Microcontact printing for co-patterning cells and viruses for spatially controlled substrate-mediated gene delivery

Abstract

Spatial organization of gene expression is a crucial element in the development of complex native tissues, and the capacity to achieve spatially controlled gene expression profiles in a tissue engineering construct is still a considerable challenge. To give tissue engineers the ability to design specific, spatially organized gene expression profiles in an engineered construct, we have investigated the use of microcontact printing to pattern recombinant adeno-associated virus (AAV) vectors on a two dimensional surface as a first proof-of-concept study. AAV is a highly safe, versatile, stable, and easyto-use gene delivery vector, making it an ideal choice for this application. We tested the suitability of four chemical surfaces (-CH 3 , -COOH, -NH 2 , and -OH) to mediate localized substrate-mediated gene delivery. First, polydimethylsiloxane stamps were used to create microscale patterns of various selfassembled monolayers on gold-coated glass substrates. Next, AAV particles carrying genes of interest and human fibronectin (HFN) were immobilized on the patterned substrates, creating a spatially organized arrangement of gene delivery vectors. Immunostaining studies reveal that -CH 3 and -NH 2 surfaces result in the most successful adsorption of both AAV and HFN. Lastly, HeLa cells were used to analyze viral transduction and spatial localization of gene expression. We find that -CH 3 , -COOH, and -NH 2 surfaces support complete uniform cell coverage with high gene expression. Notably, we observe a synergistic effect between HFN and AAV for substrate-mediated gene delivery. Our flexible platform should allow for the specific patterning of various gene and shRNA cassettes, resulting in spatially defined gene expression profiles that may enable the generation of highly functional tissue.

Live-cell Microarray Surface Coatings Supporting Reverse Transduction by Adeno-Associated Viruses

“…Although GAPS and amine silane coatings allow for localized AAV deposition, the density of nondenatured virus deposition is low, highlighted by low fluorescence values on the intensity plots (Figure 2, DE). Low levels of AAV2 adsorption on amine surfaces is consistent with our previous studies (16). Here, edge effects can be observed where more virus is deposited along the edges of the spots.…”

“…In contrast, the nitrocellulose coating yields bright virus staining, indicating a greater amount of nondenatured AAV is immobilized in the spotted area (Figure 2F). Our previous work indicates a hydrophobic methyl (-CH3) surface is able to mediate high levels of AAV2 adsorption (16), suggesting the hydrophobic character of nitrocellulose may support more effective AAV immobilization. Interestingly, a higher distribution of AAV is observed along the center axis of the spot, likely attributable to the geometry of the microarray pin.…”

Enhanced gene delivery in porcine vasculature tissue following incorporation of adeno-associated virus nanoparticles into porous silicon microparticles

“…Prior work using alkanethiols (displaying various chemical functional groups) patterned onto gold surfaces demonstrated AAV2 can adsorb to many surface chemistries, with the methyl surface yielding highest virus attachment. [15] Differences in outcome between pSi and alkanethiol-gold surfaces may be due to topographical differences.…”

Reverse transduction can improve efficiency of AAV vectors in transduction‐resistant cells

“…AAV‐based reverse transduction has also been used in various studies relevant to tissue engineering. For example, transduction efficiency resulting from AAV2 patterned onto different substrate coatings, including extracellular matrix (ECM) components, has been evaluated (McConnell, Gomez, & Suh, ; McConnell, Slater, Han, West, & Suh, ). Understanding the interaction between AAV vectors and ECM proteins is particularly relevant because the latter are present in all tissues and organs, as well as being frequently incorporated into scaffolds for tissue regeneration.…”