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• W2086174979 abstract "Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. Adeno-associated virus (AAV) is a single-stranded DNA virus belonging to the Parvoviridae family. AAV-derived vectors are promising tools for human gene therapy applications because of their absence of pathogenicity, episomal localization, and stable transgene expression. However, significant limitations to the clinical use of AAV are its promiscuity and its susceptibility to neutralization by human antibodies. Both of these limitations are determined by the nature of amino acid residues exposed at the surface of the capsid. Two main strategies are generally employed to improve AAV vectors: (i) mutagenizing capsid residues to facilitate binding, entry, and/or intracellular trafficking through a rational approach based on the knowledge of virus biology1Wu P Xiao W Conlon T Hughes J Agbandje-McKenna M Ferkol T et al.Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism.J Virol. 2000; 74: 8635-8647Crossref PubMed Scopus (328) Google Scholar,2Zhong L Li B Mah CS Govindasamy L Agbandje-McKenna M Cooper M et al.Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses.Proc Natl Acad Sci USA. 2008; 105: 7827-7832Crossref PubMed Scopus (425) Google Scholar,3Lochrie MA Tatsuno GP Christie B McDonnell JW Zhou S Surosky R et al.Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization.J Virol. 2006; 80: 821-834Crossref PubMed Scopus (158) Google Scholar,4Aslanidi GV Rivers AE Ortiz L Govindasamy L Ling C Jayandharan GR et al.High-efficiency transduction of human monocyte-derived dendritic cells by capsid-modified recombinant AAV2 vectors.Vaccine. 2012; 30: 3908-3917Crossref PubMed Scopus (34) Google Scholar,5Li C Diprimio N Bowles DE Hirsch ML Monahan PE Asokan A et al.Single amino acid modification of adeno-associated virus capsid changes transduction and humoral immune profiles.J Virol. 2012; 86: 7752-7759Crossref PubMed Scopus (75) Google Scholar,6Aslanidi GV Rivers AE Ortiz L Song L Ling C Govindasamy L et al.Optimization of the capsid of recombinant adeno-associated virus 2 (AAV2) vectors: the final threshold?.PLoS One. 2013; 8: e59142Crossref PubMed Scopus (71) Google Scholar and (ii) utilizing a process of directed evolution, a high-throughput method of introducing molecular modifications into the AAV capsids, manipulating both diversity and selection in a vastly hastened emulation of natural evolution.7Müller OJ Kaul F Weitzman MD Pasqualini R Arap W Kleinschmidt JA et al.Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.Nat Biotechnol. 2003; 21: 1040-1046Crossref PubMed Scopus (312) Google Scholar,8Maheshri N Koerber JT Kaspar BK Schaffer DV Directed evolution of adeno-associated virus yields enhanced gene delivery vectors.Nat Biotechnol. 2006; 24: 198-204Crossref PubMed Scopus (399) Google Scholar,9Li W Asokan A Wu Z Van Dyke T DiPrimio N Johnson JS et al.Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.Mol Ther. 2008; 16: 1252-1260Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar,10Asuri P Bartel MA Vazin T Jang JH Wong TB Schaffer DV Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells.Mol Ther. 2012; 20: 329-338Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar,11Yang L Jiang J Drouin LM Agbandje-McKenna M Chen C Qiao C et al.A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection.Proc Natl Acad Sci USA. 2009; 106: 3946-3951Crossref PubMed Scopus (167) Google Scholar Although both methodologies have been successful in creating vectors with improved transduction capabilities, their utility is limited by our understanding of the AAV life cycle and by the technical boundaries of the protocols for directed evolution. The strategy adopted in this project integrates both approaches in one unified design. Using detailed knowledge of AAV capsid structures12Xie Q Bu W Bhatia S Hare J Somasundaram T Azzi A et al.The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.Proc Natl Acad Sci USA. 2002; 99: 10405-10410Crossref PubMed Scopus (475) Google Scholar,13DiPrimio N Asokan A Govindasamy L Agbandje-McKenna M Samulski RJ Surface loop dynamics in adeno-associated virus capsid assembly.J Virol. 2008; 82: 5178-5189Crossref PubMed Scopus (37) Google Scholar,14Agbandje-McKenna M Kleinschmidt J AAV capsid structure and cell interactions.Methods Mol Biol. 2011; 807: 47-92Crossref PubMed Scopus (135) Google Scholar,15DiMattia MA Nam HJ Van Vliet K Mitchell M Bennett A Gurda BL et al.Structural insight into the unique properties of adeno-associated virus serotype 9.J Virol. 2012; 86: 6947-6958Crossref PubMed Scopus (135) Google Scholar,16Govindasamy L DiMattia MA Gurda BL Halder S McKenna R Chiorini JA et al.Structural insights into adeno-associated virus serotype 5.J Virol. 2013; 87: 11187-11199Crossref PubMed Scopus (57) Google Scholar and the sequence of 150 naturally occurring variants, we have conducted a process of “virtual family shuffling” in silico to help derive a combinatorial capsid library whereby only variable regions (VRs) on the surface of the structure are modified. We demonstrated the utility of the library by generating an AAV2-derived vector containing only four mutations, through a combination of rational mutagenesis and directed evolution, with enhanced transduction of murine liver of 20-fold better than the wild-type (wt) AAV2, a level comparable to AAV8. Comparison of the AAV VP3 structure among various serotypes has revealed highly homologous sequences interspersed with more evolutionary divergent areas. These amino acid stretches are commonly designated as VRs I through IX (also known as “loops”).17Govindasamy L Padron E McKenna R Muzyczka N Kaludov N Chiorini JA et al.Structurally mapping the diverse phenotype of adeno-associated virus serotype 4.J Virol. 2006; 80: 11556-11570Crossref PubMed Scopus (137) Google Scholar Incidentally, VRs are localized at the surface of the assembled capsid and are assumed to be responsible for the capsid interaction with cell surface receptors and other host factors. Because of their location, VRs are also predicted to be less critical for capsid assembly. Therefore, the guiding principle of the library's design was to modify only surface VRs while keeping the backbone sequence unchanged to maintain the integrity of the assembling scaffold. All candidate positions for mutagenesis, in the AAV2 background, were selected from the alignment of 150 AAV naturally occurring variants (see Supplementary Figure S1) and then evaluated on a three dimensional (3D) model of the AAV2 capsid.12Xie Q Bu W Bhatia S Hare J Somasundaram T Azzi A et al.The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.Proc Natl Acad Sci USA. 2002; 99: 10405-10410Crossref PubMed Scopus (475) Google Scholar Only those residues that were clearly exposed to the surface were selected for modification. To reduce the incidence of incompatible residues, the selection of the introduced substitutions was restricted to those appearing in a given position in at least one of the 150 analyzed homologues. Adding to the rational design strategy, four more positions were modified: surface Y444F and Y500F to increase the transduction efficiency by reducing targeting to the proteasome for degradation2Zhong L Li B Mah CS Govindasamy L Agbandje-McKenna M Cooper M et al.Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses.Proc Natl Acad Sci USA. 2008; 105: 7827-7832Crossref PubMed Scopus (425) Google Scholar and surface R585 and R588 to de-target binding to heparan sulfate proteoglycan,18Opie SR Warrington Jr, KH Agbandje-McKenna M Zolotukhin S Muzyczka N Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding.J Virol. 2003; 77: 6995-7006Crossref PubMed Scopus (277) Google Scholar the primary receptor for AAV2.19Summerford C Samulski RJ Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.J Virol. 1998; 72: 1438-1445Crossref PubMed Google Scholar Detailed composition of each VR, including nucleotide sequence and resulting amino acid diversity, is shown in Figure 1. To achieve the desired composition, in some cases, more than one nucleotide needed to be changed per codon, resulting in additional amino acid diversity. The AAV2 capsid gene fragment incorporating all substitutions was assembled from synthetic oligonucleotides and inserted into a plasmid vector containing the AAV2 genome from which the corresponding wt sequence had been removed. The plasmid library, with an estimated complexity of 1 × 108, was analyzed by sequencing random clones and was found to accurately reflect the library's design, including expected nucleotide substitution positions and types. However, this library failed to produce high-titer packaged virus. An apparent explanation was the presence of an alternative open reading frame coding for the assembly-activating protein (AAP).20Sonntag F Schmidt K Kleinschmidt JA A viral assembly factor promotes AAV2 capsid formation in the nucleolus.Proc Natl Acad Sci USA. 2010; 107: 10220-10225Crossref PubMed Scopus (241) Google Scholar As the AAP open reading frame overlaps with coding regions for VRs I and II, their mutagenesis would potentially interfere with AAP structure and function, likely preventing many capsid variants from being assembled. Disruption of AAP, however, was not the only reason for the low viral library complexity from the initial design. The high diversity rendered the library nonfunctional as some of the substitutions were incompatible within the context of the 3D structure. For example, both plasmid and encapsidated viral DNA showed the expected complexity when a single VR-VIII library was packaged, with all variable positions incorporating nucleotides as designed (Supplementary Figure S2, panels CapLib3A). However, when the same VR-VIII was combined with VR-III through VR-VII plasmid libraries, the packaged virions incorporated mostly wt AAV2 genomes despite having expected diversity at the plasmid level (Supplementary Figure S2, panels CapLib3B). The presence of a multitude of simultaneous substitutions increased the likelihood of combinations that are incompatible with capsid folding and/or assembly, resulting in a dramatic decrease in the fraction of viable variants and a disproportionately strong selection for wt sequences that were originally present as trace contaminants. As single VRs libraries appeared to produce both expected diversity and high-titer viral stocks, a new strategy was undertaken to generate as much diversity as possible by selecting for compatibility with capsid folding for each individual VR (as exemplified for VR-VIII, Supplementary Figure S2, panels CapLib3A). This was achieved by packaging separate viral libraries derived from individual VRs (less complex VRs I and VI were combined into a single library, as well as III and V) while keeping other VRs unmodified. After sequence analysis of the constructed individual libraries, the viral DNA revealed no diversity (wt sequence only) in VR-II (as expected due to the overlapping AAP open reading frame), low diversity in VRs III, VI, and IX, and high diversity in all other VRs. The recovered encapsidated viral DNAs for each VR were then used to create a final library by combining preselected structure-compatible variants. Schematic representation of the library design and construction steps is depicted in Figure 2. The resulting sequence includes 136 degenerate positions and encodes 59 variable amino acid positions, each with 2–16 permutations resulting in a theoretical complexity (total number of possible amino acid combinations) of 4 × 1045. 3D models of the VP3 monomer and the assembled capsid are shown in Figure 3a with variable amino acid positions colored according to VR identity.Figure 3Variable regions (VRs) on the AAV2 capsid surface. (a) Adeno-associated virus (AAV2) capsid full capsid (assembled from 60 VP3 monomers shown to the right), displaying the location of the eight VRs, colored as described below the image of the monomer. (b) LiA and LiC full capsids variants and their respective monomers. The mutated residues and their positions are shown by color and by number, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A plasmid library was generated, with an estimated complexity of 9.2 × 107, as deduced by extrapolating colony counts and correcting for positive clones. The transformation step is the complexity bottleneck, as the number of molecules involved in the previous steps is several orders of magnitude higher. The library quality was assessed by sequencing a random sample of 21 clones in which all corresponding 21 protein sequences were found to be unique. Each differed from the reference AAV2 sequence by 24 –41 amino acid substitutions. Substitutions were distributed among five to seven VRs per sequence. VRs II and III were wt in all cases. Due to wt contamination during library assembly, positions 444 and 500 exhibited Y/F polymorphism, with 1/21 and 8/21 containing wt Tyr residues in the respective positions. Extrapolating colony counts and sequencing random individual clones always overestimates the library complexity due to the fact that a fraction of the variant genomes fails to form viable capsids. To assess the library's complexity under more stringent conditions, the encapsidated viral DNA was subjected to next-generation sequencing. The distance between VRs, as well as their small size, would not allow accurate reconstruction of full-length sequence contigs from short reads. Therefore, despite its lower throughput and high error rate, the PacBio circular consensus sequencing platform was selected as the only one with a compatible read length. Because no software support existed to analyze our particular dataset, a dedicated code (caplib) was developed to process and analyze sequencing data. From the 19,455 original circular consensus sequencing reads, the caplib software generated a list of 840 corrected reads and translated them into protein sequences analyzed as described below. As a convenient way of illustrating the complexity of each permutated VR, Shannon entropy21Shannon CE The mathematical theory of communication. 1963.MD Comput. 1997; 14: 306-317PubMed Google Scholar was computed from an alignment of the 840 individual protein sequences. As evident from Figure 4a, entropy is highest in VRs I, IV, and VII, moderate in VRs V, VI, and VIII, very low in VR-IX, and almost nonexistent in VR-III (VR-II has no entropy as expected, being derived from the wt sequence only). A comparison of the 840 protein sequences identified 699 distinct sequences, including 41 that were present in more than one copy (Figure 4b). Each sequence had up to 43 mutations (substitutions), with an average of 19.16 (Figure 4c). Figure 4d shows the frequency distribution and interdependence of mutagenized VRs. A majority (75%) of sequences have mutations in three to five VRs (average 3.76). Almost half of the sequences (414 or 49.3%) have one of the three mutant VR combinations I–IV–V–VII (179), I–IV–V–VI–VII (134), or I–IV–VII (101). Two-thirds of the sequences (557 or 66.3%) have mutations in the three VRs I, IV, and VII. More than 4/5 (677 or 80.6%) have mutations in at least VRs I and IV. A comparison of expected and observed percentages of mutant residues in the plasmid and viral libraries, respectively, at each variable amino acid position is shown in Figure 4e. Comparing theoretical and plasmid libraries shows the effect of mutations in individual VRs on virion assembly, as the combined plasmid library was constructed from packaged viral DNA originating from individual VR libraries. Mutations in VRs IV, VI, and VIII alone seem not to interfere significantly with capsid assembly, whereas mutations in VRs III and IX are strongly selected against. Comparing plasmid and viral libraries allows us to evaluate the effect of combining multiple mutated VRs. Mutant VRs I, IV, and V seem to tolerate additional mutations in other VRs well (very little difference can be seen between numbers from plasmid and viral libraries), unlike VR VIII, and to a lesser extent in VRs VI, VII, and IX. A consensus of the 840 sequences is shown in Supplementary Figure S3, aligned with the nine sequences that were found in more than four copies (only regions with mutations are displayed). In these sequences, VRs III, VIII, and IX were completely wt, which suggests that these regions might be most sensitive to changes affecting the efficiency of virion assembly (in the presence of mutations in other VRs in the case of VR VIII as shown in Figure 4e). Interestingly, the consensus sequence itself was not identified among the 840 analyzed sequences. Amino acid composition at each variable position is shown in Supplementary Figure S4 (variable positions only) and Figure 1 (amino acid sequences in logo style). Although some level of diversity is visible at most positions (only position 383 has a single identity), in many cases, a single amino acid is predominant. However, 14 positions in which no single variant occurs in more than 50% of the sequences can be found, in VRs I (position 263), IV (450–461), and VII (548–550, 555, and 556). The observed complexity of variable amino acid positions compared to theoretical values is illustrated in Supplementary Figure S5. Experimental complexities vary between 1 and 14 with an average of 5.93, compared to a range of 1–16 and an average of 6.79 for theoretical complexities. Only position 383 has an observed complexity of 1, probably the result of sampling bias as only 14 sequences out of 840 have a mutation in VR III. Complexities of the 8 VRs were also computed (see Supplementary Table S1). Interestingly, they were in most cases smaller than expected. It appears that very few combinations are compatible with capsid assembly, or that different combinations result in dramatic differences in assembly efficiency, in effect reducing complexity at the VR level. Note however that the actual VR complexities are probably much higher than that observed in the sample, as many sequences occurred as single copies. As for the complexity of the library itself, although the small sample size does not allow us to determine it with confidence, we can derive a rough estimate from available data. The total number of possible VR combinations, based on the observed complexities (which are underestimated as explained above) is 2.0 × 1014. Applying the lowest observed to expected ratio obtained with amino acid combinations in VRs (18/699) would give 5.0 × 1012, still a much higher number than the limit of 9.2 × 107 described above (plasmid library complexity). During viral production, 3 × 109 cells were transfected, and from previous observations, we assume that at least 20% (6 × 108 cells) received and expressed a copy of the library plasmid, which implies that the transfection scale was not a significant limiting factor for library complexity, On the other hand, Figure 4e shows significant differences in the occurrence of mutant amino acid positions between the plasmid and viral libraries, indicating that large numbers of variants were excluded during virus production. Since this diversity loss is due to combining VRs from the sublibraries into the final library, a value can be estimated by multiplying the viral to plasmid ratios of average mutant positions within regions originating from the sublibraries (Supplementary Table S2). As much as 99% of the diversity existing in the plasmid library was lost during virus production. After adjusting according to the ratio between observed complexity and sample size in the sequencing data (699/840), we can conservatively estimate an upper limit of 8 × 105 for the actual complexity of the library. Liver is an important gene therapy target to treat many inherited disorders.22Sands MS AAV-mediated liver-directed gene therapy.Methods Mol Biol. 2011; 807: 141-157Crossref PubMed Scopus (64) Google Scholar Several serotypes target liver preferentially, with the highest transduction efficiency shown by AAV823Gao GP Alvira MR Wang L Calcedo R Johnston J Wilson JM Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.Proc Natl Acad Sci USA. 2002; 99: 11854-11859Crossref PubMed Scopus (1235) Google Scholar,24Nakai H Fuess S Storm TA Muramatsu S Nara Y Kay MA Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice.J Virol. 2005; 79: 214-224Crossref PubMed Scopus (254) Google Scholar in murine liver, with expression levels two orders of magnitude higher than those of AAV2. However, this does not translate to primates, in which the efficiency of AAV8 is dramatically lower,25Hurlbut GD Ziegler RJ Nietupski JB Foley JW Woodworth LA Meyers E et al.Preexisting immunity and low expression in primates highlight translational challenges for liver-directed AAV8-mediated gene therapy.Mol Ther. 2010; 18: 1983-1994Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,26Lisowski L Dane AP Chu K Zhang Y Cunningham SC Wilson EM et al.Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.Nature. 2014; 506: 382-386Crossref PubMed Scopus (299) Google Scholar although AAV8 is still being considered one of the best candidate vectors for human liver-directed gene therapy. Because AAV2 was used as the library's backbone, we asked whether a novel capsid variant could be isolated targeting the liver with an efficiency comparable to that of AAV8. After three rounds of in vivo selection, 41 clones were sequenced. An alignment of the recurrent sequences’ VRs is shown in Figure 5a. None of the seven liver-selected sequences could be found among the 840 translated sequence reads, which indicates that their abundance is not the result of a preexisting prevalence in the original library but is most likely the result of genuine selection. Interestingly, the K507T substitution in LiC was not included into the library's design. A comparison of motif enrichment factors in the liver-selected variants is summarized in Supplementary Table S3. Motifs with the highest enrichment were NSEGGSLTQSSLGFS in VR-IV (found in variants LiA, LiD, and LiG as shown in Figure 5a) and the single amino acid T at position 507 in VR-V (found in LiC only). In addition, motif SASGASN in VR-I (found in LiA, LiC, and LiE) also shows significant enrichment, although at a lower level. Therefore, variants LiA and LiC seem to be the most strongly selected. This was confirmed by a preliminary screening in which liver-selected capsid variants were packaged with a luciferase-expressing vector and intravenously injected into mice. Luciferase activity was restricted to the liver area in all cases, and variants LiA and LiC had the highest activity (data not shown). LiA and LiC were therefore selected for further characterization. 3D models of variant capsids LiA and LiC are shown in Figure 3b,c. LiA and LiC, as well as controls AAV2 and AAV8, were packaged with luciferase-expressing barcoded vector pTR-UF50-BC (Supplementary Figure S7). The dynamics of luciferase expression in the liver were considerably different for all serotypes and variants: AAV8 mediated a fast initial increase followed by a slow leveling off (Figure 5b,c). LiA followed a similar trend at a lower level of expression. LiC, on the other hand, provided steady gain converging with the levels of AAV8 after about 30 days. At day 40, the AAV8 and LiC-mediated luciferase expression exceeded that of AAV2 by 20-fold. Consistent with luciferase expression, hFIX transgene expression driven by ApoE-hAAT promoter followed the same pattern (Figure 5d,e). In the latter case, LiC and AAV8 were also compared to AAV2-M3 (triple tyrosine to phenylalanine mutant at positions 444, 500, and 730), which showed a significantly lower expression level (Figure 5d). To examine biodistribution of viral genomes in various tissues and organs, a cocktail of four barcoded vectors (Supplementary Figure S7) at equimolar ratios was injected via tail vein in three mice. Total DNA isolated from mice 23 days after injection was used to quantify viral genomes copy numbers in various tissues as well as the relative amount of each variant (Figure 5f). Both LiC and LiA targeted liver with high specificity. Recent advances in clinical trials involving AAV vectors, concomitant with a more favorable regulatory climate, stimulated efforts to derive vehicles with higher transduction efficiencies, higher target tropism, and lower immunogenicity. One of the most prolific approaches so far has been the isolation of novel AAV serotypes. In spite of its apparent success, characterization of new serotypes, one at a time, is very laborious. An alternative methodology has been to generate combinatorial capsid libraries of high complexity in the hope of randomly creating a new variant that meets the demands of a particular application. However, the various strategies available to generate capsid libraries suffer from sequence bias or limited diversity. Finally, a rational approach of mutagenizing amino acid residues based on understanding of the capsid structure may have its practical limits as many favorable mutations apparently cannot be combined to produce additive effects.9Li W Asokan A Wu Z Van Dyke T DiPrimio N Johnson JS et al.Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.Mol Ther. 2008; 16: 1252-1260Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar In the current report, we sought to combine all strategies listed above (utilizing naturally existing serotypes, applying directed evolution, and rational mutagenesis) in one unified approach. A bioinformatic analysis of the existing sequence database of 150 AAV naturally occurring variants was used to create a consensus AAV capsid template whereby nine VRs incorporated alternative residues present in a given position in at least one of the serotypes. This in silico–derived algorithm was converted into a combinatorial plasmid DNA library using the only feasible approach, de novo DNA synthesis. Even though the plasmid library was found to reflect faithfully the initial design, a new selection trait of 3D structural compatibility had to be introduced to isolate assembly-competent variants within each individual VR. Subsequently, the successful VR families were interbred to derive a combined viral library which was characterized in depth using next-generation sequencing. The prevalence of mutations at each variable position in the final viral library differed, in many cases dramatically, from theoretical values that would be expected in the absence of any selective pressure during capsid assembly (Figure 4e). This is expected, as AAV2 is the result of a long natural selection, therefore AAV2 amino acids are likely to be favored, especially in the context of an AAV2 backbone. However, three positions were suppos" @default.
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• W2086174979 title "Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants" @default.
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