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.

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.

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.

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.

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.

Local 3D matrix confinement determines division axis through cell shape.

How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via ß1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of ß1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.