-
Computationally designed adeno-associated virus(AAV) Rep 78 is
efficiently maintained within anadenovirus vectorVarsha Sitaramana,
Patrick Hearingb, Charles B. Wardc, Dmitri V. Gnatenkoa, Eckard
Wimmerb, Steffen Muellerb,Steven Skienac, and Wadie F. Bahoua,1
Departments of aMedicine, bMolecular Genetics and Microbiology,
and cComputer Sciences, Stony Brook University, Stony Brook, NY
11794
Edited by Thomas Shenk, Princeton University, Princeton, NJ, and
approved July 20, 2011 (received for review February 21, 2011)
Adeno-associated virus (AAV) is a single-stranded parvovirus
retain-ing the unique capacity for site-specific integration into a
transcrip-tionally silent region of the human genome, a
characteristicrequiring the functional properties of the Rep 78/68
polypeptide inconjunction with AAV terminal repeat integrating
elements. Pre-vious strategies designed to assemble these genetic
elements intoadenoviral (Ad) backbones have been limited by the
general in-tolerability of AAV Rep sequences, prompting us to
computationallyreengineer the Rep gene by using synonymous codon
pair recoding.Rep mutants generated by using de novo genome
synthesis main-tained the polypeptide sequence and endonuclease
properties ofRep 78, while dramatically enhancing Ad replication
and viral titeryields, characteristics indistinguishable from
adenovirus lackingcoexpressed Rep. Parallel approaches using domain
swaps encom-passing WT and recoded genomic segments, coupled with
iterativecomputational algorithms, collectively established that 3′
cis-actingRep genetic elements (and not the Rep 78 polypeptide)
retain dom-inant-acting sequences inhibiting Ad replication. These
data provideinsights into the molecular relationships of AAV Rep
and Ad replica-tion, while expanding the applicability of
synonymous codon pairreengineering as a strategy to effect
phenotypic endpoints.
codon pair bias | gene therapy | hybrid virus | systems
biology
Adeno-associated virus (AAV) is a nonpathogenic single-stranded
parvovirus that displays the unique capacity for site-specific
integration into the transcriptionally silent AAVS1 regionof the
human genome located on 19q13.42 (1, 2). The small 4.7-kbAAV genome
encodes three structural capsid (VP1–VP3) andfour nonstructural
replication (Rep) proteins translated from twoORFs, and
transcriptionally regulated by p5 (Rep 78 and Rep 68),p19 (Rep 52
and Rep 40), and p40 (VP1–VP3) promoters(reviewed in ref. 3).
Productive AAV infection requires helperfunctions generally
supplied by adenovirus (Ad) or herpesvirus,and latency likely
occurs by nonhomologous deletion/substitutionevents (4–6),
resulting in head-to-tail stably integrated con-catemers (7, 8).
AAV-mediated site-specific integration requiresAAV Rep 78/68
delivered in trans (9), a cis-acting Rep-bindingelement found
within the flanking terminal repeats (TRs) (10),and a restricted
33-bp cellular sequence withinAAVS1 (5). Recentdata have implicated
a 138-bp integration efficiency element (i.e.,p5IEE) within the p5
promoter as being sufficient and necessaryfor efficient Rep
78/68-mediated site-specific integration (11).The incorporation of
these AAV integrating elements into
larger-capacity hybrid viruses represents a logical strategy
forsite-specific genetic replacement therapies of large
transgenes.Although the AAV integrating elements (i.e., TRs or
p5IEE) arereadily incorporated into herpesvirus (12, 13) or Ad
vectors (14–16), Rep 78/68 is poorly tolerated. Moderate success
has beenachieved with the use of complex homologous
recombinationstrategies (17) and helper-dependent (18) or tightly
regulated(19) expression systems, although the latter two
approaches areadditionally restricted by the helper-dependent
nature of Rep
78-containing Ad. The mechanism of Rep 78/68-mediated
Adinhibition remains incompletely elucidated (20, 21), althoughRep
78 is known to inhibit Ad replication (22), and colocalizes toAd
replication centers to prevent their maturation (23). Fur-thermore,
the complexity of these relationships is highlighted bythe lack of
Rep 78/68-associated Ad inhibition when delivered intrans, such as
in Rep 78/68-expressing cell lines (24).In this article, we have
reengineered the AAV Rep gene by
modifying synonymous codon pairs to phenotypically affect
thereplicative properties of Ad-expressing Rep 78. Although
codonbias (i.e., the preferential use of synonymous codons
duringtranslation) is well recognized, codon pair bias (CPB)
representsa second, independent bias present at multiple
phylogeneticlevels from microorganisms to humans (25–27). Similar
to codonbias, synonymous codons can be paired in multiple ways to
en-code two contiguous amino acids, with evidence for strong CPBas
evidenced by disproportionate over- and under-representationof
codon pairs (28). Previous strategies to modify CPBs havebeen
developed as novel approaches to synthesize attenuatedpoliovirus
(26) and influenza virus (27), although not for char-acterization
and amelioration of cis-inhibitory signals relevant tocomplex viral
interrelationships (e.g., between Ad and AAV).Two computationally
recoded Repmutants differing in their CPBscores (but with preserved
amino acid sequence) considerablyenhanced Ad replication and viral
titer yields while preservingcritical Rep78 endonuclease (i.e.,
excision) capacity. Iterativecomputational algorithms coupled with
genomic domain swapsspecifically established that a dominant,
cis-acting genetic ele-ment(s) was localized to a 3′-Rep sequence,
and that these in-hibitory effects could be ameliorated by
genetically restructuringcodon pairs. These data provide a unique
application of synon-ymous codon pair reengineering to modulate
biological systems.
ResultsComputational Reengineering of AAV Rep Gene. We
constructeda first-generation Ad carrying the AAV2 Rep78 coding
sequenceunder a tightly regulated tetracycline-inducible promoter
withinthe background of an E1/E3-deleted (ΔE1ΔE3) Ad5 virus.
Thisvirus was additionally modified to replace the Ad5 fiber
knobwith that of Ad35 (Ad5/35 chimer) as a strategy for
efficientinfectivity of hematopoietic stem cells (29). Although the
iden-tical tetracycline-inducible Rep expression cassette was
pre-viously used for the successful construction of
helper-dependent
Author contributions: V.S., P.H., C.B.W., E.W., S.M., S.S.,
andW.F.B. designed research; V.S.and P.H. performed research;
C.B.W., E.W., S.M., and S.S. contributed new reagents/analytic
tools; V.S., P.H., D.V.G., E.W., and W.F.B. analyzed data; and
V.S., P.H., and W.F.B.wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1To whom correspondence
should be addressed. E-mail: [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102883108/-/DCSupplemental.
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Ad (19), we noticed that the same construct was incapable
ofreplication in the context of a ΔE1ΔE3 Ad, demonstrating nosigns
of viral growth despite multiple passages in 293 cells overthe
course of 50 d. This effect was noted not only with thisconstruct
(which was designed to solely express Rep78, and notRep 68, Rep 52,
or Rep 40) (19), but also seen using WT Rep. Itremained unclear why
a first-generation ΔE1ΔE3 Ad expressingRep78/68 was not
viable—although consistent with previousobservations (16, 17,
20)—and presumably explained by “leaky”or higher expression levels
of Rep 78 in the context of a ΔE1ΔE3backbone compared with that of
a helper-dependent Ad vector.As an alternative explanation, we
hypothesized that coex-
pression of AAV Rep sequences within the Ad5 backbone
nega-tively modulated Ad replication via dominant-acting
inhibitorysequences. Accordingly, we recoded the 1,866-bp Rep
genomicsegment to precisely preserve the amino acid sequence of the
Rep78 polypeptide while disrupting any cis-acting sequences that
couldputatively inhibit Ad function. Two distinct mutantRep genes
weredesigned by changingCPB for synonymous recoding ofRep 78
(26,
27). A previously developed computational algorithm was
appliedto generate a scrambled (s) Rep mutant by shuffling
synonymouscodon pairs while maintaining codon use and the free
energy offolded RNA to prevent large changes in secondary structure
(26).In parallel, we designed a maximally codon-deoptimized (d)
Repgene (by incorporating under-represented codon pairs) to
assessthe effect of attenuated Rep 78 translation on Ad replication
(Fig.1 A and B and Fig. S1). Both dRep and sRep were generated
byusing de novo genome synthesis and precisely maintained the
na-tive Rep 78 polypeptide sequence.
Recoded AAV Rep Is Sufficient for Replication and Generation
ofΔE1ΔE3 Ad. Infectious plasmid (p) clones containing dRep,sRep, or
WT Rep constructs within the fiber-modified ΔE1ΔE3Ad5/35 chimer
were used for assessment of Ad replication.Following transfection
of 293 cells, complete cytopathic effect(CPE) was observed with
both pAd/sRep and pAd/dRep withinthe first passage and by 15 d
after transfection, in sharp contrastto that seen using pAd/wtRep.
These results paralleled those
5’ 3’-OH
Wild-type AAV genome
-0.4 -0.2 0.0 0.20
2000
4000
6000
8000
10000
dRep78 sRep78 wtRep78
Deoptimized
Scrambled
0.2 0.6 1.0 1.4 1.8
Per
cent
Iden
tity
100
50
50
100
Size (kb)
TR P5 P40 setisecilpS04/86peR91P
P5 P19 P40
2.2
Codon pair bias score
10-1
100
101
102
103
104
105
Day 2 Day 15
Ad/sRep Ad/wtRepAd/AAV/BDD Ad/dRep
Rep
A
B C
Cap
Fig. 1. Characterization of codon-modified AAV Rep. (A)
Annotated schema of the 4,679-bp AAV genome is shown, delineating
the predominant Rep andCap ORFs (47) (Upper), along with an
expanded view (Lower) depicting scrambled and deoptimized homology
alignments to WT Rep nucleotides 1 to 2,400.Sequence identity plots
encompassing recoded base pairs 321 to 2,186 were generated by
using Vista genomic tools (48); the dashed line is set at 75%
identityto WT Rep and areas of white shading depict segments
displaying less than 70% identity. TR, AAV inverted TRs; promoters
(p5, p19, and p40) and Rep 68/40alternate splice sites are shown.
Complete sequences and alignments are provided in Fig. S1. (B) The
calculated CPB score for wtRep 78, sRep 78, and dRep 78are shown
compared with those of fully annotated human genes found in the
RefSeq release 22 database (N = 23,731). Each circle represents the
CPB forindividual genes as a function of amino acid length;
underrepresented codon pairs give negative scores, whereas a
positive CPB indicates the predominantincorporation of
overrepresented codon pairs. The peak distribution of the human
gene set has a positive CPB of 0.07. (C) Ad replication of distinct
constructswas quantified by qPCR by using Ad-specific primers and
DpnI-digested nuclear DNA isolated at designated time points. Ad
genome accumulation wascalculated from triplicate wells (45), and
results are expressed as the mean ± SEM from three complete sets of
experiments.
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obtained by quantitative PCR (qPCR; Fig. 1C), which
demon-strated progressive Ad genome accumulation (for pAd/sRep
andpAd/dRep only) nearly identical to that of the ΔE1ΔE3 Ad/AAV/BDD
hybrid virus lacking the Rep gene (14, 30) (Fig. 2A).These initial
observations collectively established that geneticallyrecoded Rep
fundamentally altered replicative properties of Adwhen carried in
cis. As confirmation, we generated two addi-tional constructs that
substituted the strong constitutively actinghuman small nuclear RNA
HU1-1 promoter (31) in place of thetetracycline-inducible promoter,
again observing complete CPEwithin 15 d after transfection of 293
cells. Furthermore, pro-ductive viral titer concentrations for all
dRep or sRep-expressingviruses were nearly identical, and
comparable to those seen withAd/AAV/BDD (Table 1). These results
contrast sharply with ourconsistent inability to generate Ad when
expressed with wtRep.
Preserved Endonuclease Function of Rep 78. Plasmid transfection
in293 cells established that Rep 78 protein expression was
nearlyidentical between wtRep and sRep with approximately 50%
re-duction of dRep (Fig. 2B), patterns predicted based onCPB
scores.Functional competence of WT and mutant proteins was
estab-lished by an endonuclease assay that evaluates Rep 78’s
ability tocleave the RBS within the folded AAV TRs (32). These
assayswere performed by using Ad/AAV/BDD hybrid virus as
substrate(30), and were initially documented by using plasmid
transfections,and subsequently confirmed by using intact Ad
coinfections (videinfra). Cleavage of the AAV TR by Rep 78 during
Ad DNA rep-lication is expected to release approximately 14 kb
dimeric andapproximately 7 kb monomeric excision products (16)
(Fig. 2A).Transfection of 293 cells with shuttle plasmids
expressing thevarious Rep genes, followed by Ad/AAV/BDD infection,
demon-strated identical monomeric and dimeric excision products for
allthree Rep genes, enhanced in the presence of doxycycline
andcomparable to Rep 78 delivered in trans from an AAV2
plasmidcontaining Rep/Cap genes and endogenous promoters (Fig.
2C).AttenuatedRep 78 protein expression from the dRep gene
product
was associated with diminished generation of monomeric and
di-meric excisional products compared with wtRep and sRep,
asexpected based on the protein expression patterns.We subsequently
used cesium chloride-purified Ad/sRep and
Ad/dRep virus coinfections with Ad/AAV/BDD as
second-tieredconfirmation for Rep 78 endonuclease function in the
context ofproductive and stable (passage 4) Ad generation. Similar
to theresults identified earlier, excision products of the expected
sizewere evident by using both Ad/sRep and Ad/dRep (Fig. 2D),
andidentical to those generated by using a HeLa-derived cell
linestably expressing the AAV Rep 78 and Cap proteins (24).
Asexpected, no excision products were seen in the absence of AAVRep
78. Comparable amounts of dimeric and monomeric excisionproducts
using eitherAd/sReporAd/dRep (comparedwith plasmidtransfections;
Fig. 2C) are presumably explained by logarithmicviral replication
which compensates for deoptimized Rep 78polypeptide expression.
Similarly, we demonstrated that a singlebackbone Ad incorporating
genetically recoded Rep (dRep) andAAV integrating elements retained
the capacity for stable self-excision over three sequential
passages (Fig. S2). These data col-lectively established that
replication competence ofRep-expressingAd could be rescued by using
genetically recoded Rep sequences,and were most consistent with
cis-acting inhibitory sequenceswithin the context of ΔE1ΔE3 Ad and
unrelated to expressionlevels or functional properties of the Rep
78 polypeptide.
Delineation of Rep Inhibitory Sequences That Block ΔE1ΔE3
AdReplication. To more specifically localize the
sequence-specificRep genetic segment(s) that inhibit Ad
replication, we generatedfour Rep chimers encompassing distinct
combinations of wtRep orsRep (Fig. 3A). SRep genetic sequences were
specifically chosen(over dRep) to recapitulate growth
characteristics unrelated toattenuated Rep 78 polypeptide
expression. All constructs wereinserted downstream of the
tetracycline-inducible promoter, andproductive Ad viral replication
was subsequently evaluated in 293cells as delineated earlier. Of
the four constructs evaluated, only
Ad/ReppCMV tTS
A
AdpPF4 BDD AAV TR Ad/AAV/BDDp5IEE
pPF4 BDD AAV TRp5IEE
pPF4 BDD AAV TRp5IEE
pPF4BDDp5IEE
C
dRep or sRep
AdpTK IRES-EGFPRep
D
M
Ad/AAV/BDD excisional monomer (M) Ad/AAV/BDD excisional dimer
(D)
Dox + + + - - -
*
D*
M
D
TRE
~ 7 Kb ~ 14 Kb
Rep 78
GAPDH
B
Fig. 2. Functional analyses of genetically reco-ded Rep 78. (A)
Schema of genetic constructs,viruses, and predicted monomeric and
dimericforms resulting from productive Rep 78 cleavageof Ad/AAV/BDD
[based on previously character-ized models (16, 30)]. ψ, Ad left
end packagingsequence (0.4 kb); pCMV, CMV core promoter(0.8 kb);
tTS, tetracycline (Tet)-controlled tran-scriptional silencer (1.3
kb); TRE, Tet responseelement (0.3 kb); pTK, thymidine kinase
pro-moter (0.2 kb); IRES-EGFP, internal ribosomeentry site with
enhanced GFP (2.0 kb); Ad, Adbase pairs 3,330 to 3,940; BDD, human
B-domaindeleted factor VIII (4.6 kb) (30); pPF4, PF4 pro-moter (1.1
kb) (14); p5IEE (135 bp) and AAV TR(145 bp each plus G–C tail) are
shown. (B)Immunodetection of Rep 78 was established bytransfection
of pFLAG/wtRep, pFLAG/sRep, andpFLAG/dRep in 293 cells, followed by
detectionby using 1:1,000 anti-FLAG (Rep 78) or 1:1,000anti-GAPDH
MAbs as control (10 μg lysates wereloaded per lane). (C) Excision
assays were per-formed by transfecting 293 cells with
individualpAd/Rep plasmids, followed by Ad/AAV/BDD in-fection (MOI
of 50), in the presence (+) or ab-sence (−) of doxycycline (1
μg/mL) for 24 h beforeevaluation of dimeric (D) or monomeric (M)
Ad/AAV/BDD excision products, generated only with functional Rep 78
endonuclease cleavage at the right TR (16).Genomic blots were
performed by using 1 μg DNA per lane, and detected by using the
approximately 750-bp PF4/BDD junction fragment as probe (A). pIM45
isan AAV plasmid expressing WT Rep and Cap genes, used as positive
control for excision. Faint, low-level excision in the absence of
doxycycline is presumablya result of leaky Rep 78 expression. (D)
Excision assays using viral coinfections (MOI of 50) were performed
in 293 (Ad/sRep or Ad/dRep) or C12 cells with Ad/AAV/BDD in the
presence of 1 μg/mL doxycycline for 24 h, and at 48 h, Hirt DNA was
isolated for genomic analysis as previously. C12 are HeLa-derived
stablecell lines that express Rep and Cap upon Ad coinfection, and
are used as positive controls. In C and D, parent Ad/AAV/BDD virus
is depicted by an asterisk.
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Ad/s(wt3) Rep encompassing the approximately 550 bp 3′-se-quence
fromWTRep (nucleotides 1,623–2,186) failed to replicateas long as
50 d after transfection. The other three constructscontaining
3′-scrambled sequences [Ad/s(wt1) Rep, Ad/s(wt2)Rep, and
Ad/s(wt1,2) Rep] demonstrated complete CPE, and viralyields for all
three replicating Ad chimers were nearly identicaland comparable to
those of Ad/dRep, Ad/sRep, and Ad/AAV/BDD (Table 1). These data
provided strong presumptive evidencethat dominant-acting inhibitory
sequences were contained withina restricted genomic segment
encompassing 3′ WT Rep sequen-ces, and that genetically recoded Rep
sequences encompassingthis region sufficiently relieved this
effect.
Delimitation of Rep Inhibitory Sequences Using Combinatorial
GroupTesting. In a final approach, we applied combinatorial group
testing
and balanced Gray codes to both confirm and further delimit
thesequence-specific Rep inhibitory signal in wtRep (Fig. 3B).
Giventhe binary nature of the phenotypic endpoint (i.e., Ad
replication/no replication), we predicted that the replicative
characteristics ofa limited number of WT/scrambled Rep chimers
could be appliedto further delineate distinctRep inhibitory
sequences. Accordingly,we synthesized four chimers, each with 24 −
2 (n = 14) combina-tions of WT or scrambled Rep sequences
encompassing equal-sized (∼135 bp) segments (Fig. S3). These 14
interwoven segmentsof sRep and wtRep thereby provided unique
signatures whosegrowth characteristics would more precisely delimit
Rep inhibitorysequences (note that two homogeneous sRep and wtRep
sequenceswere omitted as experimental controls). The replicative
charac-teristics of these four constructs (Ad/Rep 1, Ad/Rep II,
Ad/Rep III,and Ad/Rep IV) were subsequently studied in ΔE1ΔE3 Ad.
Ad/Rep II,Ad/Rep III, andAd/Rep IV replicated efficiently in 293
cells,althoughAd/Rep I showed no signs of viral replication as long
as 50d after transfection (Fig. 3 C and D and Table 1). These
growthpatterns were entirely concordant with (and served as
independentvalidation of) domain swaps delineated earlier.
Furthermore,growth patterns specifically delimited the 3′-terminal
Rep in-hibitory signals to a discrete 135-bp genomic segment
spanning bp1,782 to 1,916, a region encompassing both the p40
promoter andRep 68/Rep 40 splice site, and previously identified as
critical forRep-dependent p40 promoter activity (33). We then
generatedcomplementation chimers encompassingWT135-bp sequences
onthe background of sRep (wt135/sRep) or 135-bp sRep on
thebackground of WT (s135/wtRep). Both viruses were capable
ofgrowth with slightly attenuated Ad/s135/wtRep viral titers
(Table1), reaffirming the computational predictions and confirming
thata discrete, recoded 135-bp genomic segment was sufficient in
re-lieving Rep-mediated Ad inhibition.
DiscussionWe have genetically recoded the AAV Rep gene by using
syn-onymous codon pair reengineering to overcome Rep’s
inhibitoryeffects on Ad replication. Two computationally
redesignedmutants with distinct CPB scores dramatically enhanced
Adreplication and viral titer yields to levels nearly identical to
thoseof Rep-negative Ad. Distinct complementary approaches
applied
Wild-type Scrambled
Scrambled
ScrambledWild-type
Wild-typeScrambled
Wild-type Scrambled
321 AfeI 2186
S (wt1) Rep
S (wt2) Rep
S (wt3) Rep
S (wt1,2) Rep
A Viability+
+
+
-
BstBI
B Viability-+
+
+
Wild-type sequence
Ad/Rep IIAd/Rep I
Ad/Rep IIIAd/Rep IV
Scrambled Sequence
*
C2 2 2210 10 1010
Ad/Rep II
KbDay
** ****
p
2.0
3.0
1.0
Ad/Rep IAd/Rep IV Ad/Rep III
D
10-1
100
101
102
103
104
105
Ad/sRep Ad/AAV/BDD Ad/Rep IIAd/Rep IVAd/Rep IIIAd/Rep
IAd/wtRep
Day 2 Day 5 Day 10 Day 15
Aden
oviru
s D
NA
( ng/
ngAc
tin)
Fig. 3. Delineation of Rep inhibitory sequences. (A)Chimeric Rep
genes were assembled by poly-nucleotide domain swaps encompassing
discretesegments of WT or scrambled sequences, and viabil-ity
established by Ad replication and titer determi-nations (16) (Table
1 shows detailed viral titers). (B)Four distinct Rep chimers each
containing 14 discrete(132–135 bp) segments of WT or scrambled
sequen-ces were synthesized, and Ad replication (i.e., viabil-ity)
was studied in HEK 293 cells (complete nucleotidesequences are
provided in Fig. S3). Note that thecolumns can be permuted in any
of 14! (∼8.7 × 1010)combinations with equivalent ability to
identify crit-ical domains, provided the sequence is
encompassedwithin one of 14 segments. To minimize the effect
ofsignals on boundaries, columns were ordered tominimize
transitions, in effect creating a balancedGray (binary) code whose
distinct genetic signaturesand phenotypic growth patterns can be
applied fordelineation of the critical Rep inhibitory
segment(delineated by the asterisk, and encompassing WTRep
sequences 1,782–1,916). (C) Southern blot anal-ysis was performed
using DpnI/SbfI double-digestedHirt DNA isolated at day 2 or day 10
from 293 cellstransfected with pAd/Rep I, pAd/Rep II, pAd/Rep
III,or pAd/Rep IV, and detected using Ad base pairs 1–194 as probe.
Note the diminution of DpnI-sensitiveinput plasmid (p) with
concomitant appearance of DpnI-resistant replicated Ad (asterisk)
at D10 for all constructs except Ad/Rep I. (D) Ad genome
accumulationwas determined by qPCR by using viruses isolated at
distinct time points; results are the mean ± SEM from three
complete sets of experiments.
Table 1. Viral titers
Virus Titer* (pfu/mL × 108)
Ad/sRep 9.50 ± 0.50Ad/dRep 9.50 ± 0.25Ad/wtRep No
growthAd/HU1-1/sRep 7.50 ± 0.75Ad/HU1-1/dRep 8.00 ±
0.50Ad/HU1-1/wtRep No growthAd/s(wt1) Rep 8.13 ± 0.12Ad/s(wt2) Rep
8.75 ± 0.25Ad/s(wt1,2) Rep 8.13 ± 0.12Ad/s(wt3) Rep No growthAd/Rep
I No growthAd/Rep II 11.5 ± 0.75Ad/Rep III 10.4 ± 0.37Ad/Rep IV
9.38 ± 0.12Ad/s135/wtRep 2.98 ± 0.50Ad/wt135/sRep 8.00 ±
1.00Ad/AAV/BDD 10.5 ± 0.50
*Titers were calculated by serial dilution and plaque assay in
HEK 293 pack-aging cells, and are reported in pfu/mL as the mean ±
SEM from two distinctdeterminations.
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to further delineate Rep inhibitory sequences included: (i)
do-main swaps encompassing WT and scrambled genomic segments,(ii)
the application of a combinatorial sequence algorithm spe-cifically
designed to sublocalize discrete genomic signals basedon Ad
replicative growth characteristics, and (iii) final validationwith
complementation chimers. These collective strategies pro-duced
concordant conclusions, establishing that 3′-terminal Repsequences
(restricted to discrete genetic elements encompassingbase pairs
1,782–1,916) retain cis-acting inhibitory signals whoseeffects can
be relieved by genetically reengineering codon pairs.Strategies to
computationally redesign large genetic elements
as a means of effecting specific biological functions have
maturedin parallel with technological advances that provide
efficientsynthesis of sizable DNAs (34, 35). We have previously
usedcodon-pair deoptimization methodologies to synthesize
attenu-ated poliovirus (26) and influenza virus (27), and report
here (forthe first time to our knowledge) the application of this
strategyfor identification and strategic amelioration of
cis-inhibitorysignal sequences relevant to complex viral functions.
Surpris-ingly, there is a paucity of experimental evidence focusing
on themechanism(s) or the evolutionary pressures for selective
CPB.Our collective experiences clearly demonstrate that
geneticrecoding strategies designed to usurp this evolutionary
processhave broad applications to modulate diverse biological
systems.What is the mechanism whereby genetically modified Rep
ameliorates Ad replication? Productive AAV infection in
thepresence of Ad causes coordinate induction of p5, p19, and
p40promoters mediated by Rep gene products in trans. To date,
itremains unestablished if these Rep 78 binding interactions
func-tion in concert with other cellular transcription factors.
Optimalinduction of p19 and p40 are dependent on the presence of
mul-tiple cis-acting elements acting in concert (33), and in the
case ofp40, it is intriguing that these elements have beenmapped
(by usingdeletion mutagenesis) to a 90-bp sequence that overlaps
with the135-bp genetic element identified by our data (33).
Multipletranscriptional consensus sequences (i.e., AP1, SP1, GGT)
arefound in the p40 promoter overlap region (Fig. S1),
althoughprevious data are more consistent with complex
DNA-proteinjuxtapositioning of various Rep genetic elements
collectively in-volving p5, p19, and p40 (33). Our data with the
use of positive andnegative complementation chimers further
dissected the criticalfunction of the discrete 135-bp segment in
modulating Ad repli-cation, suggesting the presence of a complex
model involvingnondiscrete, juxtapositioned Rep elements (33).
Alternative ex-planations involving DNA secondary structure, poorly
delineatedconsequences of CPB bias, or the presence of cryptic
AAV-encoded miRNAs remain plausible (36). Given the complexity
ofthese interactions, we speculate that the identification of
thesegenetic elements would not have been elucidated by using
routinemutagenesis strategies involving limited Rep segments, but
ratherrequired the broad-based computational recoding strategy
thatextends beyond mutagenesis of candidate genomic
regions.Insertional mutagenesis remains a fundamental concern
for
long-term gene replacement strategies (37, 38) and for
geneticreprogramming of pluripotent stem cells (39). AAV
site-specificintegration is a unique example of an evolutionarily
developedeukaryotic system that is capable of minimizing adverse
eventsassociated with insertional mutagenesis by targeting a
transcrip-tionally silent region of the human genome. This effect
is clearlycomplicated, and involves not just the Rep
78–Rep-bindingelement–AAVS1 trimolecular complex, but includes
incompletelycharacterized host cell recombination proteins (40).
Furthermore,AAVS1 is located within a gene-dense region of the
genome, withevidence that the majority of viral/cellular junctions
are foundwithin the contiguous MBS85 gene (41, 42). An explanation
forthe relatively benign nature of the disrupted integration site
hasbeen proposed that incorporates partial duplication of the
targetlocus, presumably resulting in a preserved functional copy
of
MBS85 (6). We did not alter the Rep 78 polypeptide or
itsfunctional properties, thereby preserving the DNA binding
(43),ATPase (32), helicase (44), and endonuclease (32) activities
es-sential for targeted AAV site-specific integration
strategies.Identification of genetically modified Rep that is
readily toler-
ated within the Ad genome has implications for gene
therapystrategies at two levels: (i) facilitated assembly of a
single-back-bone delivery system retaining the requisite genetic
elementsnecessary for site-specific integration, and (ii)
generation of one-step packaging systems for recombinant AAV (rAAV)
viral pro-duction (16). Indeed, our initial feasibility studies
confirmed theviability of such a hybrid virus that was stable and
retained self-excision capacity over three passages. rAAV is widely
used asa gene transfer vector retaining the capacity for long-term
extra-chromosomal persistence and transgene expression from
non-integrated genomes (rAAVs are unable to accommodate Repbecause
of size and toxicity constraints) (31). Nonetheless, cur-rent
strategies for rAAV generation and packaging remain cum-bersome,
requiring multiple plasmids and/or helper viruses forproduction of
clinical grade material. Given its high titer and in-fectivity, Ad
carrying an AAV TR-flanked transgene on the samebackbone with sRep
(or dRep) and Cap genes provides the req-uisite elements for
one-step rAAV production.
Materials and MethodsReagents, Ad Substrates, and Cell Lines.
HEK 293 cells, HeLa cells, and HeLa-derived C12 cells expressing
AAV Rep and Cap genes (24) were maintained asadherent monolayers in
DMEM containing 10% FCS. Ad/AAV/BDD was gener-ated from the parent
Ad/AAV hybrid virus expressing human B-domain–deletedfactor VIII
[FVIIIΔ761–1639 (BDD)] (14, 30), and was specifically modified to
in-corporate the platelet factor 4 (PF4) promoter upstream of BDD
(14), and tan-dem 135-bp p5IEEs integrating elements upstream of
the 145-bp AAV TR (11).
Molecular Genetic Studies. Codon pair deoptimized and scrambled
AAV Repsequences were designed by using computational algorithms
(vide infra) (26,27), and synthesized de novo (GenScript). All Rep
constructs were designedwith flanking (i.e., unique) SbfI and SwaI
sites, and a uniquely designed AfeIsite (base pair 981) was
incorporated to facilitate genetic cloning and ma-nipulation.
Constructs were generated by using standard molecular techni-ques,
and fully sequenced to ensure proper assembly. Epitope-tagged Rep
78polypeptide was generated by PCR amplification of sRep, dRep, and
wtReplacking the stop codon, and expressed in-frame with a
C-terminal (3×) FLAGpolypeptide (DYKDDDK) within pCMV-3Tag-3A
(Stratagene) for cellulartransfection and immunodetection (vide
infra). Viral characterization andreplication was established by
qPCR or by genomic blot analysis using DpnI/SbfI-restricted Hirt
DNA and alkaline phosphatase-labeled Ad base pairs 1 to194 as
probe. As DpnI requires dam methylated substrates, it
specificallydigests transfected DNA of bacterial origin, to the
exclusion of hemi/unme-thylated replicating viral DNA. qPCR was
performed on nuclear DNA using Ad-specific primers (Table S1);
quantifications were determined from triplicatewells (45), and
standardized to β-actin to ensure cross-sample comparisons.
Ad Assembly and Characterization. The tetracycline-inducible
Rep78 expres-sion cassette was provided by F. Mavilio (Italian
Institute of Technology, Unitof Molecular Neuroscience, Istituto
Scientifico H. San Raffaele, Milan, Italy)and A. Recchia
(Department of Biomedical Sciences, University of Modenaand Reggio
Emilia, Modena, Italy). All plasmid (p) constructs used for
Adgeneration were assembled in shuttle vectors derived from
pAd/AAV-EGFP-Neo (16), and were generated by homologous
recombination in BJ5183 cellsbetween the shuttle vectors and
pTG3602 ΔE3-F5/35, which contains theintact WT Ad 5 genome (46),
specifically modified to replace the Ad5 fiberknob with that of
Ad35 (29). E1/E3-deleted (ΔE1ΔE3) Ad was generated andtitered in
293 cells as previously described (30).
Rep 78-mediated excision assays were performed in 293 cells with
plasmidtransfections or Ad coinfections, by using the TR within
Ad/AAV/BDD as thesubstrate for endonuclease function. Plasmid
transfections by using pAd/Repshuttle vectors were performed in 293
cells supplemented (or not) with 1 μg/mL doxycycline, followed at
24 h with Ad/AAV/BDD superinfection (50 pfu/cell); excision assays
with viral coinfections were demonstrated by usingidentical
multiplicities of infection (MOIs; 50 pfu/cell). In both
situations,Hirt DNA was isolated 48 h after infection for genomic
analyses by usingdigoxigenin-labeled probes (16, 30).
14298 | www.pnas.org/cgi/doi/10.1073/pnas.1102883108 Sitaraman
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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102883108/-/DCSupplemental/pnas.201102883SI.pdf?targetid=nameddest=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102883108/-/DCSupplemental/pnas.201102883SI.pdf?targetid=nameddest=ST1www.pnas.org/cgi/doi/10.1073/pnas.1102883108
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Immunoblot Analysis. Rep 78 immunodetection was established in
293 cellstransfected with FLAG-tagged Rep plasmids (pFLAG/wtRep,
pFLAG/dRep, orpFLAG/sRep), and at 48 h, protein-solubilized lysates
were prepared for 4% to15% SDS/PAGE and immunoblotting as
previously described (14). Immunode-tection was performed by
enhanced chemiluminescence by using anti-FLAG M2(1:1,000;
Sigma)andanti-GAPDHMAB374 (1:1,000;Millipore) as a loading
control.
Computational Algorithms. The CPB score for the 1,866-bp Rep
sequence wascalculated as the arithmetic mean of individual codon
pair scores (26),updated to incorporate the most recent annotation
of the National Centerfor Biotechnology Information RefSeq data set
(version 22; March 5, 2007).We applied our computational algorithm
to manipulate the CPB of the1,866-bp Rep 78 coding region spanning
base pairs 321 to 2,186 withoutmodifying the initiator methionine.
This recoded segment was designed toprecisely maintain the
polypeptide sequence and the codon use (i.e., thefrequency of use
of each existing codon), thereby “shuffling” existingcodons to
manipulate the CPB. The algorithm uses a mathematical formulafor
simulated annealing suitable for full-length optimization, and
isdesigned to prevent manipulation of regions with large secondary
structuressuch as hairpin or stem loops (26).
We developed a sequence design procedure using concepts from
discretecombinatorial group testing to locate Rep inhibitory
sequences that manifesteither inhibitory or facilitatory phenotypic
functions related to Ad replica-tion [i.e., a balanced Gray
(binary) code]. In this model, four different se-quence designs
(each of which contained discrete 132–135-bp segments ofWT or
scrambled Rep nucleotide sequences) allow for 24 combinations of
WTor scrambled sequences, each with a distinct interwoven pattern
of chimericgenomic segments. These 16 combinations (minus the two
homogenous WTand scrambled combinations) generate columns that can
be permuted in 14!orderings, each of which maintains the structure
to locate critical sequences.We reduced the effect of signals on
boundaries by ordering the columns tominimize the number of
transitions, thereby creating a series of orderedmatrices in which
neighboring regions differ in exactly one of the
fourcomputationally generated mathematical designs.
ACKNOWLEDGMENTS. WethankDr. Jizu Zhi for generation ofVista
plots, andDrs. Nicholas Muzyczka and Paul Freimuth for helpful
discussions. This workwas supported by New York State Stem Cell
Board Grants C024317 and N09S-006 (to W.F.B.); National Institutes
of Health Grants AI075219 (to E.W.) andAI41636 (to P.H.); and
National Science Foundation Grant IIS-1017181 and In-telligence
Community Postdoctoral Fellowship HM1582-07-BAA-0005 (to S.S.).
1. Samulski RJ, et al. (1991) Targeted integration of
adeno-associated virus (AAV) intohuman chromosome 19. EMBO J
10:3941–3950.
2. Kotin RM, et al. (1990) Site-specific integration by
adeno-associated virus. Proc NatlAcad Sci USA 87:2211–2215.
3. Berns KI, Giraud C (1996) Biology of adeno-associated virus.
Curr Top Microbiol Im-munol 218:1–23.
4. McCarty DM, Young SM, Jr., Samulski RJ (2004) Integration of
adeno-associated virus(AAV) and recombinant AAV vectors. Annu Rev
Genet 38:819–845.
5. Linden RM, Winocour E, Berns KI (1996) The recombination
signals for adeno-asso-ciated virus site-specific integration. Proc
Natl Acad Sci USA 93:7966–7972.
6. Henckaerts E, et al. (2009) Site-specific integration of
adeno-associated virus involvespartial duplication of the target
locus. Proc Natl Acad Sci USA 106:7571–7576.
7. Duan D, et al. (1998) Circular intermediates of recombinant
adeno-associated virushave defined structural characteristics
responsible for long-term episomal persistencein muscle tissue. J
Virol 72:8568–8577.
8. Duan D, Yan Z, Yue Y, Engelhardt JF (1999) Structural
analysis of adeno-associatedvirus transduction circular
intermediates. Virology 261:8–14.
9. Surosky RT, et al. (1997) Adeno-associated virus Rep proteins
target DNA sequences toa unique locus in the human genome. J Virol
71:7951–7959.
10. McCarty DM, et al. (1994) Identification of linear DNA
sequences that specifically bindthe adeno-associated virus Rep
protein. J Virol 68:4988–4997.
11. Philpott NJ, Gomos J, Berns KI, Falck-Pedersen E (2002) A p5
integration efficiencyelement mediates Rep-dependent integration
into AAVS1 at chromosome 19. ProcNatl Acad Sci USA
99:12381–12385.
12. Johnston KM, et al. (1997) HSV/AAV hybrid amplicon vectors
extend transgene ex-pression in human glioma cells. Hum Gene Ther
8:359–370.
13. Wang Y, et al. (2002) Herpes simplex virus type
1/adeno-associated virus rep(+) hybridamplicon vector improves the
stability of transgene expression in human cells by site-specific
integration. J Virol 76:7150–7162.
14. Damon AL, et al. (2008) Altered bioavailability of
platelet-derived factor VIII duringthrombocytosis reverses
phenotypic efficacy in haemophilic mice. Thromb
Haemost100:1111–1122.
15. Fisher KJ, Kelley WM, Burda JF, Wilson JM (1996) A novel
adenovirus-adeno-associ-ated virus hybrid vector that displays
efficient rescue and delivery of the AAV ge-nome. Hum Gene Ther
7:2079–2087.
16. Sandalon Z, Gnatenko DV, Bahou WF, Hearing P (2000)
Adeno-associated virus (AAV)Rep protein enhances the generation of
a recombinant mini-adenovirus (Ad) utilizingan Ad/AAV hybrid virus.
J Virol 74:10381–10389.
17. Carlson CA, Shayakhmetov DM, Lieber A (2002) An adenoviral
expression system forAAV rep78 using homologous recombination. Mol
Ther 6:91–98.
18. Wang H, Lieber A (2006) A helper-dependent capsid-modified
adenovirus vectorexpressing adeno-associated virus rep78 mediates
site-specific integration of a 27-kilobase transgene cassette. J
Virol 80:11699–11709.
19. Recchia A, Perani L, Sartori D, Olgiati C, Mavilio F (2004)
Site-specific integration offunctional transgenes into the human
genome by adeno/AAV hybrid vectors. MolTher 10:660–670.
20. Casto BC, Armstrong JA, Atchison RW, HammonWM (1967) Studies
on the relationshipbetween adeno-associated virus type 1 (AAV-1)
and adenoviruses. II. Inhibition ofadenovirus plaques by AAV; its
nature and specificity. Virology 33:452–458.
21. Casto BC, Atchison RW, Hammon WM (1967) Studies on the
relationship betweenadeno-associated virus type I (AAV-1) and
adenoviruses. I. Replication of AAV-1 incertain cell cultures and
its effect on helper adenovirus. Virology 32:52–59.
22. Timpe JM, Verrill KC, Trempe JP (2006) Effects of
adeno-associated virus on adeno-virus replication and gene
expression during coinfection. J Virol 80:7807–7815.
23. WeitzmanMD, Fisher KJ, Wilson JM (1996) Recruitment of
wild-type and recombinantadeno-associated virus into adenovirus
replication centers. J Virol 70:1845–1854.
24. Clark KR, Voulgaropoulou F, Johnson PR (1996) A stable cell
line carrying adenovirus-inducible rep and cap genes allows for
infectivity titration of adeno-associated virusvectors. Gene Ther
3:1124–1132.
25. Gutman GA, Hatfield GW (1989) Nonrandom utilization of codon
pairs in Escherichiacoli. Proc Natl Acad Sci USA 86:3699–3703.
26. Coleman JR, et al. (2008) Virus attenuation by genome-scale
changes in codon pairbias. Science 320:1784–1787.
27. Mueller S, et al. (2010) Live attenuated influenza virus
vaccines by computer-aidedrational design. Nat Biotechnol
28:723–726.
28. Tats A, Tenson T, RemmM (2008) Preferred and avoided codon
pairs in three domainsof life. BMC Genomics 9:463.
29. Shayakhmetov DM, Papayannopoulou T, Stamatoyannopoulos G,
Lieber A (2000)Efficient gene transfer into human CD34(+) cells by
a retargeted adenovirus vector.J Virol 74:2567–2583.
30. Gnatenko DV, et al. (2004) Expression of therapeutic levels
of factor VIII in hemophiliaA mice using a novel
adeno/adeno-associated hybrid virus. Thromb Haemost 92:317–327.
31. Gnatenko DV, et al. (1999) Human factor VIII can be packaged
and functionally ex-pressed in an adeno-associated virus
background: Applicability to haemophilia Agene therapy. Br J
Haematol 104:27–36.
32. Im DS, Muzyczka N (1990) The AAV origin binding protein
Rep68 is an ATP-dependent site-specific endonuclease with DNA
helicase activity. Cell 61:447–457.
33. McCarty DM, Christensen M, Muzyczka N (1991) Sequences
required for coordinateinduction of adeno-associated virus p19 and
p40 promoters by Rep protein. J Virol 65:2936–2945.
34. Mueller S, Papamichail D, Coleman JR, Skiena S, Wimmer E
(2006) Reduction of therate of poliovirus protein synthesis through
large-scale codon deoptimization causesattenuation of viral
virulence by lowering specific infectivity. J Virol
80:9687–9696.
35. Gibson DG, et al. (2010) Creation of a bacterial cell
controlled by a chemically syn-thesized genome. Science
329:52–56.
36. Pfeffer S, et al. (2004) Identification of virus-encoded
microRNAs. Science 304:734–736.
37. Hacein-Bey-Abina S, et al. (2003) LMO2-associated clonal T
cell proliferation in twopatients after gene therapy for SCID-X1.
Science 302:415–419.
38. Cavazzana-Calvo M, et al. (2010) Transfusion independence
and HMGA2 activationafter gene therapy of human β-thalassaemia.
Nature 467:318–322.
39. Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K
(2008) Induced pluripotentstem cells generated without viral
integration. Science 322:945–949.
40. Daya S, Cortez N, Berns KI (2009) Adeno-associated virus
site-specific integration ismediated by proteins of the
nonhomologous end-joining pathway. J Virol 83:11655–11664.
41. Dutheil N, Shi F, Dupressoir T, Linden RM (2000)
Adeno-associated virus site-specifi-cally integrates into a
muscle-specific DNA region. Proc Natl Acad Sci USA
97:4862–4866.
42. Tan I, Ng CH, Lim L, Leung T (2001) Phosphorylation of a
novel myosin binding sub-unit of protein phosphatase 1 reveals a
conserved mechanism in the regulation ofactin cytoskeleton. J Biol
Chem 276:21209–21216.
43. Chiorini JA, et al. (1994) Sequence requirements for stable
binding and function ofRep68 on the adeno-associated virus type 2
inverted terminal repeats. J Virol 68:7448–7457.
44. Wonderling RS, Kyöstiö SR, Owens RA (1995) A maltose-binding
protein/adeno-associated virus Rep68 fusion protein has DNA-RNA
helicase and ATPase activities.J Virol 69:3542–3548.
45. Gnatenko DV, et al. (2003) Transcript profiling of human
platelets using microarrayand serial analysis of gene expression.
Blood 101:2285–2293.
46. Chartier C, et al. (1996) Efficient generation of
recombinant adenovirus vectors byhomologous recombination in
Escherichia coli. J Virol 70:4805–4810.
47. Srivastava A, Lusby EW, Berns KI (1983) Nucleotide sequence
and organization of theadeno-associated virus 2 genome. J Virol
45:555–564.
48. Mayor C, et al. (2000) VISTA: Visualizing global DNA
sequence alignments of arbitrarylength. Bioinformatics
16:1046–1047.
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