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WO1999012575A1 - Procede de transformation $i(in vivo) utilisant des vecteurs lipidiques - Google Patents

Procede de transformation $i(in vivo) utilisant des vecteurs lipidiques Download PDF

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Publication number
WO1999012575A1
WO1999012575A1 PCT/US1998/018615 US9818615W WO9912575A1 WO 1999012575 A1 WO1999012575 A1 WO 1999012575A1 US 9818615 W US9818615 W US 9818615W WO 9912575 A1 WO9912575 A1 WO 9912575A1
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Prior art keywords
lipid
dna
cells
gene
expression
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PCT/US1998/018615
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English (en)
Inventor
Howard J. Federoff
Mary Haak-Frendscho
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Promega Corporation
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Priority to EP98944788A priority Critical patent/EP1011735A1/fr
Priority to AU92243/98A priority patent/AU9224398A/en
Publication of WO1999012575A1 publication Critical patent/WO1999012575A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • 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

Definitions

  • the invention is directed to clinical and therapeutic in vivo transformation of animal cells utilizing DNA or RNA molecules combined with lipid vehicles to express gene products encoded on the DNA or RNA molecules.
  • non-viral vectors for in vitro transfection have been described in the prior art. Most of these non-viral vectors are lipid-based compounds. As a group, they generally suffer from limited transfection ability and/or cytotoxicity.
  • a commercially-available lipid formulation for in vitro transfection consists of a 1 to 1 formulation of N- ⁇ l-(2,3-dioleyl)propyl ⁇ -N,N,N- trimethylammonium bromide and dioleylphosphatidylethanolamine (DOPE). This formulation suffers from the above-noted cytotoxicity and also inhibits protein kinase C activity, an undesirable side effect. Examples of lipid-based agents for in vitro transfection of animal cells are presented in U.S. Patent No. 5, 171,678; U.S. Patent No. 5,279,833; and U.S. Patent 5,527,928.
  • the patent also describes the in vitro transfection of NIH 3T3 cells utilizing lipids of the above compound which contain plasmid DNA.
  • compositions containing the above compound are commercially marketed by the Promega Corporation, Madison, Wisconsin, under the trademarks "Tfx"- 10, “Tfx”-20 and “Tfx”-50 reagents for the transfection of eukaryotic cells (product nos. E1811, E2381, E2391, and E2400.)
  • Tfx eukaryotic cells
  • All of the "Tfx” reagents available from Promega contain the same concentration of Nantz et al. 's above-noted compound and varying concentrations of DOPE, the concentration of DOPE being designated by the number following the "Tfx” trademark.
  • Tfx concentration of DOPE
  • in vitro transfection is performed in rapidly dividing cells in order to maximize uptake of the exogenous DNA.
  • Methods for the in vivo transfection of quiescent cells are of particular interest to gene therapy.
  • In vivo gene transfer into central nervous system (CNS) cells represents a valuable tool for examining gene product function as well as for modeling gene therapy. See, for example, Andersen and Breakefield (1995), Bergold et al. (1993), Brooks et al. (1997), Davidson et al. (1993), During et l. (1994) Federoff (1995), Federoff et al. (1992), Geller and Breakefield (1988), Geller et al. (1993), Geschwind et al. (1996), Glorioso et al. (1994), Ho et al. (1995), Kaplitt et al.
  • Fig. 1 is a schematic of World Precision Instruments' "UltraMicrosyringe” Pump.
  • the pump is designed to dispense down to picoliter volumes from a microliter syringe.
  • the pump utilizes a stepper motor-driven lead screw having an ultrafine pitch.
  • the lead screw drives the microsyringe plunger.
  • the pump is controlled by a microprocessor controller which is programmable for volume, rate, and syringe size.
  • Figs. 2A, 2B, 2C, 2D, 2E, and 2F X-gal histochemistry of HSVlac- infected tissue.
  • Sections (40 ⁇ m in width) representative of the injection site (Figs. 2B and 2E), a site anterior of the injection (Figs. 2 A and 2D) and a site posterior of the injection (Figs. 2C and 2F) were processed in a developing solution containing X-gal to visualize expression of j8-galactosidase.
  • Animals were either injected with a microprocessor-controlled pump (Figs. 2A, 2B, and 2C) or manually (Figs. 2D, 2E,and 2F). All sections were counterstained with thionin and acquired at a magnification of 2.5x. Expression of 3-galactosidase is evident along the needle track (Fig. 2E, arrow) in an animal injected manually.
  • FIG. 3A, 3B, 3C, 3D, 3E, and 3F - X-gal histochemistry of Adlac- infected tissue Sections (40 ⁇ m in width) representative of the injection site
  • a site anterior of the injection (Figs. 3 A and 3D) and a site posterior of the injection (Figs. 3C and 3F) were processed in a developing solution containing X-gal to visualize expression of jS-galactosidase.
  • Animals were either injected with a microprocessor-controlled pump (Figs. 3A, 3B, and 3C) or manually (Figs. 3D, 3E, and 3F). All sections were counterstained with thionin and acquired at a magnification of 2.5x. Expression of /3-galactosidase is evident anterior to (Fig. 3D) and along the needle track (Fig. 3E, arrow) in an animal injected manually.
  • Figs. 4A, 4B, 4C, 4D, 4E, and 4F X-gal histochemistry of Tfx-20- transduced tissue.
  • Sections (40 ⁇ m in width) representative of the injection site (Figs. 4B and 4E), a site anterior of the injection (Figs. 4 A and 4D) and a site posterior of the injection (Figs. 4C and 4F) were processed in a developing solution containing X-gal to visualize expression of /3-galactosidase. Animals were either injected with a microprocessor-controlled pump (Figs. 4A, 4B, and 4C) or manually (Figs. 4D, 4E, and 4F).
  • Figs. 6A, 6B, 6C, 6D, 6E, and 6F Expression of / 3-galactosidase for all gene transfer vehicles.
  • 3-galactosidase expression was measured quantitatively for all of the gene transfer vehicles and both modes of delivery described in the Examples.
  • tissue punches were harvested from the injection site (including the needle track), and from the corresponding anatomical location on the contralateral side of the injection.
  • Activity levels of jS-galactosidase were measured as a function of relative light units.
  • a first embodiment of the invention, and an invention claimed herein, is therefore drawn to a method of in vivo transformation of animal cells, including in vivo transformation of human cells, which comprises intimately associating a DNA or RNA molecule encoding a gene product of interest with a lipid composition comprising a fusogenic lipid and a cationic lipid of Formula I or Formula II:
  • R is a fatty acid alkyl or alkenyl group (i.e. , lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, palmitolyl, oleyl, linolyl, linolenyl, and arachidonyl) and X is an anion, to yield a lipid/DNA transformation reagent.
  • the transformation reagent is then injected into an animal host, preferably a mammalian host, at a selected injection site, whereby cells surrounding the injection site are transformed to express the gene product of interest encoded by the DNA or RNA molecule.
  • the method can be practiced on any type of animal, without limitation. Mammalian hosts are preferred.
  • lipid composition wherein the fusogenic lipid is dioleylphosphatidylethanolamme (DOPE) and the
  • Formula I or Formula II cationic lipid is the compound wherein R is oleyl
  • the preferred Formula I compound is N,N,N' ,N'-tetramethyl-N,N'-bis-(2- hydroxyethyl)-2,3-di(oleyloxy)-l ,4-butanediammonium iodide
  • the preferred Formula II compound is N,N-[bis(2-hydroxyethyl)-N-methyl-N-(2,3- di(tetradecanoyloxy)propyl] ammonium iodide.
  • the fusogenic lipid While DOPE is the preferred fusogenic lipid, other lipid compounds now known to, or later discovered to, facilitate fusion of lipid vesicles with animal cell membranes can be incorporated into the lipid composition and utilized in the present invention. It is preferred that the fusogenic lipid have a net neutral charge.
  • the lipid composition comprises an aqueous solution containing a concentration of from about 0.1 to about 10 mM of the Formula I or Formula II cationic lipid and from about 1 to about 100 mM of the fusogenic lipid. Most preferred is when the concentration of the cationic lipid is about 1.0 mM.
  • a second embodiment of the claimed invention is directed to the in vivo transformation of quiescent mammalian cells, particularly CNS cells.
  • the method proceeds in the same fashion as above, with the exception that the transformation reagent is injected into a central nervous system site such as the brain (via intracranial injection) or the spinal column of a mammalian host.
  • a central nervous system site such as the brain (via intracranial injection) or the spinal column of a mammalian host.
  • the present method readily induces the expression of exogenous gene products in non-replicating, quiescent mammalian cells. (See the Examples for illustrative experiments utilizing mice and exogenous DNA's encoding /3-galactosidase.)
  • transformation denotes a desired goal wherein an in vivo animal cell is induced to express, transiently or otherwise, a functional gene product encoded by a DNA or RNA sequence introduced into the animal host along with the transformation reagent described above.
  • the cationic lipids of Formula I and Formula II and the fusogenic lipid DOPE can be synthesized by prior art methods. See, for example, Nantz et al. , U.S. Patent No. 5,527,928, incorporated herein by reference. And, as noted above, compositions containing DOPE and the preferred Formula I and Formula II cationic lipid are available commercially from the Promega Corporation.
  • the DNA or RNA encoding the gene product of interest which is to be transformed into the mammalian host may be in the form of linear polynucleotide fragments or in the form of circulized molecules such as DNA or RNA plasmids.
  • the DNA or RNA may be single-stranded, double-stranded, mismatched, hetero- duplexed, right- or left-handed, contain methylated or non-natural nucleotide bases, or may be otherwise manipulated in ways known to the art.
  • the DNA or RNA may be derived from any source, either by isolation from natural sources or by synthetic means, without limitation.
  • intimately associating it is meant that the DNA or RNA to be transformed into the animal host is brought into intimate contact with the lipid composition described above by thorough mixing, sonication, and the like. It is much preferred that the DNA or RNA and the lipid composition be intimately associated with each other by forming liposomes of the lipid composition which contain the DNA or RNA within their lamellar or vesicle structure. Liposome formation is extensively described in the relevant prior art and need not be discussed in great detail here.
  • liposomes either multilamellar vesicles (MLV's), sonicated vesicles, or otherwise, can be formed from DOPE and the cationic lipids of Formula I or Formula II by mixing together separate chloroform solutions of the two lipid components and then evaporating the chloroform solvent at a constant temperature of about 37 °C. The resulting films are placed under high vacuum to remove any remaining traces of chloroform. The lipid mixture is then re-dissolved in distilled water and thoroughly mixed to yield a suspension of multilamellar vesicles (MLVs). The suspension may be sonicated to thoroughly disperse the suspended liposomes.
  • MUV's multilamellar vesicles
  • Adding the desired DNA or RNA to be transformed to the liposome suspension, followed by thorough mixing, causes the polynucleotide to form complexes with the suspended liposomes. It is believed this is due, in large part, to the electronic charge polarity between the net cationic nature of the liposome (due to the cationic lipid of Formula I or Formula II) and the net anionic nature of the polynucleotide due to its abundance of phosphate groups.
  • the underlying physical phenomena whereby the DNA or RNA molecules become associated with, incorporated into, or otherwise complexed with the liposomes of the lipid composition to yield the transformation reagent is not critical to a complete understanding of the present invention.
  • the transformation reagent in combination with a suitable pharmaceutically-acceptable diluent, is then injected into an animal host.
  • the injection site may be intramuscular, parenchymal, intracranial, sub-dermal or sub- buccal, nasal, intravenous or intraarterial, or otherwise parenteral.
  • the device includes a housing to support a microliter syringe and a connection to microprocessor control means (not shown). Within the housing is a stepper motor operationally linked to the lead screw, which is, in turn, operationally linked to reciprocating means for driving the plunger of the syringe. Operation of the stepper motor is controlled by the microprocessor control means.
  • the device shown in Fig. 1 also includes means for mounting the device upon a stereotaxic from for precise alignment of the syringe with the injection site.
  • the device shown in Fig. 1 is completely programmable for delivery volume, delivery rate, etc.
  • the method can be used for gene therapy in animals, including humans, without encountering the unknown risks associated with the use of viral vectors.
  • the method successfully transforms quiescent cells in vivo. To Applicants' knowledge, such a transformation has never been demonstrated in vivo using non- viral delivery means.
  • the invention has utility for the study of gene expression in such cells and also for clinical and therapeutic applications wherein a desired gene product is expressed in situ in a living animal subject.
  • Herpes Amplicon Virus - Cell lines Herpes Amplicon Virus - Cell lines:
  • RR1 cells used for packaging the amplicon constructs are a BHK-derived cell line engineered to stably express the HSV IE3 gene, Paterson and Everett (1990).
  • the NIH 3T3 line (ATCC 1658), as well as the RR1 line, were maintained in Dulbecco's modified Eagles Medium (DMEM) with 10% (v/v) fetal bovine serum (FBS), penicillin (100 ⁇ /ml) and streptomycin (100 ⁇ g/ml).
  • DMEM Dulbecco's modified Eagles Medium
  • FBS fetal bovine serum
  • penicillin 100 ⁇ /ml
  • streptomycin 100 ⁇ g/ml
  • Bioactive geneticin G418; 400 ⁇ g/ml, Gibco BRL, Gaithersberg, Maryland
  • HSVlac Amplicon DNA
  • Viral supernatants were clarified by centrifugation prior to repassage on RR1 cells. This second viral passage was harvested as above and concentrated overnight by ultracentrifugation in a 25% sucrose/PBS gradient. Viral pellets were resuspended in PBS (Ca 2+ and Mg 2+ - containing) and stored at -80°C for future use. Amplicon titers were determined by plating 3T3 cells in a 24-well plate at a density of 1 x 10 5 cells/well followed by infection with dilutions of concentrated viral stocks.
  • a replication-defective human adenovirus serotype 5 (Ad5)-derived adenoviral vector was used for in vivo gene transfer.
  • the replication-defective adenoviral vector (AdCMVlacz) has been deleted of sequences in the El A and
  • AdCMVlacz contains the enhancer/promoter of the cytomegalovirus (CMV) and the E. coli-de ⁇ ved lacz transcription unit with an SV40 polyadenylation signal.
  • CMV cytomegalovirus
  • High titer adenoviral stocks were generated by amplification in HEK 293 cells and concentrated by CsCl gradient ultracentrifugation. All animals received 1 x 10 5 p.f.u. (plaque-forming units, diluted in PBS) of Adlac in a volume of 1 ⁇ l.
  • Construction of stereotaxic-mounted, microprocessor-driven syringe pump World Precision Instruments' Model UMP "UltraMicroPump" brand microsyringe driver, Fig, 1, in conjunction with Model UMC-1 microprocessor- based controller, is designed to deliver picoliter injection volumes using microliter syringes.
  • the syringe is activated by a stepper motor driving a specially designed lead screw with ultrafine pitch.
  • the microprocessor is completely programmable for delivery volume (number of steps per delivery) and rate (number of steps per time interval). These parameters may also be programmed for sample withdrawal.
  • the injector unit was mounted on a precision small animal stereotaxic frame micromanipulator (ASI Instruments, Warren, Michigan) at a 90° angle using a mount for the injector designed by ASI.
  • mice were anesthetized with 3% halothane in 70% N 2 0 and 30% O 2 in an induction chamber and maintained at 2% halothane during stereotactic intracerebral injections. After positioning in an ASI murine stereotactic apparatus, the skull was exposed via a midline incision, and burr holes were drilled over the designated coordinates (bregma, +0.5 mm; lateral -2.0 mm; and deep, -3.0 mm).
  • a 33 GA steel needle was gradually advanced to the desired depth, and 1 ⁇ l of "Tfx" reagent/DNA mixture (Example 1) or Formula II reagent/DNA mixture (Example 2), virus (Comparative Examples 1 and 2) or naked DNA (Comparative Example 3) was infused by hand or by a microprocessor-controlled pump over 10 min. The injections delivered by the pump were at a constant rate of 110 nl/min. The needle was removed slowly over an additional 10 minute period.
  • mice were euthanized by cervical dislocation, the brains were immediately harvested and chilled on ice. The brains were placed into a Jacobowitz brain block (Zivic Miller, Allison Park, Pennsylvania) and 1 mm brain slices at the level of the injection site on both the ipsilateral and contralateral sides were prepared for quantification of /3-galactosidase activity.
  • Jacobowitz brain block Zivic Miller, Allison Park, Pennsylvania
  • Example 2 was performed in identical fashion as in Example 1 with the exception that a compound of Formula II wherein R is myristyl was used as the cationic lipid. The results were comparable to those for Example 1 , discussed below.
  • the plasmid-based HSV amplicon vector is packaged within a HSV virion particle.
  • the HSVlac construct which places the / 3-galactosidase gene under the transcription control of the HSV IE 4/5 promoter, was packaged, purified and concentrated.
  • One ⁇ l of HSVlac, containing 1 x 10 5 infectious particles was unilaterally delivered to the striatum (coordinates; 0.5 mm anterior of bregma; 2.0 mm lateral of midline; 3.0 mm down from the surface of the skull) of mice using either the standard slow manual delivery method or the microprocessor delivery method. All animals survived the gene transfer procedure and four days later were euthanized.
  • Figures 2A through 2F are representative tissue sections taken from microprocessor (Figs 2A, 2B, and 2C) and manually (Figs. 2D, 2E, 2F) HSVlac-injected mice. Inspection of the photomicrographs reveals that gene expression is more focal in mice injected using the microprocessor ( Figure 2B) than in those done manually ( Figure 2E). Substantial expression is noted along the injection path (arrow) in the manually-injected sections; no such injection path staining is observed in the sections from microprocessor injected mice (Fig. 2B).
  • Fig. 6 A quantitation of /3-galactosidase expression revealed that microprocessor-injected animals had greater levels of gene product than manually-injected animals. Moreover, inter-animal variability was greater in manually-injected mice, as was the propensity of virus to direct gene expression on the contralateral side (again, see Fig. 6A). This is likely due to some entry of virus into the CSF in the manually injected mice. Transduced cells are observed in ependyma and parenchymal tissues adjacent to the ventricle in manually injected mice. (See Fig. 5A).
  • Ad vectors constructed by recombination of a heterologous transcription unit in place of the EIA gene grow to high titer and have been used for many CNS gene transfer studies. See, for example, Akli et al. (1993), Andersen and Breakefield (1995), Bajocchi et al. (1993), Brody and Crystal (1994).
  • the Adlac vector utilizes the hCMV promoter to drive /3-galactosidase expression.
  • One ⁇ l of Adlac, containing 1 x 10 5 plaque forming units was unilaterally delivered to the striatum of mice using manual or microprocessor methods as described above. After four days, animals were euthanized and brain tissues were analyzed histochemically and enzymatically for
  • Comparative Example 3 Naked DNA Injection: Plasmid DNA, which has previously been shown to be an ineffective means for achieving gene expression in the CNS, was evaluated using the microprocessor delivery method. Ten ⁇ g of HSVlac in a volume of 1.0 ⁇ l was injected into the striatum of mice. Four days later these animals were euthanized and /3-galactosidase expression was analyzed. No histochemically detectable cells could be identified after a maximum of four hours in the X-gal reagent. However, minimal expression could be detected using chemiluminescent substrate. Although above the background, the extent of gene expression for naked DNA is quite low, particularly when compared with the other vehicles for gene delivery described above (see Fig. 6E).
  • Example 1 shows that the method described herein can be used to efficiently transform animal cells, even non-replicating animal cells, in vivo. While the extent of expressed gene product is less than that seen with the conventional herpes simplex and adenovirus vectors, the use of a DNA/lipid transformation reagent obviates many risks which are inherent in the use of viral vectors. There was only three-fold less expression seen in mice injected with the "Tfx "-20 reagent compared to the most efficient vehicle used (the HSVlac virus vector). Both the "Tfx" -10 reagent and the "Tfx "-20 reagent contain the same preferred cationic lipid of Formula I.
  • Tfx has an increased content of the fusogenic lipid DOPE as compared to “Tfx”-10. This difference likely underlies the greater gene transfer efficiency noted with “Tfx”-20 and suggests that studies directed toward optimizing the content of DOPE will improve the gene transfer efficiency of the present invention.
  • Example 1 further shows that transformation efficiency can be manipulated by altering the ratio of the fusogenic lipid and the cationic lipid of Formula I.
  • the efficiency of the method described herein can be optimized to match or even to exceed the transformation efficiency of the HSV and adenovirus vectors.
  • Another advantage of the present invention is its distinct promise for effective long-term transformation and its use in chronic gene therapy.
  • the antigenicity of viral vectors makes them extremely ill-suited for chronic gene therapy. While a first in vivo treatment using a viral vector may be highly effective in introducing exogenous polynucleotides, the efficacy of subsequent treatments drops precipitously as the host organism mounts an immuno-response to the antigens presented by the vector.
  • the extremely low antigenicity of the lipid transformation compositions as compared to viral vectors eliminates or greatly ameliorates this problem. Because the lipid compositions are much less antigenic than viral vectors, the animal host is far less likely to develop an immune response to the DNA/lipid injection. Therefore, the transformation compositions as described herein can be injected repeatedly without an immediate loss in transformation efficiency due to a powerful immune response by the host animal. This represents a vast improvement over prior art in vivo transfection methods.
  • Another advantage of the present invention is its ability to transform quiescent cells.
  • the Examples presented above were conducted on mice CNS cells, cells which do not replicate. By affording transformation of quiescent cells, the invention allows gene therapy to be utilized against a host of afflictions of the central nervous system.
  • a defective HSV-1 vector expresses escherichia coli beta-galactosidase in cultured peripheral neurons, Science, 241: 1667-1669.
  • Herpes simplex virus vector system analysis of its in vivo and in vitro cytopathic effects, J Neurosci Methods, Accepted for publication:

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Abstract

Cette invention se rapporte à un procédé in vivo servant à la transformation d'animaux et utilisant à cet effet un réactif de transformation contenant un mélange constitué par un ADN ou un ARN codant un produit génique recherché et par une composition lipidique contenant un lipide fusogénique et un lipide cationique.
PCT/US1998/018615 1997-09-08 1998-09-08 Procede de transformation $i(in vivo) utilisant des vecteurs lipidiques WO1999012575A1 (fr)

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EP98944788A EP1011735A1 (fr) 1997-09-08 1998-09-08 Procede de transformation $i(in vivo) utilisant des vecteurs lipidiques
AU92243/98A AU9224398A (en) 1997-09-08 1998-09-08 Method of (in vivo) transformation utilizing lipid vehicles

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US60/058,135 1997-09-08

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010555A1 (fr) * 1994-09-30 1996-04-11 The Regents Of The University Of California Reactifs cationiques de transport
WO1997013743A1 (fr) * 1995-09-27 1997-04-17 Univ California Cytofectines cationiques polyfonctionnelles, formulations et procedes pour generer des complexes de transfection actifs cytofectines/polynucleotides
WO1997014442A1 (fr) * 1995-10-18 1997-04-24 Merlin Technologies, Inc. Induction hormonale immunomodulee de reponses immunitaires des muqueuses
WO1997041834A1 (fr) * 1996-05-08 1997-11-13 Nika Health Products Limited Virosomes cationiques en tant que systeme d'apport de materiel genetique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010555A1 (fr) * 1994-09-30 1996-04-11 The Regents Of The University Of California Reactifs cationiques de transport
WO1997013743A1 (fr) * 1995-09-27 1997-04-17 Univ California Cytofectines cationiques polyfonctionnelles, formulations et procedes pour generer des complexes de transfection actifs cytofectines/polynucleotides
WO1997014442A1 (fr) * 1995-10-18 1997-04-24 Merlin Technologies, Inc. Induction hormonale immunomodulee de reponses immunitaires des muqueuses
WO1997041834A1 (fr) * 1996-05-08 1997-11-13 Nika Health Products Limited Virosomes cationiques en tant que systeme d'apport de materiel genetique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BENNETT ET AL: "CATIONIC LIPID-MEDIATED GENE DELIVERY TO MURINE LUNG: CORRELATION OF LIPID HYDRATION WITH IN VIVO TRANSFECTION ACTIVITY", THE JOURNAL OF MEDICINAL CHEMISTRY, vol. 40, 5 December 1997 (1997-12-05), pages 4069 - 4078, XP002088280 *
BROOKS, ANDREW I. ET AL: "Reproducible and efficient murine CNS gene delivery using a microprocessor-controlled injector", J. NEUROSCI. METHODS (1998), 80(2), 137-147 CODEN: JNMEDT;ISSN: 0165-0270, 30 April 1998 (1998-04-30), XP002088281 *
BUCHBERGER, B. ET AL: "DOSPER liposomal transfection reagent: a reagent with unique transfection properties", BIOCHEMICA (1996), (2), 7-10 CODEN: BIOCFE;ISSN: 0946-1310, XP002088278 *
FELGNER ET AL: "ENHANCED GENE DELIVERY AND MECHANISM STUDIES WITH A NOVEL SERIES OF CATIONIC LIPID FORMULATIONS", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, 1994, pages 2550 - 2561, XP002088279 *

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