High Efficiencies of Gene Transfer with Immobilized Recombinant Retrovirus: Kinetics and Optimization Claudel Olivier

Recombinant Retroviruses Currently used in the majority of gene therapy clinical trials. Have potential for permanent genetic modification, broad host range, and low immunogenicity. The efficiency of gene transfer is still too low to be of clinical significance.

Limitations Slow diffusion and fast decay of retroviral particles Low titers Low levels of expression of retroviral receptors on target cells

The presence of transduction inhibitors in the viral supernatant The inability of recombinant retrovirus to transduce nondividing cells due to cell cycle dependence of retroviral transduction Loss of viral activity following internalization into the cell cytoplasm

Increasing Transduction Efficiency flow-through method Centrifugation of retroviral particles to the cell monolayer Concentration of retroviral supernatant Complexation of retroviruses with liposomes

Phosphate depletion to regulate the viral receptors on the surface of the target cells Design of the retroviral envelope to improve its stability Increase binding affinity Design of recombinant retroviruses with nuclear localization signals that promote entry of the nucleoprotein complex into the nucleus of nondividing cells

Fibronectin assisted gene transfer (FAGT) Immobilized retroviral particles on the extra cellular matrix molecule fibronectin Improved gene transfer to T-lymphocytes, hematopoietic stem cells and showed promising results In FAGT, the virus is first captured on the surface of FN-coated plates by diffusion from the bulk of the solution, and gene transfer takes place on the surface upon addition of the target cells

Mathematical Model

Equations were solved numerically by using standard computer algorithms The system of ordinary differential equations was solved using the Petzold- Gear BDF method.

Results The rate of bioactivity loss of immobilized retrovirus is a strong function of temperature Binding of retrovirus to FN is independent of retroviral activity

PB significantly decreases the efficiency of FAGT. PB inhibits binding of retrovirus to FN. Ultra filtration of retrovirus stocks yields 10-fold enhancement in FAGT.

Conclusions FAGT depends strongly upon time and temperature of virus incubation in FN- coated plates. PB inhibits FAGT mainly by inhibiting binding to FN Ultra filtration of retrovirus yields 10-fold enhancement of efficiency of FAGT.