Synthetic Biology: Emerging Concepts to Design and Advance Adeno-Associated Viral Vectors for Gene Therapy

Abstract

Three recent approvals and over 100 ongoing clinical trials make adeno-associated virus (AAV)-based vectors the leading gene delivery vehicles in gene therapy. Pharmaceutical companies are investing in this small and nonpathogenic gene shuttle to increase the therapeutic portfolios within the coming years. This prospect of marking a new era in gene therapy has fostered both investigations of the fundamental AAV biology as well as engineering studies to enhance delivery vehicles. Driven by the high clinical potential, a new generation of synthetic-biologically engineered AAV vectors is on the rise. Concepts from synthetic biology enable the control and fine-tuning of vector function at different stages of cellular transduction and gene expression. It is anticipated that the emerging field of synthetic-biologically engineered AAV vectors can shape future gene therapeutic approaches and thus the design of tomorrow's gene delivery vectors. This review describes and discusses the recent trends in capsid and vector genome engineering, with particular emphasis on synthetic-biological approaches.

Keywords: AAV; adeno‐associated virus; capsid modifications; engineering; gene delivery; molecular switches; vector design.

Figure 1

Genomic organization of AAV. The AAV capsid contains an ≈4.7 kb ssDNA genome comprised of inverted terminal repeats (ITRs) flanking the regulatory regions and coding sequences for AAV replication. The two promoters p5 and p19 drive the expression of two rep transcripts, which are alternatively spliced and result in the production of two large (Rep68 and Rep78) and two small (Rep40 and Rep52) proteins. The p40 promoter controls the expression of the cap transcript. Alternative splicing, nonconventional start codons, and the use of alternative reading frames result in five gene products: the three capsid proteins (VP1, VP2, and VP3), the assembly‐activating protein (AAP), and the membrane‐associated accessory protein (MAAP).

Figure 2

Model of AAV infection. Upon binding to its cellular receptors, AAV is internalized by endocytosis. Acidification of the endosome results in conformational changes of the AAV capsid and the exposure of a phospholipase A2 (PLA2) domain. After trafficking to the Golgi apparatus and escape to the cytoplasm, AAV enters the nucleus via the nuclear pore complex. In the nucleus, the AAV genome is uncoated and converted to dsDNA. In the absence of helper functions, the dsDNA is integrated into the genome to establish a latent infection. The presence of a helper virus stimulates the replication and production of progenitor AAV virions. In contrast to wild‐type AAV, rAAV lacking the genetic information for the Rep proteins maintains long‐term transgene expression through circularization and high‐molecular‐weight circular concatemer formation.

Figure 3

rAAV production in mammalian producer cell lines. Typically, human embryonic kidney cells are transfected with three plasmids. The vector plasmid (left) contains the expression cassette for the gene of interest (GOI) flanked by the AAV ITRs (I). Regulatory sections such as enhancers and promoters (E/P), polyadenylation sites (pA), and additional posttranscriptional regulatory elements (dark turquoise) fine‐tune the transgene expression. The rep‐cap plasmid (middle) produces the viral proteins, but is not packaged due to the lack of ITRs. The adenoviral helper function is provided by co‐transfection of a pHelper plasmid (right) or by infection with, e.g., adenovirus.

Figure 4

Directed evolution of tailored AAV variants. a) Capsid libraries are generated by the insertion of randomized peptides into exposed loop structures, by error‐prone polymerase chain reaction (PCR), by capsid shuffling, or by rational design. These libraries are subjected to a selection process based on their capability to transduce target cells in vitro or target tissues in vivo. Optionally, neutralizing antibodies (NAbs) are included to select capsids with both tailored tropism and the ability to evade NAbs. b) Generation of AAV‐DJ. The cap genes of eight AAV serotypes were fragmented, reassembled to a library of shuffled cap genes, and inserted into an ITR‐carrying rep‐cap plasmid for viral library production. Five rounds of amplification on a human hepatoma cell line in the presence of IVIg resulted in enrichment of a single variant, AAV‐DJ, with superior transduction efficiency.^{[ 83 ]} c) Cre recombination‐based AAV targeted evolution (CREATE) allows the selective recovery of library sequences from Cre‐expressing target cells. For the generation of a CNS‐targeted AAV variant, a peptide library was cloned into an AAV9 vector plasmid harboring a floxed polyadenylation sequence (pA). Cre‐mediated inversion of pA results in a template for sequence recovery by PCR. Two rounds of selection in transgenic mice expressing Cre in astrocytes led to the isolation of CNS‐targeted AAV‐PHP.B from brain and spinal cord tissue.^{[ 88 ]} d) Identification of the ancestral capsid variant Anc80L65 for gene delivery. The in silico reconstruction of the evolutionary AAV lineage enabled the design and synthesis of a cap library covering the probable sequence space of an ancestral AAV variant. Screening of assembly and transduction efficiency led to the identification of Anc80L65, which is characterized by broad tissue tropism and resistance to antisera raised against other serotypes.^{[ 89 ]}

Figure 5

Rational engineering of the AAV capsid. Peptides or small protein domains can be inserted into surface‐exposed loops. Larger protein domains are preferably fused to the N‐terminus of VP2. The resulting fusion construct is provided in trans to the rep‐cap plasmid (pRC). Optionally, the start codon of VP2 is mutated in pRC (asterisk), which prevents the incorporation of unmodified VP2 into the capsids. Desirable capsid features from other AAV serotypes can be transferred by domain swapping.

Figure 6

Covalent coupling of ligands to surface‐displayed protein domains. a) Covalent coupling of ssDNA‐tagged antibodies to HUH tag‐displaying AAV‐DJ. The HUH tag was inserted into the variable region VI (position T456 of AAV‐DJ) of receptor‐blinded VP2 (vp2ΔHSPG) and expressed in trans to the receptor‐blinded rep‐vp1/3 plasmid harboring a mutated start codon for vp2 (white star) for the production of HUH tag‐displaying AAV‐DJ. Monoclonal antibodies were covalently labeled with a HUH tag‐specific ssDNA sequence by click chemistry. An activated tyrosine within the HUH tag (‐O^–) acts as nucleophile and attacks the phosphorous atom (yellow dot) in the ssDNA thereby generating a covalent phosphotyrosine linkage. b) Split intein‐mediated covalent coupling of single‐chain variable fragments (scFvs) to AAV2. NpuC was fused to the N‐terminus of receptor‐blinded VP2 and expressed in trans to the receptor‐blinded rep‐vp1/3 plasmid for the production of NpuC‐displaying AAV2. NpuN was fused to the carboxy‐terminus of an scFv and separately produced in mammalian cell culture. The scFv was covalently coupled to Npu‐AAV2 by NpuC/NpuN‐mediated trans‐splicing thereby releasing the Npu intein.

Figure 7

Synthetic‐biological switches for the control of AAV transduction. a) The interaction of proteins of interest (POI) can be controlled by chemically induced dimerization. In the presence of rapamycin, human FK‐binding protein (FKBP) binds to the FKBP‐rapamycin binding (FRB) domain of mTOR, thereby mediating the dimerization of attached POIs. This principle has been used to control the binding of FKBP‐displaying AAV2 to an EGFR‐specific DARPin fused to FRB. Rapalog‐controlled EGFR binding led to the transduction of EGFR‐expressing target cells. b) Sequence‐specific proteases can be used to release peptides or protein domains. The introduction of negatively charged peptide sequences into the HSPG‐binding loops of AAV blocked transduction of target cells. Insertion of MMP‐specific cleavage sites enabled the MMP‐triggered removal of the negatively charged peptides, which restored the infectivity of AAV. c) Optogenetic switches are used to control the interaction of POIs. Upon red‐light illumination (660 nm), phytochrome B (PhyB) fused to a nuclear localization signal (NLS) binds to PIF‐displaying AAV, supporting nuclear import, and thereby enhances transduction.

Figure 8

Strategies for controlling the expression of AAV‐delivered transgenes. a) The expression of vectored genes of interest (GOI) can be controlled at the transcriptional level by appropriately selecting the type of promoter. The choice of promoter strength and tissue‐specificity provides control of the transcription in target cells. In addition, synthetic promoters that can be controlled by chemicals or light enable the temporal and spatial fine‐tuning of gene expression. Posttranscriptional miRNA‐ or ribozyme‐based switches in the untranslated region (UTR) represent an additional possibility for controlling the level of gene products. b) Example of chemically induced AAV transgene expression. The system consists of two AAV vectors. The first produces a split transcription factor composed of a DNA‐binding zinc finger homeodomain (ZFHD) fused to three repeats of FKBP, and the p65 activator domain of NFκB fused to FRB. The second vector expresses the GOI under the control of a synthetic promoter with upstream ZFHD‐binding repeats (zinc finger binding site, ZBS). In the presence of rapamycin (+ Rap), FRB binds to ZBS‐bound FKBP and thereby recruits the p65 activating domains to the promotor and stimulates the expression of the GOI. Withdrawal of rapamycin (‐ Rap) leads to the dissociation of FRB‐p65 and the downregulation of GOI expression. c) Translational control of AAV vector expression. Incorporation of miRNA targets into the UTR recruits the RNA‐induced silencing complex (RISC) to the mRNA and inhibits translation in cells expressing the miRNA. Differential miRNA expression profiles can be used to restrict the production of the protein of interest (POI) to target tissues. Alternatively, the incorporation of ribozymes into the UTR can be used to control the translation. Ribozyme self‐cleavage destabilizes the mRNA and effectively reduces the level of translation. Addition of antisense oligos inactivates the ribozyme and enables translation.

Figure 9

Hybrid AAV vectors for gene delivery. a) Cross‐packaging of an AAV genome into human bocavirus. Cross‐packaging is achieved by triple‐transfection of producer cells with AAV vector plasmid, a plasmid encoding the rep (NS/NP1) and cap genes of bocavirus (BoV pRC), and an adenoviral helper plasmid harboring the AAV rep gene (pHelper). The resulting hybrid viral particle has a packaging capacity of ≈5.5 kb.^{[ 213 ]} b) AAV‐phage hybrid (AAVP) for the transduction of mammalian cells. A mammalian expression cassette flanked by AAV‐ITRs is inserted into the intergenic region of the filamentous phage fd‐tet. An integrin‐binding RGD‐4C peptide is incorporated into the coat protein pIII. Upon uptake by integrin‐expressing cells, the AAV‐ITRs mediate concatemer formation and the stable expression of the transgene.^{[ 215 ]} c) AAV‐exosome hybrid. During AAV production, AAVs encapsulated by or associated with exosomes are released from producer cells. These hybrids are less prone to NAb inactivation and possess enhanced transduction efficiency compared to “naked” AAVs.^{[ 229} , ²³⁰ , ^{231 ]}