A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells

Article (PDF Available)inMolecular Therapy 17(11):1888-96 · July 2009with127 Reads
DOI: 10.1038/mt.2009.128 · Source: PubMed
Abstract
Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 x 10(4) purified rAAV particles per cell (range: 3.7 x 10(4) to 9.6 x 10(4)) from suspension cultures of recombinant baculovirus-infected insect cells.
original article
1888 www.moleculartherapy.org vol. 17 no. 11, 1888–1896 nov. 2009
© The American Society of Gene & Cell Therapy
Scalable methods of recombinant adeno-associated virus
(rAAV) production have gained much recent interest as
the field of rAAV-mediated gene therapy approaches the
clinic. In particular, the production of rAAV vectors in
insect cells via the use of recombinant baculovirus tech-
nology has proven to be an efficient and scalable means
of rAAV production. Here, we describe a method for the
production of rAAV serotypes 1 and 2 in insect cells using
a simplified baculovirus-AAV expression vector system
coupled with particle purification via affinity chromato-
graphy. The number of separate baculovirus constructs
required for rAAV production was reduced by genetically
modifying the AAV rep gene to allow expression of the
AAV-encoded replication enzymes, Rep78 and Rep52,
from a single mRNA species and combining the modified
rep gene with an AAV cap gene expression cassette in a
single baculovirus construct. Additionally, we describe
lysis, binding, and elution conditions compatible with a
commercially available affinity medium (AVB Sepharose
High Performance) used to purify rAAV particles to near
homogeneity in a single chromatography step. Using
the described method, we obtained an average yield of
7 × 10
4
purified rAAV particles per cell (range: 3.7 × 10
4
to 9.6 × 10
4
) from suspension cultures of recombinant
baculovirus–infected insect cells.
Received 19 February 2009; accepted 13 May 2009; published online
16 June 2009. doi:10.1038/mt.2009.128
INTRODUCTION
As the eld of recombinant adeno-associated virus (rAAV)–
mediated gene therapy progresses, the need for scalable meth-
ods of rAAV production becomes of growing importance to the
translation of successful preclinical investigations to human clini-
cal trials. Baculovirus-mediated production of rAAV vectors in
insect cells is especially well suited for the production of large
quantities of rAAV (reviewed in refs. 1 and 2). e baculovirus-
insect cell rAAV production strategy takes advantage of the e-
ciency of viral infection coupled with the high cell density and
scalability achievable with Spodoptera frugiperda Sf9 insect cells
grown in serum-free suspension culture. Successful baculovirus-
mediated production of recombinant AAV vectors in stirred-tank
bioreactors and disposable, multi-liter “wave devices has been
described,
3–5
and rAAV produced via the baculovirus system has
been administered in a Phase II human clinical trial for the treat-
ment of lipoprotein lipase deciency.
6
In the baculovirus-mediated rAAV production strategy, as
originally congured by Urabe et al.,
7
Sf9 insect cells are infected
with three dierent recombinant baculovirus constructs: one
recombinant baculovirus, designated Bac-Rep, expresses the
major AAV replication enzyme, Rep78, and its amino-truncated
form, Rep52, from two separate transcription units via par-
tial duplication of rep coding sequences, a second recombinant
baculovirus, designated Bac-VP, expresses the AAV virion coat
proteins from a modied AAV cap gene, and a third recombi-
nant baculovirus bears the gene of interest anked by the AAV
inverted terminal repeat (ITR) elements, which provide cis-acting
elements required for rescue, replication, and packaging of trans-
gene sequences. Although eective, the Bac-Rep construct dem-
onstrates genetic instability upon serial passage due to tandem
duplication of homologous regions of the rep78 and rep52 genes,
5,8
thus hindering amplication of Bac-Rep stocks for large-scale
rAAV production.
We have sought to simplify the production of rAAV vectors
using the baculovirus-mediated production strategy in such a
way that would increase stability of rep-expressing baculovirus
constructs, reduce the number of separate, recombinant baculo-
viruses required for AAV production, and increase the overall
robustness of the system by maintaining genetic linkage between
the AAV rep and cap open reading frames. To achieve this goal,
the AAV type 2 rep gene was genetically modied to encode a
bifunctional mRNA transcript that directs the synthesis of the
AAV Rep78 and Rep52 polypeptides from a single mRNA spe-
cies via a “leaky scanning” mechanism of translational initia-
tion (reviewed in refs. 9–11), thus allowing expression of the
AAV Rep and Cap proteins from the same recombinant baculo-
virus genome without destabilizing intramolecular duplication
of rep coding sequences. In the leaky scanning mechanism of
Correspondence: Robert M Kotin, Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Building 10, Room 7N260, Bethesda,
Maryland 20892, USA. E-mail: kotinr@nhlbi.nih.gov
A Simplified Baculovirus-AAV Expression
Vector System Coupled With One-step Affinity
Purification Yields High-titer rAAV Stocks
From Insect Cells
Richard H Smith
1
, Justin R Levy
1
and Robert M Kotin
1
1
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA
Molecular erapy vol. 17 no. 11 nov. 2009 1889
© The American Society of Gene & Cell Therapy
Baculovirus-mediated rAAV Production
translational initiation, 40S ribosomal subunits load onto the
capped 5-end of an mRNA transcript and scan the message in
a 5-to-3 direction in search of a suitable initiation codon. If a
suboptimal translational initiation signal is encountered (e.g.,
an AUG codon occurring within a weak translational initiation
sequence context or a non-AUG codon occurring within a strong
translational initiation sequence context), a portion of ribosomal
subunits initiate translation at this site, while the remainder of 40S
subunits continues scanning to the next translational initiation
signal, thus resulting in full-length and specic amino-truncated
forms of a given protein. e leaky scanning mechanism was uti-
lized by Urabe et al.
7
to achieve stoichiometric expression levels
of the three AAV capsid proteins (VP1, VP2, and VP3) from a
single species of recombinant baculovirus–encoded cap mRNA,
and, more recently, by Hermens et al.
12
who have described the
use of suboptimal rep78 start codons to mediate leaky scanning
of recombinant baculovirus–encoded rep mRNA transcripts. In
an alternative approach to obtain expression of overlapping AAV
polypeptide sequences in insect cells, Chen
13
utilized strategic
placement of a synthetic, insect promoter–containing intron to
facilitate Rep78/52 and VP1/2/3 expression from either a single
recombinant baculovirus containing both intron-modied genes
or from separate recombinant baculoviruses.
Consistent with our interest in the development of rAAV-
based therapeutics for the treatment of human muscular disor-
ders, particularly Duchenne muscular dystrophy, the AAV type 1
cap gene was chosen for the proof-of-principle characterization of
the consolidated rep- and cap-expressing baculovirus construct,
as this AAV serotype demonstrates highly ecient transduction
of muscle tissue.
14–16
RESULTS
Modification of the AAV rep gene and consolidation
of AAV rep and cap gene expression to a single
recombinant baculovirus
To obtain expression of the AAV Rep78 and Rep52 proteins from a
single baculovirus construct while avoiding destabilizing genomic
duplication of rep coding sequences,
5,8
the AAV rep gene was modi-
ed to allow expression of the Rep78 and Rep52 polypeptides from
a single mRNA species via an mRNA leaky scanning mechanism.
9–11
e AUG initiation codon of the rep78 open reading frame, the adja-
cent proline codon, and nine downstream AUG triplets occurring
before the start codon of the rep52 open reading frame were altered
via synthetic gene synthesis (Figure 1). e rep78 initiation codon
and proximal anking nucleotides were mutated to an inecient
translation initiation signal composed of a CUG triplet presented in
the context of a Kozak consensus sequence.
17
AUG triplets occurring
between the initiation codon of the rep78 open reading frame and the
AUG initiation codon of the rep52 open reading frame were altered
to bear either a silent mutation (in the case of out-of-frame AUG
codons), or to encode a conservative amino acid substitution (in the
case of in-frame AUG codons). e modied rep gene along with a
serotype-specic AAV cap gene bearing a non-AUG-initiated VP1
open reading frame (see ref. 7) were cloned in opposite transcriptional
orientations into a prokaryotic transfer plasmid for bacmid- mediated
generation of an Autographa californica multiple nuclear polyhedrosis
virus–based chimeric baculovirus-AAV expression vector.
Analysis of rep and cap gene expression during rAAV
production in Sf9 insect cells
To characterize the temporal occurrence and relative abun-
dance of the AAV Rep and Cap proteins during recombinant
baculovirus–mediated rAAV production in insect cells using a
consolidated rep- and cap-expressing baculovirus construct, a
time-course analysis was performed in which Sf9 cells grown in
suspension culture were co-infected with Bac-RepCap1, a bacu-
lovirus-AAV chimera bearing modied AAV type 2 rep and AAV
type 1 cap genes, and Bac-GFP, a recombinant baculovirus bear-
ing an ITR-anked enhanced green uorescent protein reporter
gene.
7
Sf9 cells were sampled at various times postinfection and
HSV tk
pA
VP1
VP2
VP3
Rep78
UAA
AUG
ACG
ACG
AUG
UAA
CUG
Rep52
SV40
pA
P
P10
P
Ph
cap rep
a
b
Figure 1 Schematic representation of the rep and cap transcription
units within the chimeric baculovirus construct, Bac-RepCap, and
modifications to the AAV type 2 rep78 open reading frame. (a) The
Rep and Cap proteins of AAV are expressed from divergent baculo virus
late promoters. Messenger RNA transcripts are represented by wavy
lines. All pertinent codons are indicated in a left-to-right orientation
for convenience. The modified AAV serotype 2 rep gene is under the
transcriptional control of the baculovirus polyhedrin promoter (P
Ph
). The
bifunctional rep mRNA transcript utilizes a CUG triplet embedded within
a Kozak consensus sequence to direct synthesis of Rep78 polypeptides.
A portion of ribosomal subunits fail to initiate translation at the non-
standard start codon and scan to the next AUG start codon to initi-
ate translation of Rep52 polypeptides. The AAV cap gene is under the
transcriptional control of the baculovirus p10 promoter (P
P10
). The three
capsid proteins (VP1, VP2, and VP3) are translated from a single mRNA
species, as described by Urabe et al.
7
Sequences directing 3-mRNA pro-
cessing are derived from simian virus 40 (SV40 pA) and the herpes sim-
plex virus thymidine kinase gene (HSV tk pA) as indicated. (b) The DNA
sequence encoding the amino-terminus of Rep78 is shown. Translation
of Rep78 polypeptides initiates at a CUG (leucine) codon placed within
the context of a Kozak consensus sequence (specifically gccgccCUGg).
A codon encoding proline at position 2 of Rep78 was altered to encode
alanine to accommodate the Kozak consensus sequence. Four in-frame
and five out-of-frame AUG triplets were altered to yield either a conser-
vative amino acid change or a silent mutation, respectively. Nucleotide
changes and amino acid substitutions (highlighted) are indicated by
lower-case font. A BglII recognition site used for cloning purposes is
underlined. AAV, adeno-associated virus.
1890 www.moleculartherapy.org vol. 17 no. 11 nov. 2009
© The American Society of Gene & Cell Therapy
Baculovirus-mediated rAAV Production
analyzed for AAV protein expression by western blot analysis
(Figure 2). Consistent with transcriptional regulation by the
baculovirus polyhedrin promoter, a member of the “very late
temporal class of baculovirus promoters,
18
expression of the
AAV Rep78 and Rep52 proteins demonstrated a delayed onset
(Figure 2a). Minimal levels of Rep78 and Rep52 polypeptides
were detected at 24 hours postinfection, but were relatively abun-
dant by the 48-hour time-point, and persisted throughout the
remaining 96-hour time-course. e AAV Cap proteins, under
the transcriptional control of the baculovirus p10 promoter, dem-
onstrated an earlier onset of expression relative to the AAV Rep
proteins. AAV Cap proteins were detectable at 24 hours postin-
fection, peaked at the 48-hour time-point, and slowly decreased
during the remaining 48 hours of the time-course (Figure 2b).
e overall progression of baculovirus infection was followed by
western blot analysis of the A. californica multiple nuclear poly-
hedrosis virus capsid protein, VP39, and the ubiquitous cellular
protein, β-tubulin (Figure 2c).
Analysis of replicative-form transgene sequences
To determine the ability of the consolidated Bac-RepCap
baculovirus construct to mediate rescue and replication of
AAV ITR-flanked transgene sequences, Sf9 cells were co-
infected with Bac-RepCap1 and Bac-GFP, sampled at 24-hour
intervals postinfection, and analyzed for the presence of
rAAV replicative-form DNA intermediates by agarose gel
electrophoresis and ethidium bromide staining (Figure 3).
Concomitant with the appearance of abundant levels of the
AAV Rep proteins (see Figure 2), DNA bands consistent with
monomeric and dimeric replicative-form rAAV-GFP genomes
were first detected at 48 hours postinfection (Figure 3, lane
4). In a control experiment, rescue and replication of rAAV
transgene sequences was not observed in the absence of Bac-
RapCap1 co-infection (Figure 3, lane 2).
Expression stability of the Bac-RepCap
construct upon serial passage
Previous reports have noted genetic instability of the rst-
generation rep-expressing baculovirus construct (Bac-Rep) upon
serial passage.
5,8
is instability was attributed to duplication of
rep coding sequences within a single baculovirus genome.
8
To
examine the stability of Rep and Cap protein expression medi-
ated by the consolidated Bac-RepCap baculovirus construct,
which expresses Rep78 and Rep52 from a single open read-
ing frame without intramolecular duplication of rep coding
sequences, Sf9 cells were inoculated with a plaque-titered pas-
sage 3 Bac-RepCap1 stock at a multiplicity of infection (MOI) of
0.1 plaque-forming units per cell to generate a P4 stock, which
was harvested at 3 days postinfection. e P4 stock was further
serially propagated at 3-day intervals by volumetric inocula-
tion (1/100th culture volume) of fresh Sf9 suspension cultures
to obtain a total of eight serial passages. Passage samples were
analyzed for Rep and Cap protein expression by western blot
analysis (Figure 4). Stable expression of the AAV Rep and Cap
proteins was observed to passage 7. is level of stability will
support sucient baculovirus stock amplication for an MOI 1
inoculation of large-scale, stirred-tank bioreactor preparations
of rAAV in insect cells.
VP39
β-Tubulin
Hours
postinfection
- Uninfected
- 24
- 48
- 72
- 96
c
260
MW
(kd)
Hours
postinfection
160
110
VP1
VP2
VP3
Rep78
Rep52
- Uninfected
- 24
- 48
- 72
- 96
80
60
50
40
30
20
15
Hours
postinfection
- Uninfected
- 24
- 48
- 72
- 96
ab
Figure 2 Western blot analysis of Rep and Cap protein expression
in Sf9 insect cells during Bac-RepCap1-mediated rAAV production.
Sf9 insect cells (3.6 × 10
7
) grown in suspension culture were infected
with Bac-RepCap1 and Bac-GFP at an MOI of 1 each. Samples were
taken at 24-hour intervals and subjected to western blot analysis. (a) Rep
protein expression detected with anti-Rep monoclonal antibody 303.9
(b) Cap protein expression detected with an anti-AAV capsid protein
rabbit polyclonal antiserum (c) Monoclonal antibody–mediated detec-
tion of the baculovirus capsid protein VP39 and the ubiquitous cellular
protein β-tubulin. AAV, adeno-associated virus; rAAV, recombinant
adeno- associated virus; uninfected, uninfected Sf9 cell control.
12.2
kbp
6.1
3.1
2.0
1.0
RF
D
RF
M
0.5
123456
Uninfected
Bac-GFP (48 hpi)
24 48
Bac-GFP
+
Bac-Rep/Cap
72 (hpi)96
Figure 3 Bac-RepCap-mediated rescue of vector genome sequences.
Sf9 insect cell were infected with Bac-RepCap1 and Bac-GFP at an MOI
of 1 each. Episomal DNA sequences were isolated from culture samples
taken at 24-hour intervals and subjected to electrophoresis on a 0.8%
agarose–TBE gel. Replicative-form vector genomic intermediates were
visualized by ethidium bromide staining. An image negative is shown.
AAV, adeno-associated virus; GFP, green fluorescent protein; hpi, hours
postinfection; MOI, multiplicity of infection; RF
D
, replicative-form AAV-GFP
dimer (5.30 kbp); RF
M
, replicative-form AAV-GFP monomer (2.65 kbp);
TBE, Tris–borate–EDTA; uninfected, uninfected Sf9 cell control.
Molecular erapy vol. 17 no. 11 nov. 2009 1891
© The American Society of Gene & Cell Therapy
Baculovirus-mediated rAAV Production
Characterization of rAAV production
and affinity column purification
To evaluate Bac-RepCap-mediated rAAV production in insect
cells and purication of vector particles by AVB Sepharose
anity chromatography, suspension cultures of Sf9 cells were
co-infected with Bac-RepCap1 and a recombinant baculovirus
bearing an ITR-anked GFP reporter gene at an MOI of 1 plaque-
forming unit/cell for each baculovirus construct (total MOI = 2).
Recombinant baculovirus–infected cells were collected at ~ 72
hours postinfection and subjected to detergent extraction. e
infected-cell extracts were combined with the corresponding
culture medium and treated with Benzonase nuclease to digest
non-encapsidated DNA. Nuclease-treated material was loaded
onto a 10 mm × 100 mm chromatography column packed with
AVB Sepharose High Performance anity medium. Following
a phosphate-buered saline (PBS, pH 7.4) column wash, bound
material was eluted with low-pH glycine–HCl buer (pH 2.7)
and collected as 1-ml fractions into sample tubes containing
one-tenth volume neutralization buer. An example chromato-
gram of a recombinant AAV-1 preparation is shown in Figure 5.
Elution of the anity column with low-pH glycine–HCl buer
yielded a sharp peak (Figure 5b) that, by peak integration, rep-
resented ~0.08% of total UV
280
-absorbing material. A leading-
edge sub-peak was reproducibly observed in fraction 6. Upon
examination of this fraction by a variety of methods (data not
shown), we were unable to denitively identify a salient feature
that distinguishes vector in this fraction from that of the major
elution peak.
To characterize further the material eluting from the AVB
Sepharose anity medium, samples of each fraction were subjected
to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and
silver staining (Figure 6a). ree protein bands, corresponding to
the theoretical molecular weights of the AAV-1 VP1, -2, and -3
capsid proteins (81.4, 66.2, and 59.6 kd, respectively), represented
the majority of the eluting material (Figure 6a, fraction 7), and
coincided with the major UV absorption peak of the column chro-
matogram. Densitometry of the silver-stained gel image indicated
a purity of >90%. e peak and shoulder elution fractions (frac-
tions 6–8) represented an ~67-fold volumetric concentration of
rAAV-1 particles in a single step. Western blot analysis of a dupli-
cate polyacrylamide gel using an anti-AAV capsid antiserum con-
rmed the identity of the AAV capsid proteins (Figure 6b). Taken
in combination with the peak integration analysis of the chro-
matogram, these data indicate a one-step bulk purication factor
of >1000-fold. In agreement with the protein staining and immu-
noblot results, quantitative, real-time PCR analysis of the AVB
column elution fractions using vector-specic primers mapped
Rep78
Rep52
VP1
Uninfected
P4
P5
P6
P7
P8
VP2
VP3
VP39
β-Tubulin
Figure 4 Western blot analysis of Rep and Cap protein expression
during serial passage. Sf9 insect cells in suspension culture (30 ml
volume, 1.2 × 10
6
cells/ml) were inoculated with a passage 3 (P3)
stock of Bac-RepCap1 (MOI = 0.1). At 3-day intervals, this stock was
further passaged by inoculation of fresh Sf9 cells with a 0.01× volume
of culture supernatant from the previous passage. Samples were taken
at the completion of each passage, and equal amounts of total cellular
extract (10 µg) were analyzed by western blot analysis with antibodies
to the indicated protein. Passage number is indicated above each lane.
Identities of the AAV Rep and Cap proteins are indicated at left. AAV,
adeno-associated virus; β-tubulin, endogenous cellular protein; MOI,
multiplicity of infection; uninfected, uninfected Sf9 cell extract; VP39,
baculovirus-encoded capsid protein.
0
050 100
Elution volume (ml)
150
AAV
200 250
1,000
Absorbance (mAU, 280 nm)
2,000
3,000
4,000
a
0
225 230
12345678910 1112 13 14
235 240
Elution volume (ml)
245 250 255
100
200
300
400
500
600
Absorbance (mAU, 280 nm)
b
Figure 5 Affinity chromatography. (a) A 200 ml culture of Bac-
RepCap1- and Bac-GFP-infected Sf9 insect cells (2.4 × 10
8
total cells
at the time of inoculation) was processed for affinity chromatography
as described in Materials and Methods. Nuclease-treated material was
loaded onto a Tricorn 10/100 column packed with AVB Sepharose High
Performance chromatography medium, and the column was washed
with phosphate-buffered saline (pH 7.4). Beginning at ~230 ml of
total elution volume (x-axis), bound rAAV particles were eluted from the
affinity column with the application of 50 mmol/l glycine–HCl buffer (pH
2.7). Glycine buffer-eluted material was fractionated into tubes contain-
ing 100 µl of 1 mol/l Tris–HCl (pH 8.0) for elution buffer neutralization.
The graph represents the column elution profile as detected by absor-
bance at 280 nm. (b) Enlarged partial chromatogram showing the rAAV
elution peak in relation to the fraction number, which is indicated at the
bottom of the graph. rAAV, recombinant adeno-associated virus.
1892 www.moleculartherapy.org vol. 17 no. 11 nov. 2009
© The American Society of Gene & Cell Therapy
Baculovirus-mediated rAAV Production
the peak of rAAV vector genomes to fraction 7 (Figure 6c), and
indicated a nuclease-resistant, genome- containing particle titer
of 1.7 × 10
13
particles/ml for the peak fraction. To examine the
morphology of the rAAV particles, samples of the peak column
fraction were negatively stained with uranyl acetate and viewed by
transmission electron microscopy. Electron micrographs revealed
dense clusters of non-enveloped, icosahedral particles with a
diameter of ~20–25 nm that are characteristic of the Parvoviridae
family (Figure 6d).
Repeated preparations of rAAV-GFP produced using the con-
solidated Bac-RepCap baculovirus system were characterized in
terms of yield, concentration, particle-to-transducing unit ratio,
and puried particle recovery using a combination of quantita-
tive, real-time PCR analysis of vector genome content and ow
cytometric analysis of rAAV-mediated GFP expression in vector-
transduced human embryonic kidney cells (HEK-293A cells)
(Table 1). An example of rAAV-mediated GFP expression in
HEK-293A cells is shown in Figure 7. To examine the utility of
the consolidated baculovirus production strategy for AAV sero-
types other than type 1, a Bac-RepCap2 baculovirus, which con-
tains the codon-modied AAV type 2 rep gene in combination
with an ACG-initiated AAV type 2 cap open reading frame, was
constructed and characterized in terms of end-point rAAV pro-
duction parameters following AVB Sepharose anity purication
(Table 1, preparations 5 and 6). Examining all preparations,
vector yield ranged from 7.6 × 10
12
to 2.3 × 10
13
puried, nuclease-
resistant particles with an average production of 7.2 (±2.5) × 10
4
particles per cell (n = 6).
DISCUSSION
In this paper, we have described the construction and charac-
terization of second-generation rep- and cap-expressing chime-
ric baculo virus constructs for the production of rAAV particles
in insect cells. Additionally, we have evaluated the utility of a
commercially available anity medium, AVB Sepharose High
Performance, for the purication of rAAV virions from insect
cell extracts. We have used a “leaky scanningstrategy of trans-
lational initiation to facilitate consolidation of individual rep- and
cap- expressing baculovirus constructs into a single recombinant
baculovirus.
Rep modification
e Rep78 initiation codon and 10 additional downstream triplets
within the unique portion of the rep78 open reading frame were
mutated to facilitate expression of the Rep78 and Rep52 poly-
peptides from a single species of bifunctional mRNA transcript.
Peabody
19
demonstrated that, in reticulocyte lysates, wheat germ
extracts, and recombinant simian virus 40–transduced CV1
14.4
21.5
31
45
66.2
97.4
116.3
200
MW
(kd) 1234567
Fraction number
8910 11 12 13 14
M
Crude
Flow through
a
15
20
30
40
50
60
80
110
160
260
MW
(kd) 1 234567
Fraction number
8910 11 12 13 14
Crude
Flow through
b
0.0
01234567
Fraction number
8910 11 12 13 14 15
0.2
0.4
0.6
0.8
1.0
NRP/ml `× 10
13
1.2
1.4
1.6
1.8
2.0
c
100 nm
d
Figure 6 Characterization of baculovirus-produced rAAV-1 purified from insect cell extracts by affinity chromatography. (a) Samples of the
crude lysate (0.2 µg), column flow-through material (0.2 µg), and 15 µl of each column elution fraction were separated by SDS-PAGE on a 4–12%
polyacrylamide gradient gel, and the separated proteins were visualized by silver staining. (b) For western blot analysis, a polyacrylamide gel similar to
that described above was blotted to nitrocellulose and probed with an anti-AAV capsid protein-specific polyclonal rabbit antiserum. (c) Quantitative,
real-time PCR analysis was used to determine the number of nuclease-resistant particles (NRP) in each column fraction. (d) Transmission electron
micrograph of rAAV-1 particles eluted within the peak AVB affinity column fraction (fraction 7) and negatively stained with a 1% uranyl acetate solu-
tion. Bar = 100 nm. A digital enlargement of a portion of the micrograph (bottom left insert) shows morphological detail. PAGE, polyacrylamide gel
electrophoresis; rAAV, recombinant adeno-associated virus; SDS, sodium dodecyl sulfate.
Molecular erapy vol. 17 no. 11 nov. 2009 1893
© The American Society of Gene & Cell Therapy
Baculovirus-mediated rAAV Production
monkey kidney cells, most triplets diering from the canonical
AUG initiation codon by a single nucleotide can function, with
varying degrees of eciency, as start codons when presented in
the context of a Kozak consensus sequence (CCG/ACCAUGG;
ref. 17). ACG and CUG were found to be among the most e-
cient non-AUG initiation codons. As demonstrated in this report,
a CUG triplet presented in a Kozak sequence context functioned
eciently as a translational initiation codon for the rep78 open
reading frame within Sf9 insect cells. In addition to alteration of
the rep78 initiation codon, the CCG codon encoding proline at
amino acid position 2 of Rep78 was mutated to a GCG (alanine)
codon in order to support the Kozak consensus sequence context
of the adjacent nonstandard rep78 initiation codon. An alanine
residue occurs naturally at the equivalent position of the homo-
logous Rep78 protein of AAV serotype 5 (ref. 20), suggesting that
this amino acid substitution is compatible with Rep function. To
facilitate ecient ribosomal scanning to the AUG initiation codon
of the Rep52-encoding portion of the rep open reading frame,
four in-frame and ve out-of-frame AUG triplets downstream of
the Rep78 initiation codon were altered to contain either a silent
mutation in the case of out-of-frame AUG triplets, or to encode a
conservative amino acid substitution of leucine for methionine in
the case of in-frame AUG triplets. An alignment of the amino acid
sequences of the Rep78 equivalents of AAV serotypes 1–8 (data
not shown) indicates that, in three of the four instances of methi-
onine replacement within the AAV serotype 2 Rep78 protein (at
amino acid positions 43, 91, and 172), a leucine residue occurs
naturally at an analogous position within the Rep78 protein of at
least one other AAV serotype. In contrast, the methionine residue
at amino acid position 103 is invariant among the Rep78 proteins
of the AAV serotypes examined; however, as demonstrated in this
report, a modied Rep78 protein bearing a leucine substitution
at this amino acid position (as well as positions 43, 91, and 172)
was able to eciently support rescue, replication, and packaging
of ITR-anked transgene sequences in insect cells.
Affinity chromatography
Scalable methods of rAAV production require equally scalable meth-
ods of rAAV purication. Commonly used ultracentrifugation-based
procedures for rAAV particle isolation, such as the use of iodixanol
or cesium chloride gradients, are limited by capacity and diculty
of scale-up. Accordingly, liquid chromatography–based protocols
ace
bdf
Figure 7 Transduction of HEK-293A cells. HEK-293A cells in 24-well cluster plates were transduced with various amounts of affinity-purified rAAV-1
and examined by fluorescence microscopy at three days post-transduction. Cell nuclei were stained with Hoechst 33342. a and b, c and d, and e
and f represent wells receiving 0, 2, and 8 µl, respectively, of the peak elution fraction (fraction 7) of the affinity column. Top row, micrographs taken
using a GFP-compatible filter set. Bottom row, micrographs taken using a Hoechst 33342-compatible filter set. rAAV, recombinant adeno-associated
virus; GFP, green fluorescent protein.
Table 1 Yield and recovery of rAAV
Prep. number Serotype Cells/prep.
a
Yield
b,c
NRP/ml
c,d
TU/ml
d,e
NRP/TU % recovery
f
NRP/cell
g
1 rAAV-1 2.4 × 10
8
2.3 × 10
13
1.7 × 10
13
1.3 × 10
11
131 37.1 9.6 × 10
4
2 rAAV-1 2.4 × 10
8
2.2 × 10
13
1.1 × 10
13
2.2 × 10
10
500 82.3 9.2 × 10
4
3 rAAV-1 2.4 × 10
8
8.8 × 10
12
6.5 × 10
12
2.0 × 10
10
325 28.9 3.7 × 10
4
4 rAAV-1 2.4 × 10
8
1.2 × 10
13
8.2 × 10
12
2.1 × 10
10
391 51.2 5.0 × 10
4
5 rAAV-2 1.2 × 10
8
7.6 × 10
12
5.2 × 10
12
2.0 × 10
10
260 ND 6.3 × 10
4
6 rAAV-2 1.2 × 10
8
1.1 × 10
13
7.8 × 10
12
1.8 × 10
10
433 ND 9.2 × 10
4
Abbreviations: ND, not done; NRP, nuclease-resistant particle; TU, transducing unit.
All vector preparations utilized Bac-GFP except for preparation number 2, which used Bac-GFP/neo. All preparations were purified by AVB affinity chromatography.
a
At the time of baculovirus inoculation.
b
Sum of nuclease-resistant particles eluted in peak affinity column fractions (fractions 6–8).
c
Determined by quantitative real-
time PCR analysis.
d
Peak fraction from affinity column.
e
Transducing units of rAAV-GFP per ml determined by flow cytometry of HEK-293A cells transduced with rAAV in
the presence of recombinant adenovirus (rAd) co-infection.
f
Total number of transducing units in peak affinity column fractions (fractions 6–8) divided by the number
of TU in the crude cell extract (×100). Assayed on HEK-293A cells in the absence of rAd co-infection.
g
Determined by dividing the yield by the total number of cells
at the time of baculovirus inoculation.
1894 www.moleculartherapy.org vol. 17 no. 11 nov. 2009
© The American Society of Gene & Cell Therapy
Baculovirus-mediated rAAV Production
for rAAV purication have gained much recent interest. Several
reports have described chromatography-based methods for the
purication of rAAV particles from mammalian cell extracts,
21–27
and a chromatographic procedure for the purication of rAAV par-
ticles from insect cell extracts has recently been described.
28
Many
of these methods, however, require multiple chromatography steps
or are limited in applicability to a subset of AAV serotypes (e.g.,
those with high anity for immobilized heparin).
We have evaluated AVB Sepharose High Performance, a
commercially available, AAV-specic anity medium, for the
purication of recombinant AAV serotypes 1 and 2 from Sf9
insect cell extracts. Advantages of the AVB Sepharose medium
include recognition of a variety of AAV serotypes (regardless of
heparin-binding eciency), a relatively high linear ow-rate (up to
150 cm/hour), and a large vector binding capacity (10
12
particles/
ml of medium).
29
One disadvantage, however, is cost of the mate-
rial which may be signicant for quantities of medium sucient
for recovery of rAAV vectors from multi-liter, bioreactor-based
preparations which, with use of the baculovirus-AAV expression
vector system, can approach 10
14
genome-containing particles per
liter of culture volume.
5
When used in conjunction with recombinant baculovirus–
infected insect cell extracts, AVB Sepharose anity medium dem-
onstrated acceptable levels of rAAV purication, concentration,
and recovery. Using binding and elution conditions described in
this report, the anity medium provided a >1000-fold bulk puri-
cation of rAAV-1 from insect cell extracts in a single chroma-
tography step, with an average recovery of 49.9%. Use of low-pH
glycine–HCl elution buer provided sharp elution peaks that
resulted in puried vector concentrations in the range of 5 × 10
12
to 2 × 10
13
particles/ml (recovered from 100 to 200 ml suspension
cultures of recombinant baculovirus–infected Sf9 cells) without
the need for additional concentration steps.
In conclusion, the use of the described recombinant baculo-
virus-AAV expression vectors in combination with a commer-
cially available AAV-specic anity medium provides a robust,
streamlined, and scalable platform for the production and puri-
cation of high-titer rAAV vectors.
MATERIALS AND METHODS
Cell culture. Suspension cultures of S. frugiperda Sf9 insect cells were
maintained with constant orbital agitation at 28 °C in polycarbon-
ate Erlenmeyer asks (Corning, Corning, NY) containing serum-free
HyClone SFX-INSECT medium (HyClone Laboratories, Logan, UT).
HEK-293A cells were cultured at 37 °C in a humidied, 5% CO
2
atmo-
sphere in tissue culture asks containing Dulbeccos modied Eagles
medium supplemented with 4.5 g/liter glucose, 100 µg/ml streptomycin,
100 U/ml penicillin G, and 10% (vol/vol) heat-inactivated fetal calf serum.
Plasmid and recombinant baculovirus construction. Recombinant
baculo viruses were constructed using the Bac-to-Bac Baculovirus
Expression System (Invitrogen, Carlsbad, CA), which uses site-specic
transposition to insert transfer vector sequences into an A. californica
multiple nuclear polyhedrosis virus–derived bacmid DNA maintained
in Escherichia coli strain DH10Bac. Bacmid DNA isolated from ampli-
ed bacterial colonies is used to transfect Sf9 cells to generate recom-
binant baculovirus. Recombinant baculovirus Bac-GFP, which bears a
cytomegalovirus immediate early promoter-driven GFP reporter gene
anked by AAV-2 ITRs, has been described previously.
7
Recombinant
baculovirus Bac-GFP/neo (SR652) was constructed by ligating the
4.4-kbp BglII fragment of pSR460A, which contains a cytomegalovirus
promoter–driven GFP reporter gene and simian virus 40 promoter
driven aminoglycoside phosphotransferase (neo
r
) gene anked by AAV-2
ITRs, between the BbsI–BamHI sites of the transfer vector, pFastBac-1
(Invitrogen). To create a modied AAV-2 rep gene expressing a bifunc-
tional Rep78- and Rep52-encoding mRNA, a synthetic gene fragment
(produced by Integrated DNA Technologies, Coralville, IA) containing
a codon-modied partial rep78 open reading frame (see Figure 1) was
excised from its plasmid backbone as a 0.75-kbp XmaI–BamHI fragment
and cloned between the XmaI–BamHI sites of pRep(1-621) (ref. 30) to
create pSR645. Plasmid pSR645 thus contains the full-length, codon-
modied rep open reading frame in a pGEM-3Z (Promega, Madison, WI)
backbone. To construct a consolidated recombinant baculovirus express-
ing the AAV-2 Rep and AAV-1 Cap proteins from the same baculovirus
genome, the 2.3-kbp BamHI–XbaI fragment of pFBAAV1VPm11, which
contains an ACG-initiated AAV-1 cap open reading frame, was cloned
between the BbsI–NheI sites of pFastBac-Dual (Invitrogen) to create
the intermediate plasmid pSR648. e 1.9-kbp BglII–XbaI fragment of
pSR645 (above) was cloned between the BamHI–XbaI sites of pSR648 to
create the baculovirus transfer vector pSR651. Plasmid pSR651 was used
to generate recombinant baculovirus Bac-RepCap1. To create a consoli-
dated baculovirus construct expressing the codon-modied AAV-2 rep
gene in combination with an AAV-2 cap expression cassette, an interme-
diate plasmid, pSR653, was constructed by cloning the 1.9-kbp BglII–XbaI
fragment of pSR645 (above) between the BamHI–XbaI sites of pFastBac-
Dual. e ACG-initiated AAV-2 cap gene of pFBDVPm11 (ref. 7) was
PCR amplied (using primers: 5-GCCCCCGGGGGATCCTGTTAAG
ACGGC-3 and 5-GCCGCTAGCTTACAGATTACGAGTCAGGTATC
TG-3), digested with NheI–XmaI, and cloned between the NheI–XmaI
sites of pSR653, to generate the transfer vector pSR657. Plasmid pSR657
was used to generate recombinant baculovirus Bac-RepCap2. Baculovirus
stocks were titered by plaque assay on Sf9 monolayers.
Expression time-course, genome rescue, and stability. To examine Bac-
RepCap-mediated expression of the AAV Rep and Cap proteins during
rAAV production and to evaluate Rep-mediated rescue of vector genomes,
3.6 × 10
7
Sf9 suspension cells were collected by low-speed centrifugation
and resuspended in a 1.8 ml combined inoculum volume of Bac-RepCap1
and Bac-GFP virus stocks (MOI = 1 for each recombinant baculovirus). e
inoculated cells were incubated at room temperature on a rocking platform
for 1 hour. e infected cells were then diluted in serum-free medium to a
nal density of 1.2 × 10
6
cells/ml and further incubated at 28 °C. Samples
(1 ml) were taken at 24-hour intervals. Infected cells were pelleted by brief
centrifugation, the medium was discarded, and the cell pellets were stored
at −80 °C. For western blot analysis, thawed cell pellets were lysed by the
addition of 0.4 ml of NuPAGE sample buer (Invitrogen), and then
processed with a QIAshredder Mini Spin Column (Qiagen, Valencia, CA)
to reduce viscosity. Equal volumes were loaded onto polyacrylamide gels
for western blot analysis as described below. To analyze rescue of vector
genomes, episomal DNA was isolated from thawed, infected-cell pellets
using a QIAprep Spin Miniprep Kit (Qiagen) following the manufacturer’s
suggested protocol for plasmid recovery from bacterial cells. Equal sample
volumes were separated on a 0.8% agarose gel in Tris–borate–EDTA
buer. DNA was visualized by ethidium bromide staining. To examine
Bac-RepCap stability during serial passage, Sf9 suspension cells (30 ml
culture; cell density = 1.2 × 10
6
cells/ml) were inoculated with a passage 3
stock of Bac-RepCap1 at an MOI of 0.1, and then incubated at 28 °C for an
additional 3 days to generate a P4 stock. e P4 stock was serially propa-
gated at 3-day intervals (for a total of eight passages) by volumetric (1:100)
inoculation of Sf9 suspension cultures with a previous passage supernatant.
Samples (1 ml) were taken at each passage, and the cells were pelleted by
brief centrifugation. e infected-cell pellets were lysed by the addition of
1 ml of 1× NuPAGE sample buer and then processed with a QIAshredder
Molecular erapy vol. 17 no. 11 nov. 2009 1895
© The American Society of Gene & Cell Therapy
Baculovirus-mediated rAAV Production
device. Portions of each lysate were passed over a MicroSpin G-25 column
(GE Healthcare, Piscataway, NJ), and the total protein content was deter-
mined using BCA Protein Assay Reagent (ermo Scientic, Waltham,
MA). Equal amounts of total protein were analyzed by polyacrylamide gel
electrophoresis and western blot analysis as described below.
rAAV production in Sf9 cells and purification by affinity chromatography.
For anity column purication of Bac-RepCap1-produced rAAV using
AVB Sepharose High Performance medium, 2.4 × 10
8
Sf9 suspension cells
were collected by centrifugation at 300 g for 10 minutes and resuspended
in a sucient combined inoculum volume of Bac-RepCap1 and Bac-GFP
virus stocks to yield an MOI of 1 for each recombinant baculovirus con-
struct (total MOI = 2). Aer 1 hour of incubation at room temperature
on a rocking platform, the infected cells were diluted into an Erlenmeyer
ask containing 200 ml of serum-free medium (nal cell density of 1.2 ×
10
6
cells/ml) and further incubated at 28 °C with constant orbital agita-
tion. Bac-RepCap2-mediated rAAV preparations were performed in a
similar fashion, but with 1.2 × 10
8
total Sf9 cells in a nal culture volume
of 100 ml.
At ~72 hours postinfection, recombinant baculovirus–infected cells
were collected by centrifugation at 300 g for 10 minutes, and the culture
medium was retained for further processing. e infected-cell pellet was
resuspended in 10 ml of TNT extraction buer [20 mmol/l Tris–HCL
(pH 7.5), 150 mmol/l NaCl, 1% Triton X-100, 10 mmol/l MgCl
2
], and
incubated at room temperature for 10 minutes to lyse the outer cell
membrane. Insoluble material was pelleted at 2,100 g for 10 minutes, and
the supernatant was added back to the original culture medium. Benzonase
nuclease (Sigma, St Louis, MO) was added to a nal concentration of
20 U/ml, and the crude material was incubated at 37 °C for 1 hour. Just
before column loading, the crude nuclease-treated material was ltered
through a 0.2 µm polyethersulfone membrane. Anity chromatography
was performed using an AKTA-FPLC chromatography system equipped
with a Tricorn 10/100 column packed with an ~8 ml bed volume of AVB
Sepharose High Performance anity medium (all from GE Healthcare,
Piscataway, NJ). e packed column was equilibrated with PBS, pH
7.4 before use. Crude material was loaded in sequential applications using
a 50 ml or, in some instances, a 150 ml “Superloop (GE Healthcare) at
a ow rate of 2 ml/min. is ow rate was maintained throughout the
chromatographic procedure. e column was washed with PBS (pH 7.4)
until the OD
280
approached baseline. Bound material was eluted from the
column by application of 50 mmol/l glycine–HCl (pH 2.7) elution buer.
Elution fractions (1 ml) were collected into tubes containing 100 µl (one-
tenth fraction volume) of 1 mol/l Tris–HCl (pH 8.0) to neutralize the low-
pH elution buer. Fractions were stored at 4 °C for further analysis.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis, western blot
analysis, and silver staining.
Protein separations were performed using
NuPAGE Gel System reagents (Invitrogen). Proteins were separated by
electrophoresis on 4–12% NuPAGE bis–Tris polyacrylamide gradient gels
using 3-[N-morpholino] propane sulfonic acid–sodium dodecyl sul-
fate running buer. For sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis analyses, anity column fraction samples were passed over
MicroSpin G-25 columns to remove excess glycine. For silver staining,
xed gels were stained using the SilverXpress silver stain kit (Invitrogen)
according to the manufacturers protocol. For western blot analysis, poly-
acrylamide gels were soaked briey in 1× NuPAGE Transfer Buer before
electrophoretic transfer to nitrocellulose lters using the iBlot Dry Blotting
System (Invitrogen). Aer transfer, the nitrocellulose lters were blocked
for a minimum of 1 hour in PBST (PBS, pH 7.4, with 0.05% Tween-20)
containing 5% nonfat dry milk. Blots were incubated with the appropri-
ate primary detection antibody (diluted in PBST-5% nonfat dry milk) for
1.5 hours with gentle agitation, washed three times with PBST, and then
incubated an additional 1.5 hours with a horseradish peroxidase–conju-
gated secondary antibody diluted in PBST-5% nonfat dry milk. Aer three
washes in PBST, the lters were incubated with chemiluminescent detec-
tion reagent (SuperSignal West Dura Extended Duration Substrate, Pierce,
Rockford, IL) for 3 minutes, covered in plastic and exposed to X-ray lm.
Filters were stripped for reprobing using Restore Western Blot Stripping
Buer (Pierce) according to the manufacturers instructions. Rep pro-
teins were detected using an anti-Rep monoclonal antibody (clone 303.9;
American Research Products, Belmont, MA) at a 1:200 dilution. An anti-
AAV-5 capsid antiserum
27
that recognizes multiple AAV serotypes was
used at a 1:2,000 to 1:2,500 dilution. A monoclonal antibody recogniz-
ing the baculovirus VP39 capsid protein (kind gi of Loy Volkman) and
a monoclonal antibody recognizing β-tubulin (Boehringer Mannheim,
Mannheim, Germany) were both used at a 1:500 dilution. Secondary
detection antibodies were used at a 1:2,000 dilution.
Quantification of rAAV. Nuclease-resistant rAAV genome titers were
determined by quantitative, real-time PCR analysis of anity column
fractions using the cytomegalovirus promoter-specic oligonucleotide
pair 5-TCCGCGTTACATAACTTACGG-3 and 5-GGGCGTACTTGG
CATATGAT-3. Quantitative, real-time PCR analysis was performed with
a primary denaturation step of 3 minutes at 95 °C, followed by 40 cycles of
95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds on an
iCycler iQ RT-PCR thermocycler using iQ SYBR Green Supermix reagent
(both from BioRad Laboratories, Hercules, CA). Biological titers of rAAV
expressing a GFP reporter gene were determined by ow cytometric analysis
of serially diluted rAAV on HEK-293A cells in the presence of adenovirus
co-infection. Briey, HEK-293A cells were seeded into 24-well cluster plates
at a density of 1 × 10
5
cells per well and allowed to attach overnight. e next
day, the medium was removed and replaced with 0.5 ml per well of Dulbeccos
modied Eagles medium-10% fetal calf serum containing recombinant
adenovirus, AdCMVLacZ (Quantum Biotechnologies, Montreal, Canada),
diluted to an MOI of 1 plaque-forming unit per cell. e HEK-293A cells
were then inoculated with a tenfold serial dilution of rAAV and incubated
at 37 °C for 24 hours. e cells were harvested for ow cytometric analysis
by rst transferring the tissue culture supernatant of each well to individual
microfuge tubes, followed by the addition of 0.2 ml of a 0.05% trypsin–
0.53 mmol/l EDTA solution to each well and incubation for 5 minutes at
37 °C. e detached cells were removed and added to the corresponding
tissue culture supernatant. Each sample was ltered using a 5 ml polystyrene
Falcon tube with cell-strainer cap (Becton Dickinson, Franklin Lakes, NJ)
before ow cytometry. e number of GFP-positive cells was determined
using a Guava EasyCyte ow cytometer (Guava Technologies, Hayward,
CA) at an excitation wavelength of 488 nm. For uorescence microscopy,
HEK-293A cells were seeded into 24-well cluster plates at 1.2 × 10
5
cells per
well, allowed to attach overnight, and then inoculated with various amounts
of rAAV in the absence of adenovirus. At 3 days post-transduction, cell
nuclei were stained by the addition of the cell-permeable dye Hoechst 33342
to the culture medium (5 µmol/l nal concentration) followed by incuba-
tion at 37 °C for 3 hours. Transduced cells were photographed using a Zeiss
Axiovert uorescence microscope (Carl Zeiss Microimaging, ornwood,
NY) equipped with a digital CCD camera.
Transmission electron microscopy. Before electron microscopy, 50 µl ali-
quots of the peak AVB column fractions were dialyzed against two 0.5-l
volumes of 20 mmol/l bis–Tris (pH 6.0), 10 mmol/l NaCl buer using a
mini-dialysis unit (Slide-A-Lyzer MINI Dialysis unit, 20K MWCO, regen-
erated cellulose membrane; ermo Scientic). Two to ve microliters of
each sample were spotted onto formvar-coated, carbon-stabilized copper
grids (200-mesh) that had been plasma-discharged just before use. Aer
1-minute incubation, the samples were negatively stained by drop-wise
application of a 1% uranyl acetate solution. Excess staining solution was
removed by adsorption to lter paper, and the samples were allowed to
air-dry. Grids were examined using a JEM1200EX transmission electron
microscope (JEOL, Tokyo, Japan) at a magnication setting of 50,000× and
an accelerating voltage of 80 kV.
1896 www.moleculartherapy.org vol. 17 no. 11 nov. 2009
© The American Society of Gene & Cell Therapy
Baculovirus-mediated rAAV Production
ACKNOWLEDGMENTS
We thank Loy Volkman for providing the anti-VP39 monoclonal antibody.
We also thank Mathew P. Daniels and the National Heart, Lung, and
Blood Institute (NHLBI) Electron Microscopy Core Facility for assistance
with the transmission electron microscopy procedure. This work was
supported by the Intramural Research Program of the NHLBI, National
Institutes of Health. The authors declare no conflict of interest.
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    • "24. Hauck B, Murphy SL, Smith PH, et al. 2009. Undetectable transcription of cap in a clinical AAV vector: implications for preformed capsid in immune responses. "
    [Show abstract] [Hide abstract] ABSTRACT: Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cell yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based, scalable production system for high per particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.
    Full-text · Article · Jul 2015
    • "rAAVs serotype 1 and 2 were generated as described (Tang et al. 2009), and purified by AVB Sepharose affinity chromatography (Smith et al. 2009). For each virus preparation, the genomic titer was determined by Real-Time PCR (1.0–6.0 × 10 12 viral genomes (vg)/ml, TaqMan Assay, Applied Biosystems). "
    [Show abstract] [Hide abstract] ABSTRACT: Cortical spreading depression is a slowly propagating wave of near-complete depolarization of brain cells followed by temporary suppression of neuronal activity. Accumulating evidence indicates that cortical spreading depression underlies the migraine aura and that similar waves promote tissue damage in stroke, trauma, and hemorrhage. Cortical spreading depression is characterized by neuronal swelling, profound elevation of extracellular potassium and glutamate, multiphasic blood flow changes, and drop in tissue oxygen tension. The slow speed of the cortical spreading depression wave implies that it is mediated by diffusion of a chemical substance, yet the identity of this substance and the pathway it follows are unknown. Intercellular spread between gap junction-coupled neurons or glial cells and interstitial diffusion of K(+) or glutamate have been proposed. Here we use extracellular direct current potential recordings, K(+)-sensitive microelectrodes, and 2-photon imaging with ultrasensitive Ca(2+) and glutamate fluorescent probes to elucidate the spatiotemporal dynamics of ionic shifts associated with the propagation of cortical spreading depression in the visual cortex of adult living mice. Our data argue against intercellular spread of Ca(2+) carrying the cortical spreading depression wavefront and are in favor of interstitial K(+) diffusion, rather than glutamate diffusion, as the leading event in cortical spreading depression. © The Author 2015. Published by Oxford University Press.
    Full-text · Article · Apr 2015
    • "rAAVs serotype 1 and 2 were generated as described (Tang et al., 2009), and purified by AVB Sepharose affinity chromatography (Smith et al., 2009). For the virus preparation, the genomic titer was determined by Real-Time PCR (∼1.0 × 10 12 viral genomes (vg)/ml, TaqMan Assay, Applied Biosystems). "
    [Show abstract] [Hide abstract] ABSTRACT: Astrocytic endfeet are specialized cell compartments whose important homeostatic roles depend on their enrichment of water and ion channels anchored by the dystrophin associated protein complex (DAPC). This protein complex is known to disassemble in patients with mesial temporal lobe epilepsy and in the latent phase of experimental epilepsies. The mechanistic underpinning of this disassembly is an obvious target of future therapies, but remains unresolved. Here we show in a kainate model of temporal lobe epilepsy that astrocytic endfeet display an enhanced stimulation-evoked Ca(2+) signal that outlast the Ca(2+) signal in the cell bodies. While the amplitude of this Ca(2+) signal is reduced following group I/II metabotropic receptor (mGluR) blockade, the duration is sustained. Based on previous studies it has been hypothesized that the molecular disassembly in astrocytic endfeet is caused by dystrophin cleavage mediated by Ca(2+) dependent proteases. Using a newly developed genetically encoded Ca(2+) sensor, the present study bolsters this hypothesis by demonstrating long-lasting, enhanced stimulation-evoked Ca(2+) signals in astrocytic endfeet.
    Full-text · Article · Feb 2015
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