Adeno-Associated Virus-Mediated Dorsal Root Ganglion Toxicity in the New Zealand White Rabbit.

Recombinant adeno-associated virus (AAV)-mediated degeneration of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) has been observed in non-human primates (NHPs) following intravenous (IV) and intrathecal (IT) delivery. Administration of recombinant AAV encoding a human protein transgene via a single intra-cisterna magna (ICM) injection in New Zealand white rabbits resulted in histopathology changes very similar to NHPs: mononuclear cell infiltration, degeneration/necrosis of sensory neurons, and nerve fiber degeneration of sensory tracts in the spinal cord and of multiple nerves. AAV-associated clinical signs and incidence/severity of histologic findings indicated that rabbits were equally or more sensitive than NHPs to sensory neuron damage. Another study using human and rabbit transgene constructs of the same protein demonstrated comparable changes suggesting that the effects are not an immune response to the non-self protein transgene. Rabbit has not been characterized as a species for general toxicity testing of AAV gene therapies, but these studies suggest that it may be an alternative model to investigate mechanisms of AAV-mediated neurotoxicity and test novel AAV designs mitigating these adverse effects.

Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

Membrane-bound MMP-14 protease-activatable adeno-associated viral vectors for gene delivery to pancreatic tumors.

Adeno-associated virus' (AAV) relatively simple structure makes it accommodating for engineering into controllable delivery platforms. Cancer, such as pancreatic ductal adenocarcinoma (PDAC), are often characterized by upregulation of membrane-bound proteins, such as MMP-14, that propagate survival integrin signaling. In order to target tumors, we have engineered an MMP-14 protease-activatable AAV vector that responds to both membrane-bound and extracellularly active MMPs. This "provector" was generated by inserting a tetra-aspartic acid inactivating motif flanked by the MMP-14 cleavage sequence IPESLRAG into the capsid subunits. The MMP-14 provector shows lower background transduction than previously developed provectors, leading to a 9.5-fold increase in transduction ability. In a murine model of PDAC, the MMP-14 provector shows increased delivery to an allograft tumor. This proof-of-concept study illustrates the possibilities of membrane-bound protease-activatable gene therapies to target tumors.

N-terminal serine/threonine motif has diverse and important effects on behavior of multiple AAV serotypes.

Adeno-associated virus (AAV) is a promising gene therapy vector, but questions remain regarding mechanisms of basic viral functions. We previously showed that a serine/threonine (S/T) triplet motif and its flanking residues, located in the overlapping N-terminus of VP1/VP2 and highly conserved across most AAV serotypes, are critical for viral transcript production in vitro. Here we generate a panel of S/T triplet mutants in AAV serotypes 2, 4, and 9 and characterize their behaviors in vitro and in vivo using next generation sequencing. We show that S/T triplet mutations can significantly hinder some stages of transduction in a serotype-dependent manner in vitro. Interestingly, these defects are largely overcome in C57BL/6 mice, with only one mutant displaying altered behavior in vivo. Taken together, our results identify a short N-terminal capsid motif with diverse roles across several AAV serotypes which better informs engineering efforts to improve AAV as a vector for gene therapy.

Frustration and Direct-Coupling Analyses to Predict Formation and Function of Adeno-Associated Virus.

Adeno-associated virus (AAV) is a promising gene therapy vector because of its efficient gene delivery and relatively mild immunogenicity. To improve delivery target specificity, researchers use combinatorial and rational library design strategies to generate novel AAV capsid variants. These approaches frequently propose high proportions of nonforming or noninfective capsid protein sequences that reduce the effective depth of synthesized vector DNA libraries, thereby raising the discovery cost of novel vectors. We evaluated two computational techniques for their ability to estimate the impact of residue mutations on AAV capsid protein-protein interactions and thus predict changes in vector fitness, reasoning that these approaches might inform the design of functionally enriched AAV libraries and accelerate therapeutic candidate identification. The Frustratometer computes an energy function derived from the energy landscape theory of protein folding. Direct-coupling analysis (DCA) is a statistical framework that captures residue coevolution within proteins. We applied the Frustratometer to select candidate protein residues predicted to favor assembled or disassembled capsid states, then predicted mutation effects at these sites using the Frustratometer and DCA. Capsid mutants were experimentally assessed for changes in virus formation, stability, and transduction ability. The Frustratometer-based metric showed a counterintuitive correlation with viral stability, whereas a DCA-derived metric was highly correlated with virus transduction ability in the small population of residues studied. Our results suggest that coevolutionary models may be able to elucidate complex capsid residue-residue interaction networks essential for viral function, but further study is needed to understand the relationship between protein energy simulations and viral capsid metastability.

Adeno-associated virus characterization for cargo discrimination through nanopore responsiveness.

Solid-state nanopore (SSN)-based analytical methods have found abundant use in genomics and proteomics with fledgling contributions to virology - a clinically critical field with emphasis on both infectious and designer-drug carriers. Here we demonstrate the ability of SSN to successfully discriminate adeno-associated viruses (AAVs) based on their genetic cargo [double-stranded DNA (AAVdsDNA), single-stranded DNA (AAVssDNA) or none (AAVempty)], devoid of digestion steps, through nanopore-induced electro-deformation (characterized by relative current change; ΔI/I0). The deformation order was found to be AAVempty > AAVssDNA > AAVdsDNA. A deep learning algorithm was developed by integrating support vector machine with an existing neural network, which successfully classified AAVs from SSN resistive-pulses (characteristic of genetic cargo) with >95% accuracy - a potential tool for clinical and biomedical applications. Subsequently, the presence of AAVempty in spiked AAVdsDNA was flagged using the ΔI/I0 distribution characteristics of the two types for mixtures composed of ∼75 : 25% and ∼40 : 60% (in concentration) AAVempty : AAVdsDNA.

Constructing and evaluating caspase-activatable adeno-associated virus vector for gene delivery to the injured heart.

Adeno-associated virus (AAV) is a promising vector for gene therapy, but its broad tropism can be detrimental if the transgene being delivered is harmful when expressed ubiquitously in the body, i.e. in non-target tissues. Delivering the transgene of interest to target cells at levels high enough to be therapeutically effective while maintaining safety by minimizing delivery to off-target cells is a prevalent challenge in the field of gene therapy. We have developed a protease activatable vector (provector) platform based on AAV9 that can be injected systemically to deliver therapeutic transgenes site-specifically to diseased cells by responding to extracellular proteases present at the disease site. The provector platform consists of a peptide insertion into the virus capsid which disrupts the virus' ability to bind to cell surface receptors. This peptide contains a blocking motif (aspartic acid residues) flanked on either side by cleavage sequences that are recognized by certain proteases. Exposure to proteases cleaves the peptides off the capsid, activating or "switching ON" the provector. In response to the activation, the provectors regain their ability to bind and transduce cells. Here, we have designed a provector that is activated by cysteine aspartic proteases (caspases), which have roles in inflammation and apoptosis and thus are elevated at sites of diseases such as heart failure, neurodegenerative diseases, and ischemic stroke. This provector demonstrates a 200-fold reduction in transduction ability in the OFF state compared to AAV9, reducing the virus' ability to transduce off-target healthy tissue. Following exposure to and proteolysis by caspase-3, the provector shows a 95-fold increase in transduction compared to the OFF state. The switchable transduction behavior was found to be a direct result of the peptide insertion ablating the ability of the virus to bind to cells. In vivo studies were conducted to characterize the biodistribution, blood circulation time, neutralizing antibody formation, and targeted delivery ability of the caspase-activatable provector in a model of heart failure.

Display of Self-Peptide on Adeno-Associated Virus Capsid Decreases Phagocytic Uptake in Vitro.

Adeno-associated virus (AAV) vectors are currently investigated as gene transfer agents for the treatment of a variety of diseases. However, activation of the host immune response upon vector administration limits the use of AAV in the clinical setting. To decrease host detection of AAVs, we tested the CD47-based "don't-eat-me" signal in the context of the AAV capsid. We genetically incorporated the bioactive region of CD47, named "self-peptide" (SP), onto the surface of the AAV2 capsid. AAV mutants were structurally and functionally characterized for vector production, SP and linker incorporation into the capsid, transduction efficiency, and phagocytic susceptibility. We demonstrate that utilizing linkers improves the AAV2 capsid's tolerance to SP insertion. Notably, the SP significantly decreases the phagocytic susceptibility of AAV2 in vitro. Collectively, these results suggest that display of the SP motif on the AAV capsid surface can inhibit phagocytosis of the vector in vitrovia the "don't-eat-me" signaling.

An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription.

Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.

Site-Specific Post-translational Surface Modification of Adeno-Associated Virus Vectors Using Leucine Zippers.

Adeno-associated virus (AAV) is widely favored as a gene therapy vector, tested in over 200 clinical trials internationally. To improve targeted delivery a variety of genetic capsid modifications, such as insertion of targeting proteins/peptides into the capsid shell, have been explored with some success but larger insertions often have unpredictable deleterious impacts on capsid formation and gene delivery. Here, we demonstrate a modular platform for the integration of exogenous peptides and proteins onto the AAV capsid post-translationally while preserving vector functionality. We decorated the AAV capsid with leucine-zipper coiled-coil binding motifs that exhibit specific noncovalent heterodimerization. AAV capsids successfully display hexahistidine tagged-peptides using this approach, as demonstrated through nickel column affinity. This protein display platform may facilitate the incorporation of biological moieties on the AAV surface, expanding possibilities for vector enhancement and engineering.

A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPR-Cas9-mediated somatic recombination.

Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single "workhorse" mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras G12D allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic KrasG12D allele at the endogenous locus, and insertion deletion ("indel") and frameshift mutations of targeted tumor suppressor alleles. By using a single "workhorse" mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we present a facile autochthonous platform for interrogation of the PDAC genome.

Protease-Activatable Adeno-Associated Virus Vector for Gene Delivery to Damaged Heart Tissue.

Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, its adoption as a safe and effective delivery vector for certain diseases relies on altering its tropism to deliver transgenes to desired cell populations. To this end, we have developed a protease-activatable AAV vector, named provector, that responds to elevated extracellular protease activity commonly found in diseased tissue microenvironments. The AAV9-based provector is initially inactive, but then it can be switched on by matrix metalloproteinases (MMP)-2 and -9. Cryo-electron microscopy and image reconstruction reveal that the provector capsid is structurally similar to that of AAV9, with a flexible peptide insertion at the top of the 3-fold protrusions. In an in vivo model of myocardial infarction (MI), the provector is able to deliver transgenes site specifically to high-MMP-activity regions of the damaged heart, with concomitant decreased delivery to many off-target organs, including the liver. The AAV provector may be useful in the future for enhanced delivery of transgenes to sites of cardiac damage.

Longer Inactivating Sequence in Peptide Lock Improves Performance of Synthetic Protease-Activatable Adeno-Associated Virus.

Adeno-associated viruses (AAVs) are promising gene therapy vectors but may exhibit off-target delivery due to broad tissue tropism. We recently developed a synthetic protease-activatable AAV vector, named provector, that transduces cells preferentially in environments rich in matrix metalloproteinases (MMPs) which are elevated in a variety of diseases, including various cancers and heart diseases. The provector displays peptide locks made up of MMP recognition sites flanking an inactivating sequence (IS) composed of four aspartic acid residues (D4). When present, the IS prevents AAV from binding cell receptors and no transduction occurs (OFF state). High levels of MMPs cleave the recognition sequences and release the IS from the capsid surface, restoring cell receptor binding (ON state). The AAV9 provector prototype is not optimal as it displays baseline OFF transduction at 5-10% of that of the wild-type capsid, which can lead to off-target delivery. We hypothesized that changes to the IS may decrease OFF state transduction. We created a provector panel with IS of lengths 0 (D0) to 10 (D10) aspartic acid residues and characterized this panel in vitro. Notably, we find that the D10 provector has an OFF transduction of less than 1% of wild-type capsid and an ON/OFF transduction ratio of 27, the best outcome achieved for any provector thus far. In summary, our results enable us to define new design rules for the provector platform, specifically that (1) the IS is necessary for provector locking and (2) increasing the number of aspartic acid residues in this sequence improves locking.

Physical, chemical, and synthetic virology: Reprogramming viruses as controllable nanodevices.

The fields of physical, chemical, and synthetic virology work in partnership to reprogram viruses as controllable nanodevices. Physical virology provides the fundamental biophysical understanding of how virus capsids assemble, disassemble, display metastability, and assume various configurations. Chemical virology considers the virus capsid as a chemically addressable structure, providing chemical pathways to modify the capsid exterior, interior, and subunit interfaces. Synthetic virology takes an engineering approach, modifying the virus capsid through rational, combinatorial, and bioinformatics-driven design strategies. Advances in these three subfields of virology aim to develop virus-based materials and tools that can be applied to solve critical problems in biomedicine and biotechnology, including applications in gene therapy and drug delivery, diagnostics, and immunotherapy. Examples discussed include mammalian viruses, such as adeno-associated virus (AAV), plant viruses, such as cowpea mosaic virus (CPMV), and bacterial viruses, such as Qß bacteriophage. Importantly, research efforts in physical, chemical, and synthetic virology have further unraveled the design principles foundational to the form and function of viruses. This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Reverse transduction can improve efficiency of AAV vectors in transduction-resistant cells.

Reverse transduction, also known as substrate-mediated gene delivery, is a strategy in which viral vectors are first coated onto a surface that subsequently comes into contact with mammalian cells. The cells internalize the surface-attached vectors, resulting in transgene expression. We hypothesized that forcing the interaction between cells and adeno-associated virus (AAV) vectors through a reverse transduction format would increase in vitro gene delivery efficiencies of the vectors in transduction-resistant cells. We tested this hypothesis by comparing the gene delivery efficiencies of three AAV serotypes using either standard or reverse transduction approaches. Our study reveals reverse transduction of AAV7 and AAV9 can significantly improve their delivery efficiencies. In contrast, AAV2 does not perform better under the reverse transduction format. Interestingly, increased vector uptake by cells does not provide a complete explanation for the increased transduction efficiency. Our findings offer a simple and practical method for improving transduction outcomes in vitro in cell types less permissive to a particular AAV vector.

Reprogramming the Activatable Peptide Display Function of Adeno-Associated Virus Nanoparticles.

We harnessed an intrinsic activatable peptide display behavior shared by several parvoviruses, including the adeno-associated virus (AAV), in order to design protein-based nanodevices that can carry out an exogenous functional output in response to stimulus detection. Specifically, we generated truncated viral capsid subunits that, when combined with native capsid components into mosaic capsids, can perform robust activatable peptide display. By modulating the ratio of subunits in the mosaic capsid, properties of the activatable peptide display function can be optimized. Interestingly, the truncated subunits can form homomeric capsids not observed in nature, but at the price of losing the ability to carry out activatable peptide display. Collectively, our results demonstrate the importance of capsid mosaicism when activatable peptide display is desired and help explain why the wild-type AAV capsid exists as a mosaic of different subunits. This proof-of-concept study illustrates a strategy for reprogramming a particular conformational output behavior of AAV in pursuit of the long-term vision of creating stimulus-responsive nanodevices.

Adeno-Associated Virus (AAV) Vectors: Rational Design Strategies for Capsid Engineering.

Adeno-associated virus (AAV) consists of a simple genome, infects mammalian cells, displays nonpathogenicity in humans, and spans an array of serotypes and variants bearing distinct tissue tropisms. These attributes lend AAV tremendous promise as a gene delivery vector, further substantiated by its extensive testing in human clinical trials. Rational design approaches to capsid engineering leverage current scientific knowledge of AAV to further modulate, enhance and optimize the performance of the vectors. Capsid modification strategies include amino acid point mutations, peptide domain insertions, and chemical biology approaches. Through such efforts, insights regarding AAV capsid sequence-structure-function relationships can be learned. Developments over the last 5 years in rational design-based capsid engineering approaches will be presented and discussed.

Stimulus-responsive viral vectors for controlled delivery of therapeutics.

Virus-based therapies have gained momentum as the next generation of treatments for a variety of serious diseases. In order to make these therapies more controllable, stimulus-responsive viral vectors capable of sensing and responding to specific environmental inputs are currently being developed. A number of viruses naturally respond to endogenous stimuli, such as pH, redox, and proteases, which are present at different concentrations in diseases and at different organ and organelle sites. Additionally, rather than relying on natural viral properties, efforts are underway to engineer viruses to respond to endogenous stimuli in new ways as well as to exogenous stimuli, such as temperature, magnetic field, and optical light. Viruses with stimulus-responsive capabilities, either nature-evolved or human-engineered, will be reviewed to capture the current state of the field. Stimulus-responsive viral vector design considerations as well as gaps in current research efforts will be identified.