Therapy (2010) 17, 1175–1180 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 www.nature.com/gt ORIGINAL ARTICLE Self-complementary AAV mediates gene targeting and enhances delivery for double-strand break repair

ML Hirsch1, L Green1, MH Porteus2,3 and RJ Samulski1,4 1UNC Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA; 3Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA and 4Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Adeno-associated virus (AAV) mediates gene targeting in ITR sequences were also investigated in a plasmid repair by providing exogenous DNA for allelic replacement context. No correction was noted in the absence of a DSB, through . In comparison to other however, a modest inhibitory effect correlated with the methods of DNA delivery or alternative DNA substrates, AAV increasing presence of ITR sequences. Similarly, signaling gene targeting is reported to be very efficient, perhaps due to cascades stimulated upon recombinant AAV had its single-stranded DNA genome, the inverted terminal repeats no effect on plasmid-mediated DSB repair. Noteworthy, was (ITRs), and/or the consequence of induced cellular signals on the 20-fold additional enhancement in reporter correction infection or uncoating. These viral attributes were investigated using scAAV vectors, over ss versions, to deliver both the in the presence and absence of an I-Sce endonuclease- repair substrate and the endonuclease. In this case, homo- induced double-strand break (DSB) within a chromosomal logous recombination repaired the defective reporter in 4% of defective reporter in embryonic kidney cells. Gene cells without any selection. This report provides novel insights correction was evaluated using self-complementary (sc) AAV, regarding the recombination substrates used by AAV vectors which forms a duplexed DNA molecule and results in earlier in promoting homologous recombination and points to the and robust expression compared with conventional initial steps in vector optimization that could facilitate their use single-strand (ss) AAV genomes. An scAAV repair substrate in gene correction of genetic disorders. was modestly enhanced for reporter correction showing no Gene Therapy (2010) 17, 1175–1180; doi:10.1038/gt.2010.65; dependency on ssAAV genomes for this process. The AAV published online 13 May 2010

Keywords: adeno-associated virus; gene targeting; self-complementary AAV; double-strand break; gene conversion

Introduction or alternate virus-mediated recombination.1,6,7 AAV is a single-strand (ss) DNA dependovirus of the family, Gene targeting is the process in which exogenously Parvoviridae. The 4.7 kb genome is flanked by 145 delivered nucleic acids are used to modify a partially nucleotide inverted terminal repeats (ITRs). In the homologous target by homologous recombination.1–3 absence of selection, AAV-induced homologous recom- This ability is largely conserved across kingdoms, and bination levels have been as high as 1%.8 Several reports early work in embryonic stem cells showed gene have characterized this process and note that the knockout technology using plasmid DNA for repair.1,4 efficiency of the event depends on several factors The transition of this powerful approach to human including (1) type of target mutation, (2) length of repair genetic correction for therapeutic applications, however, homology, (3) position of the site of repair on the AAV is limited by the relative inefficiency of the repair process genome with respect to the viral ITRs, (4) cell cycle and (about one in a million) in addition to challenges for (5) chromosomal context.9–12 effective DNA delivery. AAV is efficient for gene targeting perhaps due to Following the of the adeno-associated virus nuclear localization of a high number of genomes, the (AAV) genome and subsequent applications for recombinogenic ITRs and/or the ss nature of the AAV therapeutic gene addition in humans,5 the recombinant vector template.9 Consistent with the third hypothesis virus was noted to be enhanced several orders of was the observation that duplexed AAV genomes, magnitude for gene targeting, compared with plasmid from smaller than wild-type packaging cassettes, when mixed with ss genomes, did not contribute significantly to gene targeting.9 However, the importance of an ss Correspondence: Dr RJ Samulski, UNC Gene Therapy Center, repair substrate is logical, as no helicase would be University of North Carolina at Chapel Hill, Campus Box 7352, necessary for replication protein A binding, followed by Chapel Hill, NC 27599-7352, USA. E-mail: [email protected] heteroduplex formation, strand exchange or synthesis- Received 31 January 2010; revised 23 February 2010; accepted 23 dependent strand annealing, and subsequent opposite February 2010; published online 13 May 2010 strand repair. Targeting by scAAV M Hirsch et al 1176 Although ss genome dependency for AAV gene a ss-gfpR sc-gfpR targeting represents an attractive hypothesis, it remains .25 kb 1 kb .25 kb 1 kb at odds with the observations that both increased time and transduced genomes enhance this process.8,13 In human cell cultures, ssAAV2 initiates transgene syn- 3.4 kb 4 kb thesis soon after infection (green fluorescent protein (Gfp) fluorescence is detectable as early as 6 h), confirm- ing that at least some AAV genomes are not ss (data not shown (DNS)). In fact, the duplexed genomes are a natural part of AAV transduction and are generated in two ways: (1) synthesis of the complementary strand using ITR priming or (2) by opposite polarity genome annealing (both + and À are packaged at similar 14,15 b AAV2-gfpR efficiencies). Although the chosen mechanism ap- 0.8 pears cell line and specific, it is also a function of 0.7 ss the multiplicity of infection. At higher multiplicity 0.6 sc 0.5 + DSB of infections, AAV genomes likely anneal, yet may still 0.4 undergo second-strand synthesis, and both processes 0.3 would increase over time shifting the equilibrium away 0.2 15 % Gfp+ cells 0.1 - DSB from ss and toward duplexed genomes. These con- 0 siderations suggest that all of the above-mentioned AAV 0123456789 gene targeting experiments most likely produced a Day post-infection significant mixture of AAV vector DNA templates (that is, ss or duplexed) making it virtually impossible to Figure 1 AAV gene targeting is stimulated by a local double- 8,12 strand break. (a) Southern blot analysis of purified single-strand (ss) assign targeting efficiency to a particular substrate. or (sc) AAV2-gfp repair (R) genomes. The grouped columns show More importantly, circular DNA substrates unrelated to consecutive twofold dilutions. An illustration depicts a possible AAV virion delivery, or the presence of the ITRs, showed conformation of the genomes and in each case gfpR homology is nearly identical results from a direct comparison of ss identical (50 homologous arm is 0.25 kb and the 30 homologous arm and duplexed DNA for mammalian gene targeting.16 is 1 kb). The ssAAV genome also contains 2.1 kb of stuffer DNA Similar to AAV second-strand synthesis, this report also sequence located downstream of the gfp repair homology for a total genome size of 3.4 kb. The scAAV genome contains stuffer DNA notes synthesis of a complementary strand for the M13 sequence as well, 0.46 kb upstream and 0.29 kb downstream of the produced ss circular DNA even in the absence of a gfp repair homology (final genome size of about 4 kb). (b) The 16 mammalian replication origin. defective gfp 293 cell line was infected with either ss or sc AAV2-gfp repair (R) at a gcp/cell ratio of 10 000. Simultaneously, cells were also transduced by another vector, either ssAAV2-factor9 repair (no double-strand break (no ÀDSB)) or ssAAV2-Sce (+DSB) at 10 000 Results gcp per cell. Gfp+ cells were quantitated by flow cytometry at the indicated times. To investigate whether ss or duplexed AAV genomes are preferred in gene targeting, we used our previously described self-complementary (sc) context.17 Such parti- cles harbor a single DNA molecule with a hinge (mutant the nature of the repair genome (Figure 2b). However, a ITR) at one end and near-perfect complementarity modest increase was noted for scAAV over ssAAV, with thereon.17 Theoretically, these molecules snap back on targeting values of 0.004 and 0.002%, respectively. From themselves forming duplexed DNA. Physiologically, these experiments, Gfp+ cells were isolated by fluores- both in cell lines and in vivo, scAAV results in earlier cence-activated cells sorting and sequence analysis of ten and dramatically enhanced transgene synthesis clones from either group (ss or sc) suggested error-free compared with conventional ssAAV.17,18 homologous recombination as the mechanism of repair Using a human embryonic kidney cell line containing (DNS). a chromosome defective gfp reporter harboring a 35 bp Many reports using various technologies agree that a insertion,19 we performed a head to head comparison of local double-strand break (DSB) increases targeted gene ssAAV2 versus scAAV2 repair substrates (Figure 1). The correction.21–26 Thus, due to the inadequate targeting promoter-less, truncated gfp repair homology of ssAAV2- efficiency of AAV for human therapy applications, we gfp repair13 was cloned into an scAAV2 plasmid17 and investigated the enhancement of this process by a DSB transduction by either virus alone does not result in GFP generated by a -derived endonuclease, I-SceI (Sce). synthesis in unmodified human embryonic kidney Conveniently, the 35 bp insert of the defective gfp cell line cells.13 Production, characterization and quantitation of harbors the Sce recognition sequence and cleavage at all recombinant AAV vectors were as described.20 this site results in 4 nt 30 overhangs.19 Co-transduction Southern blot analysis of both the ss and scAAV2-gfp of an ssAAV2-CMV-Sce,13 along with ssAAV2-gfp repair repair genomes was used as an additional control (10 000 gcp per cell of each virus), resulted in an (Figure 1a). The purity of the respective viral genomes approximate 100-fold increase in the amount of cells was nearly 100%. positive for Gfp (Figure 1b). Consistent to the results Given the results of others13 and preliminary experi- without the break, scAAV2-gfp repair resulted in a ments (DNS), we chose a ratio of 10 000 genome- twofold enhancement in Gfp+ cells. Transduction by containing particles (gcps) per cell. Gfp+ cells after ssAAV2-Sce along with a nonhomologous ssAAV2 target transduction by either virus were scarce, regardless of repair vector yielded no false positives in 105 cells (DNS).

Gene Therapy Targeting by scAAV M Hirsch et al 1177 a AAV2-Sce 0.35 Day ss sc 0.3 0.25 2 0.2 5 0.15 0.1 % Gfp+ cells 0.05 0 012 AAV ITRs b 5 4.5 AAV2-Sce Figure 3 AAV inverted terminal repeat sequences do not enhance 4 plasmid-mediated double-strand break repair. The defective gfp 293 3.5 3 cell line (35 bp insertion) described in the text was transfected with 2.5 ss plasmids containing identical gfp repair homology and either 0, 1 or 2 sc 2 AAV inverted terminal repeat sequences (ITRs). At the same time, 1.5 cells were also given a plasmid expressing the Sce endonuclease. At 1 % Gfp+ cells 0.5 the indicated time points, Gfp+ cells were quantitated by flow 0 cytometry. 0246810 Day post-infection Because the ss nature of the AAV genome did not c AAV2-Sce enhance repair, compared with the duplexed version, we - ss - sc also investigated the role of the ITRs in this process. To determine if the AAV ITR sequence stimulates DSB Sce repair, we cloned the identical gfp repair homology into plasmid constructs containing no ITR, one ITR27 or between two ITRs.28 These substrates were compared for their ability to repair the defective gfp cell line containing the 35 bp insertion. Co- of either repair plasmid, along with a plasmid expressing Sce13 at a ratio of 4:1, respectively, showed a slight decrease in Figure 2 Self-complementary AAV mediates efficient endonu- Gfp+ cells inversely correlated with increasing number clease delivery for increased AAV-mediated double-strand break of ITRs present (Figure 3). Although the double-strand repair. (a) Southern analysis of AAV2 capsid isolated single-strand (ss) or (sc) Sce genomes. (b) The defective gfp 293 cell line described plasmid context of the ITRs is different from that of viral in the text was co-transduced by (sc) AAV2-gfp repair (R) along with genomes, these data suggest that the ITRs solely do not either ss or scAAV2-Sce (10 000 genome-containing particles for stimulate targeted DSB repair of the host chromosome, each virus). Gfp+ cells were quantitated by flow cytometry at the despite the likelihood of being processed to a DSB by indicated times. (c) Western blot analysis of the Sce protein. Two host repair enzymes as reported.29 This result is perhaps days after co-transduction of the vectors mentioned in a, cell lysates consistent with a report describing no contribution of were prepared and analyzed for Sce by western blotting using an antibody to the N-terminal HA tag. The lanes marked with a AAV ITRs to chromosome integration (in a double-strand 30 symbol ‘À’ represent cells that were transduced only by plasmid context) in vivo. scAAV-gfpR. A nonspecific band is common to all lanes and serves As the ss nature of the AAV genome appears not as a loading control. required for efficient AAV targeting, nor do the isolated ITR sequences placed next to repair homology, it was hypothesized that AAV-induced signaling cascades In nearly all cases, transduction by scAAV results in originating from particle binding, trafficking and/or rapid and robust transgene expression when compared uncoating influence the efficiency of the event. For with the ss equivalent.17 Therefore, we evaluated this example, AAV transduction induces a DNA damage observation for Sce by packaging it in the sc format.17 response that arrests cells at the G2/M checkpoint31 and Genome purity of the ss and scAAV2-Sce variants was results in the altered expression of cell-cycle regulators near 100% as determined by Southern blot analysis including p21WAF and several cyclin proteins.32 In (Figure 2a). Gfp+ cells were quantitated following addition, empty AAV capsids elicited similar changes co-transduction of scAAV2-gfp repair and either ss or in the expression of these regulators, suggesting that scAAV2-Sce (10 000 gcp per cell of each virus). Delivery signaling cascades, initiated from binding or entry, of Sce in the sc context resulted in a therapeutic level of altered regulation as these particles do not accumulate reporter activity (3–4%) that was increased 2000-fold in the nucleus and do not contain an AAV genome over ssAAV2-gfp repair gene targeting in the absence of (DNS).32 Therefore, the ability of a plasmid, containing an induced break (Figure 2b). The dramatic effect of two ITR sequences flanking the gfp repair homology scAAV2-Sce compared with the ss version correlated to mentioned above, to repair a chromosomal DSB was increased Sce abundance (Figure 2c). In addition, the investigated during the infection of an ssAAV vector that targeting efficiency likely underrepresents the actual has no target or repair homology. For these experiments, value, as it is not normalized to AAV2 co-transduction. Gfp+ cells were quantitated 3 days after a co-transfection In other experiments using the same capsid, cells and then transduction scheme, or vice versa. The gcp/cell ratio, this value was approximated at 80–85% co-transfection event used both the ITR repair plasmid (DNS). and the CMV-SceI expression plasmid given either 12 h

Gene Therapy Targeting by scAAV M Hirsch et al 1178 a Transfection Infection genomes serving as templates for homologous recombi- nation is that in addition to the mechanisms of 0.4 chromosomal repair induced by ss genomes, such as 0.35 gene conversion, duplexed genomes can also undergo 0.3 double crossover events. Observations of increased 0.25 stability of the scAAV genomes could also result in 0.2 increased targeting efficiencies.18

%Gfp+ 0.15 Perhaps more promising from a therapeutic stand- 0.1 point is the ability to increase AAV targeting efficiencies 13 0.05 using a local DSB (Figure 1). As the Sce site is not found 0 in the human genome, much effort is focused on the NT 0 102 103 104 [gcp] generation of designer (that is Zn-finger endonucleases, homing endonucleases and so on) that pRepair + pSce can be tailored for recognition and cleavage at a specific chromosomal site.19,33,34 Although the efficiencies of b Infection Transfection these enzymes for target cleavage varies, gene correction 0.4 values have been reported near 20%.34 However, 0.35 concerns remain as to the toxicity of designer endo- 0.3 nucleases as DSBs can induce apoptosis and, depending 0.25 on the binding efficiency of the endonuclease, off-target 0.2 cutting may occur. Therefore, transient expression of

%Gfp+ 0.15 these molecules is preferred, as well as obligate nuclease 0.1 heterodimerization domains.35,36 As such, the direct 0.05 application of this technology to in vivo applications is 0 unlikely. Thus, ex vivo manipulations of human stem cells 0 10e3 10e4 0 10e3 10e4 [gcp] represents a more attractive approach, especially given 37 T = 1 h T = 12 h the ability to direct their differentiation. Therefore, it seems possible to repopulate an individual with their pRepair + pSce own cells that have been genetically modified with the greatest barrier being selection of the relatively rare event Figure 4 AAV transduction does not affect plasmid DSB repair. or achieving a high enough correction efficiency to avoid (a) The defective gfp 293-cell line mentioned in the text was co-transfected with gfp repair and Sce expression plasmids. After the difficulties and problems associated with clonal 12 h of transfection, the cells were infected with AAV2-TTR-F9 at isolation and outgrowth. For this purpose, use of AAV the indicated gcps per cell. This virus has no significant homology packaged with an sc genome may be preferable as the to the defective gfp target. (b) The same cells were first infected with capsid can be tailored for transduction of many cell types AAV2-TTR-F9 at the indicated gcps and then transfected with the and unwanted vector genome integration is inherently gfp repair and Sce expression plasmids at either 1 or 12 h after minimal and can be further reduced.38,39 infection. In both a and b, Gfp+ cells were quantitated by flow cytometry 48 h after the initial treatment. Materials and methods

before or after ssAAV2-TTR-F9 infection at the indicated Maintenance of cells multiplicity of infections (Figure 4). In either regimen, no A human embryonic kidney cell line, having a defective significant effect of plasmid-mediated DSB correction of gfp reporter integrated into the chromosome, was used the host chromosome was observed upon nonhomo- for all experiments, unless indicated, and was described logous AAV infection at different gcp/cell ratios. previously.19 All cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and 1 Â Penn/Strep Discussion (DMEM+; Sigma, St Louis, MO, USA). Cells were maintained at 37 1C in a 5% CO2 humidified incubator. Herein, we tested a standing hypothesis that the ss nature of the AAV genome is necessary for AAV gene AAV production and characterization correction.9 This assertion is sensible as an ssAAV All vectors used in this study were produced using a genome could directly compete for annealing at its triple transfection protocol followed by CsCl gradient chromosomal target site without requiring the assistance centrifugation and dialysis in 1 Â phosphate-buffered of a helicase for strand unwinding (as would be saline solution as previously described.20 The ssAAV necessary for duplexed repair substrates). However, the production plasmids for CMV-Sce and the gfpR homo- results herein show that scAAV genomes also mediate logy were also previously described.13 To create the sc gene correction, and at a level slightly increased AAV versions of these plasmids, we cloned the identical compared with the ss equivalent. Although significant, sequence elements (CMV-Sce or gfpR) into the pHpa-Trs- the twofold increase in targeting efficiency of sc over SK plasmid previously described.17 In all cases, these ssAAV was modest, and could simply be explained by recombinant AAV genomes were packaged in the AAV the fact that the sc genomes actually have two copies serotype 2 capsids. Virus titer was determined by of repair homology in opposite polarities on a single Southern dot blot and Southern alkaline gel electro- molecule.17 Another possible explanation for scAAV phoresis as previously described.20

Gene Therapy Targeting by scAAV M Hirsch et al 1179 rAAV transduction References A total 50 000 293 cells were seeded in a 24-well plate 24 h before transduction. The next day, the indicated 1 Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, amount of virus particles was diluted in 50 ml of DMEM+ Thompson S et al. Targetted correction of a mutant HPRT gene in before well addition. At the indicated time point (usually mouse embryonic stem cells. Nature 1987; 330: 576–578. 72 h), cells were harvested by gentle aspiration, washed 2 Thomas KR, Capecchi MR. Introduction of homologous DNA in phosphate-buffered saline and resuspended in sequences into mammalian cells induces mutations in the phosphate-buffered saline for immediate flow cytometric cognate gene. Nature 1986; 324: 34–38. analysis. In the experiments described herein, the 3 Thomas KR, Folger KR, Capecchi MR. 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