The goal of this project is to develop gene therapy approaches as potential treatments for both hemophilia A and B. Although these disease are similar clinically, they are caused by distinct genes whose proteins display different challenges for effective gene expression. In the case of factor VIII, it is a large protein/cDNA that has proved difficult to express in any system and must be produced by cells that have ready access to the circulation. For example, its' physical size (at 4.5 kb for the smallest cDNA construct) all but rule-out AAV as a delivery vector. This leaves retroviruses as the only stable gene transfer system, but factor VIII has an inhibitory effect on retrovirus expression. For factor IX, gene expression has not been problematic, but because it has a higher steady state concentration and larger volume distribution, effective gene therapy strategies will require 50-100 fold higher gene expression than for factor VIII. Because of these observations, the specific aims of this project are: 1. Construct retroviral vectors for the transfer and expression of factor VIII. We have successfully completed this aim by determining that the introduction of an MLV intron before the factor VIII cDNA increases vector titer. We have further isolated both ecotropic and amphotropic factor VIII producers cell clones with titers greater than 1 X 10 5 cfu/ml and are analyzing the expression properties of these vectors. 2. Produce viral vectors that efficiently transfer and express factor IX in primary cells. To achieve this goal we produced and analyzed retroviral vectors and adeno-associated virus (AAV) vectors that efficiently express factor IX. We subsequently compared the relative gene transfer efficiencies and factor IX expression properties of retroviral vectors and AAV vectors, and found the retroviral vectors to be the better choice.
