New Evidence That ECM Can Act as a Barrier to AAV Gene Therapy
ECM as a barrier to gene therapy
An in vitro study found that the extracellular matrix (ECM) can limit the diffusion of adeno-associated virus (AAV) vectors and transduction. The effects differed by AAV serotype and the tissue source of the ECM. The study did not assess treatment outcomes in patients or test ways to overcome this delivery barrier.
In AAV-based gene therapy, vectors must reach their target cells. ECM, which forms part of the tissue microenvironment, may physically impede particle movement or bind particles, thereby limiting delivery. The researchers examined how tissue-specific ECM composition and AAV serotype affect diffusion and transduction.
Diffusion and transduction vary by AAV type and tissue
The team fluorescently labeled AAV2, AAV6, and AAV8 and used imaging to track their diffusion in hydrogels made from decellularized extracellular matrix (dECM) derived from porcine lung, liver, and small-intestinal submucosa. The comparators were similarly sized, 20 nm polyethylene glycol (PEG)-coated nanoparticles.
AAV6 and AAV8 diffused significantly more slowly than the comparator nanoparticles in dECM from all three tissues. AAV2 diffused significantly more slowly in lung and liver dECM, but showed no significant difference from the nanoparticles in small-intestinal submucosa dECM. Among the three serotypes, AAV2 diffused fastest, while the relative order of AAV6 and AAV8 varied by tissue.
Transduction was assessed in two-dimensional cultures and three-dimensional spheroid models of A549 cells. In 2D cultures overlaid with lung dECM at 6 mg/mL, the proportion of green fluorescent protein (GFP)-positive cells was measured after 48 hours. For AAV2 and AAV6, it fell to about half the level seen without dECM. The same reduction was not observed for AAV8. However, even without dECM, AAV8 produced a lower proportion of GFP-positive cells than the other two serotypes.
The proportion of transduced cells should be distinguished from GFP fluorescence intensity per cell. In the presence of lung dECM, GFP fluorescence intensity in viable cells decreased with AAV6 but increased with AAV2 and AAV8. In the AAV6 experiment using three-dimensional spheroids surrounded by lung dECM, both the proportion of transduced cells among viable cells and GFP fluorescence intensity per cell decreased. These results cannot be generalized to the responses of all three serotypes in three-dimensional conditions.
Evaluating vectors with ECM in mind and remaining questions
The findings provide a basis for considering interactions with ECM when designing next-generation AAVs. Based on the difference from similarly sized nanoparticles, the researchers interpreted ECM binding as a possible partial explanation for reduced diffusion. They did not directly establish the amount of binding to specific ECM components or a causal link from reduced diffusion to lower transduction.
The dECM hydrogel is a model for comparing tissue-specific compositional differences; it does not reproduce the structure of living tissue. Cell viability also decreased in the three-dimensional dECM condition, and changes in cell properties caused by cell–ECM interactions may have affected transduction. Modification of the capsid, the virus's outer protein shell, to reduce ECM binding was proposed as a direction for future research. This study did not test whether the barrier could be overcome or treatment outcomes in patients improved.
Source: Original study, paper DOI.
PURPOSE: The extracellular matrix (ECM) is a major component of the tissue microenvironment which may pose a barrier to the distribution of AAV in target organs, preventing delivery of therapeutic cargo. We sought to address this potential barrier to AAV gene therapy by furthering our understanding of AAV-ECM interactions. We hypothesized that both the AAV serotype and ECM composition will impact AAV transport and gene delivery. METHODS: AAV2, AAV6, and AAV8 viral vectors were fluorescently labeled to allow for visualization of their diffusion through the ECM. Lung, liver, and small intestinal submucosal dECM hydrogels were formulated as models of the ECM with tissue-specific biomolecular content. We then characterized AAV and nanoparticle diffusion within decellularized ECM using fluorescent video microscopy and multiple particle tracking. Additionally, we evaluated AAV transduction in dECM-incorporated 2D and 3D spheroid tissue culture models. RESULTS: All AAV displayed reduced diffusivity through ECM as compared to similarly sized nanoparticles. AAV2 diffusion was least affected by the presence of ECM across tissue types as compared to AAV6 and AAV8. AAV transduction in dECM incorporated CONCLUSIONS: These results suggest binding of AAV to the ECM may decrease their therapeutic effect in target tissues throughout the body. The barrier function of the ECM should be considered in development of AAV for gene therapy applications.
This study provides a basis for comparing AAV candidates both in cell-only conditions and in conditions that include ECM derived from the target tissue. For example, when testing a vector targeting lung tissue, an evaluation design could separately measure diffusion in lung dECM, the proportion of transduced cells, and GFP fluorescence intensity per cell. These measures did not always change in the same direction in this study. That is why slower diffusion alone makes it difficult to judge transduction or expression patterns uniformly. However, these in vitro findings alone cannot determine which serotype is suitable for patients or predict improved clinical efficacy.