Femtosecond laser treatment enhances DNA transfection efficiency in vivo
- Shaw-Wei D Tsen†1,
- Chao-Yi Wu†1,
- Avedis Meneshian5,
- Sara I Pai6,
- Chien-Fu Hung1, 2 and
- T-C Wu1, 2, 3, 4Email author
© Tsen et al; licensee BioMed Central Ltd. 2009
Received: 10 November 2008
Accepted: 01 April 2009
Published: 01 April 2009
Gene therapy with plasmid DNA is emerging as a promising strategy for the treatment of many diseases. One of the major obstacles to such therapy is the poor transfection efficiency of DNA in vivo.
In this report, we employed a very low power, near-infrared femtosecond laser technique to enhance the transfection efficiency of intradermally and intratumorally administered DNA plasmid.
We found that femtosecond laser treatment can significantly enhance the delivery of DNA into the skin and into established tumors in mice. In addition, we found that both laser power density as well as duration of laser treatment are critical parameters for augmenting DNA transfection efficiency. The femtosecond laser technique employs a relatively unfocused laser beam that maximizes the transfected area, minimizes damage to tissue and simplifies its implementation.
This femtosecond new laser technology represents a safe and innovative technology for enhancing DNA gene transfer in vivo.
Gene therapy continues to evolve as an attractive approach for the treatment of many diseases (for reviews, see [1–11]). In particular, the use of plasmid DNA for gene therapy has several advantages which can circumvent the limitations and potential risks associated with viral vector-based DNA delivery. It is relatively safe, stable, and inexpensive to manufacture, making it attractive for application in the clinical arena. Furthermore, in contrast to viral vectors, DNA vaccines do not elicit anti-vector immune responses in the vaccinated patient, and, therefore, are well suited for indications likely to require multiple administrations in order to achieve and maintain target immune responses.
The ideal approach for enhancing DNA vaccine potency is by improving the transfection efficiency with minimal tissue damage. Several physical techniques including electroporation and ultrasound have been employed in an effort to improve gene transfection efficiency. However, several safety concerns have been raised with the application of these approaches in humans (for reviews, see [12, 13]). Therefore, continued exploration for new methods of enhancing DNA transfection efficiency while minimizing side effects is essential for generating potent DNA vaccination strategies and also for gene therapy using plasmid DNA.
Femtosecond laser treatment represents a novel and attractive method for in vivo gene delivery because the lasers are convenient to operate, relatively non-invasive, and have been shown to significantly enhance gene transfection efficiency without detectable tissue damage in mice [14, 15]. They have been applied toward in vitro genetic modification of cells (for a review, see ) and have recently been found to improve intradermal and intramuscular delivery of DNA in mice [14, 15]. Therefore, in the current study, we employed femtosecond laser treatment in an effort to improve the transfection efficiency of DNA encoding luciferase that was administered intradermally as well as intratumorally in mice, with the hope of finding an innovative technology that may be used both for DNA vaccination as well as for plasmid DNA gene therapy in the clinical setting.
Plasmids and Cell Lines
The construct encoding firefly luciferase, termed pcDNA3-Luc, was a kind gift from Dr. Hyam Levitsky, Johns Hopkins University. TC-1, an E6/E7-expressing tumor cell line, was derived from primary epithelial cells of C57BL/6 mice and co-transformed with HPV-16 E6 and E7 and c-Ha-ras oncogenes as previously described .
Intradermal gene transfection experiments
C57BL/6 mice (3 per group) were anesthetized by isofluorane inhalation, shaved, and injected intradermally (depth = 0.5 mm) with 10 μg/mouse of pcDNA3-Luc in a total volume of 50 μl. This was followed immediately by either femtosecond laser treatment at a laser power density of 0.04 GW/cm2 for 80 sec or no treatment.
Nude mice were similarly injected intradermally with 10 μg/mouse of pcDNA3-Luc in a total volume of 50 μl, followed immediately by either femtosecond laser treatment at various laser power densities for 80 sec. In addition, a group of nude mice received treatment at a laser power density of 0.04 GW/cm2 for different laser treatment time durations. Nude mice without laser treatment were used as control. The intensity of the luminescence in the mice was monitored by bioluminescent imaging 16 hours after DNA administration.
Intratumoral gene transfection experiments
Female C57BL/6 mice (7 per group) were subcutaneously challenged in the abdominal wall with 1 × 105 TC-1 tumor cells/mouse. When tumors reached a diameter of ~0.7 cm, the mice were anesthetized by isoflurane inhalation, shaved, and injected intratumorally (depth = 1.0 mm) with a single dose of 10 μg/mouse of pcDNA3-Luc in a total volume of 50 μl, which was followed immediately by either femtosecond laser treatment at a laser power density of 0.04 GW/cm2 for 110 sec or no treatment. The intensity of the luminescence in the mice was monitored by bioluminescent imaging 16 hours after DNA injection.
In vivo bioluminescence imaging
To monitor luminescent intensity, the mice were injected with 0.2 ml of 15 mg/ml beetle luciferin (potassium salt, Promega) per mouse. After 10 minutes, the mice were imaged using the IVIS 200 system (Xenogen Corp, Alameda, CA). An integration time of 1 minute was used for luminescence image acquisition.
All data expressed as means ± SE are representative of at least two different experiments. Comparisons between individual data points were made using the Student's t test. A p value < 0.05 was considered statistically significant.
Femtosecond lasers enhance the transfection efficiency of luciferase-encoding plasmid DNA administered intradermally
Enhancement in transfection efficiency using femtosecond laser treatment depends on the laser power density and duration of laser treatment
Femtosecond laser treatment can enhance the transfection efficiency of luciferase-expressing DNA injected intratumorally
In this study, we found that femtosecond lasers can significantly enhance DNA transfection efficiency into the skin and into established tumors in mice, and that both the laser power density used as well as the laser treatment duration are important parameters that determine the enhancement in gene transfection afforded by the femtosecond laser. Using these parameters, we have optimized the laser system for maximum intradermal and intratumoral DNA transfection efficiency.
Our data are consistent with previous reports that femtosecond lasers can enhance intradermal DNA delivery . However, ours is the first report demonstrating the use of femtosecond lasers to enhance intratumoral DNA delivery. Compared to these previous studies, our femtosecond laser treatment employs significantly reduced laser focusing, which offers several advantages. First, it greatly increases the transfected area and thus maximizes the number of cells that are transfected. Second, the laser energy is greatly reduced, thereby minimizing damage to healthy host tissue. Moreover, it is a relatively simple system to implement. Such characteristics make this technique especially promising for future clinical applications.
Our results suggest that femtosecond laser treatment may improve the efficacy of DNA vaccination and/or gene therapy using plasmid DNA. Such a laser technology can be used to deliver DNA encoding antitumor genes including proapoptotic, immunostimulatory, and/or antiangiogenic factors to effect eradication of tumors through apoptosis, immune-mediated mechanisms, and nutrient deprivation. In future studies, it will be important to explore the employment of laser treatment in combination with gene therapy using plasmid DNA encoding these factors for the control of disease in preclinical models.
However, the eventual clinical translation of the femtosecond laser technique would require several important considerations. Although our results demonstrated significantly improved transfection of subcutaneous tumors using laser treatment, many tumors clinically are less accessible and are located deep within body cavities. Novel adaptations must therefore be made to make these tumors accessible, such as the employment of lasers with longer wavelengths that have greater penetration depth in tissue, or percutaneous laser delivery devices which can allow laser delivery to deeper structures through minimally invasive means. In addition, the size of the laser beam can be further increased to allow the transfection of larger numbers of tumor cells, and the portability of the laser system must be improved in order to make it more clinically accessible.
Our data provide a platform on which a new femtosecond laser-based DNA delivery strategy can be developed. We have determined that laser power density and duration of laser treatment play a crucial role for optimizing the transfection efficiency of DNA, and that these parameters vary depending on the location of delivery (intradermal versus intratumoral). Further optimization of these parameters will be necessary for future application of the femtosecond laser system in humans and in a broader range of tumor sites. It is our hope that such a technology will aid gene therapy with plasmid DNA and DNA vaccination for the treatment of human disease.
We gratefully acknowledge Ms. Archana Monie for assistance in preparation of the manuscript. We would also like to thank IMRA America, Inc for providing the IMRA μJewel D-400 laser instrument for our study. This work was supported by the National Cancer Institute SPOREs (P50 CA098252 and P50 CA96784-06), the 1 RO1 CA114425-01, the NCDGG (1U19 CA113341-01) and the NIH/NIAID 1 UO1 AI070346-01.
- Azzouz M: Gene Therapy for ALS: progress and prospects. Biochimica et biophysica acta. 2006, 1762 (11–12): 1122-1127.View ArticlePubMedGoogle Scholar
- Bainbridge JW, Tan MH, Ali RR: Gene therapy progress and prospects: the eye. Gene therapy. 2006, 13 (16): 1191-1197.View ArticlePubMedGoogle Scholar
- Cotrim AP, Baum BJ: Gene therapy: some history, applications, problems, and prospects. Toxicologic pathology. 2008, 36 (1): 97-103.View ArticlePubMedGoogle Scholar
- Coutelle C, Themis M, Waddington SN, Buckley SM, Gregory LG, Nivsarkar MS, David AL, Peebles D, Weisz B, Rodeck C: Gene therapy progress and prospects: fetal gene therapy – first proofs of concept – some adverse effects. Gene therapy. 2005, 12 (22): 1601-1607.View ArticlePubMedGoogle Scholar
- Foster K, Foster H, Dickson JG: Gene therapy progress and prospects: Duchenne muscular dystrophy. Gene therapy. 2006, 13 (24): 1677-1685.View ArticlePubMedGoogle Scholar
- Herweijer H, Wolff JA: Progress and prospects: naked DNA gene transfer and therapy. Gene therapy. 2003, 10 (6): 453-458.View ArticlePubMedGoogle Scholar
- Shirakawa T, Fujisawa M, Gotoh A: Gene therapy in prostate cancer: past, present and future. Front Biosci. 2008, 13: 2115-2119.View ArticlePubMedGoogle Scholar
- Torras J, Cruzado JM, Herrero-Fresneda I, Grinyo JM: Gene therapy for acute renal failure. Contributions to nephrology. 2008, 159: 96-108.View ArticlePubMedGoogle Scholar
- Wolkowicz R, Nolan GP: Gene therapy progress and prospects: novel gene therapy approaches for AIDS. Gene therapy. 2005, 12 (6): 467-476.View ArticlePubMedGoogle Scholar
- Yamamoto T, Tsunetsugu-Yokota Y: Prospects for the therapeutic application of lentivirus-based gene therapy to HIV-1 infection. Current gene therapy. 2008, 8 (1): 1-8.View ArticlePubMedGoogle Scholar
- Yechoor V, Chan L: Gene therapy progress and prospects: gene therapy for diabetes mellitus. Gene therapy. 2005, 12 (2): 101-107.View ArticlePubMedGoogle Scholar
- Wells DJ: Gene therapy progress and prospects: electroporation and other physical methods. Gene therapy. 2004, 11 (18): 1363-1369.View ArticlePubMedGoogle Scholar
- Newman CM, Bettinger T: Gene therapy progress and prospects: ultrasound for gene transfer. Gene therapy. 2007, 14 (6): 465-475.View ArticlePubMedGoogle Scholar
- Zeira E, Manevitch A, Khatchatouriants A, Pappo O, Hyam E, Darash-Yahana M, Tavor E, Honigman A, Lewis A, Galun E: Femtosecond infrared laser-an efficient and safe in vivo gene delivery system for prolonged expression. Mol Ther. 2003, 8 (2): 342-350.View ArticlePubMedGoogle Scholar
- Zeira E, Manevitch A, Manevitch Z, Kedar E, Gropp M, Daudi N, Barsuk R, Harati M, Yotvat H, Troilo PJ: Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization. Faseb J. 2007, 21 (13): 3522-3533.View ArticlePubMedGoogle Scholar
- Yao CP, Zhang ZX, Rahmanzadeh R, Huettmann G: Laser-based gene transfection and gene therapy. IEEE transactions on nanobioscience. 2008, 7 (2): 111-119.View ArticlePubMedGoogle Scholar
- Lin KY, Guarnieri FG, Staveley-O'Carroll KF, Levitsky HI, August JT, Pardoll DM, Wu TC: Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer research. 1996, 56 (1): 21-26.PubMedGoogle Scholar
- Hung CF, Tsai YC, He L, Coukos G, Fodor I, Qin L, Levitsky H, Wu TC: Vaccinia virus preferentially infects and controls human and murine ovarian tumors in mice. Gene therapy. 2007, 14 (1): 20-29.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.