Efficiency of Protease-Activatable Virus Nanonodes Tuned Through Incorporation of Wild-Type Capsid Subunits

Ho, Michelle; Judd, Justin; Kuypers, Brianna E.; Yamagami, Momona; Wong, Fergus F.; Suh, Junghae

doi:10.1007/s12195-014-0334-y

Cited by 16 publications

(17 citation statements)

References 30 publications

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“…The PAVs are less efficient compared to wt, which has been previously observed. 17,18 Both of the tested PAVs exhibit 2-fold lower TI values in the unlocked state (i.e., MMP treatment condition) compared to wt. These findings indicate PAV-D4 and PAV-E4 are able to regain transduction abilities in the presence of MMP7, albeit at a lower-than-wt efficiency.…”

Section: Resultsmentioning

confidence: 99%

Role of Tetra Amino Acid Motif Properties on the Function of Protease-Activatable Viral Vectors

Robinson

Judd

et al. 2016

ACS Biomater. Sci. Eng.

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Protease-activatable viruses (PAV) based on adeno-associated virus have previously been generated for gene delivery to pathological sites characterized by elevated extracellular proteases. “Peptide locks”, composed of a tetra-aspartic acid motif flanked by protease cleavage sequences, were inserted into the virus capsid to inhibit virus-host cell receptor binding and transduction. In the presence of proteases, the peptide locks are cleaved off the capsid, restoring the virus’ ability to bind cells and deliver cargo. Although promising, questions remained regarding how the peptide locks prevented cell binding. In particular, it was unclear if the tetra-amino acid (4AA) motif blocks receptor binding via electrostatic repulsion or steric obstruction. To explore this question, we generated a panel of PAVs with lock designs incorporating altered 4AA motifs, each wielding various chemical properties (negative, positive, uncharged polar, and hydrophobic) and characterized the resultant PAV candidates. Notably, all mutants display reduced receptor binding and decreased transduction effciency in the absence of proteases, suggesting simple electrostatics between heparin and the D4 motif do not play an exclusive role in obstructing virus-receptor binding. Even small hydrophobic (A4) and uncharged polar (SGGS) motifs confer a reduction in heparin binding compared to the wild type. Furthermore, both uncharged polar N4 and Q4 mutants (comparable in size to the D4 and E4 motifs respectively, but lacking the negative charge) demonstrate partial ablation of heparin binding. Collectively, these results support a possible dual mechanism of PAV lock operation, where steric hindrance and electrostatics make nonredundant contributions to the disruption of virus-receptor interactions. Finally, because of high virus titer production and superior capsid stability, only the negatively charged 4AA motifs remain viable design choices for PAV construction. Future studies probing the structure–function relationship of PAVs will further expand its promise as a gene delivery vector able to target diseased tissues exhibiting elevated extracellular proteases.

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Section: Resultsmentioning

confidence: 99%

Role of Tetra Amino Acid Motif Properties on the Function of Protease-Activatable Viral Vectors

Robinson

Judd

et al. 2016

ACS Biomater. Sci. Eng.

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“…334 By controlling the ratio of assembled wild-type viral capsid to protease-activatable subunits, the overall transduction level of protease-activatable viruses (PAVs) increased. 335 Using error-prone polymerase chain reaction (EP-PCR), Asuri et al created a library of AAV capsid genes with point mutations, which resulted in a viral variant that was more efficient in delivering genetic payloads to human stem cells. 336 The vector is further enhanced by conjugative delivery to ZFNs: the induced DSB facilitated HDR repair of the delivered transgene, thereby enabling gene targeting.…”

Section: Increasing the Specificity Of Gene Correctionmentioning

confidence: 99%

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

Li¹,

Yang²,

Hong³

et al. 2020

Sig Transduct Target Ther

1,470

869

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Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

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“…Protease activity was measured prior to each experiment to reduce variability due to enzyme storage, as previously described. 12,44 Briefly, the activity of 5 nM MMP on 5 mM of the fluorogenic substrate Mca-PLGL-Dpa-AR (Calbiochem, Burlington, MA) was measured using a Tecan M1000 plate reader in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM CaCl 2 , and 0.005% Brij-35. The amount of enzyme added to each proteolysis reaction was standardized to the initial reaction velocity, as previously described.…”

Section: Structure Determination Model Building and Refinementmentioning

confidence: 99%

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

et al. 2019

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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.

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Efficiency of Protease-Activatable Virus Nanonodes Tuned Through Incorporation of Wild-Type Capsid Subunits

Cited by 16 publications

References 30 publications

Role of Tetra Amino Acid Motif Properties on the Function of Protease-Activatable Viral Vectors

Role of Tetra Amino Acid Motif Properties on the Function of Protease-Activatable Viral Vectors

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

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

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