Delivery of macromolecules into cells

Details Delivery of macromolecules into cells Delivery of macromolecules into cells

2001-07-30

2003-08-05

Casler, Brian L. (Department: 3763)

Surgery

Means for introducing or removing material from body for...

Infrared, visible light, ultraviolet, x-ray or electrical...

C604S501000

Reexamination Certificate

active

06603998

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to methods and apparatus for delivery of macromolecules into cells. More specifically, the present invention provides methods and apparatus for delivering substances, such as macromolecules, e. g. polynucleotide vaccines (DNA vaccine and/or RNA vaccine) and protein-based vaccines, into selected cells in epidermal tissue with reduced sensation (reduced pain).
BACKGROUND ART
The first DNA vaccination procedure in the prior art was called naked DNA vaccination because a liquid solution of DNA was injected into the muscle of mice with no additives to enhance transfection. This method does transfect a few cells and does induce an immune response to the expressed antigen in mice. However, in humans and primates, the method does not work well.
In the prior art, an improvement in DNA vaccine efficiency was obtained by the use of a biolistic method for DNA delivery. The biolistic method is done by coating metal microbeads with DNA and shooting the particles into skin after accelerating the particles to a chosen velocity. This method works much better than naked DNA. Part of the reason is that the DNA coated particles are injected into the skin to a depth that increases the chance of transfecting Langerhans cells. However, the biolistic method has some disadvantages. First, it causes some skin damage that may scar in some individuals. Second, in spite of the increased efficiency, more efficiency is needed. Third, the ballistic particle remains inside the patient after treatment. In this respect, it would be desirable if a method for delivering DNA to biological cells were provided which does not cause skin damage that results in scarring. Also, it would be desirable if a method for delivering DNA to biological cells were provided which does not leave a residue of ballistic particles in cells that are treated. As a matter of interest, the following U.S. patents disclose biolistic methods: U.S. Pat. Nos. 5,036,006 and 5,478,744.
A number of additional approaches to delivering macromolecules to biological cells are disclosed in the prior art and are represented by the following U.S. patents or other publications as follows.
U.S. Pat. No. 5,019,034 of Weaver et al discloses a process for electroporation of tissues in which electrodes are placed on top of the tissue surface, such as skin, of a patient. Molecules that are used for treating the skin are placed in reservoirs on top of the skin surface, and the treatment molecules must penetrate into the skin tissues transdermally. That is, the treatment molecules must pass from outside the skin to inside the skin. Not only is the surface layer of the skin relatively impermeable, if the layers of the skin to be treated are near the basal lamina of the epidermis, then the treatment molecules must traverse considerable skin tissue before the cells to be treated are reached by the treatment molecules. Such a treatment method is inefficient for treating cells near the basal lamina. Rather than using electrodes that are placed on the skin surface and have treatment molecules pass through the skin transdermally to treat biological cells near the basal lamina of the epidermis, it would be desirable if an electroporation method were provided for delivering molecules to biological cells in the epidermis, near the basal lamina, without having the treatment molecules pass through the skin transdermally.
U.S. Pat. No. 5,273,525 of Hofmann discloses an apparatus for electroporation of drugs and genetic material into tissues which employs a hollow hypodermic needle for placing the drugs and genetic material in the vicinity of the tissues to be electroporated. Whenever a hollow hypodermic is employed in a tissue, the tissue is cut with a circular cut by the hollow hypodermic needle. As a result, when a patient receives hypodermic injection, the patient has considerable pain. To avoid such a circular cut, and to avoid the considerable pain involved, it would be desirable if a method for delivering molecules to biological cells were provided which does not employ a hypodermic needle.
U.S. Pat. No. 5,318,514 of Hofmann discloses an applicator for the electroporation of drugs and genes into cells. The applicator includes a plurality of needle electrodes which can be penetrated into the skin of a patient. Material to be electroporated into the skin is retained in a fluid reservoir which wets an open cell foam elastomer carrier for the fluid. Because the material to be electroporated is retained in a fluid, in both the reservoir and the open cell foam elastomer, careful control of the amount of the material at the electrode surfaces is difficult. It is difficult to control how much fluid flows down from the reservoir and the open cell foam elastomer to the surfaces of the needle electrodes, and, thereby, it is difficult to control how much of the treatment molecules is actually present on the surfaces of the electrodes
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as the electroporation process is being carried out on the patient. Moreover, the presence of the fluid medium can have a flushing or washing effect on the tissues that are electroporated in such a way that the electroporation process is interfered with. In these respects, it would be desirable if an electroporation method for delivering molecules to biological cells were provided which does not employ a fluid medium that flows down onto the electrodes as the electroporation process is being carried out on the patient.
Other disclosures relating to the use of electroporation to mediate gene transfer into epidermal cells are found in an article by Reiss et al entitled “DNA-mediated gene transfer into epidermal cells using electroporation” in Biochem. Biophys. Res. Commun., Vol. 137, No. 1, (1986), pages 244-249 and in an article by Titomirov entitled “In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA” in Biochim. Biophys. Acta., Vol. 1088, No. 1, (1991), pages 131-134.
U.S. Pat. No. 5,389,069 of Weaver discloses a method and apparatus for in vivo electroporation of tissues which employs a hollow cylindrical needle for providing treating substances deep into tissues. As mentioned above, avoiding the use hollow cylindrical needles would be desirable to avoid the pain involved therewith.
U.S. Pat. Nos. 5,580,859 and 5,589,466, both of Felgner et al, disclose a method of delivering macromolecules into muscles and skin of a patient by an injection method. Their method does not employ electroporation.
U.S. Pat. No. 5,697,901 of Eriksson discloses gene delivery into tissues by the use of a gene-carrying fluid medium that is pressurized in conjunction with hollow microneedles. Problems of control and flushing using fluid media have been discussed hereinabove. An electroporation step is not employed in the Eriksson patent. As a matter of interest, Eriksson addresses the subject of pain in two respects. There is a statement that the hollow microneedle system can be used for treating pain. There is a statement that pain in wounds can be relieved by cooling. It is noted by the present inventors herein that Eriksson does not discuss his treatment method per se as being of a pain free or reduced pain treatment method. The present inventors theorize that the pressurized fluid injection method that is employed by Eriksson is not conducive to a pain free or reduced pain treatment method. In this respect, it would be desirable to provide a gene therapy treatment method that employs micro-sized needles, but that does not employ a pressurized fluid injection step for injecting fluid into a patient.
In an article by Henry et al entitled “Microfabricated Microneedles: A Novel Approach to Transdermal Drug Delivery” in Journal of Pharmaceutical Sciences, Vol. 87, No. 8, August 1998, pages 922-925, there is a disclosure that an array of microneedles are employed to penetrate the epidermis to leave micro-sized perforations to facilitate transdermal permeability of fluid-carried treatment agents into the microperforated epidermis. Because the microneedles are inserted only

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