Pendrin stimulates a chloride absorption pathway to increase CFTR-mediated chloride secretion from Cystic Fibrosis airway epithelia.

Cystic Fibrosis (CF), an inherited multi-system disease, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that disrupt its ability to secrete anions from epithelia. Recovery of functional anion secretion may be curative for CF, so different components of the ion transport machinery have become attractive therapeutic targets. Several members of the SLC26 ion transporter family have been linked to epithelial ion flux, some through putative functional interactions with CFTR. Using a small-scale qPCR screen, we confirmed that the anion transporter SLC26A4 (pendrin) is downregulated in CF. Upregulation of pendrin using interleukins IL-4 or IL-13 increased Cl- secretion through CFTR in human bronchial epithelial cell (HBEC) derived epithelia differentiated in vitro and measured in the Ussing Chamber. Inhibition or knockdown of pendrin prevented this increased secretion. Increased CFTR activity was not driven by increases in CFTR protein or upstream regulatory pathway components. When basolateral Cl- absorption through NKCC1 was inhibited, a pendrin-dependent Cl- absorption pathway allowing CFTR to continue secreting Cl- from the epithelium was revealed. Although CFTR is often considered the bottleneck in the transepithelial Cl- transport pathway, these studies indicate that basolateral Cl- permeability becomes limiting as CFTR activity increases. Therefore, an increase of epithelial Cl- absorption via pendrin might have additional therapeutic benefit in combination with CFTR modulators.

Gene transfer to the CNS is efficacious in immune-primed mice harboring physiologically relevant titers of anti-AAV antibodies.

Central nervous system (CNS)-directed gene therapy with recombinant adeno-associated virus (AAV) vectors has been used effectively to slow disease course in mouse models of several neurodegenerative diseases. However, these vectors were typically tested in mice without prior exposure to the virus, an immunological scenario unlikely to be duplicated in human patients. Here, we examined the impact of pre-existing immunity on AAV-mediated gene delivery to the CNS of normal and diseased mice. Antibody levels in brain tissue were determined to be 0.6% of the levels found in systemic circulation. As expected, transgene expression in brains of mice with relatively high serum antibody titers was reduced by 59-95%. However, transduction activity was unaffected in mice that harbored more clinically relevant antibody levels. Moreover, we also showed that markers of neuroinflammation (GFAP, Iba1, and CD3) and histopathology (hematoxylin and eosin (H&E)) were not enhanced in immune-primed mice (regardless of pre-existing antibody levels). Importantly, we also demonstrated in a mouse model of Niemann Pick Type A (NPA) disease that pre-existing immunity did not preclude either gene transfer to the CNS or alleviation of disease-associated neuropathology. These findings support the continued development of AAV-based therapies for the treatment of neurological disorders.

Systemic administration of AAV8-α-galactosidase A induces humoral tolerance in nonhuman primates despite low hepatic expression.

In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal. However, this effect was not robust, and sustained α-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.

Preexisting immunity and low expression in primates highlight translational challenges for liver-directed AAV8-mediated gene therapy.

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.

Crossing paths with Notch in the hyper-network.

The development of complex and diverse metazoan morphologies is coordinated by a surprisingly small number of evolutionarily conserved signaling mechanisms. These signals can act in parallel but often appear to function as an integrated hyper-network. The nodes defining this complex molecular circuitry are poorly understood, but the biological significance of pathway cross-talk is profound. The importance of such large-scale signal integration is exemplified by Notch and its ability to cross-talk with all the major pathways to influence cell differentiation, proliferation, survival and migration. The Notch pathway is, thus, a useful paradigm to illustrate the complexity of pathway cross-talk: its pervasiveness, context dependency, and importance in development and disease.