Engineering the AAV capsid to optimize vector–host-interactions

Available online at www.sciencedirect.com

ScienceDirect Engineering the AAV capsid to optimize vector–host-interactions Hildegard Bu¨ning1,2,3,4, Anke Huber2,3,4, Liang Zhang2,3,4, Nadja Meumann2,3,4 and Ulrich Hacker5 Adeno-associated viral (AAV) vectors are the most widely used delivery system for in vivo gene therapy. Vectors developed from natural AAV isolates achieved clinical benefit for a number of patients suffering from monogenetic disorders. However, high vector doses were required and the presence of preexisting neutralizing antibodies precluded a number of patients from participation. Further challenges are related to AAV’s tropism that lacks cell type selectivity resulting in off-target transduction. Conversely, specific cell types representing important targets for gene therapy like stem cells or endothelial cells show low permissiveness. To overcome these limitations, elegant rational design- as well as directed evolution-based strategies were developed to optimize various steps of AAV’s host interaction. These efforts resulted in next generation vectors with enhanced capabilities, that is increased efficiency of cell transduction, targeted transduction of previously nonpermissive cell types, escape from antibody neutralization and off-target free in vivo delivery of vector genomes.These important achievements are expected to improve current and pave the way towards novel AAV-based applications in gene therapy and regenerative medicine. Addresses 1 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany 2 Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany 3 German Center for Infection Research (DZIF), Partner Sites BonnCologne and Hannover-Braunschweig, Germany 4 Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany 5 University Cancer Center Leipzig (UCCL), Leipzig, Germany Corresponding author: Bu¨ning, Hildegard ([email protected])

Current Opinion in Pharmacology 2015, 24:94–104 This review comes from a themed issue on New technologies Edited by Andrew H Baker and Adrian J Thrasher

http://dx.doi.org/10.1016/j.coph.2015.08.002 1471-4892/Published by Elsevier Ltd.

(AAV), discovered 50 years ago as contamination in adenoviral stocks [1]. Natural evolution has created a portfolio of AAV variants with 13 being classified as human or non-human primate serotypes [2,9]. Some serotypes show considerable differences in tropism (tissue preference) compared to AAV serotype 2 (AAV2), the prototype AAV vector [1]. Consequently, improved transduction efficiencies were achieved for muscle (AAV1), heart (AAV1, AAV6), liver (AAV8, AAV9) or brain (AAVrh10) when using these serotypes as vectors [3]. In addition, switching to a serotype with lower prevalence amongst humans represents an effective strategy to overcome the hurdle of pre-existing immunity towards AAV2. As of yet, AAV vectors (AAV1, AAV-2, AAV-6, AAV-8 and AAV-9) have been utilized in more than 120 clinical trials in particular for long-term modification of post mitotic tissues (http://www.abedia.com/wiley/). For example, clinical benefit was achieved in the treatment of hemophilia, Leber’s congenital amaurosis or lipoprotein lipase deficiency [4]. In addition, trials proved safety for both local and systemic (by intravenous injection) administration of AAV vectors [4]. AAV vectors are also a frequently used gene transfer system in basic research both in vitro and in models ranging from mice to large animals. AAV as member of the Parvoviridae are non-enveloped single-stranded DNA viruses (Figure 1). The wild-type AAV genome encodes for a family of multifunctional proteins (Rep proteins), for three different capsid proteins (VP1, VP2 and VP3) and the Assembly Activating Protein (AAP), required to initiate capsid assembly [1]. The genome is flanked by inverted terminal repeats (ITRs), which serve as packaging signal and origin of replication. These elements are maintained in vector genomes, while all viral open reading frames (ORFs) are replaced by the transgene expression cassettes (Figure 1). The viral/vector genome is packaged into a capsid that is arranged as an icosahedra with protrusions at the 3-fold and pores at the 5-fold symmetry axes [5]. The protrusions representing the most exposed regions of the capsids are critical sites of AAV’s host interaction as they are involved in receptor binding and contain immunological epitopes [6]. The pores on the other hand form channels through which viral/vector genomes are funneled into the capsids during progeny or vector production [7].

Introduction The AAV vector system is based on a helper virusdependent parvovirus, the Adeno-Associated Virus Current Opinion in Pharmacology 2015, 24:94–104

As indicated in Figure 1, the three capsid proteins share the so-called common VP3 region that forms the actual www.sciencedirect.com

AAV capsid modification Bu¨ning et al. 95

Figure 1

ITR

ITR REP

CAP/AAP

VP1 VP2 VP3 common VP3 region

ITR

Transgene expression cassette

ITR 3ʹ 5ʹ

single-stranded

self complementary Current Opinion in Pharmacology

Schematic representation of AAV using AAV2 as example. Adeno-Associated Viruses (AAV) are composed of a single-stranded DNA genome and a protein capsid of 25 nm. The viral genome is flanked by inverted terminal repeats (ITR), which serve as packaging signal and origin of replication. The rep open reading frame (ORF) encodes a family of multifunctional proteins (Rep proteins) responsible for transcription, replication and packaging of viral genomes, while information for the Assembly Activating Protein (AAP) and the viral capsid proteins (VP = viral proteins) are contained within the cap/aap ORF. Capsid assembly is initiated by AAP in the nucleus [56]. The common VP3 region of each of the three capsid proteins contribute in 1 (VP1 (90 kDa)): 1 (VP2 (72 kDa)): 10 (VP3 (60 kDa)) ratio to the icosahedral capsid. The N0 -terminal extensions of 65 amino acids for VP2 and further 137 amino acids for VP1 are buried inside the capsid and contain functional domains such as nuclear localization signals or a phospholipase A2 homology domain (in the VP1 unique region) [15]. Protrusions are formed at the 3-fold and pores at the 5-fold symmetry axes [7]. Marked are the tip of the highest (blue, 453) and second-highest (green, 587) protrusions and the N0 -terminus of VP2. These positions accept peptide insertions without disturbing capsid assembly and are thus explored in cell entry targeting (see below). Upon AAV’s use as vector, viral ORFs are replaced by the transgene expression cassette containing the gene of interest flanked by control elements. In AAV vectors either single-stranded DNA genomes of approximately 4.7 kB or self-complementary vector genomes (half of coding sequence) are packaged. Selfcomplementary genomes deliver the sense and anti-sense version on a single DNA separated by an additional ITR [1]. Upon release of vector genomes a DNA double-strand is formed that serves as template for transcription overcoming thereby the limitation of second-strand synthesis.

capsids. The N0 -terminal extensions of VP2 and VP1 are buried inside the capsid [8]. They contain domains required for viral infectivity [8] that become exposed through the above-mentioned pores during cell infection (Figure 2). Comparison of AAV1-9 revealed that serotypes are remarkably similar in structure (95-99%), while differing in amino acid sequence [9]. Since these differences have developed during natural evolution, they are mainly located on exposed capsid regions/protrusions that harbor — as mentioned — residues critically influencing viral infectivity [9].

the host, to improve efficiency of transduction, re-direct AAV particles towards novel, non-natural receptors or facilitate AAV’s escape from host immune responses. In this way, host–vector interactions are tailored to optimize efficiency of gene transfer, lower vector dose and reduce the amount of ‘foreign’ components presented to the immune system. As an important spin-off, this research area in addition contributes to decipher AAV’s infection biology by identifying barriers towards transduction with natural occurring serotypes.

AAV and its journey to the nucleus Here, we will summarize the basic principles and discuss recent progress made in the engineering of this vector system. Traditionally, these attempts are mainly focussing on the viral capsid, that is the predominant interface with www.sciencedirect.com

Primates are natural hosts for AAV with liver and spleen being the predominant sites of infection [2]. Biodistribution studies revealed a similar tropism for AAV vectors [10]. Knowledge on AAV’s host interactions on the Current Opinion in Pharmacology 2015, 24:94–104

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Figure 2

(a) HSPG

FGFR

αvβ5 or α5β1

Rac1 PI3K

(b)

early endosome

Clathrin-coated pit

PI3K

microtubuli

(c)

(e)

pH Nucleus

(d) late endosome

NPC

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Cell infection by AAV2. Microscopically, multiple cell surface contacts, each lasting for approximately 62 ms, are detected, which mirror AAV2’s interaction with HSPG, its primary receptor [57,58]. This low-affinity binding not only increases the contact time between virus and cell, but primes the capsid — through a conformation change — for binding to the internalization receptors avb5 or alternatively a5b1 integrin [7,59,60]. This interaction induces clathrin-coated pit formation and uptake through endocytosis, and in addition, a rearrangement of the cytoskeleton. AAV2 is then transported within endosomes along the cytoskeleton. Acidification of the endosomal compartment, a feature of endosomal maturation, induces a second conformational change, which results in relocation of the N-termini of VP1 (and possibly VP2). Domains contained within these N0 -termini mediate endosomal release and nuclear delivery of AAV. Details of the uncoating process are as of yet unknown. Progeny production requires the presence of a helper virus. In the absence of a helper virus, AAV establishes a latent infection. In case of AAV vectors, transgene expression is regulated by the co-delivered promoter once a double-stranded DNA genome has been formed.

cellular level is still incomplete. However, although AAV serotypes differ in receptors used for cell binding and uptake, a common scheme for the following steps of cell infection can be assumed (Figure 2): Initial cell contact is mediated by low-affinity binding to glycans [11] thereby increasing cellular contact time. This interaction is stabilized by co-receptor binding and primes the capsid for binding to internalization receptors [7]. Uptake is mediated by endocytosis, although additional entry routes have been observed [12,13]. AAV-containing endosomes are then moved from the periphery towards the microtubuli organization center located in the vicinity of the cell nucleus [14]. As endosomal vesicles maturate a drop in pH leads to exposure of Current Opinion in Pharmacology 2015, 24:94–104

functional domains required for endosomal release and nuclear delivery such as nuclear localization sequences and a phospholipase A2 (PLA2) homology domain located at the N0 -terminus of VP1 [15,17,18]. Once released from the endosome, capsids are targets for ubiquitination that mark particles for degradation [19]. Those particles escaping degradation accumulate in the nuclear area and are transported — in part as intact particles — into the nucleus. Genome release may occur at the nuclear pore, during nuclear entry or within the nucleus [16]. Once released single-stranded vector genomes have to be converted into a double-stranded DNA conformation before transcription is initiated. In order to enhance efficiency of this process, so-called www.sciencedirect.com

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self-complementary AAV vector genomes were developed, that form a double-stranded DNA upon being released from the viral capsid (Figure 1) [1].

The viral capsid as target structure The viral capsid protects viral/vector genomes against enzymatic or physical destruction and recognition by the host’s immune system. It also contains domains responsible for receptor binding, penetration from the endosome into the cytosol, transport to and presumably into the nucleus and uncoating [1,18,20]. As a consequence, engineering approaches aiming to tailor AAV vectors for application in gene therapy, regenerative medicine, but also basic research mainly focus on the viral capsid. Both, knowledge-based rational and directed evolutionbased approaches have been applied. Rational design-based approaches that alter AAV–host interaction

Infection biology studies for example have identified a cellular enzyme, EGFR-PTK (epidermal growth factor receptor protein tyrosine kinase), which marks AAV2 particles for proteasomal degradation through phosphorylation of tyrosine residues [21]. Aiming to inhibit loss of vector particles through this mechanism, a panel of mutants were designed by changing solvent exposed tyrosines (Y) to phenylalanin (F) [19]. Subsequent studies identified Y444, Y500 and Y730 as critical residues in AAV2 capsids. Substitution of Y to F resulted in improved AAV2 transduction efficiencies in permissive and nonpermissive cells both in cell culture and in vivo. Success of the strategy was preserved for other serotype capsids [22] and for alternative targets of phosphorylation [23], arguing for proteasomal degradation as a general barrier limiting efficiency of AAV vectors. Identification of residues involved in receptor binding does not only help to decipher early steps in cell infection, but is also critical for re-directing vector tropism towards a defined target cell type, a strategy termed cell entry targeting. Mapping of the first receptor binding site for AAV dates back to 2003, when two groups independently identified positively charged residues in the AAV2 capsid required for binding to heparan sulphate proteoglycan (HSPG), the primary receptor of this serotype, by a rational design-based approach [24,25]. These residues (arginine (R) at position 484, 487, 585 and 588 as well as lysine (K) at position 527 and 532) form a basic patch between two neighboring protrusions into which the negatively charged sugar residues fit [7]. Site-directed mutagenesis of even one single residue resulted in an up to 6 orders of magnitude lowered infectivity on AAV2 permissive cell lines compared to wild type [24,25]. Surprisingly, in sharp contrast to the negative influence of impaired HSPG binding on AAV2’s infectivity in cell culture, these AAV2 capsids showed enhanced cardiac tropism www.sciencedirect.com

when administered in vivo [24,26,27]. Interestingly, this unexpected novel in vivo tropism can be reversed by cell entry targeting as demonstrated recently [28]. Specifically, AAV2 particles carrying mutations in the HSPG binding motif (R585A, R588A) and additionally equipped with Design Ankyrin Repeat Proteins (DARPins) specific for Her2/neu or CD4 were able to precisely target human Her2/neu-positive tumors or human CD4-positive lymphocytes in vivo, without any detectable off-target activity [28]. As illustrated by this example, in which the antibody-like specificity of DARPins had been employed to direct AAV2 vectors to a cell type of choice, rational design-based strategies are frequently explored in cell entry targeting. They are defined as genetic or non-genetic targeting strategies dependent on whether the ligand is directly inserted into the viral capsid or not [29]. Non-genetic targeting approaches rely on the use of bi-specific linkers or adaptors that bridge between the viral capsid and the receptor of choice. Classical linkers are bi-specific antibodies that were successfully explored to improve transduction of AAV2 non-permissive cells [30]. Later on the strongest non-covalent binding of two molecules, namely the interaction of biotin with avidin was adapted for cell entry targeting approaches. Initially, biotin was introduced through chemical modification of purified viral vector particles followed by decoration with streptavidin-conjugated ligands of known specificity [31]. This successful strategy was further simplified by directly introducing a biotin acceptor side into the capsid protein sequence [32]. An alternative strategy employed the high affinity of Staphylococcus aureus protein A towards immunoglobulins for non-genetic targeting [33]. As an elegant solution to overcome the challenge of complex-destabilization, a covalent modification strategy was more recently added to the portfolio of non-genetic targeting strategies. Specifically, a cysteine-containing peptide sequence that is modified by a cellular enzyme to an aldehyde was introduced into the AAV2 capsid protein sequences. Through this reactive group ligands equipped with a hydrazide or hydroxylamine are covalently linked thus introducing a modification that cannot dissociate [34]. Another interesting variation of the targeting concept relies on the idea to use a chemical switch, thereby limiting target receptor binding to a specific predefined condition [35]. In contrast to non-genetic targeting approaches, genetic cell entry targeting strategies are based on novel protein sequences, conferring binding capacity, that are directly incorporated into the capsid structure. Therefore, sites that accept peptide ligands without interfering with capsid assembly and vector genome packaging, and displaying the peptide in a manner that allows target receptor binding had to be identified. In earlier times when the 3D structure Current Opinion in Pharmacology 2015, 24:94–104

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of the AAV capsid was not available and knowledge on capsid biology was limited, this was a difficult task taken the fact that established strategies for high throughput selection screens of AAV mutants were also lacking. Nevertheless, using rational design approaches, the N0 -terminus of VP2 and the tips of the protrusions at the 3-fold symmetry axis were identified as the as of yet most promising positions [29,36]. These sites were initially identified and explored for cell entry targeting in the context of AAV2, but homologous sites showed promise also in the context of other serotypes such as AAV8 or AAV9 [37,38]. In line with results obtained for non-genetic targeting approaches, insertion of receptor-binding ligands conferred AAV targeting vectors with the ability to transduce cell types permissive for the parental serotypes with improved efficiency and — even more important — opened up the avenue for modification of non-permissive cells types ([6,29,39] and Table 1). Using these vectors as tools, changes in the vector–cell interaction imposed by targeting a novel, non-natural ligand, are currently investigated in detail. These investigations focus as of yet on the AAV2 capsid position 587 located at the tip of the second highest protrusion [13,40–43]. This is maybe caused historically as this position was the first that enabled production of AAV targeting vectors with reasonable titers [44]. Insertion of peptides at this position separates R585 and R588, which are part of AAV2’s HSPG binding motif [41]. Despite this modification, only some of the targeting vectors transduced cells independently of HSPG, while others showed a reduced binding or even binding affinities comparable to AAV2. Subsequent detailed analyses of the peptide inserts revealed that peptides with an overall positive charge could restore binding to HSPG [41]. This is in particular true for sequences that contain two positively charged amino acids separated by two further amino acids (‘BXXB’ motif) or peptides which together with R588 of the AAV2 backbone form this BXXB motif, which is a classical glycan binding motif. In contrast, HSPG-independent cell transduction was demonstrated in the presence of peptides containing bulky amino acids or being characterized by an overall neutral or negative charge [13,41]. The ability of HSPG-binding is related to distinct features of the respective AAV targeting vectors: AAV2 targeting vectors that transduce cells independently of HSPG showed a detargeting from liver and a tropism defined by the novel peptide sequence [13,41]. In contrast, AAV2 targeting vectors that bind HSPG accumulated in the liver and showed a broad, non-specific tropism, very similar to AAV2, the parental serotype [13,41]. Nevertheless, AAV2 targeting vectors with positive charged peptides clearly differ from AAV2 with regard to the parts of the capsid involved in HSPG binding (protrusions versus basic patch) [7,13,41] and with regard Current Opinion in Pharmacology 2015, 24:94–104

to the affinity of HSPG binding [13]. It is therefore unlikely that the HSPG-induced conformational change — discussed as a key step in AAV2’s natural infection [7] — takes place in case of a peptide-mediated glycan binding. This hypothesis is support by experiments with the HSPG-binding mutant D5, which enters cells with comparable efficiency to AAV2, but through a clathrinindependent route, arguing against the use of avb5 or a5b1 as internalization receptors [13]. Usage of the clathrin-dependent entry path, however, is a critical parameter defining efficiency and thus success of targeting approaches [13]. This is likely due to the dependency on the acidification-triggered conformational change of the capsid for endosomal release of AAV particles and nuclear delivery of their genetic payload (Figure 2). Directed evolution approaches to optimize vector–host interaction

AAV targeting vectors developed by rational design, that is by insertion of peptides with known specificity, were in particular successful in situations where the availability of a receptor limited transduction efficiency (pre-entry barrier). In situation were knowledge about barrier/s imposed by given target cells or on receptors that can be targeted are lacking, directed evolution approaches offer an elegant technical solution. These approaches use libraries of mutants that are screened for those variants that produce progeny in a given target cell (upon codelivery of a helper virus owning to AAV’s helper virus dependency) [6]. Consequently, mutants selected by this approach are inherently characterized by features allowing to overcome pre-entry and post-entry barriers. Technically, mutants are enriched through iterative rounds of selection. Finally, selected viral variants are sequenced to map the capsid modification and to develop the vector counterpart. To produce the large number of randomly generated capsid mutants of a library, three different strategies for random mutagenesis are applied: Insertion of oligonucleotides with random sequence (AAV peptide display), DNA shuffling and Error prone PCR (EP) (Figure 3). EP libraries are classically applied to identify variants that transduce cells despite the presence of neutralizing antibodies raised against the parental serotype [45,46]. Commonly, the number of mutations per capsid is low [45]. Therefore, multiple rounds of mutagenesis [46] and/or mutagenesis of shorter regions [47] are performed to obtain a viral pool with sufficient diversity. Besides identification of variants that transduce cells in the presence of serum concentration capable of neutralizing the parental serotype, EP library selections both confirmed known and helped to map novel immunogenic epitopes. More comprehensive analyses regarding the immune escape phenotype of selected variants revealed at least two mechanisms: (a) amino acid changes interfering with antibody binding and (b) enhanced infectivity [45–47]. www.sciencedirect.com

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Table 1 Examples of AAV capsid variants selected by AAV peptide display (information of variants developed prior to 2008 is provided in [6,29,39])

Vector AAVEARVRPP

Serotype AAV2

Modification QRGQRGEARVRPPA QAA

Selected on Chronic myelogenous leukemia (CML) cell line (K562) Human CD34+ PBPC cell Human CD34+ PBPC cell Human CD34+ PBPC cell Human CD34+ PBPC cell Primary breast cancer cell Primary breast cancer cell (in vivo selection)

AAV LS1

AAV2

QRGQRGNDVRSANAQAA

AAV LS2

AAV2

QRGQRGNESRVLSA QAA

AAV LS3

AAV2

QRGQRGNRTWEQQ A QAA

AAV LS4

AAV2

QRGQRGNSVQSSWAQAA

AAVRGDLGLS AAVESGLSQS

AAV2

QRGQRGRGDLGLSAQAA

AAV2

QRGQRGESGLSQSA QAA

AVPRSTSDP

AAV2

QRGQRGPRSTSDPAQAA

Lung (in vivo selection)

AAV2PSVSPRP

AAV2

QRGQRGPSVSPRPAQAA

Heart (in vivo selection)

AAV2VNSTRLP

AAV2

QRGQRGVNSTRLPAQAA

Heart (in vivo selection)

AAV1.9-3SKAGRSP AAV-C4

AAV1/9 hybrid AAV2

SKAGRSP (at position 590)

AAV-D10

AAV2

QRGNAAASRGATTTAAQAA

AAV-r3.45

AAV2

QRGNLATQVGQKTAQAA (additional mutation: V719M)

AAVNDVRSAN

AAV2

QRGQRGNDVRSANAQAA

AAVGPQGKNS

AAV2

QRGQRGGPQGKNSAQAA

AAVSLRSPPS

AAV9

GQAGSLRSPPSAQAA

AAVRGDLRVS AAV-7m8

AAV9

GQAGRGDLRVSAQAA

Human dermal fibroblast (HDF) Melanoma cell line (BLM) Melanoma cell line (BLM) Primary rat neuronal stem cells (NSC) Low-passage human melanoma cell lines Low-passage glioblastoma cell lines Human coronary artery endothelial cells (HCAEC) HCAEC

AAV2

588LGETTRP

AAV-Kera2

AAV2

QRGNAAAPRGDLAPAAQAA

AAV2/8BP2

AAV8

PERTAMSLP

QRGNAAAPRGTNGPAAQAA

Intravitreal injection Primary human keratinocytes Subretinal injection

Enhanced transduction CML cell lines, primary CML, primary human haematopoietic progenitor cells Human CD34+ PBPC cell Human CD34+ PBPC cell Human CD34+ PBPC cell Human CD34+ PBPC cell Primary breast cancer cell Tumor tissue (breast cancer) and heart; moderate detargeting from liver Lung and enhanced transduction of further tissues Preferentially heart, but also further tissues; detargeting from liver and kidney Preferentially heart, but also further tissues; detargeting from liver and kidney HDF

References [61]

[62] [62] [62] [62] [63] [63]*

[63]

[64]

[64]

[65]

Primary melanoma cells

[66]

Primary melanoma cells

[66]

Rat, murine, human NSC and further cell types

[67]

Melanoma cell lines

[40]

Glioblastoma cell lines

[40]

HCAEC, HUVEC (endothelial cells in situ), non-EC cell lines HCAEC, non-EC cell lines Various retinal cell types (intravitreal administration) Human and murine keratinocyte Retina, ON-bipolar cells

[37]

[37] [68] [54] [55]

* Cardiac transgene expression is avoided by combining cell entry targeting and micro-RNA technology [69].

In line with the observation that EP library selection may identify mutants with enhanced infectivity, is the recent report on the AAV2 capsid variant EP1.9 (R459G) that demonstrated a 3-fold increase in human pluripotent stem cell transduction over the parental serotype [48]. www.sciencedirect.com

Interestingly, EP1.9 was more efficient than mutants selected by DNA shuffling strategies or AAV peptide display libraries. This finding is unexpected, given the fact that modifications introduced by EP are rather small compared to DNA shuffling or peptide insertion. Current Opinion in Pharmacology 2015, 24:94–104

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Figure 3

Capsid shuffling

Display Ligand Ligand Ligand Ligand

Ligand L iga nd

nd

Ligand

d

and

an an

d

Lig

Lig

d

Lig

Lig

an d

Lig

nd

Lig

an d

an

Ligand L iga nd

Lig

and

Lig

d

Ligand L iga

Ligand

Ligand Liga

Lig

an d

an

nd

Ligand

d

Ligand Lig a

Lig

an

Capsid

nd

Ligand

an d

Lig

and

Ligand

Ligand

Ligand L iga

and

d

Lig

an

Ligand

Ligand L iga nd

Ligand L ig

Ligand L iga nd

Genome

Lig

Error Prone

Lig

an

d

Lig an d

Current Opinion in Pharmacology

The three strategies of random mutagenesis currently applied for directed evolution approaches. (a) In error-prone PCR based mutagenesis point mutations are introduced into the cap ORF by a polymerase defective in proof-reading activity and in conditions that favor mutagenesis. Production of virions exploring the mutagenized cap ORF as templates results in a library of mutants with amino acids substitutions in their capsid proteins. Commonly, the number of mutations per capsid is low. (b) DNA shuffling is a strategy in which the cap ORF of different AAV serotypes are fragmented by nucleases followed by a ligation step. This results in a new and random combination of capsid sequences. (c) AAV peptide display libraries are based on a single serotype backbone. At an exposed position on the capsid surface, an oligonucleotide with random sequence is introduced genetically. Viruses from this library differ in the peptide displayed at this site.

DNA shuffling libraries are based on the idea that advantageous feature of different serotypes can be combined to optimize the vector–host interaction. Technically, this is achieved by random shuffling of capsid DNA sequences. First described in 2008 [49], it has become a frequently applied strategy, both ex vivo and in vivo, to select for variants with enhanced transduction efficiencies (Table 2). Iterative rounds of in vivo selection requires isolation of viral genomes from target organs followed by PCR-mediated amplification and subsequent cloning and production of a sublibrary that is then subjected to the next round of selection. This procedure, although sophisticated, has the major advantage to directly select variants in the context of the later in vivo application. In addition to their improved efficiency, an immune escape phenotype represents an important advantage of these variants [49,50]. Consequently, through DNA shuffling AAV’s toolbox was greatly expanded (Table 2), more recently for variants with in vivo tropism for human hepatocytes [51]. The latter were selected in a humanized mouse model utilizing adenoviral helper function to amplify Current Opinion in Pharmacology 2015, 24:94–104

variants accumulated in human, but not mouse hepatocytes. In contrast to the DNA shuffled libraries, capsid variants in AAV peptide display libraries share a common backbone and thus the basic feature of a single serotype, which is modified to display a peptide of random sequence. AAV peptide display selections opened the area of directed evolution techniques [52,53] and yielded a huge number of peptides useful for AAV cell entry targeting. Like DNA shuffling libraries, AAV peptide display libraries were explored ex vivo and in vivo (Table 1) to identify variants with enhanced transduction efficiency. If the natural ligand–receptor interaction of AAV peptide display variants has been destroyed, tropism of such variants is defined by the specificity of the peptide ligand. Otherwise, tropism is expanded. Thus, in marked contrast to DNA shuffling libraries, AAV peptide display selection in principle allows not only selection of variants with improved transduction efficiency, but also with a re-directed and restricted tropism. Compared to the initial libraries www.sciencedirect.com

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Table 2 Examples of AAV capsid variants selected by AAV DNA shuffling libraries Vector

Information on clone

Selected on

Enhanced transduction

References

AAV-cA2 AAV-DJ

AAV1/2/6/8/9 chimera AAV2/8/9 chimera

Liver and heart Different cell lines

[50] [49]

Chimeric-1829

AAV1/2/8/9 chimera Additional mutations at nt 763, 1285 and 2099 AAV1/6/7/8 chimera

HEK293 Human hepatoma cell line in the presence of antibodies Hamster melanoma cell line (CS1)

CS1, mouse melanoma cell lines, high passage human melanoma cell lines

[70]

Higher efficiency than AAV6 in heart and muscle; similar efficiency to AAV9 in heart, but weaker in muscle Human airway epithelia

[71]

Retina Mu¨ller glia cells Astrocytes Glioma cell lines and primary human glioblastoma cells Crossing seizure-compromised blood–brain barrier; detargeting from liver Crossing seizure-compromised blood–brain barrier; detargeting from liver; transduction of oligodendrocytes and neurons Human hepatocytes

[73] [74] [74] [75]

AAVM42

AAV2.5T

ShH10 ShH13 ShH19 AAV_U87R7-C5 AAV-32

AAV-83

AAV-LK03

AAV2/5 chimera: AAV2 (aa1-128)/AAV5 (aa129-725)-A581T Closely related to AAV6

AAV1/2/rh10/rh8 chimera

In vivo panning in muscle

Organotypic human airway epithelial cells Human Human Human Human

astrocytes astrocytes astrocytes U87 glioma cells

AAV1/8 chimera Additional amino acid substitutions AAV1/3/8/9 chimera Additional amino acid substitutions

In vivo panning in brain

AAV2/3/4 chimera Common VP3 region based on AAV3

Humanized mouse model

In vivo panning in brain

that contained HSPG-binding and non-binding peptides, a remarkable preference for variants with integrin-binding motifs was observed when an AAV2 display library depleted for mutants that bind to HSPG was used [54]. One of the variants, which transduced target cells (primary human keratinocytes) with remarkable efficiency in monocultures, mixed and organotypic cultures was used to establish comparative gene analysis, a microarraybased bioinformatic approach, as strategy to map receptors engaged by targeting vectors [54]. The candidate receptor, avb8, was later confirmed by competition and drug inhibition studies and represents a novel candidate for targeting approaches in skin gene therapy [54]. Functionality of peptides selected by AAV2 peptide display across serotypes was proven for endothelial cell targeting, one of the main target cell types in AAV-based cell entry targeting approaches [37], and for the ESGLSQS peptide selected by in vivo AAV2 peptide display in a xenograft tumor model [38]. Variations of the initial strategy were more recently explored. Specifically, instead of adding a peptide with random sequence to AAV8 capsid proteins, a stretch of nine amino acids was replaced by a random sequence of the same length [55]. www.sciencedirect.com

[72]

[76]

[76]

[51]

In addition, variants of this library were equipped with a marker gene expression cassette enabling detection of infected cells in vivo thus enabling collection of these specific cells for sublibrary production [55].

Conclusion Natural isolates of AAV possess a number of features such as high stability, low immunogenicity and lack of pathogenicity that fostered their development towards one of the most promising vector systems. In particular, upon AAV’s application in human clinical trials, limitations such as low efficiency and specificity of gene delivery, prevalence of neutralizing antibodies and lack of tropism for certain cell types became obvious. Today, an impressive portfolio of strategies to address these problems has evolved resulting in important technological improvements and consequently, a greatly expanded (and still expanding) toolbox. These new generation AAV vectors are expected to improve current gene therapy strategies and will help to translate novel strategies into clinical application.

Conflict of interest statement Nothing declared. Current Opinion in Pharmacology 2015, 24:94–104

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Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SPP1230, SFB670 and KFO286), the Center for Molecular Medicine Cologne (CMMC) and the Institute of Experimental Hematology (Hannover Medical School).

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