Methods and reagents for identifying synthetic genetic elements

• US 20030157477A1
• Filed: 09/12/2001
• Published: 08/21/2003
• Est. Priority Date: 03/12/1999
• Status: Abandoned Application

• Current Assignee: GPC Biotech Inc. (GPC AG - Genome Pharmaceuticals Corp.)
• Original Assignee: GPC Biotech Inc. (GPC AG - Genome Pharmaceuticals Corp.)
• Inventors: Gyuris, Jeno
• Application Number:
  US09/950,983
• Publication Number:
  US 20030157477A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/505 involving T-cells
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention relates to a selection method that allows fast recovery and identification of functional gene fragments which selectively inhibit growth, e.g., are cytostatic or cytotoxic, of particular cell-types, such as transformed cells. The strategy relies, in part, on the ability of small gene fragments to encode dominant-acting synthetic genetic elements (SGEs), e.g., molecules which interfere with the function of genes from which they are derived. SGEs which can be identified by the subject method include, but are not limited to, inhibitory antisense RNA molecules, ribozymes, nucleic acid decoys, and small peptides.

Claims

1. A method for identifying agents which selectively confer a desired phenotype on a target cell, comprising:

(i) transfecting subtractive cells with a library of expression vectors comprising a variegated population of coding sequences for potential synthetic genetic elements (SGEs);

(ii) isolating, from the subtractive cells, those SGE vectors of the SGE library which do interfere with the detection of the desired phenotype in the target cell;

(iii) transfecting target cells with the sub-population of SGE vectors isolated in step (ii); and

(iv) isolating those SGE vectors which confer the desired phenotype to the target cell.

14. An isolated SGE identified according to the method of claim 1.

18. The method of claim 1, comprising the further step of formulating, in a pharmaceutical preparation, one or more SGEs that are identified as lethal to the target cell.

19. A method for identifying a compound having a selective biological effect on a target cell comprising,

(i) providing a drug screening assay for detecting agents which inhibit or potentiate the function of a target gene or target gene product corresponding to a SGE identified by the method according to claim 1;

and

(ii) testing, in the drug screening assay, the ability of a test compound to modulate the function of the target gene or target gene product.

20. The method of claim 19, comprising the further step of formulating a pharmaceutical preparation comprising one or more agents identified as able to modulate the function of the target gene or target gene product

21. A method for modulating a phenotype of a target cell comprising contacting the target cells with an agent identified by the method of claim 19 able to modulate the function of the target gene or target gene product.

2. A method for identifying agents with selective antiproliferative activity for a target cell, comprising:

(i) transfecting subtractive cells with a library of expression vectors comprising a variegated population of coding sequences for potential synthetic genetic elements (SGEs);

(ii) isolating those SGE vectors of the SGE library that are not antiproliferative to the subtractive cells;

(iii) transfecting target cells with the non-antiproliferative SGE vectors isolated in step (ii); and

(iv) isolating those SGE vectors that are antiproliferative to the target cell.

3. The method of claim 2, wherein the target and subtractive cells are eukaryotic cells.

4. The method of claim 3, wherein the target and subtractive cells are mammalian cells.

5. The method of claim 3, wherein at least one of the target and subtractive cells are human cells.

6. The method of claim 2, wherein the target cell is a transformed cell, and subtractive cell is an untransformed cell.

7. The method of claim 2, wherein the target cell is infected by a virus, and subtractive cell is a cell not infected with the virus.

8. The method of claim 2, wherein the expression vectors are viral vectors.

9. The method of claim 8, wherein the viral vectors are retroviral vectors.

10. The method of claim 2, wherein the SGE library is generated from a normalized cDNA library.

11. The method of claim 2, wherein the SGE library is generated from a subtractive cDNA library.

12. The method of claim 2, wherein the SGE is a sense oriented sequence encoding a peptide.

13. The method of claim 2, wherein the SGE is an antisense-oriented sequence encoding an antisense RNA.

15. A synthetic oligonucleotide having a nucleotide sequence from about 12 nucleotides to all of the nucleotide sequence of an antisense RNA encoded by an SGE identified according to the method of claim 13.

16. An isolated peptide encoded by an SGE identified according to the method of claim 12.

17. An peptidomimetic corresponding to a peptide encoded by an SGE identified according to the method of claim 12.

22. The method of claim 2, wherein the antiproliferative activity is the result of cell death.