Cell targeted phagemid rescued by preselected landscape phage.

We have developed a gene delivery system that utilizes a cell-binding helper phage preselected from a landscape phage display library, and a phagemid harboring a marker gene and all regulatory elements (origins of replication and promoter-enhancer cassettes) necessary for replication of the phagemid and expression of the marker gene in the targeted cell. All the proteins required for encapsulation of the phagemid DNA and cell targeting are provided by the phage helper and are separate from the phagemid. Therefore, the resultant Phagemid Infective Particles (PIPs) are able to bind and infect target cells and express the marker gene from within the cell. Our approach, shown here for glioma cells, differs from others in that a phagemid expressing a model marker or particular therapeutic gene can be easily exchanged for a phagemid expressing a different therapeutic gene. Also, a different helper phage, selected from a phage display library, such as the f8-8-mer landscape library used here, can target any cell type and direct the encapsulation of any therapeutic gene encoding phagemid. Because of its versatility, the PIPs system may be readily used for optimization of the gene-delivery strategies applied to specific cell and tissue targets.

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy.

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)-mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 x 10(12) vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.