RESUMO
Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts.
Assuntos
Proteínas de Arabidopsis/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Núcleo Celular/genética , Técnicas de Transferência de Genes , Optogenética/métodos , Fitocromo B/química , Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Núcleo Celular/ultraestrutura , Dependovirus/genética , Dependovirus/metabolismo , Dependovirus/efeitos da radiação , Dictyostelium/genética , Dictyostelium/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Luz , Nanopartículas/química , Nanopartículas/efeitos da radiação , Sinais de Localização Nuclear , Fitocromo B/genética , Fitocromo B/metabolismo , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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-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.
Assuntos
Dependovirus/química , Nanopartículas/química , Nanotecnologia/métodos , Peptídeo Hidrolases/química , Vírus/química , Sequência de Aminoácidos , Capsídeo/química , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Nanomedicina/métodos , Plasmídeos/química , Engenharia de Proteínas/métodos , Propriedades de Superfície , Temperatura , TransgenesRESUMO
Delivering genes from surfaces, called substrate-mediated gene delivery or reverse transduction, is a useful method to achieve spatial localization of gene delivery. We tested the compatibility of adeno-associated virus (AAV) vectors with various cell adhesive proteins to mediate gene delivery from surfaces. Our studies demonstrate that AAV vectors can be successfully adsorbed on collagen I, elastin, and laminin substrates leading to robust gene delivery to overlying cells. Notably, AAV immobilization on laminin yields the highest efficiency of gene expression. This increased gene expression cannot be explained by increases in the levels of virus deposition, transcriptional activity of cells, or virus vector uptake into cells. Further refinement of our knowledge of AAV interactions with extracellular matrix proteins may have important implications in a variety of applications ranging from tissue engineering to in vivo gene therapy.