal microscope.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS
This article and the naming of the novel AAV clones is dedicated to the memory of Paul H. Patterson (PHP) who
passed away during the preparation of this manuscript. We wish to thank Laura Rodriguez and Pat Anguiano for
administrative assistance, Alex Balazs and Stijn Cassenaer and the entire Gradinaru and Patterson laboratories for
helpful discussions and Andrea Choe for helpful comments on the manuscript. We thank the U. Penn vector core
for the AAV2/9 rep-cap plasmid, A. Balazs and David Baltimore for the AAV genome plasmid, and the Biological
Imaging Facility, supported by the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation, for
use of imaging equipment. This work was supported by the Hereditary Disease foundation and the Caltech-City of
Hope Biomedical Initiative (to PHP), and by grants to VG: NIH Director's New Innovator IDP20D017782-01;
NIH/NIA 1R01AG047664-01; Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research;
Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular
Technology Initiative. Work in the Gradinaru Laboratory is also funded by the following awards (to VG): NIH
BRAIN 1U01NS090577; NIH/NIMH 1R21MH103824-01; Pew Charitable Trust; Kimmel Foundation; Human
Frontiers in Science Program; Caltech-GIST; Caltech-City of Hope Biomedical Initiative. Work in the Pasca
Laboratory is supported by a US National Institute of Mental Health (NIMH) 1R01MH100900 and
1R01MH100900-02S1, the NIMH BRAINS Award (R01MH107800), MQ Fellow Award and the Donald E. and
Delia B. Baxter Foundation Scholar Award (to S.P.P.).
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Figure 1. Cre-dependent recovery of AAV capsid sequences from transduced target cells
(
a
) An overview of the CREATE selection process. PCR is used to introduce diversity (full
visual spectrum vertical band) into a capsid gene fragment (yellow). The fragment is cloned
into the rAAV genome harboring the remaining capsid gene (gray) and is used to generate a
library of virus variants. The library is injected into Cre transgenic animals and PCR is used
to selectively recover capsid sequences from Cre
+
cells. (
b
) The rAAV-Cap-in-cis-lox rAAV
genome. Cre inverts the polyadenylation (pA) sequence flanked by the lox71 and lox66
sites. PCR primers (half arrows) are used to selectively amplify Cre-recombined sequences.
(
c
) PCR products from Cre recombination-dependent (top) and -independent (bottom)
amplification of capsid library sequences recovered from two Cre
+
or Cre
−
mice are shown.
Schematics (bottom) show the PCR amplification strategies (see
Supplementary Fig. 1
for
details). (
d
) Schematic shows the AAV genes within the Rep-AAP AAV helper plasmid and
the proteins encoded by the
cap
gene. Stop codons inserted in the
cap
gene eliminate VP1,
VP2 and VP3 capsid protein expression. (
e
) DNase-resistant AAV vector genomes (vg)
produced with the split AAV2/9 rep-AAP and rAAV-Cap-in-cis-lox genome (top) as
compared to the vg produced with standard AAV2/9 rep-cap helper and rAAV-UBC-
mCherry genome (middle) or with the AAV2/9 rep-AAP and rAAV-UBC-mCherry genome
(bottom).
N
=3 independent trials per group; mean ± s.d.; **
p
<0.01, ***
p
<0.001; one-way
ANOVA and Tukey multiple comparison test. (
f
) Cloning the 7-mer capsid library into the
rAAV-ΔCap-in-cis vector. (
g
) The AAV9 surface model shows the location of the 7-mer
inserted between AA588-589 (magenta). Sites encoded with the PCR-generated library
fragment (AA450-592) are shown in yellow.
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Figure 2. AAV-PHP.B mediates efficient gene delivery throughout the CNS after intravenous
injection in adult mice
(
a-f
) ssAAV9:CAG-GFP or ssAAV-PHP.B:CAG-GFP, at 1×10
12
vg/mouse or 1×10
11
(
a
,
right), was intravenously injected into adult mice. Images show GFP expression 3 weeks
after injection. (
a
) Representative images of GFP IHC in the brains of mice given AAV9
(left) or AAV-PHP.B (middle and right). (
b)
Native GFP fluorescence in the cortex (left) or
striatum (right) in 50 μm maximum intensity projection (MIP) confocal images. (
c)
GFP
fluorescence in the PARS-cleared
24
lumbar spinal cord. (
d)
GFP fluorescence in the retina
(left: 20 μm MIP, transverse section; right: whole-mount MIP).
(e
,
f)
GFP fluorescence in 3D
MIP images of PARS-cleared tissue from AAV-PHP.B transduced cortex and striatum (
e
)
and indicated organs from mice transduced with AAV9 (top) or AAV-PHP.B (bottom) (
f
).
Arrows highlight GFP
+
nerves. Asterisks in the image of the pancreas highlight GFP
+
islet
cells. Major tick marks in 3D projections are 100 μm. (
g)
AAV biodistribution in the
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indicated brain regions (top) and organs (bottom) 25 days after intravenous injection of
1×10
11
vg into adult mice.
N
=3 for AAV-PHP.B and
n
=4 for AAV9; mean ± s.d.; **
p
<0.01,
***
p
<0.001, unpaired t tests corrected for multiple comparisons by the Holm-Sidak method.
Scale bars: 1 mm (
a
,
c
(left)); 50 μm
(b
,
c
(right),
d
,
e
). Major tick marks in 3D projections
in
c
,
e
,
f
are 100 μm.
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