In Fantastic Voyage, miniaturized scientists injected into a man's bloodstream destroy a blood clot in his brain. Had they needed to destroy a brain tumor, they might not have been so successful. The blood-brain barrier blocks molecules in the blood from entering neural tissue—including beneficial molecules, such as drugs and gene therapy agents. Now Spencer and Verma report that ligands to receptors in the blood-brain barrier help fused gene products cross into brain tissue in a recent article in Proceedings of the National Academy of Sciences.
Gaucher's disease is a lysosomal storage disorder caused by deficiency of the glucocerebrosidase enzyme, which is involved in fatty acid degradation. The most common form of Gaucher's disease, type I, affects organs and the skeletal system and can be treated by enzyme replacement therapy. Types II and III are less common and affect the central nervous system. Because it cannot breach the blood-brain barrier, enzyme replacement therapy is an ineffective treatment for type II and III Gaucher's disease.
Endothelial cells ensheath brain capillaries. These cells and the astrocytic processes that contact them comprise the blood-brain barrier. Small organic molecules can cross the barrier, but anything larger must be shuttled in by receptors. The blood-brain barrier contains low-density lipoprotein receptors (LDLRs), transferrin receptors and insulin-like growth factor receptors. In most tissues, LDLRs bind and endocytose apolipoproteins, targeting them for lysosomal degradation. On the capillary surface of the blood-brain barrier, LDLRs bind apolipoproteins and shuttle them into brain tissue.
The authors designed a lentiviral vector that induces peripheral organs to continuously produce and secrete its contents in vivo. They subcloned enhanced green fluorescent protein (GFP) or glucocerebrosidase into the vector with and without the LDLR-binding domain of apolipoprotein B. Two weeks after peripheral treatment, adult mouse liver and spleen showed glucocerebrosidase expression regardless of the inclusion of the apolipoprotein B LDLR-binding domain. Cell division in liver and spleen allowed continuous expression and a secretory signal in the vector allowed continuous secretion of the recombinant proteins. The authors detected GFP and glucocerebrosidase in brain only when mice were treated with lentivirus containing apolipoprotein B fusion protein, suggesting that LDLR binding shuttled gene products across the blood-brain barrier.
Expression of the apolipoprotein fusion protein varied in different types of brain cells. As shown by coimmunofluorescence, recombinant protein was expressed in all calbindin-positive Purkinje cells in the cerebellum, but only approximately 55% of calbindin-positive interneurons in the cortex. Approximately 30% of all NeuN-positive neurons expressed recombinant protein, with NeuN-positive striatal neurons showing the most and NeuN-positive hippocampal neurons showing the least recombinant protein. Roughly 84% of glial fibrillary acidic protein-positive astrocytes showed recombinant protein expression. Fusing ligands from other blood-brain barrier receptors to recombinant proteins may alter the pattern of recombinant protein uptake in brain cells.
In addition to treating those with Gaucher's disease, this technique may also help people with other currently untreatable lysosomal storage disorders, including Tay-Sachs and Niemann-Pick diseases.