Computationally designed adeno-associated virus (AAV) Rep 78 is efficiently maintained within an adenovirus vector Varsha 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 retaining the unique capacity for site-specific integration into a transcriptionally silent region of the human genome, a characteristic requiring the functional properties of the Rep 78/68 polypeptide in conjunction with AAV terminal repeat integrating elements. Previous strategies designed to assemble these genetic elements into adenoviral (Ad) backbones have been limited by the general intolerability of AAV Rep sequences, prompting us to computationally reengineer the Rep gene by using synonymous codon pair recoding. Rep mutants generated by using de novo genome synthesis maintained the polypeptide sequence and endonuclease properties of Rep 78, while dramatically enhancing Ad replication and viral titer yields, characteristics indistinguishable from adenovirus lacking coexpressed Rep. Parallel approaches using domain swaps encompassing WT and recoded genomic segments, coupled with iterative computational algorithms, collectively established that 3′ cis-acting Rep genetic elements (and not the Rep 78 polypeptide) retain dominant-acting sequences inhibiting Ad replication. These data provide insights into the molecular relationships of AAV Rep and Ad replication, while expanding the applicability of synonymous codon pair reengineering as a strategy to effect phenotypic endpoints. codon pair bias

| gene therapy | hybrid virus | systems biology

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deno-associated virus (AAV) is a nonpathogenic singlestranded parvovirus that displays the unique capacity for sitespecific integration into the transcriptionally silent AAVS1 region of the human genome located on 19q13.42 (1, 2). The small 4.7-kb AAV genome encodes three structural capsid (VP1–VP3) and four nonstructural replication (Rep) proteins translated from two ORFs, 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 helper functions generally supplied by adenovirus (Ad) or herpesvirus, and latency likely occurs by nonhomologous deletion/substitution events (4–6), resulting in head-to-tail stably integrated concatemers (7, 8). AAV-mediated site-specific integration requires AAV Rep 78/68 delivered in trans (9), a cis-acting Rep-binding element found within the flanking terminal repeats (TRs) (10), and a restricted 33-bp cellular sequence within AAVS1 (5). Recent data have implicated a 138-bp integration efficiency element (i.e., p5IEE) within the p5 promoter as being sufficient and necessary for 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 for site-specific genetic replacement therapies of large transgenes. Although the AAV integrating elements (i.e., TRs or p5IEE) are readily incorporated into herpesvirus (12, 13) or Ad vectors (14– 16), Rep 78/68 is poorly tolerated. Moderate success has been achieved with the use of complex homologous recombination strategies (17) and helper-dependent (18) or tightly regulated (19) expression systems, although the latter two approaches are additionally restricted by the helper-dependent nature of Rep

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78-containing Ad. The mechanism of Rep 78/68-mediated Ad inhibition remains incompletely elucidated (20, 21), although Rep 78 is known to inhibit Ad replication (22), and colocalizes to Ad replication centers to prevent their maturation (23). Furthermore, the complexity of these relationships is highlighted by the lack of Rep 78/68-associated Ad inhibition when delivered in trans, 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 the replicative properties of Ad-expressing Rep 78. Although codon bias (i.e., the preferential use of synonymous codons during translation) is well recognized, codon pair bias (CPB) represents a second, independent bias present at multiple phylogenetic levels from microorganisms to humans (25–27). Similar to codon bias, synonymous codons can be paired in multiple ways to encode two contiguous amino acids, with evidence for strong CPB as evidenced by disproportionate over- and under-representation of codon pairs (28). Previous strategies to modify CPBs have been developed as novel approaches to synthesize attenuated poliovirus (26) and influenza virus (27), although not for characterization and amelioration of cis-inhibitory signals relevant to complex viral interrelationships (e.g., between Ad and AAV). Two computationally recoded Rep mutants differing in their CPB scores (but with preserved amino acid sequence) considerably enhanced Ad replication and viral titer yields while preserving critical Rep78 endonuclease (i.e., excision) capacity. Iterative computational algorithms coupled with genomic domain swaps specifically established that a dominant, cis-acting genetic element(s) was localized to a 3′-Rep sequence, and that these inhibitory effects could be ameliorated by genetically restructuring codon pairs. These data provide a unique application of synonymous codon pair reengineering to modulate biological systems. Results Computational Reengineering of AAV Rep Gene. We constructed

a first-generation Ad carrying the AAV2 Rep78 coding sequence under a tightly regulated tetracycline-inducible promoter within the background of an E1/E3-deleted (ΔE1ΔE3) Ad5 virus. This virus was additionally modified to replace the Ad5 fiber knob with that of Ad35 (Ad5/35 chimer) as a strategy for efficient infectivity of hematopoietic stem cells (29). Although the identical tetracycline-inducible Rep expression cassette was previously used for the successful construction of helper-dependent

Author contributions: V.S., P.H., C.B.W., E.W., S.M., S.S., and W.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. 1

To 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 of replication in the context of a ΔE1ΔE3 Ad, demonstrating no signs of viral growth despite multiple passages in 293 cells over the course of 50 d. This effect was noted not only with this construct (which was designed to solely express Rep78, and not Rep 68, Rep 52, or Rep 40) (19), but also seen using WT Rep. It remained unclear why a first-generation ΔE1ΔE3 Ad expressing Rep78/68 was not viable—although consistent with previous observations (16, 17, 20)—and presumably explained by “leaky” or higher expression levels of Rep 78 in the context of a ΔE1ΔE3 backbone compared with that of a helper-dependent Ad vector. As an alternative explanation, we hypothesized that coexpression of AAV Rep sequences within the Ad5 backbone negatively modulated Ad replication via dominant-acting inhibitory sequences. Accordingly, we recoded the 1,866-bp Rep genomic segment to precisely preserve the amino acid sequence of the Rep 78 polypeptide while disrupting any cis-acting sequences that could putatively inhibit Ad function. Two distinct mutant Rep genes were designed by changing CPB for synonymous recoding of Rep 78 (26,

Recoded AAV Rep Is Sufficient for Replication and Generation of ΔE1ΔE3 Ad. Infectious plasmid (p) clones containing dRep,

sRep, or WT Rep constructs within the fiber-modified ΔE1ΔE3 Ad5/35 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 within the first passage and by 15 d after transfection, in sharp contrast to that seen using pAd/wtRep. These results paralleled those

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Fig. 1. Characterization of codon-modified AAV Rep. (A) Annotated schema of the 4,679-bp AAV genome is shown, delineating the predominant Rep and Cap 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% identity to 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 68/40 alternate 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 78 are shown compared with those of fully annotated human genes found in the RefSeq release 22 database (N = 23,731). Each circle represents the CPB for individual genes as a function of amino acid length; underrepresented codon pairs give negative scores, whereas a positive CPB indicates the predominant incorporation of overrepresented codon pairs. The peak distribution of the human gene set has a positive CPB of 0.07. (C) Ad replication of distinct constructs was quantified by qPCR by using Ad-specific primers and DpnI-digested nuclear DNA isolated at designated time points. Ad genome accumulation was calculated from triplicate wells (45), and results are expressed as the mean ± SEM from three complete sets of experiments.

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27). A previously developed computational algorithm was applied to generate a scrambled (s) Rep mutant by shuffling synonymous codon pairs while maintaining codon use and the free energy of folded RNA to prevent large changes in secondary structure (26). In parallel, we designed a maximally codon-deoptimized (d) Rep gene (by incorporating under-represented codon pairs) to assess the effect of attenuated Rep 78 translation on Ad replication (Fig. 1 A and B and Fig. S1). Both dRep and sRep were generated by using de novo genome synthesis and precisely maintained the native Rep 78 polypeptide sequence.

obtained by quantitative PCR (qPCR; Fig. 1C), which demonstrated progressive Ad genome accumulation (for pAd/sRep and pAd/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 genetically recoded Rep fundamentally altered replicative properties of Ad when carried in cis. As confirmation, we generated two additional constructs that substituted the strong constitutively acting human small nuclear RNA HU1-1 promoter (31) in place of the tetracycline-inducible promoter, again observing complete CPE within 15 d after transfection of 293 cells. Furthermore, productive viral titer concentrations for all dRep or sRep-expressing viruses were nearly identical, and comparable to those seen with Ad/AAV/BDD (Table 1). These results contrast sharply with our consistent inability to generate Ad when expressed with wtRep. Preserved Endonuclease Function of Rep 78. Plasmid transfection in 293 cells established that Rep 78 protein expression was nearly identical between wtRep and sRep with approximately 50% reduction of dRep (Fig. 2B), patterns predicted based on CPB scores. Functional competence of WT and mutant proteins was established by an endonuclease assay that evaluates Rep 78’s ability to cleave the RBS within the folded AAV TRs (32). These assays were 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 (vide infra). Cleavage of the AAV TR by Rep 78 during Ad DNA replication is expected to release approximately 14 kb dimeric and approximately 7 kb monomeric excision products (16) (Fig. 2A). Transfection of 293 cells with shuttle plasmids expressing the various Rep genes, followed by Ad/AAV/BDD infection, demonstrated identical monomeric and dimeric excision products for all three Rep genes, enhanced in the presence of doxycycline and comparable to Rep 78 delivered in trans from an AAV2 plasmid containing Rep/Cap genes and endogenous promoters (Fig. 2C). Attenuated Rep 78 protein expression from the dRep gene product

was associated with diminished generation of monomeric and dimeric excisional products compared with wtRep and sRep, as expected 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-tiered confirmation for Rep 78 endonuclease function in the context of productive and stable (passage 4) Ad generation. Similar to the results identified earlier, excision products of the expected size were evident by using both Ad/sRep and Ad/dRep (Fig. 2D), and identical to those generated by using a HeLa-derived cell line stably expressing the AAV Rep 78 and Cap proteins (24). As expected, no excision products were seen in the absence of AAV Rep 78. Comparable amounts of dimeric and monomeric excision products using either Ad/sRep or Ad/dRep (compared with plasmid transfections; Fig. 2C) are presumably explained by logarithmic viral replication which compensates for deoptimized Rep 78 polypeptide expression. Similarly, we demonstrated that a single backbone Ad incorporating genetically recoded Rep (dRep) and AAV integrating elements retained the capacity for stable selfexcision over three sequential passages (Fig. S2). These data collectively established that replication competence of Rep-expressing Ad could be rescued by using genetically recoded Rep sequences, and were most consistent with cis-acting inhibitory sequences within the context of ΔE1ΔE3 Ad and unrelated to expression levels or functional properties of the Rep 78 polypeptide. Delineation of Rep Inhibitory Sequences That Block ΔE1ΔE3 Ad Replication. To more specifically localize the sequence-specific

Rep genetic segment(s) that inhibit Ad replication, we generated four Rep chimers encompassing distinct combinations of wtRep or sRep (Fig. 3A). SRep genetic sequences were specifically chosen (over dRep) to recapitulate growth characteristics unrelated to attenuated Rep 78 polypeptide expression. All constructs were inserted downstream of the tetracycline-inducible promoter, and productive Ad viral replication was subsequently evaluated in 293 cells as delineated earlier. Of the four constructs evaluated, only

dRep or sRep Fig. 2. Functional analyses of genetically recoded Rep 78. (A) Schema of genetic constructs, Ad/Rep pCMV tTS Rep IRES-EGFP Ad TRE pTK viruses, and predicted monomeric and dimeric forms resulting from productive Rep 78 cleavage p5IEE of Ad/AAV/BDD [based on previously characterAd/AAV/BDD pPF4 BDD AAV TR Ad ized models (16, 30)]. ψ, Ad left end packaging sequence (0.4 kb); pCMV, CMV core promoter (0.8 kb); tTS, tetracycline (Tet)-controlled tran~ 7 Kb ~ 14 Kb p5IEE p5IEE p5IEE scriptional silencer (1.3 kb); TRE, Tet response pPF4 BDD AAV TR pPF4 BDD AAV TR BDD pPF4 element (0.3 kb); pTK, thymidine kinase promoter (0.2 kb); IRES-EGFP, internal ribosome Ad/AAV/BDD excisional monomer (M) Ad/AAV/BDD excisional dimer (D) entry site with enhanced GFP (2.0 kb); Ad, Ad base pairs 3,330 to 3,940; BDD, human B-domain deleted factor VIII (4.6 kb) (30); pPF4, PF4 promoter (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 by Rep 78 * transfection of pFLAG/wtRep, pFLAG/sRep, and * D pFLAG/dRep in 293 cells, followed by detection D by using 1:1,000 anti-FLAG (Rep 78) or 1:1,000 GAPDH M M anti-GAPDH MAbs as control (10 μg lysates were loaded per lane). (C) Excision assays were per+ + + - - Dox formed by transfecting 293 cells with individual pAd/Rep plasmids, followed by Ad/AAV/BDD infection (MOI of 50), in the presence (+) or absence (−) of doxycycline (1 μg/mL) for 24 h before evaluation 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 is an AAV plasmid expressing WT Rep and Cap genes, used as positive control for excision. Faint, low-level excision in the absence of doxycycline is presumably a 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 stable cell 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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Virus Ad/sRep Ad/dRep Ad/wtRep Ad/HU1-1/sRep Ad/HU1-1/dRep Ad/HU1-1/wtRep Ad/s(wt1) Rep Ad/s(wt2) Rep Ad/s(wt1,2) Rep Ad/s(wt3) Rep Ad/Rep I Ad/Rep II Ad/Rep III Ad/Rep IV Ad/s135/wtRep Ad/wt135/sRep Ad/AAV/BDD

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9.50 ± 0.50 9.50 ± 0.25 No growth 7.50 ± 0.75 8.00 ± 0.50 No growth 8.13 ± 0.12 8.75 ± 0.25 8.13 ± 0.12 No growth No growth 11.5 ± 0.75 10.4 ± 0.37 9.38 ± 0.12 2.98 ± 0.50 8.00 ± 1.00 10.5 ± 0.50

*Titers were calculated by serial dilution and plaque assay in HEK 293 packaging cells, and are reported in pfu/mL as the mean ± SEM from two distinct determinations.

Ad/s(wt3) Rep encompassing the approximately 550 bp 3′-sequence from WT Rep (nucleotides 1,623–2,186) failed to replicate as long as 50 d after transfection. The other three constructs containing 3′-scrambled sequences [Ad/s(wt1) Rep, Ad/s(wt2) Rep, and Ad/s(wt1,2) Rep] demonstrated complete CPE, and viral yields for all three replicating Ad chimers were nearly identical and comparable to those of Ad/dRep, Ad/sRep, and Ad/AAV/ BDD (Table 1). These data provided strong presumptive evidence that dominant-acting inhibitory sequences were contained within a restricted genomic segment encompassing 3′ WT Rep sequences, and that genetically recoded Rep sequences encompassing this region sufficiently relieved this effect. Delimitation of Rep Inhibitory Sequences Using Combinatorial Group Testing. In a final approach, we applied combinatorial group testing

and balanced Gray codes to both confirm and further delimit the sequence-specific Rep inhibitory signal in wtRep (Fig. 3B). Given the binary nature of the phenotypic endpoint (i.e., Ad replication/ no replication), we predicted that the replicative characteristics of a limited number of WT/scrambled Rep chimers could be applied to further delineate distinct Rep inhibitory sequences. Accordingly, we synthesized four chimers, each with 24 − 2 (n = 14) combinations of WT or scrambled Rep sequences encompassing equalsized (∼135 bp) segments (Fig. S3). These 14 interwoven segments of sRep and wtRep thereby provided unique signatures whose growth characteristics would more precisely delimit Rep inhibitory sequences (note that two homogeneous sRep and wtRep sequences were omitted as experimental controls). The replicative characteristics 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, and Ad/Rep IV replicated efficiently in 293 cells, although Ad/Rep I showed no signs of viral replication as long as 50 d after transfection (Fig. 3 C and D and Table 1). These growth patterns were entirely concordant with (and served as independent validation of) domain swaps delineated earlier. Furthermore, growth patterns specifically delimited the 3′-terminal Rep inhibitory signals to a discrete 135-bp genomic segment spanning bp 1,782 to 1,916, a region encompassing both the p40 promoter and Rep 68/Rep 40 splice site, and previously identified as critical for Rep-dependent p40 promoter activity (33). We then generated complementation chimers encompassing WT 135-bp sequences on the background of sRep (wt135/sRep) or 135-bp sRep on the background of WT (s135/wtRep). Both viruses were capable of growth with slightly attenuated Ad/s135/wtRep viral titers (Table 1), reaffirming the computational predictions and confirming that a discrete, recoded 135-bp genomic segment was sufficient in relieving Rep-mediated Ad inhibition. Discussion We have genetically recoded the AAV Rep gene by using synonymous codon pair reengineering to overcome Rep’s inhibitory effects on Ad replication. Two computationally redesigned mutants with distinct CPB scores dramatically enhanced Ad replication and viral titer yields to levels nearly identical to those of Rep-negative Ad. Distinct complementary approaches applied

321 AfeI BstBI 2186 Fig. 3. Delineation of Rep inhibitory sequences. (A) Viability Chimeric Rep genes were assembled by polyS (wt1) Rep Wild-type Scrambled + nucleotide domain swaps encompassing discrete S (wt2) Rep Scrambled Wild-type Scrambled + segments of WT or scrambled sequences, and viability established by Ad replication and titer determiS (wt3) Rep Scrambled Wild-type nations (16) (Table 1 shows detailed viral titers). (B) S (wt1,2) Rep Wild-type Scrambled + Four distinct Rep chimers each containing 14 discrete (132–135 bp) segments of WT or scrambled sequenViability * ces were synthesized, and Ad replication (i.e., viabilAd/Rep I ity) was studied in HEK 293 cells (complete nucleotide Ad/Rep II + sequences are provided in Fig. S3). Note that the Ad/Rep III + 10 Ad/Rep IV columns can be permuted in any of 14! (∼8.7 × 10 ) + combinations with equivalent ability to identify critWild-type sequence Scrambled Sequence ical domains, provided the sequence is encompassed within one of 14 segments. To minimize the effect of 10 Ad/sRep signals on boundaries, columns were ordered to Ad/Rep IV Ad/Rep III Ad/Rep I Ad/Rep II Ad/AAV/BDD 10 Ad/Rep II minimize transitions, in effect creating a balanced Kb Day Ad/Rep IV 2 10 2 10 2 10 2 10 Gray (binary) code whose distinct genetic signatures Ad/Rep III 3.0 10 Ad/Rep I * and phenotypic growth patterns can be applied for * * Ad/wtRep * * * delineation of the critical Rep inhibitory segment 10 2.0 (delineated by the asterisk, and encompassing WT 10 Rep sequences 1,782–1,916). (C) Southern blot analysis was performed using DpnI/SbfI double-digested p 10 1.0 Hirt DNA isolated at day 2 or day 10 from 293 cells transfected with pAd/Rep I, pAd/Rep II, pAd/Rep III, 10 or pAd/Rep IV, and detected using Ad base pairs 1– Day 2 Day 5 Day 10 Day 15 194 as probe. Note the diminution of DpnI-sensitive input plasmid (p) with concomitant appearance of DpnI-resistant replicated Ad (asterisk) at D10 for all constructs except Ad/Rep I. (D) Ad genome accumulation was determined by qPCR by using viruses isolated at distinct time points; results are the mean ± SEM from three complete sets of experiments.

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Table 1. Viral titers

to further delineate Rep inhibitory sequences included: (i) domain swaps encompassing WT and scrambled genomic segments, (ii) the application of a combinatorial sequence algorithm specifically designed to sublocalize discrete genomic signals based on Ad replicative growth characteristics, and (iii) final validation with complementation chimers. These collective strategies produced concordant conclusions, establishing that 3′-terminal Rep sequences (restricted to discrete genetic elements encompassing base pairs 1,782–1,916) retain cis-acting inhibitory signals whose effects can be relieved by genetically reengineering codon pairs. Strategies to computationally redesign large genetic elements as a means of effecting specific biological functions have matured in parallel with technological advances that provide efficient synthesis of sizable DNAs (34, 35). We have previously used codon-pair deoptimization methodologies to synthesize attenuated poliovirus (26) and influenza virus (27), and report here (for the first time to our knowledge) the application of this strategy for identification and strategic amelioration of cis-inhibitory signal sequences relevant to complex viral functions. Surprisingly, there is a paucity of experimental evidence focusing on the mechanism(s) or the evolutionary pressures for selective CPB. Our collective experiences clearly demonstrate that genetic recoding strategies designed to usurp this evolutionary process have broad applications to modulate diverse biological systems. What is the mechanism whereby genetically modified Rep ameliorates Ad replication? Productive AAV infection in the presence of Ad causes coordinate induction of p5, p19, and p40 promoters mediated by Rep gene products in trans. To date, it remains unestablished if these Rep 78 binding interactions function in concert with other cellular transcription factors. Optimal induction of p19 and p40 are dependent on the presence of multiple cis-acting elements acting in concert (33), and in the case of p40, it is intriguing that these elements have been mapped (by using deletion mutagenesis) to a 90-bp sequence that overlaps with the 135-bp genetic element identified by our data (33). Multiple transcriptional consensus sequences (i.e., AP1, SP1, GGT) are found in the p40 promoter overlap region (Fig. S1), although previous data are more consistent with complex DNA-protein juxtapositioning of various Rep genetic elements collectively involving p5, p19, and p40 (33). Our data with the use of positive and negative complementation chimers further dissected the critical function of the discrete 135-bp segment in modulating Ad replication, suggesting the presence of a complex model involving nondiscrete, juxtapositioned Rep elements (33). Alternative explanations involving DNA secondary structure, poorly delineated consequences of CPB bias, or the presence of cryptic AAVencoded miRNAs remain plausible (36). Given the complexity of these interactions, we speculate that the identification of these genetic elements would not have been elucidated by using routine mutagenesis strategies involving limited Rep segments, but rather required the broad-based computational recoding strategy that extends beyond mutagenesis of candidate genomic regions. Insertional mutagenesis remains a fundamental concern for long-term gene replacement strategies (37, 38) and for genetic reprogramming of pluripotent stem cells (39). AAV site-specific integration is a unique example of an evolutionarily developed eukaryotic system that is capable of minimizing adverse events associated with insertional mutagenesis by targeting a transcriptionally silent region of the human genome. This effect is clearly complicated, and involves not just the Rep 78–Rep-binding element–AAVS1 trimolecular complex, but includes incompletely characterized host cell recombination proteins (40). Furthermore, AAVS1 is located within a gene-dense region of the genome, with evidence that the majority of viral/cellular junctions are found within the contiguous MBS85 gene (41, 42). An explanation for the relatively benign nature of the disrupted integration site has been proposed that incorporates partial duplication of the target locus, presumably resulting in a preserved functional copy of 14298 | www.pnas.org/cgi/doi/10.1073/pnas.1102883108

MBS85 (6). We did not alter the Rep 78 polypeptide or its functional properties, thereby preserving the DNA binding (43), ATPase (32), helicase (44), and endonuclease (32) activities essential for targeted AAV site-specific integration strategies. Identification of genetically modified Rep that is readily tolerated within the Ad genome has implications for gene therapy strategies at two levels: (i) facilitated assembly of a single-backbone delivery system retaining the requisite genetic elements necessary for site-specific integration, and (ii) generation of onestep packaging systems for recombinant AAV (rAAV) viral production (16). Indeed, our initial feasibility studies confirmed the viability of such a hybrid virus that was stable and retained selfexcision capacity over three passages. rAAV is widely used as a gene transfer vector retaining the capacity for long-term extrachromosomal persistence and transgene expression from nonintegrated genomes (rAAVs are unable to accommodate Rep because of size and toxicity constraints) (31). Nonetheless, current strategies for rAAV generation and packaging remain cumbersome, requiring multiple plasmids and/or helper viruses for production of clinical grade material. Given its high titer and infectivity, Ad carrying an AAV TR-flanked transgene on the same backbone with sRep (or dRep) and Cap genes provides the requisite elements for one-step rAAV production. Materials and Methods Reagents, Ad Substrates, and Cell Lines. HEK 293 cells, HeLa cells, and HeLaderived C12 cells expressing AAV Rep and Cap genes (24) were maintained as adherent monolayers in DMEM containing 10% FCS. Ad/AAV/BDD was generated from the parent Ad/AAV hybrid virus expressing human B-domain–deleted factor VIII [FVIIIΔ761–1639 (BDD)] (14, 30), and was specifically modified to incorporate the platelet factor 4 (PF4) promoter upstream of BDD (14), and tandem 135-bp p5IEEs integrating elements upstream of the 145-bp AAV TR (11). Molecular Genetic Studies. Codon pair deoptimized and scrambled AAV Rep sequences were designed by using computational algorithms (vide infra) (26, 27), and synthesized de novo (GenScript). All Rep constructs were designed with flanking (i.e., unique) SbfI and SwaI sites, and a uniquely designed AfeI site (base pair 981) was incorporated to facilitate genetic cloning and manipulation. Constructs were generated by using standard molecular techniques, and fully sequenced to ensure proper assembly. Epitope-tagged Rep 78 polypeptide was generated by PCR amplification of sRep, dRep, and wtRep lacking the stop codon, and expressed in-frame with a C-terminal (3×) FLAG polypeptide (DYKDDDK) within pCMV-3Tag-3A (Stratagene) for cellular transfection and immunodetection (vide infra). Viral characterization and replication was established by qPCR or by genomic blot analysis using DpnI/ SbfI-restricted Hirt DNA and alkaline phosphatase-labeled Ad base pairs 1 to 194 as probe. As DpnI requires dam methylated substrates, it specifically digests transfected DNA of bacterial origin, to the exclusion of hemi/unmethylated replicating viral DNA. qPCR was performed on nuclear DNA using Adspecific primers (Table S1); quantifications were determined from triplicate wells (45), and standardized to β-actin to ensure cross-sample comparisons. Ad Assembly and Characterization. The tetracycline-inducible Rep78 expression cassette was provided by F. Mavilio (Italian Institute of Technology, Unit of Molecular Neuroscience, Istituto Scientifico H. San Raffaele, Milan, Italy) and A. Recchia (Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy). All plasmid (p) constructs used for Ad generation were assembled in shuttle vectors derived from pAd/AAV-EGFPNeo (16), and were generated by homologous recombination in BJ5183 cells between the shuttle vectors and pTG3602 ΔE3-F5/35, which contains the intact WT Ad 5 genome (46), specifically modified to replace the Ad5 fiber knob with that of Ad35 (29). E1/E3-deleted (ΔE1ΔE3) Ad was generated and titered in 293 cells as previously described (30). Rep 78-mediated excision assays were performed in 293 cells with plasmid transfections or Ad coinfections, by using the TR within Ad/AAV/BDD as the substrate for endonuclease function. Plasmid transfections by using pAd/Rep shuttle 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 using identical multiplicities of infection (MOIs; 50 pfu/cell). In both situations, Hirt DNA was isolated 48 h after infection for genomic analyses by using digoxigenin-labeled probes (16, 30).

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Computational Algorithms. The CPB score for the 1,866-bp Rep sequence was calculated as the arithmetic mean of individual codon pair scores (26), updated to incorporate the most recent annotation of the National Center for Biotechnology Information RefSeq data set (version 22; March 5, 2007). We applied our computational algorithm to manipulate the CPB of the 1,866-bp Rep 78 coding region spanning base pairs 321 to 2,186 without modifying the initiator methionine. This recoded segment was designed to precisely maintain the polypeptide sequence and the codon use (i.e., the frequency of use of each existing codon), thereby “shuffling” existing codons to manipulate the CPB. The algorithm uses a mathematical formula for simulated annealing suitable for full-length optimization, and is designed to prevent manipulation of regions with large secondary structures such as hairpin or stem loops (26).

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We developed a sequence design procedure using concepts from discrete combinatorial group testing to locate Rep inhibitory sequences that manifest either inhibitory or facilitatory phenotypic functions related to Ad replication [i.e., a balanced Gray (binary) code]. In this model, four different sequence designs (each of which contained discrete 132–135-bp segments of WT or scrambled Rep nucleotide sequences) allow for 24 combinations of WT or scrambled sequences, each with a distinct interwoven pattern of chimeric genomic segments. These 16 combinations (minus the two homogenous WT and 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 to minimize the number of transitions, thereby creating a series of ordered matrices in which neighboring regions differ in exactly one of the four computationally generated mathematical designs. ACKNOWLEDGMENTS. We thank Dr. Jizu Zhi for generation of Vista plots, and Drs. Nicholas Muzyczka and Paul Freimuth for helpful discussions. This work was supported by New York State Stem Cell Board Grants C024317 and N09S006 (to W.F.B.); National Institutes of Health Grants AI075219 (to E.W.) and AI41636 (to P.H.); and National Science Foundation Grant IIS-1017181 and Intelligence Community Postdoctoral Fellowship HM1582-07-BAA-0005 (to S.S.).

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PNAS | August 23, 2011 | vol. 108 | no. 34 | 14299

MICROBIOLOGY

Immunoblot Analysis. Rep 78 immunodetection was established in 293 cells transfected with FLAG-tagged Rep plasmids (pFLAG/wtRep, pFLAG/dRep, or pFLAG/sRep), and at 48 h, protein-solubilized lysates were prepared for 4% to 15% SDS/PAGE and immunoblotting as previously described (14). Immunodetection was performed by enhanced chemiluminescence by using anti-FLAG M2 (1:1,000; Sigma) and anti-GAPDH MAB374 (1:1,000; Millipore) as a loading control.