Challenges Posed by Immune Responses to AAV Vectors: Addressing Root Causes.

Host immune responses that limit durable therapeutic gene expression and cause clinically significant inflammation remain a major barrier to broadly successful development of adeno-associated virus (AAV)-based human gene therapies. In this article, mechanisms of humoral and cellular immune responses to the viral vector are discussed. A perspective is provided that removal of pathogen-associated molecular patterns in AAV vector genomes to prevent the generation of innate immune danger signals following administration is a key strategy to overcome immunological barriers.

Transduction of Pig Small Airway Epithelial Cells and Distal Lung Progenitor Cells by AAV4.

Cystic fibrosis (CF) is caused by genetic mutations of the CF transmembrane conductance regulator (CFTR), leading to disrupted transport of Cl- and bicarbonate and CF lung disease featuring bacterial colonization and chronic infection in conducting airways. CF pigs engineered by mutating CFTR develop lung disease that mimics human CF, and are well-suited for investigating CF lung disease therapeutics. Clinical data suggest small airways play a key role in the early pathogenesis of CF lung disease, but few preclinical studies have focused on small airways. Efficient targeted delivery of CFTR cDNA to small airway epithelium may correct the CFTR defect and prevent lung infections. Adeno-associated virus 4 (AAV4) is a natural AAV serotype and a safe vector with lower immunogenicity than other gene therapy vectors such as adenovirus. Our analysis of AAV natural serotypes using cultured primary pig airway epithelia showed that AAV4 has high tropism for airway epithelia and higher transduction efficiency for small airways compared with large airways. AAV4 mediated the delivery of CFTR, and corrected Cl- transport in cultured primary small airway epithelia from CF pigs. Moreover, AAV4 was superior to all other natural AAV serotypes in transducing ITGα6ß4+ pig distal lung progenitor cells. In addition, AAV4 encoding eGFP can infect pig distal lung epithelia in vivo. This study demonstrates AAV4 tropism in small airway progenitor cells, which it efficiently transduces. AAV4 offers a novel tool for mechanistical study of the role of small airway in CF lung pathogenesis in a preclinical large animal model.

Polarized AAVR expression determines infectivity by AAV gene therapy vectors.

Adeno-associated virus (AAV) has been investigated to transfer the cystic fibrosis transmembrane conductance regulator (CFTR) to airways. Inhaled AAV2-CFTR in people with cystic fibrosis (CF) is safe, but inefficient. In vitro, AAV2 transduction of human airway epithelia on the apical (luminal) side is inefficient, but efficient basolaterally. We previously selected AAV2.5T, a novel capsid that apically transduces CF human airway epithelia and efficiently restores CFTR function. We hypothesize the AAV receptor (AAVR) is basolaterally localized, and that AAV2.5T utilizes an alternative apical receptor. We found AAVR in human airway epithelia by western blot and RNA-Seq analyses. Using immunocytochemistry we did not find endogenous AAVR at membranes but overexpression localized AAVR to the basolateral membrane, where it preferentially increased transduction. Anti-AAVR antibodies blocked transduction by AAV2 from the basolateral side but not AAV2.5T from the apical side, suggesting a unique apical receptor. Finally, we found infection by AAV2 but not AAV2.5T was blocked by CRISPR knockout of AAVR in cell lines. Our data suggest the absence of apical AAVR is rate limiting for AAV2, and efficient transduction by AAV2.5T is accomplished using an AAVR independent pathway. Our findings inform the development of gene therapy for CF, and AAV vectors in general.

EGF regulation of proximal tubule cell proliferation and VEGF-A secretion.

Proximal tubule cell (PTC) proliferation is critical for tubular regeneration and recovery from acute kidney injury. Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF-A) are important for the maintenance of tubulointerstitial integrity and can stimulate PTC proliferation. We utilized HK-2 cells, an immortalized human PTC line, to characterize the EGF-dependent regulation of VEGF-A secretion and proliferation in PTCs. We demonstrate that EGF stimulates VEGF-A secretion via the EGF receptor (EGFR) and stimulates cell proliferation via activation of the VEGF receptor, VEGFR-2. EGFR activation promotes MAPK (ERK1/2) activation and HIF-1α expression, which are required for basal and EGF-stimulated VEGF-A secretion. EGF also stimulates the phosphorylation of P70S6 kinase (P70S6K), the downstream target of mTORC1. Rapamycin decreased basal and EGF stimulated HIF-1α and enhanced MAPK (ERK1/2) activation, while MAPK (ERK/12) inhibition downregulated HIF-1α expression and the phosphorylation of p70S6K. EGF stimulation of p70S6K was also independent of p-AKT Inhibition of the mTORC1 pathway with rapamycin abolished phosphorylation of p70S6K but had no effect on VEGF-A secretion, indicating that EGF-stimulated VEGF-A secretion did not require mTORC1 pathway activation. We demonstrate evidence of a complex crosstalk between the MAPK/ERK and mTORC1 pathways, wherein MAPK (ERK1/2) activation stimulates p-P70S6K, while p-P70S6K activation seems to inhibit MAPK (ERK1/2) in EGF-treated HK-2 cells. Our results suggest that EGF stimulates MAPK (ERK1/2) in HK-2 cells, which in turn increases HIF-1α expression and VEGF-A secretion, indicating that VEGF-A mediates EGF-stimulated cell proliferation as an autocrine proximal tubular epithelial cell growth factor.

Chronic stress-induced alterations of dendritic spine subtypes predict functional decrements in an hypothalamo-pituitary-adrenal-inhibitory prefrontal circuit.

Activation of the hypothalamo-pituitary-adrenal (HPA) axis plays a vital role in promoting adaptation during acute stress, but adverse effects of chronic stress may result from overactivity of this system. Recent evidence highlights a subdivision of GABAergic neurons within anterior bed nuclei of the stria terminalis (aBST) that integrates and relays inhibitory influences to HPA-effector neurons in paraventricular hypothalamus during acute stress, notably from medial prefrontal [prelimbic (PL)] and hippocampal [ventral subiculum (vSUB)] cortical fields. Here we localize the site and candidate mechanism of neuroplasticity within upstream regions of this inhibitory network after chronic variable stress (CVS). Rats bearing retrograde tracer injections in aBST underwent CVS for 14 d. Retrogradely labeled and unlabeled neurons in vSUB and PL were selected for intracellular dye filling, followed by three-dimensional imaging and analysis of dendritic arborization and spine morphometry. Whereas PL neurons displayed decreases in dendritic branching and spine density after CVS, aBST-projecting cells showed a selective loss of mature mushroom-shaped spines. In a follow-up experiment, CVS-treated and control rats were exposed to a novel restraint challenge for assay of HPA activation and engagement of aBST-projecting cortical regions. CVS animals showed enhanced HPA output and decreased Fos activation in aBST-projecting PL neurons compared with acutely stressed controls. In contrast, vSUB failed to show any significant differences in structural plasticity or functional activation patterns after CVS. These findings define a mechanism whereby synaptic destabilization in the PL → aBST pathway may dampen its ability to impart inhibitory control over the HPA axis after chronic stress exposure.