Browsing by Author "Ho, Michelle L."

Now showing 1 - 3 of 3
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    Effective Gene Delivery to Valvular Interstitial Cells Using Adeno-Associated Virus Serotypes 2 and 3
    (Mary Ann Liebert, Inc., 2015) Wong, Fergus F.; Ho, Michelle L.; Yamagami, Momona; Lam, Michael T.; Grande-Allen, K. Jane; Suh, Junghae; Systems, Synthetic, and Physical Biology Program
    Currently, curative therapies for heart valve diseases do not exist, thus motivating the need for new therapeutics, regenerative and tissue-engineered valves, and further basic research into pathological mechanisms. For studying valve diseases and developing valve therapies, effective methods to manipulate gene expression in primary valvular interstitial cells (VICs), which promote calcification in disease, would be valuable. Unfortunately, there is little information reported about effective gene delivery methods for VICs. Adeno-associated virus (AAV) is a clinically proven gene delivery vector capable of transducing many cell types and tissues, but has not yet been reported to infect valvular cells. In this study, AAV serotypes 1–9 were tested for their ability to deliver a green fluorescent protein (GFP) reporter into VICs in vitro. Flow cytometry results indicate AAV2 and AAV3 are capable of transducing VICs more efficiently than other serotypes. Furthermore, transduction efficiencies can be optimized by increasing the multiplicity of infection (MOI) and using self-complementary, double-stranded genomes, yielding up to 98% successfully transduced cells. Transduction of VICs by AAV2 or AAV3 in the presence of competing soluble heparin significantly reduces delivery efficiencies, suggesting heparan sulfate proteoglycans act as the primary VIC receptors of these two serotypes. Overall, this study establishes AAV2 and AAV3 as efficient gene delivery vehicles for primary VICs. Such effective delivery vectors for valve cells may be broadly useful for numerous applications, including the study of valvular cell biology, development of valve disease therapies, and regulation of genes for tissue engineering heart valves.
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    SCHEMA Computational Design of Virus Capsid Chimeras: Calibrating How Genome Packaging, Protection, and Transduction Correlate with Calculated Structural Disruption
    (American Chemical Society, 2013) Ho, Michelle L.; Adler, Benjamin A.; Torre, Michael L.; Silberg, Jonathan J.; Suh, Junghae
    Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.
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    Tunable Protease-Activatable Virus Nanonodes
    (American Chemical Society, 2014) Judd, Justin; Ho, Michelle L.; Tiwari, Abhinav; Gomez, Eric J.; Dempsey, Christopher; Vliet, Kim Van; Igoshin, Oleg A.; Silberg, Jonathan J.; Agbandje-McKenna, Mavis; Suh, Junghae
    We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virusﾖreceptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.