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Last Updated: March 28, 2024

Claims for Patent: 9,388,425


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Summary for Patent: 9,388,425
Title:Tunable genetic switch for regulating gene expression
Abstract: The present invention relates generally the field of genetics, in particular methods, compositions and systems for controlling the inducible expression of transgenes, while eliminating background expression of transgene expression. The present invention relates to methods of use of the compositions and systems as disclosed herein for controlling the inducible expression of transgenes while eliminating background expression of transgene expression, such as use in, for example, the in generation of transgenic animals, use in therapeutic application and use in assays. In some embodiments, the present invention relates to a system of controlled expression of RNAi molecules which target binding sites in the untranslated regions of transgene, thereby the expression of the transgene is modulated and leakiness is reduced. The compositions and methods of the present invention can be used to for therapy, prophylaxis, research and diagnostics in diseases and disorders which afflict mammalian species, generation of transgenic animals, in the study of biological processes as well as for enhance performance of agricultural crops.
Inventor(s): Collins; James J (Newton Center, MA), Deans; Tara L (Clarksville, MD)
Assignee: TRUSTEES OF BOSTON UNIVERSITY (Boston, MA)
Application Number:12/446,300
Patent Claims:1. A method of dose-dependent gene expression of a heterologous target gene in a biological process in a cell, the method comprising (a) providing a cell with a heterologous target gene for expression, wherein the heterologous target gene is inserted into the cell having a system comprising: a nucleic acid sequence encoding an RNA interference molecule (RNAi) agent and a nucleic acid RNAi target sequence, wherein the RNAi target sequence is not endogenous to the cell and is located within the 3'UTR or an intron sequence of the nucleic acid sequence encoding the heterologous target gene, wherein the RNAi molecule agent is substantially complementary to the RNAi target sequence such that gene silencing of the RNAi target sequence and the heterologous target gene occurs in the presence of, and upon complementation with the RNAi molecule agent; wherein a nucleotide sequence encoding the heterologous target gene is operatively linked to a first repressor promoter sequence, wherein the nucleotide sequence encoding the RNAi agent is operatively linked to a second repressor promoter sequence, wherein the second repressor promoter sequence is controlled by a second repressor molecule, wherein the nucleic acid sequence encoding the second repressor molecule is operatively linked to a second promoter and a first repressor promoter sequence, wherein the first repressor promoter sequence is controlled by a first repressor molecule, wherein the nucleic acid sequence encoding the first repressor molecule is operatively linked to a first promoter, wherein the first repressor molecule is inhibited by a small molecule inducer; and wherein the presence of the small molecule inducer relieves repression of the first repressor promoter sequence thereby inducing the expression of the second repressor molecule which represses the expression of the RNAi agent, thereby permitting dose-dependent gene expression of the heterologous target gene; and (b) contacting the cell with an effective amount of the small molecule inducer to induce the gene expression of the heterologous target gene in a dose-dependent manner.

2. The method of claim 1, wherein the first repressor promoter sequence comprises a Lac operator sequence (LacO) and the first repressor molecule is Lac Repressor (LacI).

3. The method of claim 1, wherein the second repressor promoter sequence comprises a Tet operator sequence (TetO) and the second repressor molecule is Tet Repressor (TerR).

4. The method of claim 1, wherein the small molecule inducer is IPTG.

5. The method of claim 1, wherein the first promoter and second promoters are selected from the group consisting of a constitutive promoter or a tissue-specific promoter.

6. The method of claim 1, wherein the first repressor molecule is Lad, or a variant or fragment thereof.

7. The method of claim 1, wherein the first repressor promoter sequence comprises a LacO site, or a variant or fragment thereof.

8. The method of claim 1, wherein the small molecule inducer is IPTG.

9. The method of claim 1, wherein the second repressor molecule is TetR, or a variant or fragment thereof.

10. The method of claim 1, wherein the target nucleotide sequence of the first repressor comprises a tet operator site (TetO) or a variant or fragment thereof.

11. The method of claim 1, wherein the RNA interference (RNAi) molecule is selected from the group consisting of; antisense oligonucleotide, oligonucleotide, siRNA, shRNA or double stranded RNA (dsRNA).

12. The method of claim 1, wherein the RNAi molecule corresponds to SEQ ID NO:1 or SEQ ID NO:2 or a variant or homologue thereof.

13. The method of claim 1, wherein the heterologous target gene is a marker gene or a fragment thereof.

14. The method of claim 1, further comprising a marker gene or a fragment thereof, operatively linked to the first repressor promoter sequence.

15. The method of claim 1, wherein the heterologous gene is selected from the group comprising: a cre recombinase (Cre) gene, a toxin molecule gene, an immunotoxin molecule gene, a gene encoding a molecule that sensitizes the cell to one or more secondary agents, an anti-apoptotic gene or a fragment thereof.

16. The method of claim 1, wherein the nucleotide construct optionally further comprises a nucleotide sequence encoding at least one marker gene which is operatively linked to the expression of the heterologous target gene or the first repressor promoter sequence, wherein gene expression of the marker gene identifies cells also expressing the target gene.

17. The method of claim 1, wherein the first or second promoter is selected from the group comprising of; 3' untranslated regions (3'UTR), repressor sequences; constitutive promoters, inducible promoter; tissue specific promoter or variants thereof.

18. The method of claim 1, wherein the first or second promoter is a selected from the group consisting of: albumin, lymphoid specific promoters, T-cell promoters, neurofilament promoter, pancreas specific promoters, milk whey promoter; hox promoters, .alpha.-fetoprotein promoter, human LIMK2 gene promoters, FAB promoter, insulin gene promoter, transphyretin promoter, alpha.1-antitrypsin promoter, plasminogen activator inhibitor type 1 (PAI-1) promoter, apolipoprotein myelin basic protein (MBP) promoter, GFAP promoter, OPSIN promoter, NSE promoter, tetracycline promoter, metallothionine promoter, ecdysone promoter, a mammalian virus promoter, steroid-responsive promoters, rapamycin responsive promoters and fragments thereof.

19. The method of claim 18, wherein the mammalian virus promoter is an adenovirus late promoter or a mouse mammary tumor virus long terminal repeat (MMTV-LTR).

Details for Patent 9,388,425

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 06/04/1986 ⤷  Try a Trial 2026-10-20
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 ⤷  Try a Trial 2026-10-20
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b Injection 103132 ⤷  Try a Trial 2026-10-20
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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