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26. Ultrasound Enhances Factor IX Gene Transfer into Mouse Livers
NAKED DNA: METHODS These results indicated that a membrane modifier, in addition to an echo contrast agent, Levovist, significantly enhanced USMT in vitro and in vivo, suggesting that the combination could be useful for the ultrasound-mediated gene therapy in the future.
26. Ultrasound Enhances Factor IX Gene Transfer into Mouse Livers Carol H. Miao,1 Andrew A. Brayman,2 Peiqing Ye,1 Pierre Mourad,2 Lawrence A. Crum.1 1 Department of Pediatrics and Medicine, University of Washington, Seattle, WA, United States; 2Center for Industrial and Medical Ultrasound, Applied Physics Laboratory, University of Washington, Seattle, WA, United States. Ultrasound (US) can greatly enhance plasmid DNA transfer efficiency under appropriate acoustic conditions; e.g., those which promote inertial cavitation activity. US contrast agents (stabilized microbubbles) can be used as cavitation nuclei, and lower the ultrasonic pressure threshold for inertial cavitation occurrence. A liver-specific, high-expressing human factor IX (HFIX) plasmid (pBS-HCRHP-FIXIA, Miao et al., Mol. Ther, 2001; 3, 9477) was mixed with contrast agent (Optison®) or PBS, and delivered to mouse livers by intrahepatic injection, with simultaneous exposure to US using various protocols. In each, the acoustic frequency was 1.13 MHz, the peak negative pressure (P–) was ~1.8 MPa, the duty factor, pulse repetition frequency (PRF) and pulse duration were variable, US exposure duration was 60 s, and the US was applied using a solid cone transducer. This P– was selected on the basis of in vitro data indicating that the pressure was sufficient to disrupt Optison® microspheres, generate free microbubbles, and to initiate moderate levels of inertial cavitation activity. In the first protocol, the livers were exposed to 20 cycle acoustic pulses at a PRF of 500 Hz. HFIX gene expression was enhanced up to 3 fold by US at days 1 and 4 post-treatment, however no further enhancement was observed with the addition of Optison® (Avg. control hFIX levels=0.95ng/ml at day 1, and 0.84ng/ml at day 4). We believe that use of this high PRF resulted in destruction of the microbubbles as they exited the needle, precluding their entry into the extracellular space. In the second protocol, the livers were exposed/sham-exposed to 500 cycle tone bursts at PRF = 1 Hz. These settings were designed to provide a long quiescent period between bursts, allowing new microbubbles and plasmids to enter and accumulate in the tissues before the next US burst. With this protocol, up to 13 fold enhancement in gene expression was achieved by applying US + Optison®, and up to 3.7 fold increment was achieved by applying US only. No increment in gene expression levels was observed in plasmid only, or plasmid + sham US control animals (0/12). In the third protocol, we explored the effect of pulse duration (10, 100, 1000, or 10000 cycles/pulse at a constant PRF of 1 Hz and with coinjection of plasmid and Optison®). Statistical analyses indicated that US mediated enhancement of transgene expression (4-14 fold) in all four groups (n=6/group) of mice was highly significant. On both days 1 and 4, short pulses produced a nominally greater level of expression than did long pulses, but on neither day was this statistically significant. These results demonstrate that therapeutic US in combination with microbubble contrast agents has the potential to promote safe and efficient nonviral gene transfer of hFIX for the treatment of hemophilia.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
27. Femtosecond Infrared Laser- A Novel Gene Delivery System for Prolonged Expression Evelyne Zeira,1 Alexandra Manevitch,2 Artium Khatchatouriants,2 Orit Papo,3 Einat Tavor,4 Merav Darash– Yahana,1 Michal Gropp,1 Alik Honigman,4 Aaron Lewis,2 Eithan Galun.1 1 The Goldyne-Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem, Israel; 2Deptartment of Ophtalmology, Hadassah Laser Center, Hebrew University, Jerusalem, Israel; 3Department of Pathology, Hadassah University Hospital, Jerusalem, Israel; 4Department of Virology, Hadassah University Hospital, Jerusalem, Israel. Introduction: A serious limitation in translating the concept of gene therapy, is the low efficiency of gene delivery. A highly efficient method for non-viral-mediated gene expression is naked DNA electroporation (EP). However, in practice it could be hazardous for patients, as it would cause tissue damage. To overcome these obstacles we developed a new method of gene transduction using laser energy. Methods: The femtosecond infrared titanium sapphire laser beam (LBGT) was used as a laser illumination source, and a BTX electroporator was used for EP. The leg muscles of Balb/c mice were exposed for comparable assessment of laser beam and EP gene transduction. The pLNC/luc construct was used for the expression of luciferase (luc). For the in vivo real time continuous luc expression, we used the biochemiluminescence CCCD system (Mol. Ther. 4:239, 2001). Results. In a dose response experiment using laser beam gene transduction (LBGT), the optimal dose was shown to be 10 μg DNA (Figure 1A). An injection of 10 μg of pLNC/luc (Figure 1B) resulted in the same luc expression at day one in both the LBGT (Lt leg) and EP (Rt leg) methods. System optimization revealed that injection of 10 μg naked DNA into muscle followed by LBGT application for 5 sec, focused to 2 mm depth upon an area of 95 mm², resulted in the highest intensity and duration of gene expression without histological or biochemical evidence muscle damage. In addition, LBGT-mediated murine erythropoietin (mEPO), resulted in elevated hematocrit levels (>22%), sustained for 8 weeks. Gene expression following LGBT was detected for >100 days. Conclusion. LBGT is a simple and reproducible method for in vivo gene expression with the potential of being utilized in clinical use.
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26. Ultrasound Enhances Factor IX Gene Transfer into Mouse Livers Carol H. Miao,1 Andrew A. Brayman,2 Peiqing Ye,1 Pierre Mourad,2 Lawrence A. Crum.1 1 Department of Pediatrics and Medicine, University of Washington, Seattle, WA, United States; 2Center for Industrial and Medical Ultrasound, Applied Physics Laboratory, University of Washington, Seattle, WA, United States. Ultrasound (US) can greatly enhance plasmid DNA transfer efficiency under appropriate acoustic conditions; e.g., those which promote inertial cavitation activity. US contrast agents (stabilized microbubbles) can be used as cavitation nuclei, and lower the ultrasonic pressure threshold for inertial cavitation occurrence. A liver-specific, high-expressing human factor IX (HFIX) plasmid (pBS-HCRHP-FIXIA, Miao et al., Mol. Ther, 2001; 3, 9477) was mixed with contrast agent (Optison®) or PBS, and delivered to mouse livers by intrahepatic injection, with simultaneous exposure to US using various protocols. In each, the acoustic frequency was 1.13 MHz, the peak negative pressure (P–) was ~1.8 MPa, the duty factor, pulse repetition frequency (PRF) and pulse duration were variable, US exposure duration was 60 s, and the US was applied using a solid cone transducer. This P– was selected on the basis of in vitro data indicating that the pressure was sufficient to disrupt Optison® microspheres, generate free microbubbles, and to initiate moderate levels of inertial cavitation activity. In the first protocol, the livers were exposed to 20 cycle acoustic pulses at a PRF of 500 Hz. HFIX gene expression was enhanced up to 3 fold by US at days 1 and 4 post-treatment, however no further enhancement was observed with the addition of Optison® (Avg. control hFIX levels=0.95ng/ml at day 1, and 0.84ng/ml at day 4). We believe that use of this high PRF resulted in destruction of the microbubbles as they exited the needle, precluding their entry into the extracellular space. In the second protocol, the livers were exposed/sham-exposed to 500 cycle tone bursts at PRF = 1 Hz. These settings were designed to provide a long quiescent period between bursts, allowing new microbubbles and plasmids to enter and accumulate in the tissues before the next US burst. With this protocol, up to 13 fold enhancement in gene expression was achieved by applying US + Optison®, and up to 3.7 fold increment was achieved by applying US only. No increment in gene expression levels was observed in plasmid only, or plasmid + sham US control animals (0/12). In the third protocol, we explored the effect of pulse duration (10, 100, 1000, or 10000 cycles/pulse at a constant PRF of 1 Hz and with coinjection of plasmid and Optison®). Statistical analyses indicated that US mediated enhancement of transgene expression (4-14 fold) in all four groups (n=6/group) of mice was highly significant. On both days 1 and 4, short pulses produced a nominally greater level of expression than did long pulses, but on neither day was this statistically significant. These results demonstrate that therapeutic US in combination with microbubble contrast agents has the potential to promote safe and efficient nonviral gene transfer of hFIX for the treatment of hemophilia.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
27. Femtosecond Infrared Laser- A Novel Gene Delivery System for Prolonged Expression Evelyne Zeira,1 Alexandra Manevitch,2 Artium Khatchatouriants,2 Orit Papo,3 Einat Tavor,4 Merav Darash– Yahana,1 Michal Gropp,1 Alik Honigman,4 Aaron Lewis,2 Eithan Galun.1 1 The Goldyne-Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem, Israel; 2Deptartment of Ophtalmology, Hadassah Laser Center, Hebrew University, Jerusalem, Israel; 3Department of Pathology, Hadassah University Hospital, Jerusalem, Israel; 4Department of Virology, Hadassah University Hospital, Jerusalem, Israel. Introduction: A serious limitation in translating the concept of gene therapy, is the low efficiency of gene delivery. A highly efficient method for non-viral-mediated gene expression is naked DNA electroporation (EP). However, in practice it could be hazardous for patients, as it would cause tissue damage. To overcome these obstacles we developed a new method of gene transduction using laser energy. Methods: The femtosecond infrared titanium sapphire laser beam (LBGT) was used as a laser illumination source, and a BTX electroporator was used for EP. The leg muscles of Balb/c mice were exposed for comparable assessment of laser beam and EP gene transduction. The pLNC/luc construct was used for the expression of luciferase (luc). For the in vivo real time continuous luc expression, we used the biochemiluminescence CCCD system (Mol. Ther. 4:239, 2001). Results. In a dose response experiment using laser beam gene transduction (LBGT), the optimal dose was shown to be 10 μg DNA (Figure 1A). An injection of 10 μg of pLNC/luc (Figure 1B) resulted in the same luc expression at day one in both the LBGT (Lt leg) and EP (Rt leg) methods. System optimization revealed that injection of 10 μg naked DNA into muscle followed by LBGT application for 5 sec, focused to 2 mm depth upon an area of 95 mm², resulted in the highest intensity and duration of gene expression without histological or biochemical evidence muscle damage. In addition, LBGT-mediated murine erythropoietin (mEPO), resulted in elevated hematocrit levels (>22%), sustained for 8 weeks. Gene expression following LGBT was detected for >100 days. Conclusion. LBGT is a simple and reproducible method for in vivo gene expression with the potential of being utilized in clinical use.
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