EP0273085A1
Expired / Lapsed
EP
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A method for internalizing nucleic acids into eukaryotic cells
Assignee: Intracel Corp
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Filing: 1986-12-29
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Granted: —
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Expires: 2006-12-29
Abstract
Abstract A piece of useful nucleic acid is linked to a cell homing factor, for instance a cell receptor such as that for the epidermal growth factor (EGF), and the resulting coupled system is used to transfect and/or transform suitably selected host cells. This technique provides internalization in high yields and constitutes a significant advance over classic transfection, e.g. with CA⁺⁺ ions.
Inventors
- 👤 André Myers
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 22:21
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; A; A61; A61P; A61P35/00; C12N15/87
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
122 words
Method for transfecting cells, i.e. for introducing into appropriate host-cells one or more selected pieces of foreign polynucleotides (nucleic acids such as DNA or RNA), which comprises the steps of : (1) making a coupled complex by chemically coupling said piece(s) of nucleic acid to a ligand having high affinity for predefined recepter sites of said host-cells, said ligand being capable of acting as a cell targetting agent toward said cells and as a cell internalization vector; (2) exposing said host-cells to the coupled complex resulting from step (1), these cells being selected for the presence thereon of a significant quantity of said receptors, for a time sufficient to achieve effective penetration into the cells and internalization of said coupled nucleic acid.
Claim 2
Dependent Claim
122 words
Method for transfecting cells, i.e. for introducing into appropriate host-cells one or more selected pieces of foreign polynucleotides (nucleic acids such as DNA or RNA), which comprises the steps of : (1) making a coupled complex by chemically coupling said piece(s) of nucleic acid to a ligand having high affinity for predefined recepter sites of said host-cells, said ligand being capable of acting as a cell targetting agent toward said cells and as a cell internalization vector; (2) exposing said host-cells to the coupled complex resulting from step (1), these cells being selected for the presence thereon of a significant quantity of said receptors, for a time sufficient to achieve effective penetration into the cells and internalization of said coupled nucleic acid.
Claim 3
Dependent Claim
21 words
The method of claim 1, wherein the polynucleotide is chosen from RNA, single stranded DNA and duplex DNA and derivatives thereof.
Claim 4
Dependent Claim
20 words
The method of claim 2, wherein the polynucleotide is a sequence containing a functional gene of interest and untranslated sequences.
Claim 5
Dependent Claim
19 words
The method of claim 3, wherein the gene codes for identifiable product expressed under control of the untranslated sequences.
Claim 6
Dependent Claim
20 words
The method of claim 3, wherein the gene codes for anti-sense RNA for blocking viral RNA or cellular RNA functions.
Claim 7
Dependent Claim
35 words
The method of claim 3, wherein the gene codes for a biologically active product selected from cytotoxins, hormones, enzymes, lectins, antibodies, antisense RNA and DNA, etc, under the driving action of a stress inducible promotor.
Claim 8
Dependent Claim
16 words
The method of claim 6, wherein the promotor is a heat-shock control sequence of eukaryotic origin.
Claim 9
Dependent Claim
27 words
The method of claim 1, wherein said cell receptor is selected among cell growth factors (e.g. EGF), synthetic and natural derivatives thereof, antibodies and cell specific markers.
Claim 10
Dependent Claim
26 words
The method of claim 3, comprising the additional steps of : (3) inducing expression of said gene of interest into useful products and isolating said products.
Claim 11
Dependent Claim
95 words
The method of claim 2, wherein the polynucleotide comprises an inducible gene coding for a product of interest and the host cell is eukaryotic and unrestricted multiplications, the method comprising the following additional steps of: (3) inoculating the transformed cell lines into immunodeficient warm-blooded animals which develop tumors in response to inoculation and allowing said tumors to grow to 10⁶-10¹⁰ cells; (4) dissociating said tumors into individual cells, cultivating said dissociated cells in culture media; (5) subjecting said cultures to stress whereby said gene is expressed; and (6) collecting and purifying said product of interest.
Claim 12
Dependent Claim
78 words
The method of claim 6 in which the gene codes for a cytotoxin, comprising effecting step (2) by injecting said coupled complex into the blood stream or a living tissue containing normal cells and tumoral cells bearing said receptor sites, whereby said coupled complex is selectively internalized in the tumoral cells, then (3) stressing local said tissue to induce the production of the cytotoxin, whereby the tumoral cells are inhibited or destroyed while leaving the normal cells unharmed.
Claim 13
Dependent Claim
24 words
The method of claim 2 involving co-transfection with at least two independent polynucleotides, said nucleotides being coupled to one or to two different ligands.
Claim 14
Dependent Claim
43 words
As a new composition of matter, a cell transforming vector consisting of a nucleic acid sequence chemically coupled to a ligand with specific affinity toward a receptor of the cellular membrane, this receptor promoting internalization of said nucleic acid sequence in high yield.
Claim 15
Dependent Claim
43 words
As a new composition of matter, a cell transforming vector consisting of a nucleic acid sequence chemically coupled to a ligand with specific affinity toward a receptor of the cellular membrane, this receptor promoting internalization of said nucleic acid sequence in high yield.
References Cited (Backward Citations)
No citation records indexed.
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