Modulation of the liver immune microenvironment by the adeno-associated virus serotype 8 gene therapy vector.

Adeno-associated viruses (AAVs) are emerging as one of the vehicles of choice for gene therapy. However, the potential immunogenicity of these vectors is a major limitation of their use, leading to the necessity of a better understanding of how viral vectors engage the innate immune system. In this study, we demonstrate the immune response mediated by an AAV vector in a mouse model.

Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency.

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells.

Genome-wide expression profiling of the retinoschisin-deficient retina in early postnatal mouse development.

Purpose: The Rs1h knockout mouse displays retinal features typical for X-linked juvenile retinoschisis (RS). Consequently, this mouse line represents an excellent model to study early molecular events in RS.

Methods: Whole genome expression profiling using DNA-microarrays was performed on total RNA extracts from retinoschisin-deficient and wild-type murine retinas from postnatal days 7, 9, 11, and 14.

Insertion and topology of normal and mutant bestrophin-1 in the endoplasmic reticulum membrane.

The vitelliform macular dystrophy type 2 (VMD2) gene mutated in Best macular dystrophy encodes a 585-amino acid putative transmembrane protein termed bestrophin-1. The vast majority of known disease-associated alterations are of the missense type, which cluster near predicted transmembrane domains (TMDs). To investigate bestrophin-1 membrane topology and to assess consequences of point mutations on membrane integration, we have analyzed the insertion of putative TMDs into the endoplasmic reticulum (ER) membrane.