Vectors for gene transfer
AssigneeNature Technology Corporation
InventorClague P. Hodgson
Life of this patent
Estimating the term…
Abstract
Improved recombinant retrotransposon vectors for gene transfer are disclosed. The synthetic vectors are truncated so as to reduce or altogether eliminate homologous recombination with retroviral helper sequences found in helper cells used to propagate the vectors, making them safer for use in humans and providing more space for therapeutic genes. The vectors transmit foreign DNA efficiently, are stable, enable abundant RNA expression from the retrotransposon transcriptional promoter, and through their diversity permit many useful applications in therapeutics and transgenics. Methods are described for rescuing tissue-specifics promoters obtaining expression in primary cells, mapping the genome and other techniques of therapeutic and transgenic utility.
Claims76
A biologically active transfer vector incorporating no viral genes comprising, linked:
- (a) a 5′ long terminal repeat (LTR) sequence derived from a VL30 retrotransposon comprising a transcription initiation site for RNA;
- (b) an encapsidation sequence positioned 3′ of the 5′ LTR;
- (c) a primer binding site sequence derived from a VL30 retrotransposon and positioned 3′ of the 5′ LTR;
- (d) a 3′ LTR sequence derived from a VL30 retrotransposon positioned 3′ of the primer binding site which includes:
- (1) sequences necessary for polyadenylation of a RNA transcript initiated in the 5′ LTR;
- (2) sequences necessary for reverse transcription of the RNA transcript from step (d)(1) into a double stranded cDNA;
- (e) a polypurine tract sequence from a VL30 retrotransposon located 5′ to the 3′ LTR;
- and
- (f) sequences within each LTR which are necessary for integration of the biologically active transfer vector into the genome of a recipient cell,
- wherein the vector sequences comprise no more than 2 kbp.
The method of claim 25 in which the donor cell is selected from the group consisting of psi2 and PA317.
A method of introducing and expressing a DNA sequence in an oviduct or embryo of egg laying species comprising:
- (a) inserting a DNA sequence encoding a protein or RNA into a vector to yield a DNA transfer vector wherein the DNA sequence is operably linked to a transcription unit in the DNA transfer vector;
- (b) introducing the DNA transfer vector into a donor cell to yield a transformed donor cell;
- (c) transcribing RNA from the DNA transfer vector in the transformed donor cell to yield RNA derived from the DNA transfer vector;
- (d) packaging the RNA derived from the DNA transfer vector into a virion to yield a virus comprising RNA derived from the DNA transfer vector;
- (e) introducing the virus into the oviduct or germ line embryo of egg laying species;
- and
- (f) identifying animals which express the protein or RNA encoded by the DNA sequence.
A method of introducing an expressing a DNA sequence in an oviduct or embryo of egg laying species comprising:
- (a) inserting a DNA sequence encoding a protein or RNA into a vector to yield a DNA transfer vector wherein the DNA sequence is operably linked to a transcription unit in the DNA transfer vector;
- (b) introducing the DNA transfer vector into a donor cell to yield of donor cell transformed with the DNA transfer vector;
- (c) introducing the transformed donor cell, or progeny of the transformed donor cell, into the oviduct or germ line embryo of egg laying species;
- and
- (d) identifying animals which express the protein or RNA encoded by the DNA sequence.
A method for reconstituting blood with genetically modified hematopoietic stem cells derived from bone marrow comprising:
- (a) inserting a preselected DNA sequence into a LTR-containing vector 3′ of the transcription initiation site in the 5′ LTR to yield a DNA transfer vector;
- (b) introducing the DNA transfer vector into a donor cell to yield a transformed donor cell;
- (c) transcribing RNA from the DNA transfer vector in the transformed donor cell to yield a transfer vector RNA;
- (d) packaging the transfer vector RNA to yield a virion with the transfer vector RNA;
- and
- (e) infecting bone marrow cells which were first treated with an agent to render the cells quiescent, and then treated with at least one cytokine to induce the proliferation of the cells with the virion.
A method of reconstituting tissues with genetically modified embryonic stem cells comprising:
- (a) inserting a preselected DNA sequence into a VL30-derived vector 3′ of the transcription initiation site in the 5′ LTR to yield a DNA transfer vector;
- (b) introducing the DNA transfer vector into a donor cell to yield a transformed donor cell;
- (c) transcribing RNA from the DNA transfer vector in the transformed donor cell to yield a transfer vector RNA;
- (d) packaging the transfer vector RNA to yield virions with the transfer vector RNA;
- (e) infecting an embryonic stem cell with the virions;
- (f) reverse transcribing the transfer vector RNA packaged in the virion in the embryonic stem cell to yield a cDNA;
- and
- (g) identifying embryonic stem cells containing the cDNA and culturing the embryonic stem cells containing the cDNA to permit their maturation.
A method of reconstituting tissues with genetically modified embryonic stem cells comprising:
- (a) inserting a preselected DNA sequence into a VL30-derived vector 3′ of the transcription initiation site in the 5′ LTR to yield a DNA transfer vector;
- (b) introducing the DNA transfer vector into a donor cell to yield a transformed donor cell;
- (c) introducing the transformed donor cell, or progeny of the transformed donor cell into an organ, a tissue, an embryo, or an animal host, and
- (d) identifying an embryonic stem cell, or progeny thereof, within the animal in which a virion, comprising vector-derived RNA, produced by the transformed donor cell, or progeny cells of the transformed donor cell, has entered the embryonic stem cell, virion RNA has been reverse transcribed, and a resulting cDNA integrated into the genome of the embryonic stem cell.
A method of increasing the resistance of a cell to infection by a retrovirus comprising introducing at least one retrotransposon-derived vector that is transcribed at a high efficiency into the cell so that RNA transcribed from the retrotransposon vector outcompetes RNA derived from the retrovirus for packaging proteins or cellular translational machinery, following infection by the retrovirus.
A method of selecting retrotransposon-derived vectors which replicate efficiently comprising:
- (a) introducing a retrotransposon-derived transfer vector into a donor helper cell to yield a transformed donor helper cell;
- (b) transcribing RNA from the DNA transfer vector in the donor helper cell to yield transfer vector RNA;
- (c) packaging the transfer vector RNA with packaging proteins provided by the helper cell into a virion to yield a virus with transfer vector RNA;
- (d) infecting a different donor helper cell with the virus;
- (e) reverse transcribing transfer vector RNA in the donor helper cell of step (d) to yield a cDNA;
- (f) transcribing RNA from the cDNA to yield transfer vector RNA;
- (g) packaging transfer vector RNA of step (f) with packaging proteins provided by the helper cell of step (d) into a virion to yield a virus with transfer vector RNA;
- and
- (h) repeating steps (d)-(g) until the vector which predominates is one which replicates more efficiently than a vector which was not repeatedly passaged through helper cells.
Dependencies are inferred from the claim text.