Claims
- 1. A trans-recoverable packaging-deficient retrovirus vector, the vector comprising a retrovirus having a genome which comprises a portion derived from the sequence of a cDNA corresponding to a protein expressed in a diseased cell and which lacks a functional copy of a gene necessary for packaging of progeny of the vector, the portion having a length less than about 3,000 nucleotide residues.
- 2. The vector of claim 1, wherein the portion is complementary to the cDNA.
- 3. The vector of claim 1, wherein the portion is homologous with the cDNA.
- 4. The vector of claim 1, wherein the cDNA corresponds to a cell surface adhesion protein of the diseased cell.
- 5. The vector of claim 4, wherein the diseased cell is a melanoma cell.
- 6. The vector of claim 5, wherein the protein is selected from the group consisting of Mel-CAM and integrin (beta)3.
- 7. The vector of claim 1, wherein the gene is selected from the group consisting of the gag gene, the pol gene, and the env gene of the retrovirus.
- 8. The vector of claim 1, wherein the retrovirus vector is derived from a retrovirus selected from the group consisting of a Molony murine leukemia virus and a Molony murine sarcoma virus.
- 9. The vector of claim 8, wherein the retrovirus vector is a PG1EN vector comprising the portion.
- 10. The vector of claim 1, wherein the vector further comprises a selectable marker.
- 11. The vector of claim 1, wherein the portion is operably linked with a promoter/enhancer region.
- 12. The vector of claim 11, wherein the portion is operably linked with an ATG codon.
- 13. The vector of claim 12, wherein the portion is operably linked with a stop codon.
- 14. The vector of claim 13, wherein the portion is operably linked with an internal ribosome entry site and a selectable marker, the internal ribosome entry site being interposed between the portion and the selectable marker.
- 15. A library comprising a plurality of the vector of claim 1, wherein at least two of the vectors collectively comprise different portions derived from the sequence of the same cDNA.
- 16. The library of claim 15, wherein the vectors collectively comprise at least 10 different portions derived from the sequence of the cDNA.
- 17. The library of claim 15, wherein the portions are generated by random cleavage of the cDNA.
- 18. The library of claim 15, wherein the portions are generated by amplification of sequential regions of the cDNA.
- 19. The library of claim 15, wherein the cDNA corresponds to Mel-CAM and wherein the portions are derived from at least one region selected from the group consisting of SEQ ID NOs: 1-9.
- 20. The library of claim 15, wherein the cDNA corresponds to integrin (beta)3 and wherein the portions are derived from at least one region selected from the group consisting of SEQ ID NOs: 10-21.
- 21. A pharmaceutical composition comprising the vector of claim 1 and a pharmaceutically acceptable carrier.
- 22. A method of generating a genetic suppressor element which suppresses an undesirable phenotype in a diseased cell, the method comprising
a) contacting a retrovirus library with a population of target cells, the library comprising a plurality of retrovirus particles, wherein individual retrovirus particles comprise a selectable marker and a fragment of an RNA which is transcribed in the diseased cell, the fragment having a length less than about 3,000 nucleotide residues and being operably linked with an ATG codon, the retrovirus particles lacking a component necessary for packaging of progeny retrovirus particles, and the target cells being susceptible to infection by the retrovirus particles; and b) performing at least one selection cycle using the population, the selection cycle comprising selecting a fraction of the target cells which express the selectable marker and which exhibit suppression of the undesirable phenotype.
- 23. The method of claim 22, wherein at least two selection cycles are performed and wherein cells of the fraction are propagated between the selection cycles.
- 24. The method of claim 22, further comprising providing the component to cells of the fraction, whereby progeny retrovirus particles comprising the genetic suppressor element are generated.
- 25. The method of claim 24, further comprising isolating the genetic suppressor element from the progeny retrovirus particles.
- 26. The method of claim 22, wherein the diseased cell is a melanoma cell.
- 27. The method of claim 26, wherein the undesirable phenotype is selected from the group consisting of:
i) expression of a cell-surface protein associated with metastasis; ii) expression of an mRNA encoding a cell-surface protein associated with metastasis; iii) cell-to-cell adhesion among the melanoma cells; iv) invasiveness of the melanoma cells; v) survival of the melanoma cells; vi) growth of the melanoma cells; and vii) proliferation of the melanoma cells, wherein the melanoma cells are located in the body of a mammal.
- 28. The method of claim 27, wherein the cell-surface protein associated with metastasis is selected from the group consisting of Mel-CAM and integrin (beta)3.
- 29. The method of claim 22, wherein the diseased cell is a solid tumor cell.
- 30. The method of claim 28, wherein the undesirable phenotype is angiogenesis.
- 31. The method of claim 22, wherein the diseased cell is located in the body of a mammal.
- 32. A genetic suppressor element which exhibits an anti-melanoma effect, the genetic suppressor element being a polynucleotide having a length of at least about 10 nucleotide residues and being derived from at least about 10 consecutive nucleotide residues of a portion of the cDNA corresponding to Mel-CAM, wherein the portion is selected from the group consisting of SEQ ID NOs: 1-9.
- 33. The genetic suppressor element of claim 32, wherein the genetic suppressor element is complementary to the portion of the cDNA.
- 34. The genetic suppressor element of claim 32, wherein the genetic suppressor element is homologous with the portion of the cDNA.
- 35. The genetic suppressor element of claim 32, wherein the genetic suppressor element has a nucleotide sequence selected from the group consisting of
a) nucleotide sequences complementary to a portion of the cDNA corresponding to Mel-CAM selected from the group consisting of SEQ ID NOs: 1-4 and 6; and b) nucleotide sequences homologous with a portion of the cDNA corresponding to Mel-CAM selected from the group consisting of SEQ ID NOs: 5 and 7-9.
- 36. A pharmaceutical composition comprising the genetic suppressor element of claim 32 and a pharmaceutically acceptable carrier.
- 37. A genetic suppressor element which exhibits an anti-angiogenesis effect in a solid tumor, the genetic suppressor element being a polynucleotide having a length of at least about 10 nucleotide residues and being derived from at least about 10 consecutive nucleotide residues of a known genetic suppressor element which inhibits expression of integrin (beta)3, the known genetic suppressor element being derived from a portion of the cDNA corresponding to integrin (beta)3 and having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10-21.
- 38. The genetic suppressor element of claim 37, wherein the genetic suppressor element is complementary to the portion of the cDNA.
- 39. The genetic suppressor element of claim 37, wherein the genetic suppressor element is homologous with the portion of the cDNA.
- 40. The genetic suppressor element of claim 37, wherein the known genetic suppressor element has a nucleotide sequence selected from the group consisting of
a) nucleotide sequences complementary to a portion of the cDNA corresponding to integrin (beta)3 selected from the group consisting of SEQ ID NOs: 12, 15, and 17-19; and b) nucleotide sequences homologous with a portion of the cDNA corresponding to integrin (beta)3 selected from the group consisting of SEQ ID NOs: 10, 11, 13, 14, 16, 20, and 21.
- 41. A pharmaceutical composition comprising the genetic suppressor element of claim 37 and a pharmaceutically acceptable carrier.
- 42. A method of inhibiting an undesirable phenotype of a human melanoma cell, the method comprising providing a genetic suppressor element to the cell, wherein the genetic suppressor element is selected from the group consisting of
a) a polynucleotide having a length of at least about 10 nucleotide residues and having a nucleotide sequence complementary to at least about 10 consecutive nucleotide residues of a portion of the cDNA corresponding to Mel-CAM, wherein the portion is selected from the group consisting of SEQ ID NOs: 1-4 and 6; b) a polynucleotide having a length of at least about 10 nucleotide residues and having a nucleotide sequence homologous with at least about 10 consecutive nucleotide residues of a portion of the cDNA corresponding to Mel-CAM, wherein the portion is selected from the group consisting of SEQ ID NOs: 5 and 7; c) a polynucleotide having a length of at least about 10 nucleotide residues and having a nucleotide sequence complementary to at least about 10 consecutive nucleotide residues of a portion of the cDNA corresponding to integrin (beta)3, wherein the portion is selected from the group consisting of SEQ ID NOs: 12 and 15; and d) a polynucleotide having a length of at least about 10 nucleotide residues and having a nucleotide sequence homologous with at least about 10 consecutive nucleotide residues of a portion of the cDNA corresponding to integrin (beta)3, wherein the portion is selected from the group consisting of SEQ ID NOs: 10, 11, 13, 14 and 16.
- 43. The method of claim 42, wherein the human melanoma cell is located in the body of a mammal.
- 44. The method of claim 42, wherein the undesirable phenotype is selected from the group consisting of:
i) expression of a cell-surface protein associated with metastasis; ii) expression of an mRNA encoding a cell-surface protein associated with metastasis; iii) cell-to-cell adhesion among the melanoma cells; iv) invasiveness of the melanoma cells; v) survival of the melanoma cells; vi) growth of the melanoma cells; and vii) proliferation of the melanoma cells, wherein the melanoma cells are located in the body of a mammal.
- 45. A method of inhibiting an undesirable phenotype of a human solid tumor cell, the method comprising providing a genetic suppressor element to the cell, the genetic suppressor element being a polynucleotide having a length of at least about 10 nucleotide residues and being derived from at least about 10 consecutive nucleotide residues of a known genetic suppressor element which inhibits expression of integrin (beta)3, the known genetic suppressor element having a nucleotide sequence derived from a portion of the cDNA corresponding to integrin (beta)3 having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10-16.
- 46. The method of claim 45, wherein the undesirable phenotype is selected from the group consisting of:
i) expression of a cell-surface protein associated with metastasis; ii) expression of an mRNA encoding a cell-surface protein associated with metastasis; iii) cell-to-cell adhesion among the melanoma cells; iv) invasiveness of the melanoma cells; v) survival of the melanoma cells; vi) growth of the melanoma cells; and vii) proliferation of the melanoma cells, wherein the melanoma cells are located in the body of a mammal.
- 47. A method of treating a human having a solid tumor, which tumor exhibits an undesirable phenotype, the method comprising administering to the human a composition comprising a genetic suppressor element, the genetic suppressor element being a polynucleotide having a length of at least about 10 nucleotide residues and being derived from at least about 10 consecutive nucleotide residues of a known genetic suppressor element which inhibits expression of integrin (beta)3, the known genetic suppressor element having a nucleotide sequence derived from a portion of the cDNA corresponding to integrin (beta)3 having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10-16, thereby treating the human having the solid tumor.
- 48. The method of claim 47, wherein the undesirable phenotype is selected from the group consisting of:
i) expression of a cell-surface protein associated with metastasis; ii) expression of an mRNA encoding a cell-surface protein associated with metastasis; iii) cell-to-cell adhesion among the melanoma cells; iv) invasiveness of the melanoma cells; v) survival of the melanoma cells; vi) growth of the melanoma cells; and vii) proliferation of the melanoma cells, wherein the melanoma cells are located in the body of a mammal.
- 49. The method of claim 47, wherein the solid tumor is an early stage solid tumor.
- 50. The method of claim 47, wherein the composition further comprises a pharmaceutically acceptable carrier.
- 51. A method of inhibiting solid tumor recurrence, which solid tumor exhibits an undesirable phenotype, the method comprising providing a composition comprising a genetic suppressor element to the solid tumor, the genetic suppressor element being a polynucleotide having a length of at least about 10 nucleotide residues and being derived from at least about 10 consecutive nucleotide residues of a known genetic suppressor element which inhibits expression of integrin (beta)3, the known genetic suppressor element having a nucleotide sequence derived from a portion of the cDNA corresponding to integrin (beta)3 having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10-16; thereby inhibiting solid tumor recurrence.
- 52. A method of prolonging remission of a solid tumor, the method comprising providing a composition comprising a genetic suppressor element to the solid tumor, the genetic suppressor element being a polynucleotide having a length of at least about 10 nucleotide residues and being derived from at least about 10 consecutive nucleotide residues of a known genetic suppressor element which inhibits expression of integrin (beta)3, the known genetic suppressor element having a nucleotide sequence derived from a portion of the cDNA corresponding to integrin (beta)3 having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10-16; thereby prolonging remission of a solid tumor.
- 53. The method of claim 52, wherein the composition further comprises a pharmaceutically acceptable carrier.
- 54. The method of claim 52, wherein the remission of the solid tumor constitutes the absence of one or more solid tumor characteristics selected from the group consisting of metastasis, invasiveness, accelerated growth, and accelerated proliferation.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of international patent application PCT/US00/07807, filed on Mar. 24, 2000. This application is also entitled to priority pursuant to 35 U.S.C. §119(e) to U.S. provisional patent application No. 60/126,479, which was filed on Mar. 26, 1999.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This research was supported in part by U.S. Government funds (National Institutes of Health grant number CA47159), and the U.S. Government may therefore have certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60126479 |
Mar 1999 |
US |
Continuations (1)
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Number |
Date |
Country |
Parent |
PCT/US00/07807 |
Mar 2000 |
US |
Child |
09967432 |
Sep 2001 |
US |