Wild type AAV, recombinant AAV, and Adenovirus super infection impact on AAV vector mobilization

Recombinant Adeno-associated viral vector (rAAV) mobilization is a largely theoretical process in which intact AAV vectors spread or “mobilize” from transduced cells and infect additional cells within, or external, of the initial host. This process can be replication independent (vector alone), or replication-dependent (de novo rAAV production facilitated by super-infection of both wild-type AAV (wtAAV) and Ad helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 Kb), rAAV-IDUA (3.7 Kb), and rAAV-NanoDysferlin (4.9 Kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and a rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be de fined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed non-replicative mobilization). In rAAV infected cells co-infected with Ad5 and wtAAV, rAAV particle production was increased > 50-fold compared to non-replicative conditions. In addition, replicative dependent rAAV vectors mobilized and resulted in >1,000 -fold transduction upon a subsequent 2nd round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization resistant vectors and the opportunity to derive better vector production systems.

analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc 24 (4.1 Kb), rAAV-IDUA (3.7 Kb), and rAAV-NanoDysferlin (4.9 Kb) generating 2.5-, 5.9-, or 10.7-fold lower 25 amounts, respectively. Surprisingly, cotransfection of a wtAAV and a rAAV plasmid resulted in a uniform 26 decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that 27 wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be 28 independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel 29 aspects of wtAAV complementation that enhance current vector production systems yet to be de fined.

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In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained 31 intact and competent for a secondary round of infection (termed non-replicative mobilization). In rAAV 32 infected cells co-infected with Ad5 and wtAAV, rAAV particle production was increased > 50-fold Introduction

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The wild type AAV2 (wtAAV2) genome was cloned into several plasmid constructs in the 1980s 9-11 , 52 and these constructs serve as the parental plasmids of most recombinant AAV (rAAV) vector constructs.

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In recombinant AAV (rAAV, also termed AAV vectors herein), the ITRs of serotype 2 (ITRs), are the only 54 viral cis elements, flanking transgenic cassettes, as they are required for minimally, rAAV genome 55 replication and capsid packaging 12 . Currently, AAV vectors are the most promising delivery method for 56 in vivo human gene therapy with successes demonstrated in clinical trials for diverse diseases and a few 57 drugs are FDA-approved and commercialized 2,9,10,[12][13][14][15] . Despite the popularity of AAV-based gene 58 therapies, there remain unanswered questions regarding nearly all aspects of wtAAV and rAAV biology, 59 in addition to the implications of the vector-induced genetic modifications in human patients 16 . pXX680 51 . To produce wtAAV, pSSV9 11 was used along with pXX680 and an additional plasmid, 145 pcDNA3.1 (to maintain consistent molar amount of total plasmids). Sixty-five h post-transfection, cells 146 were lysed and virions were purified by CsCl gradient centrifugation. Alkaline gel electrophoresis 147 followed by SYBR gold staining were used to visualize the packaged genome integrity and relative viral 148 titer by loading the same recovered volume of the preparation ( Fig. 2A), and quantitative PCR (qPCR) 149 was utilized to determine the absolute titers (Fig. 2B). When packaging the wtAAV and rAAV separately, 150 wtAAV2 preparations consistently demonstrated the highest titer at about 4.5e 11 viral genome/plate 151 (vg/plate) which was 3-10 fold greater than all rAAV2 preparations ( Fig. 2Bindividual production groups).

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In addition, wtAAV contamination in the three different rAAV preparations was investigated.

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The results of qPCR using a probe specific to cap2 sequence demonstrate that, when producing rAAV 158 vectors with a triple plasmid transfection protocol in 293 cells, one of the most commonly used 159 methods 51 , all rAAV preparations contained wtAAV particle contamination (or at least Benzonase-160 resistant cap2 sequence), the amount of which ranged from 0.8-1.7% of the intended encapsidated 161 rAAV genomes, depending on the transgenic sequence and/or size (Fig. 2C).

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Next, rAAV and wtAAV virion production was assessed following plasmid cotransfections of 163 wtAAV and rAAV plasmids. These experiments relied on the co-transfection of equal moles of pSSV9 164 and pITR2-transgenic, along with pXX680. In contrast to the production of wtAAV and rAAV seperately, 165 wtAAV2 virion production during co-production setting was decreased approximately 3-fold with titers 166 not significantly different than production of rAAV2 ( Fig. 2Bco- 198 The luciferase activity of the first infection was measured at 48 h postinfection as described in Methods.

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As expected the rAAV transduction efficiency was dose dependent with dramatic enhancement in the

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In addition, the rAAV-Luc were slightly increased in both doses of 1,000 and 10,000 vg/cell, further 238 suggesting that these replication-competent wtAAV might supply the Rep and Cap for the production of shedding 37,38,40 . Surprisingly, rAAV mobilization, another largely theoretical concern associated with 251 rAAV vectors, has been underappreciated in the AAV research community with only sparse reports over 252 several decades 10,29,31,33 . wtAAV is reported to have better yields than rAAV during production in the 253 laboratory 65 yet, in a gene therapy setting, when a cell is transduced by rAAV and co-or super-infected 254 by wtAAV and a helper virus, it is unclear whether wtAAV retains its advantage for replication and/or 255 capsid packaging thereby potentially inhibiting de novo rAAV production in patients. In the current 256 study, the reality of these concerns have been investigated in a quantitative manner in both transfection 257 and transduction contexts in cell culture with several novel findings: i) replication-independent rAAV 258 mobilization resulting in serial transduction is substantial (Figs. 3 and 6); ii) with the presence of both 259 wtAAV and rAAV, there is no obvious bias between wtAAV and rAAV for production; however, a dose-260 dependent effect exists (Figs. 4C, S2); iii) wtAAV contamination of rAAV stocks facilitates rAAV de novo production and mobilization (Figs. 4A, B, and 5); and iv) replication-dependent mobilization results 262 in >1,000 fold higher serial transduction compared to replication-independent mobilization (Figs. 4D, 6).

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The data generated herein expose the potential for AAV gene therapy treated patients to produce and 264 disseminate rAAV, and thereby highlight the need for better vector production protocols that eliminate 265 wtAAV contamination and safer, mobilization-resistant AAV vectors. Additionally, a yet to be defined 266 aspect that increases rAAV production in the presence of a wtAAV plasmid is reported (Fig. 2 for the wtAAV was observed previously 65 and consistently herein, with wtAAV producing at a 3-10 folder 273 higher titer compared to rAAV depending on the transgene (Fig. 2B). However, following co-transfection 274 of wtAAV and rAAV plasmids, rAAV vector DNA tends to replicate more efficiently in the presence of 275 wtAAV (Fig. 1C), and there is little to no bias observed toward wtAAV production ( Fig. 2A, B). This may 276 be attributed to the equal efficiency of wtAAV and rAAV genome encapsidation 67 , yet the improved 277 efficiency of rAAV vector yield may be a result of when the rep and cap genes are supplied by the wtAAV 278 genome, which is assumed to have the optimal rep/cap expression ratios during production in the 279 laboratory setting 68 . Although still unknown, this result may be related to: 1) inherent ITR 280 transcriptional activity that may fine-tune expression of known or currently undescribed AAV ORFs 69 and 281 or perhaps 2) the ability of the wtAAV helper plasmid to replicate, an activity shown to increase rAAV 282 production 68 . Since the rAAV vector production via co-transfection in the laboratory mimics the 283 production of wtAAV and rAAV with regard to replication substrates, these observations strongly suggest the likelihood of wtAAV and rAAV particle production at similar efficiencies post co-transduction 285 (Fig. 2).

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In the studies herein, it was also found that rAAV preparations produced by a common triple 287 transfection protocol 70 are contaminated with wtAAV particles (or at least Benzonase resistant wt capsid 288 sequence) (Fig. 2C). This finding is consistent with several other reports demonstrating wtAAV 289 contamination in rAAV preparations at a range of 0.01-10% 37, 57, 58, 71 . If these wtAAV particles proved to 290 be replication competent virus, it decreases the stringency required for AAV vector mobilization, 291 potentially by eliminating the requirement for subsequent (or prior) wtAAV transduction of a rAAV 292 genome harboring cell (since it was administered to the patient as a rAAV preparation contaminant).

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The data show that upon Ad infection, the wtAAV-"like" particles significantly increased in the rAAV 294 alone group, indicating that the contaminants are replication competent, consistent with a previous 295 observation (Fig. 5) 58 . A number of approaches have been used to obtain replication-competent wtAAV-296 free stocks of rAAV 57, 58 , however, based on our observations, it is not clear if these approaches have 297 gained widespread adoption for clinical rAAV production.

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In the transduction analysis, rAAV transduction efficiency was substantially increased by the 299 presence of Ad5 by an unknown mechanism, consistent with published studies 55 . Interestingly, the Ad 300 enhancement appeared to be inhibited by the addition of wtAAV, since rAAV transduction in the 301 presence of Ad and wtAAV was decreased when compared to rAAV+Ad groups (sFig. 2), this 302 phenomenon is not understood and requires additional investigation 72, 73 . 303 Notably, rAAV particle production, following cell transduction, increased up to 100-fold in cells 304 also infected with wtAAV and Ad (Fig.4A). These replication-dependent rAAV vectors, mobilized and 305 resulted in a 1,000-fold increase in transduction efficiency in subsequently infected cells (Figs. 4D, 6).

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The extent of rAAV production directly correlated to the original input of materials, and the increased 307 production of rAAV decreased the magnitude of the wtAAV production (Figs. 4C, S2). Although we did 308 look at ratios of wtAAV to rAAV in the above experiment, it should be noted that wtAAV contamination 309 in clinical rAAV preparations is likely several magnitudes less than the intended AAV vector (Fig. 2), 310 thereby conferring a production advantage to rAAV in treated patient cells. This may lead to transgene 311 specific toxicity as particular transgene products used in clinic could have detrimental effects in off-312 target cells and or healthy by-standers with the observations that tissue-specific promoter restriction 313 and regulatory mRNA targets engineered into transgenic cassettes are incomplete.

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Regarding the overall safety concern of rAAV mobilization, usually neutralizing antibodies to the AAV human spread of replication-independent and/or replication-dependent mobilized rAAV represents a 326 formal safety concern for most mammals in general, and is theoretically dependent upon many factors    Brister, JR, and Muzyczka, N (2000). Mechanism of Rep-mediated adeno-associated virus origin nicking. J 516 Virol 74: 7762-7771. 517 22.