Moderate Heat-Assisted Gene Electrotransfer as a Potential Delivery Approach for Protein Replacement Therapy through the Skin

Gene-based approaches for protein replacement therapies have the potential to reduce the number of administrations. Our previous work demonstrated that expression could be enhanced and/or the applied voltage reduced by preheating the tissue prior to pulse administration. In the current study, we utilized our 16-pin multi-electrode array (MEA) and incorporated nine optical fibers, connected to an infrared laser, between each set of four electrodes to heat the tissue to 43 °C. For proof of principle, a guinea pig model was used to test delivery of reporter genes. We observed that when the skin was preheated, it was possible to achieve the same expression levels as gene electrotransfer without preheating, but with a 23% reduction of applied voltage or a 50% reduction of pulse number. With respect to expression distribution, preheating allowed for delivery to the deep dermis and muscle. This suggested that this cutaneous delivery approach has the potential to achieve expression in the systemic circulation, thus this protocol was repeated using a plasmid encoding Human Factor IX. Elevated Factor IX serum protein levels were detected by ELISA up to 100 days post gene delivery. Further work will involve optimizing protein levels and scalability in an effort to reduce application frequency.

Download Full-text

Persistent Systemic Production of Human Factor IX in Mice by Skeletal Myoblast-Mediated Gene Transfer: Feasibility of Repeat Application to Obtain Therapeutic Levels

Blood ◽

10.1182/blood.v90.3.1075 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1075-1082 ◽

Cited By ~ 34

Author(s):

Jian-Min Wang ◽

Hong Zheng ◽

Mila Blaivas ◽

Kotoku Kurachi

Keyword(s):

Gene Transfer ◽

Human Factor ◽

Plasmid Vector ◽

Systemic Circulation ◽

Scid Mice ◽

Factor Ix ◽

Therapeutic Level ◽

Muscle Creatine Kinase ◽

Human Factor Ix

Abstract Myoblast-mediated gene transfer and its repeated applications were tested for achieving a long-term stable systemic production of human factor IX (hFIX) at a therapeutic level in SCID mice. Primary skeletal myoblasts were stably transfected with a hFIX expression plasmid vector, pdLMe4βAhIXm1, which contains a hFIX minigene under the control of a β-actin promoter with muscle creatine kinase enhancers. Myotubes derived from the myoblasts produced 1,750 ng hFIX/106 cells/24 hours in culture. hFIX secretion by the myoblasts and thereof derived myotubes were equally efficient, and myotubes were shown to have a sufficient secretory capacity to handle a substantially elevated production of hFIX. After intramuscular injection of 5, 10, and 20 × 106 myoblasts, SCID mice stably produced hFIX into the systemic circulation proportional to the number of implanted cells, and the expression levels were maintained for at least up to 10 months (end of the experiment). Additional cell injections administered to animals that originally received 10 × 106 cells approximately 2 months later elevated the systemic hFIX levels to an average of 182 ± 21 ng/mL, a therapeutic level, which persisted for at least 8 months (end of the experiment). These results indicate that long-term, stable systemic production of hFIX at therapeutic levels can be achieved by repeated application of myoblast-mediated gene transfer.

Download Full-text

Intratracheal Administration of Recombinant Human Factor IX (BeneFix™) Achieves Therapeutic Levels in Hemophilia B Dogs

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615602 ◽

2001 ◽

Vol 85 (03) ◽

pp. 445-449 ◽

Cited By ~ 7

Author(s):

K. E. Russell ◽

M. S. Read ◽

D. A. Bellinger ◽

K. Leitermann ◽

B. J. Rup ◽

...

Keyword(s):

Therapeutic Option ◽

Systemic Circulation ◽

Factor Ix ◽

Biologically Active ◽

Hemophilia B ◽

Pharmacokinetic Parameters ◽

Intratracheal Administration ◽

Human Factor Ix ◽

Post Infusion ◽

Recombinant Human Factor Ix

SummaryThe purpose of this paper was to establish proof of concept for administration of human recombinant F.IX (rF.IX) by inhalation for therapy of hemophilia B. The pharmacokinetics of intratracheal (IT) administration of rF.IX was studied in nine hemophilia B dogs randomized into 3 groups that received 200 IU/kg IT, 1000 IU/kg IT, or 200 IU/kg intravenously (IV). IT rF.IX produced therapeutic levels of F.IX antigen and activity and the pharmacokinetic parameters were consistent with a slow release from a depot site within the lungs. Bio-availability compared to IV administration was 11% for 200 IU/kg IT and 4.9% for 1000 IU/kg. The whole blood clotting time began to shorten at 2 h but F.IX bioactivity was not detected until 8 h post infusion in both IT groups. In all groups, F.IX activity was detected through 72 h post administration. These data demonstrate that biologically active rF.IX can reach the systemic circulation when given IT. Aerosolization of rF.IX may provide a needle-free therapeutic option for delivery of rF.IX to hemophilia B patients.

Download Full-text

Persistent Systemic Production of Human Factor IX in Mice by Skeletal Myoblast-Mediated Gene Transfer: Feasibility of Repeat Application to Obtain Therapeutic Levels

Blood ◽

10.1182/blood.v90.3.1075.1075_1075_1082 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1075-1082 ◽

Cited By ~ 1

Author(s):

Jian-Min Wang ◽

Hong Zheng ◽

Mila Blaivas ◽

Kotoku Kurachi

Keyword(s):

Gene Transfer ◽

Human Factor ◽

Plasmid Vector ◽

Systemic Circulation ◽

Scid Mice ◽

Factor Ix ◽

Therapeutic Level ◽

Muscle Creatine Kinase ◽

Human Factor Ix

Myoblast-mediated gene transfer and its repeated applications were tested for achieving a long-term stable systemic production of human factor IX (hFIX) at a therapeutic level in SCID mice. Primary skeletal myoblasts were stably transfected with a hFIX expression plasmid vector, pdLMe4βAhIXm1, which contains a hFIX minigene under the control of a β-actin promoter with muscle creatine kinase enhancers. Myotubes derived from the myoblasts produced 1,750 ng hFIX/106 cells/24 hours in culture. hFIX secretion by the myoblasts and thereof derived myotubes were equally efficient, and myotubes were shown to have a sufficient secretory capacity to handle a substantially elevated production of hFIX. After intramuscular injection of 5, 10, and 20 × 106 myoblasts, SCID mice stably produced hFIX into the systemic circulation proportional to the number of implanted cells, and the expression levels were maintained for at least up to 10 months (end of the experiment). Additional cell injections administered to animals that originally received 10 × 106 cells approximately 2 months later elevated the systemic hFIX levels to an average of 182 ± 21 ng/mL, a therapeutic level, which persisted for at least 8 months (end of the experiment). These results indicate that long-term, stable systemic production of hFIX at therapeutic levels can be achieved by repeated application of myoblast-mediated gene transfer.

Download Full-text

The Activation of Human Factor IX

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647849 ◽

1975 ◽

Vol 33 (03) ◽

pp. 553-563 ◽

Cited By ~ 7

Author(s):

B Østerud ◽

K Laake ◽

H Prydz

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl ◽

Apparent Molecular Weight ◽

Factor Ix ◽

Factor Vii ◽

Activate Factor ◽

Human Factor Ix ◽

Factor Xia ◽

Tissue Thromboplastin ◽

Native Factor

SummaryThe activation of factor IX purified from human plasma has been studied. Factor XIa and kallikrein separately activated factor IX to factor IXa. In both cases factor IX a had an apparent molecular weight of about 42–45000 in sodium dodecyl sul-phate-polyacrylamide disc gel electrophoresis compared with a molecular weight of about 70000 for the native factor IX. The activation by XIa required Ca2+-ions whereas Ca2+-ions did not influence the activation by kallikrein. A mixture of tissue thromboplastin and factor VII or RusselPs-viper venom alone did not activate factor IX. Trypsin activated and plasmin inactivated factor IX.

Download Full-text