Modulating gene expression by transfection or RNA-interference (RNAi) is a powerful means for studying the functions of genes. Realization of both techniques depends on delivery of the corresponding nucleic acids into cells in a tissue. The existing methods for localized delivery, e.g. microcapillary injection and electroporation, are laborious, invasive and often damaging.
Several techniques for introducing nucleic acids into cells and tissues are currently in use, including viral transformation, lipofection, electroporation, direct injection through microcapillaries and ballistic carrier particle delivery. In the latter technique, termed “biolistic”, the molecules to be delivered are carried by micron-size particles of a heavy metal that are accelerated to high speeds and launched into the target cells. Substances injected into cells using the biolistic method have included DNA, fluorescent dyes, and RNA. The particle-mediated delivery is not sensitive to permeability of the cell membrane to particular reagents and lacks the potentially deleterious effects of viruses and lipofection. It can also be particularly advantageous for live tissue applications, because it does not depend on molecular diffusion within tissue and can target cells in internal layers. Nevertheless, the application area of the particle -mediated delivery has been limited by the current design of “gene guns” used for particle acceleration.
Gene gun operation can be based on a variety of different principles. In one method, a shock wave can be generated by a chemical explosion (dry gunpowder), a discharge of helium gas under high pressure, by vaporization of a drop of water through a electric discharge at high voltage and low capacitance, or at low voltage and high capacitance. Most of the original work on this technique is described in patents by inventors from Cornell University and Agracetus, Inc. of Middleton, Wis. Another technique is detailed in U.S. Pat. No. 5,525,510, incorporated herein by reference, and falls in the class of “fluid effects” for achieving high power with little damage to the tissue. This patent describes a gene gun using the “Coanda Effect” to accelerate the projectiles. The Coanda Effect is a passive design using the geometry of the diverter of the gas stream to pull the accelerant away from the nozzle by having it follow a curved surface.
Existing gene guns, including the table-top PDS-1000 and the popular hand-held Helios (both available from Bio-Rad Laboratories of Hercules, Calif.), deliver particles to relatively large areas (cm2) with limited accuracy and reproducibility. In addition, the tissue targeted by a Helios™ gun may be damaged by the jet of gas emerging from the gun nozzle. An image of the Helios™ gun and a diagram showing the basic components of the device are provided in FIGS. 1a and 1b, respectively. Beads coated with genetic material are glued to the internal wall of the cartridge using a preparation available from the manufacturer. The gene gun uses compressed helium at pressures of 7-20 atm. Particles are accelerated by helium flow in the “acceleration channel”, which is followed by an opening cone, “barrel liner”, and a spacer, illustrated in FIG. 1b. The two latter elements are intended to vent the helium gas away from the target to minimize cell surface impact. Nonetheless, unlike the narrow holes perforated by the micron size particles, the impact of the high speed helium jet emerging from the barrel may inflict significant damage to the tissue located in front of the barrel. Therefore, the problem of stopping/diverting the flow of the gas accelerating the particles has been a major concern with the gene gun design.
With current methods, there is a trade off between penetration depth and tissue damage. The Helios™ device is limited in that the range of bead penetration into the tissue is less than ˜50 μm. To increase the penetration depth, the particles must be accelerated to a higher velocity, which can only be achieved by increasing the helium jet pulse velocity which, in turn, increases damage to the tissue.
Both in-vivo and in slice preparation would greatly benefit from a method for delivery of dyes or genetic material into the cells that lie as deep as 200-400 μm. A technique for delivery fluorescent dyes into living tissue is described by Gan, W. B., J. Grutzendler, et al. (2000), “Multicolor “DiOlistic” Labeling of the Nervous System using Lipophilic Dye Combinations,” Neuron 27(2): 219-25. A Bio-Rad gene gun was used to deliver multiple fluorescent dyes into neuronal tissue for anatomical study. The described method had limited effect, however, due to the low penetration depth of the beads. Efforts to reduce damage caused by the gas flow have led to deceleration of the particles and a resulting reduction of their penetration depth. This limits the usefulness the current technology for applications in mammalian brain tissue, where most of the cell bodies lie 100 μm or more below the surface.
Tests using an agarose gel to emulate brain tissue showed that it is possible to obtain a major increase in the depth of penetration with a more focused jet of helium, however, there was a concomitant increase in damage to the gel surface. Accordingly, the need remains for a gene gun that can reproducibly achieve large penetration depths with minimal damage. Moreover, there is demand for new techniques of localized, accurate, reproducible and non-damaging delivery of substances such as nucleic acids and dyes into live tissue. The present invention is directed to such a device and method.