With small viruses come giant responsibilities - Next-generation parvoviral vectors for human gene therapy with extended packaging capacity and enhanced safety profile

Over the last decade, the field of gene and cell therapy has experienced a major turning point and has finally begun to fully realize its potential as a very attractive, versatile and innovative platform for the development of gene-based drugs. Gene therapy encompasses a spectrum of approaches, ranging from supplying missing genes to the correction of diseases at their molecular level, that all have in common the need of a vehicle (“vector") for specific and efficient delivery of therapeutic DNA or RNA. One branch of small viruses - the parvoviruses - have gained increasing attention as such vectors due to their non-pathogenicity, ease of engineering and low genotoxicity. Particularly, the adeno-associated virus (AAV) emerged as a top candidate, culminating in the authorization of three AAV-based gene therapy products, Glybera, Luxturna and Zolgensma. However, despite all the successes using recombinant (r)AAVs, there is still a demand for more specific vectors with larger DNA cargo capacity and lower immunogenicity. This need defined the scope of this doctoral thesis, which aimed at the construction and evaluation of new parvoviral vectors (derived from bocaviruses [BoVs]) and to increase the safety of vector application in humans. The first part of this work was fueled by a seminal study by Ziying Yan and colleagues in 2013, who used parvovirus cross-genera pseudotyping to combine an oversized rAAV2 genome of 5.5 kilobases (kb) with the capsid of the human bocavirus 1 (HBoV1). As reported, the rAAV2/HBoV1 vector could be produced efficiently and potently transduced primary human airway epithelial cells (pHAE). Here, we have validated and expanded on these intriguing findings by more comprehensively exploring the upper DNA packaging limit of the HBoV1 capsid. Notably, we found that up to 6.2 kb single-stranded (ss) - or 3.2 kb self-complementary (sc) - AAV genomes can be efficiently packaged into the HBoV1 capsid, as compared to only 5.1 (ssAAV) and 2.8 kb (scAAV) for AAV2, which has important ramifications for the delivery of complex rAAV vector DNA. Next, we further expanded this system to other primate BoV serotypes - three from humans (HBoV2, 3 and 4) and one from Gorilla (GBoV) - that have not been studied as vectors before. To this end, we successfully assembled the capsid genes of HBoV2-4/GBoV and produced chimeric rAAV/BoV vectors of all studied serotypes. With the help of reporter genes, we subsequently started to study and unravel the so-far unknown tropism of the new viral vectors. Strikingly, our screens on various primary cells and cell lines revealed that BoVs (especially GBoV) have a much wider tropism in vitro than previously anticipated. We found a wide range of primary and therapeutically relevant cells to be amenable to BoV infection, including human hepatocytes, T-cells and skeletal muscle cells. In addition, we obtained the first evidence that pseudotyped rAAV/BoV vectors also differ in their reactivity to pooled human antibodies (intravenous immunoglobulin, IVIg), which implies the possibility of vector re-dosing in rAAV/BoV-treated human gene therapy patients. Finally, we aimed to increase the fitness of BoV vectors and therefore employed a high-throughput diversification method called DNA family shuffling (DFS), to create the first library of chimeric BoV capsids. As hoped for, the library was packaging-competent, increased in titer over selection rounds and acquired a unique footprint when cycled in pHAE. Despite an excellent safety record of rAAV vectors, undesirable toxicity resulting from permanent gene expression represents a clinical concern. So far, the ensuing need to gain temporal control over vector persistence or expression has been addressed by using Cre recombinase or inducible systems that necessitate complex vector re-engineering. Thus, in the second part of this work, we aimed to overcome these limitations by introducing novel rAAV vectors that harbor a kill-switch (KS) based on the bacterial CRISPR II system (clustered regularly interspaced short palindromic repeats). This approach has two major components: (i) a (g)uide RNA expressed from the rAAV vector and (ii) the CRISPR/Cas9 endonuclease, which is supplied in trans and directed by the gRNA to a target site in the vector/transgene itself. We tested our KS system extensively in vitro and show a 10- to 100-fold reduction in transgene expression (Firefly luciferase) after supplying Cas9 in trans using ss and scAAV vectors for the expression of full-length and split Cas9, respectively. Moreover, we expanded our study to an in vivo application in mice, where we could recapitulate our findings in cell culture and trigger an up to 50% reduction in transgene expression. Finally, we devised a universal approach to inactivate any rAAV vector without further modifications. Therefore, we developed and experimentally validated self-inactivating (SIN) CRISPR vectors based on split Cas9 and ssAAVs that harbor the anti-target and anti-Cas9 gRNA and hence allow concurrent targeting of both. Moreover, we utilized different RNA polymerase III promoters (Pol III) to study and eventually optimize the effect of differential gRNA expression on the kinetics of both processes. Collectively, this work has yielded original BoV helper constructs and chimeras that represent valuable new tools to investigate fundamental and applied aspects of bocaviral biology, from the discovery of antigenic domains to the construction of designer viral vectors. Concomitantly, we have implemented and validated novel concepts to increase the safety of recombinant vectors including rAAV KS or SIN constructs that can be harnessed in future work, either alone or in combination with BoV capsids, to form the next generation of parvoviral vectors

Severe Human Bocavirus 1 Respiratory Tract Infection in an Immunodeficient Child With Fatal Outcome

We report a case of lower respiratory tract infection with human bocavirus 1 (HboV1) in an immunodeficient 6-month-old boy leading to respiratory failure with fatal outcome. Polymerase chain reaction of serum/tracheal secretions revealed exceptionally high HboV1-DNA levels and immunoassays showed seroconversion indicating an acute primary HboV1 infection. All assays for other pathogens were negative, strongly suggesting that HboV1 was the causative agent in this case.Peer reviewe

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity

Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors. IMPORTANCE The family of Parvoviridae comprises a wide variety of members that exhibit a unique biology and that are concurrently highly interesting as a scaffold for the development of human gene therapy vectors. A most notable example is human bocavirus 1 (HBoV1), which we and others have recently harnessed to cross-package and deliver recombinant genomes derived from another parvovirus, the adeno-associated virus (AAV). Here, we expanded the repertoire of known HBoV1 variants by cloning 29 distinct HBoV1 capsid sequences from primary human samples and by analyzing their properties as AAV/HBoV1 gene transfer vectors. This led to our discovery of a mutational hot spot at HBoV1 capsid position 590 that accumulated in two patients during natural infection and that lowers viral loads but increases vector yields. Thereby, our study expands our current understanding of HBoV1 biology in infected human subjects and concomitantly provides avenues to improve AAV/HBoV1 gene transfer vectors.Peer reviewe

Characterization of the GBoV1 Capsid and Its Antibody Interactions

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties

Fantastic AAV Gene Therapy Vectors and How to Find Them—Random Diversification, Rational Design and Machine Learning

Parvoviruses are a diverse family of small, non-enveloped DNA viruses that infect a wide variety of species, tissues and cell types. For over half a century, their intriguing biology and pathophysiology has fueled intensive research aimed at dissecting the underlying viral and cellular mechanisms. Concurrently, their broad host specificity (tropism) has motivated efforts to develop parvoviruses as gene delivery vectors for human cancer or gene therapy applications. While the sum of preclinical and clinical data consistently demonstrates the great potential of these vectors, these findings also illustrate the importance of enhancing and restricting in vivo transgene expression in desired cell types. To this end, major progress has been made especially with vectors based on Adeno-associated virus (AAV), whose capsid is highly amenable to bioengineering, repurposing and expansion of its natural tropism. Here, we provide an overview of the state-of-the-art approaches to create new AAV variants with higher specificity and efficiency of gene transfer in on-target cells. We first review traditional and novel directed evolution approaches, including high-throughput screening of AAV capsid libraries. Next, we discuss programmable receptor-mediated targeting with a focus on two recent technologies that utilize high-affinity binders. Finally, we highlight one of the latest stratagems for rational AAV vector characterization and optimization, namely, machine learning, which promises to facilitate and accelerate the identification of next-generation, safe and precise gene delivery vehicles

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

International audienceThe discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production

Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9

Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells

Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Parvoviruses are highly attractive templates for the engineering of safe, efficient, and specific gene therapy vectors, as best exemplified by adeno-associated virus (AAV). Another candidate that currently garners increasing attention is human bocavirus 1 (HBoV1). Notably, HBoV1 capsids can cross-package recombinant (r) AAV2 genomes, yielding rAAV2/HBoV1 chimeras that specifically transduce polarized human airway epithelia (pHAEs). Here, we largely expanded the repertoire of rAAV/BoV chimeras, by assembling packaging plasmids encoding the capsid genes of four additional primate bocaviruses, HBoV2-4 and GBoV (Gorilla BoV). Capsid protein expression and efficient rAAV cross-packaging were validated by immunoblotting and qPCR, respectively. Interestingly, not only HBoV1 but also HBoV4 and GBoV transduced pHAEs as well as primary human lung organoids. Flow cytometry analysis of pHAEs revealed distinct cellular specificities between the BoV isolates, with HBoV1 targeting ciliated, club, and KRT5+ basal cells, whereas HBoV4 showed a preference for KRT5+ basal cells. Surprisingly, primary human hepatocytes, skeletal muscle cells, and T cells were also highly amenable to rAAV/BoV transduction. Finally, we adapted our pipeline for AAV capsid gene shuffling to all five BoV isolates. Collectively, our chimeric rAAV/BoV vectors and bocaviral capsid library represent valuable new resources to dissect BoV biology and to breed unique gene therapy vectors