WO1997007826A1 - Electroporation in vivo de cellules - Google Patents
Electroporation in vivo de cellules Download PDFInfo
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- WO1997007826A1 WO1997007826A1 PCT/US1996/013591 US9613591W WO9707826A1 WO 1997007826 A1 WO1997007826 A1 WO 1997007826A1 US 9613591 W US9613591 W US 9613591W WO 9707826 A1 WO9707826 A1 WO 9707826A1
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- cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Definitions
- This invention relates to electroporation of living cells in vivo for intracellular delivery of nucleic acids including genes of interest for the expression of desired protein or antisense nucleotides for the suppression or expression of certain intracellular proteins.
- Liposomes and positively charged polymers like poly-L-Lysine have been used during the past years as in vivo delivery systems for gene therapy and cancer treatment, (Nicolau et al Proc. Natl. Acad. Sci. USA 80, 1068-1072, 1983 Duzgunes N. and Feigner P.L., 1993, Methods Enzymol., 221 :303-306).
- these approaches have clearly demonstrated serious disadvantages including low efficiency in vivo and relatively low efficacy of intracellular DNA delivery.
- the nature of liposome delivery to cells in organs other than liver and lung make liposomes a relatively inefficient delivery vehicle for gene therapy for various practical applications including treatment of many solid tumors.
- Electroporation has been shown to be the most efficient method for DNA delivery in vitro (Neuman et al, 1982, EMBO J., 1 :841-845).
- the mechanism of DNA transfer in this method is based on formation of membrane pores followed by DNA electrophoresis through these pores into the cells (Neuman E. et al., 1982, EMBO J., 1 :841-845).
- the mechanism of electroporation does not demonstrate many ofthe disadvantages of other gene transfer approaches.
- the procedure can be applied to any cell type without limitations.
- Still another object of this invention is to provide treated cells in accordance with the treatment ofthe methods of this invention.
- a method of treating living cells in vivo comprises introducing foreign nucleic acids by electroporation into cells in vivo to cause suppression or expression of at least one protein in vivo.
- the method can be carried out as a means of introducing genes into cells in vivo.
- the nucleic acid introduced alters protein expression to make the cells sensitive to destruction when treated with a drug nontoxic to normal cells. This method is useful as a cancer treatment for man, animals and mammals.
- nucleic acids including DNA is passed into living cells in vivo through the cell membrane and is taken up in the cell DNA as known in the art.
- Fig. 1 is a diagrammatic illustration showing a top plan view of electrode placement for electroporation in an exposed medial lobe of a rat liver
- Fig. 2 is a diagrammatic cross-sectional view through the liver of Fig. 1 diagrammatically showing the needles and injected DNA;
- Fig. 3 is a summary of luciferase activity 48 hours after electroporation using the methods of this invention with pulses at varying field strength;
- Fig. 4 shows expression of luciferase expression after electroporation
- Fig. 5 shows the dose response curve for luciferase expression plating the dose injected into a rat liver or control (no injection of DNA, just electroporation) against luciferase activity in AU arbitrary light units
- Fig. 6 is a flow cytometry assay of ⁇ -galactosidase expression after in vivo electroporation without the gene injected;
- Fig. 7 is the same as Fig. 6 with the gene injected at 25 mg/ml.
- Each histogram of Figs. 6 and 7 plots the log of fluorescent intensity (abscissae) versus number of cells population (ordinate).
- living cells can be treated in vivo by introducing foreign nucleic acid by electroporation into cells in vivo to cause suppression or expression of at least one protein in vivo.
- the method can be used to treat living cells by introducing foreign nucleic acid such as genes into cells for gene therapy to alter a genetic characteristic ofthe cells as, for example, to correct genetic problems and disorders as in replacement gene therapy and in cancer treatment.
- the nucleic acids to be injected into cells in vivo includes nucleic acids of any size, whole or partial genes, DNA, RNA and generally any nucleic acid sequence which are injected by electroporation.
- the nucleic acids can be foreign nucleic acids or can supplement nucleic acids already present in the cells.
- cells are meant to include living cells, organs, tissues and solid cancers. Thus, malignancies can be treated, particularly solid tumors, by the methods of this invention as will be described.
- the cells are meant to include cells of man and animals including, but not limited to. all mammalian species.
- In vivo treatment is treatment in or at the body while the cells are living as, for example, electroporation of organs through the skin ofthe body or after surgical procedures to expose organs.
- electroporation can be carried out with the use of electroporation needle displays which are of extremely small size and which can be combined in cannula and endoscopic fiber optic instruments.
- Endoscopes are preferred instruments for reaching internal solid tumors since they can carry viewing optics as well as nucleic acid injection needles and electrodes allowing in vivo steps to be carried out internally without the need for surgery. It is preferred that needle penetration for injection of nucleic acids and application of an applied voltage, preferably follow a multi-needle design as, for example, shown in Figs.
- Fig. 2 shows a cross section thereof with DNA previously injected through hypodermic syringes/needles, which also apply the voltage.
- a central electrode needle surrounded by eight additional needles.
- the eight electrode needles are equispaced around a 0.5 cm radius from the center needle.
- the conductive needles can be 0.3 millimeter diameter. This electrode is inserted so that the central needle enters the center ofthe area for the quantity of DNA to be injected as shown in Fig. 1.
- the second electrode utilizes six needles that can be equispaced around 1 cm diameter.
- the electrode can be inserted so that the site ofthe DNA injection is within a circular region encompassed by the needles.
- the needles can protrude for example approximately 5 mm into the liver or other cells, organs, cancers or tissues which are to be treated. Electroporation techniques for injection into cells in vitro are well-known in the art and such known techniques can be used, see Eur. J. Cancer, Vol. 27, No. 1, pp 68-72, 1991, Mir et al., Biochemical and Biophysical Research Communications, pp 938-943, Vol. 194, No. 2, 1993, Salford et al. and Cancer, Vol. 72, No.
- the needle arrangement ofthe electrode be arranged to give the smallest overall diameter ofthe electrode needle array.
- plural needle arrays are incorporated in endoscopic instruments of known types.
- the electrodes pass through the endoscopes.
- the needles can be used to treat remote areas in the body accessed through endoscopes as, for example, treating solid tumors ofthe pancreas, brain, gastrointestinal tract. liver, skin, kidneys, gynecological tumors and cancer of other organs ofthe body.
- Electroporation in vivo for the local intracellular delivery of nucleic acid can be achieved by the local injection of any desired nucleic acids.
- Nucleic acids as used in this application include DNA, RNA and synthetic oligonucleotides.
- the nucleic acids can be injected into the tissue of interest followed by either a single or multiple rectangular pulse or exponential electric pulses with strength ranging from 200 V/cm to 1 ,500 V/cm and the time constant ranging from 1 microsecond to 100 millisecond.
- the nucleic acid is used in aqueous or other suspension, with the concentration ranging from 0.001 mg/ml to 10 mg/ml and is injected into the tissue of interest in a dose ranging from 0.001 to 10 mg/kilogram or more.
- the electric pulses must be essentially applied locally to the tissue injected with said nucleic acid of interest within period of time ranging from 5 seconds to 30 minutes after injection using needle-like electrodes from 1 mm to 5 mm in length and separated by 0.4 mm to 20 mm distance from each other.
- the multiple pulses with different parameters including electric field strength, time constant and shape can be applied to the tissue injected with the said nucleic acid for an efficient electroporation of cellular membrane and intracellular delivery by means of transmembrane electrophoresis.
- the preferred electroporation device in the present invention consists of a fiberoptic endoscopic component combined with injection needles connected to a syringe for injection and needle-like electrodes connected to an electric pulse generator.
- the injection needle and needle ⁇ like electrodes are normally within the endoscope device and can be moved in the operating position and inserted into a tissue independently under the visual control of a fiberoptic component ofthe device.
- Genes which can be transferred to tumor or cancer cells for therapeutic purposes can be any ofthe known genes for any known therapeutic purposes. Specific useful genes include: herpes virus thymidine kinase. Wagner M.J. et al. Proc. Natl. Acad. Sci. USA, Vol.
- Genes which can be injected in vivo for replacement therapy for genetic disorders include those encoding for ⁇ -glucocerebrosidase (Gaucher disease) and - ⁇ -25-hydroxylase (phenylketonuria).
- Rat livers can be electroporated in vivo with a plasmid carrying the luciferase gene site or the gene encoding for ⁇ -galactosidase as a reporter.
- the conditions described in the first example below can be used to inject the luciferase gene or the ⁇ -galactosidase into a rat liver.
- the animals can be sacrificed at various times after electroporation and luciferase activity can be measured in a liver homogenate.
- the data shown in Figs. 3-5 indicate that luciferase expression can be observed for at least 21 days after an electroporation procedure, according to the first example below.
- a protocol for gene transfer into rat liver organs is established.
- a 3 mg/kg dose of atropine is administered by subcutaneous injection into the right flank of a male rat.
- Ten minutes later a 45 mg/kg dose of sodium pentobarbital is introduced by intraperitoneal injection.
- the rat is then completely anesthetized and the liver is surgically exposed. This is preferably done by making a transfer incision starting from the mid-sagittal position, approximately 1 cm caudal to the xiphoid process, extending 3 to 4 cm toward the dorsal surface ofthe rat.
- the medial lobe (both halves) ofthe liver is exposed by drawing it out ofthe incision without causing tissue damage.
- Nucleic acid or DNA is injected into the right central lobe ofthe exposed liver tissue.
- 100 ⁇ g of DNA in 100 ⁇ l of sterile injectable saline are used.
- a time period of 1.5 minutes is allowed to elapse after injection in order to allow for diffusion of the DNA. Electrodes are inserted into the liver tissue.
- the first is comprised of a central needle surrounded by eight additional needles.
- the eight needles are equispaced around a 0.5 cm radius from the center needle. This electrode is inserted so that the central needle enters the center of the injected quantity of DNA (see Figure 1).
- the second electrode utilizes 6 needles that are equispaced around a 1 cm diameter. This electrode is inserted so that the site of nucleic and/or DNA injection is within the circular region encompassed by the needles. All needles on both electrode types protrude 5 mm. Pulses are administered to the tissue. Pulsing is slightly different for each ofthe two electrode types.
- the center needle functions as the anode and all eight needles positioned around the annulus ofthe electrode body function as the cathode. The distance between the center needle and any annular needle is 5 mm.
- the 6 needle electrode uses a rotating electric field. For this type of pulsing six 99 ⁇ second rectangular DC pulses are delivered. The electric field strength is 1000 V/cm, and the duty cycle is 1 second.
- the area that was encompassed by the electrode is marked using sutures.
- the electrode is removed, and the liver is placed back into its natural position. The incision is closed with surgical staples.
- the animal is euthanized by administering a 100 mg/kg dose of sodium pentobarbital by intraperitoneal injection. The treated tissue is then removed for analysis.
- Results are obtained which demonstrate that electroporation can be efficiently used in vivo. Pulses with the field strength up to 2 kV/cm were applied to the live tissues without affecting experimental animals but the optimal field strength for electroporation appears to be significantly lower.
- Fig. 3 summarizes the results of these experiments. Electric pulses with the field strength between 1 kV/cm and 2 kV/cm allow an efficient transfection of liver cells and a high level of luciferase expression as measured at 48 hours after electroporation. The field strength above 2 kV/cm led to necrosis ofthe electroporated tissue, possibly due to the insufficient heat dissipation. The level of expression in electroporated cells is probably higher than the one we detected experimentally because all measurements were performed in the liver tissue homogenate from a broad area covering the electroporated region. Accurate location of the electroporated area is limited by technical factors.
- luciferase expression at various times after in vivo electroporation demonstrates that the level of luciferase activity in tissue reaches the maximal value at 48 hours followed by decrease to 5-7% ofthe initial activity during the first week and remains stable for three weeks after electroporation (Fig. 4).
- Fig. 4 The efficiency ofthe reporter gene expression after in vivo electroporation strongly depends on the amount of plasmid DNA and optimal results were detected using 25 ⁇ g of DNA while an increase in this amount leads to the reduction of luciferase activity (Fig. 5).
- liver tissue was transfected with the ⁇ Gal gene as described above in the first example.
- Hepatocytes were isolated from the dissected liver tissue using Collagenase treatment.
- the isolated cells were treated with chloroquine to block lysosome activity followed by incubation with the lipophilic ⁇ Gal substrate C ]2 FDG (Molecular Probes Eugene Oregon).
- the activity ofthe reporter gene was assayed using fiowcytometry.
- Fig. 6 shows results of this experiment.
- the treatment of melanoma is achieved by an injection of 0.1-0.5 ml of plasmid DNA carrying the herpesvirus gene of thymidine kinase under the control ofthe CMV promoter with a concentration in normal saline of about 1 mg/ml followed by DNA diffusion in the tumor tissue for about 2 min.
- ganciclovir is administered to a patient at therapeutical doses of 5 mg per kilogram weight ofthe patient intravenously every twelve hours for 14 days.
- the nontoxic ganciclovir drug is converted into a toxic compound by thymidine kinase expressed in the electroporated tumor cells with cytotoxic effect on these treated cells and neighboring cells.
- the treatment of pancreatic tumors of either acinar or endocrine phenotype is achieved by providing endoscopic electroporation.
- An endoscopic device comprising a fiber optic component, injection needle and electroporation needle-like electrodes is passed through the esophagus and the front end ofthe instrument is moved to the tumor site under the control ofthe fiber optic component of said device.
- the injection needle is moved forward from the device and inserted into the tissue under visual control using a fiber optic component ofthe device.
- An injection of 0.1-0.5 ml of plasmid DNA carrying the he ⁇ esvirus gene of thymidine kinase under the control ofthe CMV promoter with concentration of about 1 mg/ml is injected into the tumor.
- Injected DNA is allowed to diffuse in the tumor tissue for 5 min.
- Six circularly arranged needle-like electrodes mounted within the device are moved at working position from said device and inserted into the tumor tissue containing the plasmid DNA.
- Eight rectangular electric pulses of 99 ⁇ sec with electric field strength of 1000 V/cm (applied tension of 9 volts) are delivered to the tumor tissue with 1 second interval through each pair of opposite electrodes. The procedure is repeated at four spaced tumor sites.
- Ganciclovir is administered to a patient at therapeutical doses, as in the second example, for the intracellular conversion ofthe nontoxic drug into the toxic compound by thymidine kinase expressed in the electroporated tumor cells with cytoxic effect on these cells, and the cells surrounding them.
- the treatment of inherited beta-glucocerebrosidase deficiency is achieved by electroporation of normal liver tissue with plasmid DNA carrying the gene of beta-glucocerebrosidase under the control of CMV promoter as described in Example 3.
- the treatment of tumors of gastrointestinal origin including tumors ofthe stomach, pancreatic tumors of ductal phenotype and tumors of duodenum is achieved through the noninvasive oral access to the tumor site using an endoscopic device as described above and electroporation procedure described in Example 3.
- RNA such as genes which cause living cells to express protein materials which are sensitive to specific drugs are preferred for use in treating tumors in accordance with this invention.
- a gene encoding the herpes virus thymidine kinase can be injected into cells at a dosage rate of 10 mg/kilogram and preferably around 0.125 mg/kilogram, as set forth above, using the electroporation parameter set forth above to have the cells produce the protein thymidine kinase.
- the treated cells which were treated by electroporation in vivo undergo a reaction between the ganciclovir and thymidine kinase to form a toxic substance which kills the transformed cells and cells surrounding them.
- ganciclovir which can be used can be Cytovene, a ganciclovir sodium sterile powder produced by Syntex Laboratories of Palo Alto, California. Dosages of about 1.25 to about 5mg per kilogram of body weight administered intravenously at a constant rate over one hour, every 12 hours, for fourteen to twenty-one days, can be used to treat tumors so as to kill some or all of such tumors.
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Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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AU68557/96A AU6855796A (en) | 1995-08-29 | 1996-08-26 | In vivo electroporation of cells |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US291195P | 1995-08-29 | 1995-08-29 | |
US60/002,911 | 1995-08-29 |
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WO1997007826A1 true WO1997007826A1 (fr) | 1997-03-06 |
WO1997007826A9 WO1997007826A9 (fr) | 1997-09-18 |
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PCT/US1996/013591 WO1997007826A1 (fr) | 1995-08-29 | 1996-08-26 | Electroporation in vivo de cellules |
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Cited By (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2765241A1 (fr) * | 1997-06-30 | 1998-12-31 | Roussy Inst Gustave | Amelioration du transfert d'acide nucleique dans les cellules d'organismes eucaryotes pluricellulaires et combinaison permettant la mise en oeuvre du procede |
FR2765242A1 (fr) * | 1997-06-30 | 1998-12-31 | Roussy Inst Gustave | Amelioration du transfert d'acide nucleique dans le muscle strie et combinaison permettant la mise en oeuvre du procede |
WO1999001175A1 (fr) * | 1997-06-30 | 1999-01-14 | Rhone-Poulenc Rorer S.A. | Dispositif permettant un transfert in vivo optimise de vecteurs d'acides nucleiques dans des tissus |
WO1999001157A1 (fr) * | 1997-06-30 | 1999-01-14 | Rhone-Poulenc Rorer S.A. | Amelioration du transfert d'acide nucleique dans les cellules d'organismes eucaryotes pluricellulaires et combinaison permettant la mise en oeuvre du procede |
WO1999001158A1 (fr) * | 1997-06-30 | 1999-01-14 | Rhone-Poulenc Rorer S.A. | Amelioration du transfert d'acide nucleique dans le muscle strie et combinaison permettant la mise en oeuvre du procede |
US6181964B1 (en) | 1997-08-01 | 2001-01-30 | Genetronics, Inc. | Minimally invasive apparatus and method to electroporate drugs and genes into tissue |
US6216034B1 (en) | 1997-08-01 | 2001-04-10 | Genetronics, Inc. | Method of programming an array of needle electrodes for electroporation therapy of tissue |
US6241701B1 (en) | 1997-08-01 | 2001-06-05 | Genetronics, Inc. | Apparatus for electroporation mediated delivery of drugs and genes |
WO2001068889A2 (fr) * | 2000-03-16 | 2001-09-20 | Malone Robert W | Procede de transfection genique et de vaccination a travers la peau par electropermeabilisation |
US6302874B1 (en) | 1998-07-13 | 2001-10-16 | Genetronics, Inc. | Method and apparatus for electrically assisted topical delivery of agents for cosmetic applications |
EP1194574A1 (fr) * | 1999-06-25 | 2002-04-10 | Genetronics, Inc. | Transfection tres efficace utilisant une faible intensite de champ electrique et une longue duree d'impulsion |
WO2003038112A2 (fr) | 2001-10-26 | 2003-05-08 | Baylor College Of Medicine | Composition et methode permettant de modifier la masse maigre et les proprietes osseuses d'un sujet |
US6654636B1 (en) | 1998-07-13 | 2003-11-25 | Genetronics, Inc. | Skin and muscle-targeted gene therapy by pulsed electrical field |
US6678556B1 (en) | 1998-07-13 | 2004-01-13 | Genetronics, Inc. | Electrical field therapy with reduced histopathological change in muscle |
US6800484B2 (en) | 1998-06-24 | 2004-10-05 | Genetronics, Inc. | High efficiency transfection based on low electric field strength, long pulse length |
US7361642B2 (en) | 2003-08-04 | 2008-04-22 | Vgx Pharmaceuticals, Inc. | Canine specific growth hormone releasing hormone |
US7517863B2 (en) | 2004-01-20 | 2009-04-14 | Vgx Pharmaceuticals, Inc. | Enhanced secretion/retention of growth hormone releasing hormone (GHRH) from muscle cells by species-specific signal peptide |
WO2010137021A2 (fr) | 2009-05-27 | 2010-12-02 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Procédé de génération de tissu conjonctif |
US7922709B2 (en) | 1998-07-13 | 2011-04-12 | Genetronics, Inc. | Enhanced delivery of naked DNA to skin by non-invasive in vivo electroporation |
US7931640B2 (en) | 1997-04-03 | 2011-04-26 | Inovio As | Method for muscle delivery of drugs, nucleic acids and other compounds |
US8058253B2 (en) | 2006-07-06 | 2011-11-15 | Vgx Pharmaceuticals, Inc. | Growth hormone releasing hormone treatment to decrease cholesterol levels |
US8188056B2 (en) | 2003-12-31 | 2012-05-29 | Vgx Pharmaceuticals, Inc. | Reducing arthritis and lameness in subjects by growth hormone releasing hormone (GHRH) supplementation |
-
1996
- 1996-08-26 WO PCT/US1996/013591 patent/WO1997007826A1/fr active Application Filing
- 1996-08-26 AU AU68557/96A patent/AU6855796A/en not_active Abandoned
Non-Patent Citations (4)
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ARCH. SURG., November 1993, Vol. 128, LYERLY et al., "Gene Delivery Systems in Surgery", pages 1197-1206. * |
CANCER RESEARCH, 01 March 1996, Vol. 56, NISHI et al., "High-Efficiency In Vivo Gene Transfer Using Intraarterial Plasmid DNA Injection Following In Vivo Electroporation", pages 1050-1055. * |
GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER, 1994, SUKHAREV et al., "Electrically-Induced DNA Transfer Into Cells. Electrotransfection In Vivo", pages 210-232. * |
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US6241701B1 (en) | 1997-08-01 | 2001-06-05 | Genetronics, Inc. | Apparatus for electroporation mediated delivery of drugs and genes |
US6216034B1 (en) | 1997-08-01 | 2001-04-10 | Genetronics, Inc. | Method of programming an array of needle electrodes for electroporation therapy of tissue |
US6800484B2 (en) | 1998-06-24 | 2004-10-05 | Genetronics, Inc. | High efficiency transfection based on low electric field strength, long pulse length |
US6678556B1 (en) | 1998-07-13 | 2004-01-13 | Genetronics, Inc. | Electrical field therapy with reduced histopathological change in muscle |
US7570992B2 (en) | 1998-07-13 | 2009-08-04 | Genetronics, Inc. | Electrical field therapy with reduced histopathological change in muscle |
US6697669B2 (en) | 1998-07-13 | 2004-02-24 | Genetronics, Inc. | Skin and muscle-targeted gene therapy by pulsed electrical field |
US7922709B2 (en) | 1998-07-13 | 2011-04-12 | Genetronics, Inc. | Enhanced delivery of naked DNA to skin by non-invasive in vivo electroporation |
US6654636B1 (en) | 1998-07-13 | 2003-11-25 | Genetronics, Inc. | Skin and muscle-targeted gene therapy by pulsed electrical field |
US6947791B2 (en) | 1998-07-13 | 2005-09-20 | Genetronics, Inc. | Method and apparatus for electrically assisted topical delivery of agents for cosmetic applications |
US6302874B1 (en) | 1998-07-13 | 2001-10-16 | Genetronics, Inc. | Method and apparatus for electrically assisted topical delivery of agents for cosmetic applications |
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EP1194574A1 (fr) * | 1999-06-25 | 2002-04-10 | Genetronics, Inc. | Transfection tres efficace utilisant une faible intensite de champ electrique et une longue duree d'impulsion |
EP1829973A2 (fr) * | 1999-06-25 | 2007-09-05 | Genetronics, Inc. | Transfection à fort rendement selon une faible intensité de champ électrique et une faible longueur de l'impulsion |
EP1194574A4 (fr) * | 1999-06-25 | 2003-04-02 | Genetronics Inc | Transfection tres efficace utilisant une faible intensite de champ electrique et une longue duree d'impulsion |
WO2001068889A3 (fr) * | 2000-03-16 | 2002-04-25 | Robert W Malone | Procede de transfection genique et de vaccination a travers la peau par electropermeabilisation |
WO2001068889A2 (fr) * | 2000-03-16 | 2001-09-20 | Malone Robert W | Procede de transfection genique et de vaccination a travers la peau par electropermeabilisation |
WO2003038112A2 (fr) | 2001-10-26 | 2003-05-08 | Baylor College Of Medicine | Composition et methode permettant de modifier la masse maigre et les proprietes osseuses d'un sujet |
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US7517863B2 (en) | 2004-01-20 | 2009-04-14 | Vgx Pharmaceuticals, Inc. | Enhanced secretion/retention of growth hormone releasing hormone (GHRH) from muscle cells by species-specific signal peptide |
US8058253B2 (en) | 2006-07-06 | 2011-11-15 | Vgx Pharmaceuticals, Inc. | Growth hormone releasing hormone treatment to decrease cholesterol levels |
WO2010137021A2 (fr) | 2009-05-27 | 2010-12-02 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Procédé de génération de tissu conjonctif |
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