Vector for gene silencing and replacement and methods of use thereof
An expression cassette for gene silencing and replacement, including, in operable communication, a promoter, an expression attenuator, a nucleotide sequence encoding a gene for a replacement target protein, and an shRNA sequence for knockdown of an endogenous variant of the target protein, wherein the promoter, the expression attenuator, the nucleotide sequence encoding the gene for the replacement target protein, and the shRNA are expressed as a single transcript. Also included are expression vectors and cells. Also included are methods of silencing and replacement of a target gene in a cell in culture by transforming the cells with the expression vectors described herein. Also included are minimal expression cassettes suitable for therapeutic methods.
1. An expression cassette for gene silencing and replacement of a target protein, comprising, in operable communication,
a promoter,
an expression attenuator wherein the expression attenuator comprises one or more 5′UTR hairpins, wherein the number and/or stability of 5′ UTR hairpins tunes the level of translation of the replacement target protein,
a nucleotide sequence encoding a gene for the replacement target protein, and
an shRNA sequence for knockdown of an endogenous variant of the target protein,
wherein the promoter, the expression attenuator, the nucleotide sequence encoding the gene for the replacement target protein, and the shRNA are expressed as a single transcript.
2. The expression cassette of claim 1 , wherein the promoter is a constitutive promoter or an inducible promoter, wherein, when the promoter is an inducible promoter, transcription of the single transcript is controlled by the presence of an inducer for the inducible promoter.
3. The expression cassette of claim 1 , wherein the expression cassette further comprises a transactivator gene for the inducible promoter, wherein the transactivator gene for the inducible promoter is operably linked to a constitutive promoter.
4. The expression cassette of claim 3 , wherein the inducible promoter is a tetracycline-responsive promoter.
5. The expression cassette of claim 1 , wherein the shRNA sequence is in the form of a precursor molecule comprising the sequence of the shRNA and a microRNA.
6. The expression cassette of claim 1 , wherein the target protein comprises a developmentally regulated protein, or a protein wherein expression of the protein interferes with normal cell division.
7. The expression cassette of claim 1 , further comprising an operably linked IRES upstream of a reporter gene.
8. An expression vector comprising the expression cassette of claim 1 .
9. The expression vector of claim 8 , comprising nucleic acid sequences from Moloney murine leukemia virus, Murine stem cell virus, Harvey murine sarcoma virus, murine mammary tumor virus, Rous sarcoma virus, adenovirus, adeno-associated virus, an SV40-type virus, a polyoma virus, Epstein-Barr virus, a papilloma virus, a herpes virus, a vaccinia virus, polio virus, a lentiviruses, or an RNA virus.
10. A cell comprising the expression vector of claim 8 .
11. The cell of claim 10 , comprising primary neuronal cells, astrocytes, oligodendrocytes, or cardiomyocytes.
12. The cell of claim 10 , comprising B35 rat neuroblastoma cells, Cos7 monkey kidney cells, HEK human embryonic kidney cells, CAD mouse Cath.a-differentiated catecholaminergic neuron-like cells, or PC12 rat pheochromocytoma neuron-like cells.
13. A method of silencing and replacement of a target gene in a cell in culture, comprising
transforming the cell with the expression vector of claim 7 , and
optionally applying to the cell an inducer when the promoter is an inducible promoter to provide expression of the single transcript, wherein expression of the shRNA silences the expression of the endogenous variant of the target protein, and expression of the coding sequence for the replacement target protein provides the replacement target protein in the cell.
14. The method of claim 13 , wherein the cell is a primary neuronal cell, an astrocyte, an oligodendrocyte, or a cardiomyocyte.
15. The method of claim 14 , wherein the transforming is done one or more weeks prior to applying the inducer.
16. The method of claim 13 , wherein the cell comprises B35 rat neuroblastoma cells, Cos7 monkey kidney cells, HEK human embryonic kidney cells, CAD mouse Cath.a-differentiated catecholaminergic neuron-like cells, or PC12 rat pheochromocytoma neuron-like cells.
17. A therapeutic method, comprising administering to a subject in need thereof the expression vector of claim 8 .
18. The method of claim 17 , wherein the subject is suffering from Amyotrophic Lateral Sclerosis Disease, Leukodystrophy Disease, Alzheimer's Disease, or Parkinson's Disease.
19. A therapeutic method, comprising administering to a subject in need thereof the cell of claim 10 .
20. The method of claim 19 , wherein the subject is suffering from Amyotrophic Lateral Sclerosis Disease, Leukodystrophy Disease, Alzheimer's Disease, or Parkinson's Disease.
21. The expression cassette of claim 1 , wherein the promoter is an inducible promoter selected from a doxycycline-inducible promoter, lac operator system, and a LoxP-stop-LoxP system.
22. The expression cassette of claim 5 , wherein the microRNA is miR-30.