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. Mice were infected intravenously with 4 × 1012 copies (cp)/kg of AAV8, and the ensuing immune response was analyzed using intravital microscopy during a period of weeks. Administration of AAV8 resulted in the infection of hepatocytes, and this infection led to a moderate, but significant, activation of the immune system in the liver. This host immune response involved platelet aggregation, neutrophil extracellular trap (NET) formation, and the recruitment of monocytes, B cells, and T cells. The resident liver macrophage population, Kupffer cells, was necessary to initiate this immune response, as its depletion abrogated platelet aggregation and NET formation and delayed the recruitment of immune cells. Moreover, the death of liver cells produced by this AAV was moderate and failed to result in a robust, sustained inflammatory response. Altogether, these data suggest that AAV8 is a suitable vector for gene therapy approaches.

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 in vitro 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. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.

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. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.

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. Quantitative real-time RT-PCR (qRT-PCR) analysis of additional time points facilitated the refinement of the temporal expression profile of differentially regulated transcripts. Differential protein expression was confirmed by Western blot analysis. RESULTS: Based on biostatistic and knowledge-based DNA-microarray analyses we have identified differentially regulated retinal genes in early postnatal stages of the Rs1h-deficient mouse defining key molecular pathways including adhesion, cytoskeleton, vesicular trafficking, and immune response. A significant upregulation of Egr1 at P11 and several microglia/glia-related transcripts starting at P11 with a peak at P14 were identified in the diseased retina. The results provided evidence that macrophage/microglia activation precedes apoptotic photoreceptor cell death. Finally, the role of Egr1 in the pathogenesis of Rs1h-deficiency was investigated, and the results indicated that activation of the MAPK Erk1/2 pathway occurs as early as P7. Analyses of Rs1h(-/Y)/Egr1(-/-) double-knockout mice suggest that Egr1 upregulation is not a prerequisite for macrophage/microglia activation or apoptosis. CONCLUSIONS: The findings imply that microglia/glia activation may be triggering events in the photoreceptor degeneration of retinoschisin-deficient mice. Furthermore, the data point to a role of Erk1/2-Egr1 pathway activation in RS pathogenesis.

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. Out of six potential TMDs, our data suggest a topological model of bestrophin-1 with four transmembrane-spanning segments and one large cytoplasmatic loop between putative TMD2 and TMD5. Consequently, a relatively hydrophobic segment containing putative TMD3 (aa 130-149) and TMD4 (aa 179-201) is located within the cytoplasm. Furthermore, we show that three out of 18 disease-associated alterations investigated (I73N, Y85H, F281del) reveal measurable effects on membrane insertion suggesting that defective membrane integration of bestrophin-1 may represent a potential disease mechanism for a small subset of Best macular dystrophy-related mutations.