阅读说明：本技术 通过适体调节的rna酶p切割调控基因表达 (RNAse P cleavage regulated Gene expression by aptamer modulation ) 是由 郭雪翠 于 2018-03-02 设计创作，主要内容包括：本公开提供了用于通过靶基因RNA的适体介导的核糖核酸酶切割来调节靶基因表达的多核苷酸构建体,以及使用所述构建体来响应于结合适体的配体的存在或不存在来调节基因表达的方法。所述多核苷酸构建体含有核糖核酸酶底物序列(例如,RNA酶P底物)和包含效应物区和适体的核糖开关,使得当适体结合配体时,发生靶基因表达。(The present disclosure provides polynucleotide constructs for modulating expression of a target gene by aptamer-mediated ribonuclease cleavage of the target gene RNA, and methods of using the constructs to modulate gene expression in response to the presence or absence of a ligand that binds to the aptamer. The polynucleotide construct contains a ribonuclease substrate sequence (e.g., an rnase P substrate) and a riboswitch comprising an effector region and an aptamer such that when the aptamer binds to the ligand, target gene expression occurs.)

1. a polynucleotide cassette for regulating expression of a target gene comprising an rnase P substrate sequence linked to a riboswitch, wherein the riboswitch comprises an effector region and an aptamer sequence, wherein the effector region comprises a sequence that is complementary to a portion of the rnase P substrate sequence.

2. The polynucleotide cassette of claim 1, wherein the aptamer binds a small molecule ligand.

3. The polynucleotide cassette of claim 1, wherein the rnase P substrate sequence comprises a sequence encoding a tRNA, a mascRNA, a MEN β tRNA-like structure, a viral tRNA-like structure, a rnase P model substrate, and a homologous sequence that can initiate cleavage by rnase P.

4. The polynucleotide cassette of claim 1, wherein the effector region comprises a sequence capable of forming a stem structure upon ligand binding to the aptamer, optionally wherein the effector region stem is 6 to 12 base pairs.

5. The polynucleotide cassette of any one of claims 1 to 4, wherein the aptamer sequence is located 5' of the RNase P substrate sequence and the effector region comprises a sequence complementary to the leader sequence of the RNase P substrate.

6. The polynucleotide cassette of claim 5, wherein the acceptor stem and riboswitch effector regions of the RNase P substrate are separated by 0,1, 2,3 or 4 nucleotides.

7. The polynucleotide cassette of claim 5, wherein the effector region further comprises a sequence complementary to a receptor stem sequence of the RNase P substrate.

8. The polynucleotide cassette of any one of claims 1 to 4, wherein the aptamer sequence is located 3 'of the rnase P substrate sequence and the effector region comprises a sequence complementary to the 3' acceptor stem of the rnase P substrate sequence.

9. The polynucleotide cassette of claim 8, wherein the effector region sequence complementary to the 3' acceptor stem of the rnase P substrate is 1 to 7 nucleotides.

10. A method of modulating expression of a target gene comprising:

a. Inserting the polynucleotide cassette of any one of claims 1 to 4 into an untranslated region (UTR) of a target gene,

b. Introducing a target gene comprising said polynucleotide cassette into a cell, and

c. The cells are exposed to a small molecule ligand that specifically binds to the aptamer in an amount effective to increase expression of the target gene.

11. The method of claim 10, wherein the aptamer sequence of the polynucleotide cassette is located 5' of the rnase P substrate sequence and the effector region comprises a sequence complementary to the leader sequence of the rnase P substrate.

12. the method of claim 11, wherein the acceptor stem and riboswitch effector regions of the rnase P substrate are separated by 0,1, 2,3, or 4 nucleotides.

13. The method of claim 10, wherein the aptamer sequence of the polynucleotide cassette is located 3 'of the rnase P substrate sequence and the effector region comprises a sequence complementary to the 3' acceptor stem of the rnase P substrate sequence.

14. The method of claim 13, wherein the effector region sequence complementary to the 3' acceptor stem of the rnase P substrate is from 1 to 7 nucleotides.

15. The method of claim 10, wherein the polynucleotide cassette is inserted into the 5' untranslated region of the target gene.

16. The method of claim 10, wherein the polynucleotide cassette is inserted into the 3' untranslated region of the target gene.

17. The method of claim 10, wherein two or more polynucleotide cassettes are inserted into the target gene.

18. The method of claim 17, wherein two or more polynucleotide cassettes comprise different aptamers that specifically bind different small molecule ligands.

19. The method of claim 17, wherein two or more polynucleotide cassettes comprise the same aptamer.

20. The method of claim 10, wherein the target gene further comprises a gene regulatory cassette that regulates target gene expression through aptamer-mediated regulation of alternative splicing.

21. the method of claim 10, wherein the target gene comprising the polynucleotide cassette is incorporated into a vector for expression of the target gene.

22. The method of claim 21, wherein the vector is a viral vector.

23. The method of claim 22, wherein the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, and a lentiviral vector.

24. A vector comprising a target gene comprising a polynucleotide cassette according to any one of claims 1-4.

25. The vector of claim 24, wherein the vector is a viral vector.

26. The vector of claim 25, wherein the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, and a lentiviral vector.

27. The vector of claim 24, wherein the target gene further comprises a gene regulatory cassette that regulates target gene expression through aptamer-mediated regulation of alternative splicing.

Technical Field

The present invention provides polynucleotide constructs for modulating gene expression by aptamer-mediated ribonuclease cleavage of RNA, and methods of using the constructs to modulate gene expression in response to the presence or absence of a ligand that binds to an aptamer. The polynucleotide construct contains a ribonuclease substrate sequence and a riboswitch comprising an effector region and an aptamer such that when the aptamer binds to the ligand, target gene expression occurs.

Background

Ribonuclease P ("rnase P") is a riboprotein complex and functions as an endoribonuclease that removes the 5' leader sequence from the precursor tRNA by catalyzing the hydrolysis of a particular phosphodiester bond to generate the mature tRNA. Rnase P is found in all interkingdom species and is composed of one RNA subunit and one (bacterial) or many (archaea and eukaryotes) proteins. Studies of rnase P substrate recognition have revealed that the enzyme recognizes the structure of the substrate rather than the major nucleotide sequence of the substrate and can cleave model substrates containing structures equivalent to the acceptor stem, T-stem, 3 'tail sequence and 5' leader sequence of the precursor tRNA.

Rnase P cleavage of target mRNA has been accomplished by expression of an External Guide Sequence (EGS), a sequence designed to form a hybrid complex with an mRNA target sequence similar to the precursor tRNA. EGS bind their mRNA targets through base-pairing interactions and direct rnase P cleavage of the mRNA targets. The use of EGS sequences to target RNAse P cleavage requires the expression of exogenous EGS sequences.

Summary of The Invention

In one aspect, the invention provides a polynucleotide cassette for regulating expression of a target gene comprising an rnase P-substrate sequence linked to a riboswitch, wherein the riboswitch comprises an effector region and an aptamer, wherein the effector region comprises a sequence complementary to a portion of the rnase P-substrate sequence. In one embodiment, the aptamer binds to a small molecule ligand.

In an embodiment of the invention, the effector region comprises a sequence capable of forming a stem structure upon ligand binding to the aptamer, wherein the effector region stem is 7 to 12 base pairs. In embodiments of the invention, the effector stem is 7, 8, 9, 10, 11, or 12 base pairs.

In one embodiment, the rnase P substrate sequence comprises a sequence encoding a precursor tRNA, or a homologous sequence. In other embodiments, the rnase P target sequence comprises a sequence encoding a tRNA, a mascRNA, a MEN β tRNA-like structure, a viral tRNA-like structure, a rnase P model substrate, and a homologous sequence that can initiate cleavage by rnase P.

In one embodiment, the aptamer sequence of the polynucleotide cassette is located 5' of the rnase P substrate sequence and the effector region comprises a sequence complementary to the leader sequence of the rnase P substrate. In one embodiment, the aptamer sequence is located 5 'of the rnase P substrate sequence and the effector region comprises all or part of the leader sequence of the rnase P substrate sequence and all or part of the 5' receptor stem sequence. In further embodiments, the acceptor stem and riboswitch effector regions of the rnase P substrate are separated by 0,1, 2,3, or 4 nucleotides. In other embodiments, the effector stem, in addition to the leader sequence (and its complement), includes one or more nucleotides of the acceptor stem of the rnase P substrate, and a sequence complementary to one or more nucleotides of the acceptor stem.

In one embodiment, the aptamer sequence of the polynucleotide cassette is located 3 'of the rnase P substrate sequence and the effector region comprises a sequence complementary to all or part of the 3' receptor stem of the rnase P substrate sequence. In a further embodiment, the sequence of the effector region complementary to the 3' acceptor stem of the rnase P substrate is from 1 to 7 nucleotides. In other words, the effector stem comprises 1 to 7 nucleotides of the receptor stem and comprises a sequence complementary to 1 to 7 nucleotides of the receptor stem.

In one embodiment, the riboswitch is located 3' of the rnase P substrate, so the effector and acceptor stems of the rnase P substrate do not overlap. In embodiments, the effector region and the acceptor stem of the rnase P substrate are immediately adjacent (i.e., do not overlap). In other embodiments, the effector region and the acceptor stem of the rnase P substrate are separated by 1, 2,3, 4, 5, or more nucleotides.

In one embodiment, the riboswitch sequence is located within the stem-loop of the rnase P substrate sequence. In one embodiment, the riboswitch sequence is located within the D stem-loop of the rnase P substrate sequence. In one embodiment, the riboswitch sequence is located within the T-stem-loop of the rnase P substrate sequence. In one embodiment, the riboswitch sequence is located in the variable loop of the rnase P substrate sequence. In one embodiment, the riboswitch sequence is located within the anticodon stem-loop of the rnase P substrate sequence.

In another aspect, the present invention provides a method of modulating expression of a target gene, comprising: (a) inserting a polynucleotide cassette of the invention into an untranslated region (UTR) of a target gene; (b) introducing a target gene comprising a polynucleotide cassette into a cell, and (c) exposing the cell to a ligand that specifically binds to the aptamer in an amount effective to increase expression of the target gene. In one embodiment, the ligand is a small molecule.

In one embodiment, the polynucleotide cassette is inserted into the 5' untranslated region of the target gene. In one embodiment, the polynucleotide cassette is inserted into the 3' untranslated region of the target gene. In one embodiment, the polynucleotide cassette is inserted into an intron of a target gene.

In one embodiment, two or more polynucleotide cassettes are inserted into the target gene. In one embodiment, two or more polynucleotide cassettes comprise different aptamers that specifically bind different small molecule ligands. In one embodiment, two or more polynucleotide cassettes comprise the same aptamer. In one embodiment, the two or more polynucleotide cassettes are in the 5 'untranslated region, the 3' untranslated region, or both of the target gene.

In one aspect, the polynucleotide cassettes of the invention are used in combination with other mechanisms for regulating expression of a target gene. In one embodiment, the polynucleotide cassettes of the invention are used in combination with gene regulatory cassettes that regulate target gene expression through aptamer-mediated regulation of alternative splicing, as described in WO 2016/126747 (which is incorporated herein by reference). In other embodiments, the polynucleotide cassettes of the invention are used in combination with gene regulatory cassettes that regulate target gene expression through aptamer-mediated regulation of self-cleaving ribozymes, as described in PCT/US2017/016303 (which is incorporated herein by reference). In other embodiments, the polynucleotide cassettes of the invention are used in combination with gene regulatory cassettes that modulate target gene expression through aptamer-mediated regulation of polyadenylation, as described in PCT/US2017/016279 (which is incorporated herein by reference). In other embodiments, the polynucleotide cassettes of the invention are used in combination with gene regulatory cassettes that regulate target gene expression through aptamer-mediated accessibility of polyadenylation signals, as described in PCT/US2018/019056 (which is incorporated herein by reference).

In one embodiment, the target gene comprising the polynucleotide cassette is incorporated into a vector for expression of the target gene. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector and a lentiviral vector.

In another aspect, the invention provides a vector comprising a target gene comprising a polynucleotide cassette of the invention. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector and a lentiviral vector.

In one embodiment, the target gene comprising the polynucleotide cassette is incorporated into an expression construct or vector comprising a non-tissue specific promoter. In other embodiments, the promoter is tissue specific.

Brief Description of Drawings

Figure 1a schematic of an embodiment wherein a polynucleotide cassette is inserted into the 3' untranslated region (UTR) of a target gene. In this embodiment, the effector sequence of the riboswitch includes a sequence complementary to the leader sequence. When no ligand is present, the effector sequence does not form a stem that includes the leader sequence. The leader sequence is accessible to rnase P and the RNA is cleaved, resulting in degradation and prevention (or reduction) of expression of the target gene.

FIG. 1b is a schematic representation of the polynucleotide cassette from FIG. 1a, wherein a ligand (♦) is bound to the aptamer. When a ligand is present, the effector sequence forms a stem that includes the leader sequence of the rnase substrate. The leader sequence is no longer accessible and cleavage of the RNA by rnase P is inhibited, resulting in increased expression of the target gene.

Northern blot analysis of mascRNA. Insertion of a sequence encoding a mascRNA into the 3' UTR of the target Gene (GFP) results in mRNA cleavage and reduced protein expression. HeLa cells transfected with the constructs shown were collected for RNA extraction 24 hours or 48 hours after transfection. DIG-labeled probes recognize human and mouse mascRNA. Lanes 1 and 4 show endogenous human mascRNA, and lanes 2 and 5 show increased expression of mascRNA produced by mouse mascRNA inserted into the 3' UTR of the GFP reporter.

FIG. 2b. insertion of mascRNA into the 5-UTR or 3' UTR reduces GFP expression. HeLa cells were transfected with the constructs shown and analyzed by flow cytometry 48 hours after transfection. The percentage (%) of GFP positive population and the Mean Fluorescence Intensity (MFI) are shown. Cells transfected with constructs with mascRNA in the 3'UTR (GFP-masc), 5' UTR (masc-GFP), or 5 'and 3' UTR (masc-GFP-masc) had a reduction in both the percentage of GFP positive cells and MFI compared to cells with control CMV-GFP constructs.

Figure 2c. insertion of mascRNA into the 3' UTR reduced luciferase target gene expression. A mouse mascRNA or a mutant mouse mascRNA (referred to herein as masc-mlp) was inserted into the 3' UTR of the luciferase gene. The predicted tRNA-like secondary structure of mouse mascRNA is shown on the left panel, where mutations in the T-loop from UUCC to AAG are indicated. The right panel shows the results of the luciferase assay. Insertion of mascRNA into the 3' UTR resulted in an 81% reduction in luciferase expression (Luci-masc), whereas cells containing mutations in the T-loop of mascRNA (Luci-masc-mlp) showed only a 20% reduction in luciferase expression.

FIG. 3a is a schematic representation of an mRNA transcript from the MG1 (SEQ ID NO:1) polynucleotide cassette.

FIG. 3b is a schematic representation of mRNA transcripts from the GM1 (SEQ ID NO:2) polynucleotide cassette.

Figure 3c aptamer-mediated regulation by rnase P cleavage of mRNA to regulate gene expression. Shows the results of the luciferase assay. HEK293 cells were transfected with the indicated luciferase control construct Con8 or constructs containing mascRNA and guanine aptamer sequences (MG1, GM1, GM2(SEQ ID NO:3) and GM3(SEQ ID NO: 4)). Transfected cells were treated with DMSO as solvent control or guanosine as aptamer ligand. In the absence of guanine treatment, cells transfected with constructs MG1, GM1, or GM2 showed reduced luciferase expression compared to cells transfected with control constructs. Guanosine treatment of the cells with MG1, GM1, or GM2 (but not GM3) increased luciferase expression. Luciferase activity was expressed as mean ± s.d. (n =3), and fold induction was expressed as the quotient of luciferase activity obtained in the presence of guanosine divided by the value obtained in the absence of guanosine.

Figure 4a. variation of the local sequence upstream (5') of the aptamer stem affects riboswitch activity in GM constructs. Luciferase activity was measured in HEK293 cells transfected with the constructs shown and treated with or without guanine. GM4(SEQ ID NO:5) has a 3 nucleotide change compared to GM1, where 3 nucleotides 5' of the aptamer stem are changed to reduce the likelihood of additional base pairing of these nucleotides.

Figure 4b distance between aptamer and rnase P substrate affects riboswitch activity in GM constructs. The position of the aptamer/effector region in the GM construct was shifted upstream (5') in one nucleotide increment compared to mascrnas that allowed 0 to 3 nucleotides of the leader sequence that were not part of the stem of the effector region (aptamer) (constructs GM5 to GM8(SEQ id no:7-10), respectively). Luciferase activity (expressed as mean ± s.d.) was measured from HEK293 cells transfected with the indicated constructs and treated with or without guanine, and (n = 3). Fold induction is expressed as the quotient of the luciferase activity obtained in the presence of guanine divided by the value obtained in the absence of guanine. The GM5 construct (without space between the two constructs) had more efficient rnase P-mediated cleavage as shown by lower basal levels of luciferase expression than the construct GM 4.

FIG. 4c. effect of length of riboswitch effector region complementary to the receptor stem of RNase substrate in MG construct. Constructs MG1 to MG4 were generated with effector region stems of 6 to 3 nucleotides (each with a total of 8bp effector region stem) complementary to the acceptor stem of the mascRNA, respectively. MG3(SEQ ID NO:12) and MG4(SEQ ID NO:13) had slightly more efficient RNase P cleavage than MG1 and more efficient induction of luciferase following treatment with guanine.

FIG. 5 Xpt-guanine aptamer modulation by human tRNA^Argas a substrate for rnase P mediated cleavage by rnase P. From control constructs Con8, GM1 (containing mascRNA) or GArg (SEQ ID NO:14) (containing human tRNA)^Arg) Transfected HEK293 cells measure luciferase activity. Transfected cells were treated with or without guanine and luciferase activity was measured and expressed as mean ± s.d. (n = 3). Fold induction is expressed as the quotient of the luciferase activity obtained in the presence of guanine divided by the value obtained in the absence of guanine.

Figures 6a and 6b riboswitch with either theophylline aptamer or adenine aptamer modulates mascRNA-rnase P mediated mRNA cleavage. Luciferase activity was measured from HEK293 cells transfected with control construct Con8, construct TheoM (SEQ ID NO:16) containing theophylline aptamer and mascRNA (FIG. 6a) or construct YM (SEQ ID NO:15) containing ydhl-A adenine aptamer and mascRNA (FIG. 6 b). Transfected cells were treated with or without 3 mM theophylline or 1 mM adenine and luciferase activity was measured and expressed as mean ± s.d. (n = 3). Fold induction is expressed as the quotient of the luciferase activity obtained in the presence of the aptamer ligand divided by the value obtained in the absence of the aptamer ligand.

Figures 7a and 7b use of a riboswitch based on rnase P substrate in combination with a second riboswitch to achieve tighter regulation of target gene expression. Luciferase activity was measured from HEK293 cells transfected with construct G15 or GM1-G15 (FIG. 7a) or construct G17 or GM1-G17 (FIG. 7 b). Transfected cells were treated with or without 500 μ M guanine and luciferase activity was measured and expressed as mean ± s.d. (n = 3). Fold induction is expressed as the quotient of the luciferase activity obtained in the presence of guanine divided by the value obtained in the absence of guanine.

Figure 7c use of a riboswitch based on rnase P substrate in combination with a second riboswitch to achieve tighter regulation of Epo target gene expression. Expression of mouse Epo protein was measured using ELISA from supernatants collected from HEK293 cells transfected with construct G17_2IR3 or construct GM1-G17_2IR 3. The concentration of Epo is expressed as the mean ± s.d. (n =3), and the fold induction is expressed as the quotient of the concentration of Epo obtained in the presence of guanine divided by the concentration of Epo obtained in the absence of guanine.

the sequence of the constructs described herein. The 3' end of the coding sequence of the target gene (e.g., firefly luciferase) is in capital letters; mascRNA or tRNA^ArgThe sequences are italicized lower case letters and are shown in grey shading; aptamer sequence is underlined in waves. The sequences forming the effector region stems are double underlined.

Detailed Description

This application claims priority from U.S. provisional application serial No. 62/466,138 filed on 3/2/2016, which is incorporated herein in its entirety. The present application is directed to providing a sequence listing of SEQ ID NOs listed below, provided herewith as an electronic document and incorporated herein by reference in their entirety.

Modulation of expression of a target gene (e.g., a therapeutic transgene) is useful or necessary in a variety of circumstances. In the context of therapeutic expression of genes, technologies that enable the regulation of transgene expression have the potential to improve safety by regulating expression levels and timing thereof. For safe and effective therapeutic applications, regulatory systems that control protein expression have a practical, and in some cases essential, role. The present invention provides polynucleotide cassettes for modulating expression of a target gene by aptamer-mediated ribonuclease cleavage of RNA of the target gene, and methods of using the polynucleotide cassettes to modulate expression of the target gene in response to the presence or absence of a ligand that binds to the aptamer. Endonucleolytic cleavage of mRNA causes, for example, loss of the 5 'cap or 3' poly a tail, resulting in degradation of mRNA by the RNA exosome pathway. Thus, the present invention provides gene regulatory cassettes that can regulate expression of a target gene by providing an aptamer ligand. Such methods are useful, for example, for studying target gene expression in cells, tissues, and organisms, or for modulating the expression level of therapeutic proteins.

A gene regulatory polynucleotide cassette refers to a recombinant DNA construct that, when incorporated into the DNA of a target gene, provides the ability to regulate expression of the target gene through aptamer/ligand mediated ribonuclease cleavage of the resulting RNA. As used herein, a polynucleotide cassette or construct is a nucleic acid (e.g., DNA or RNA) that comprises elements derived from different sources (e.g., different organisms, different genes from the same organism, etc.). The polynucleotide cassette comprises a riboswitch and a ribonuclease substrate sequence. In the present case riboswitches contain a sensor region (e.g., an aptamer) and an effector region, which together are responsible for sensing the presence of a ligand that binds to the sensor region and altering the cleavage of the ribonuclease substrate sequence by the ribonuclease. In one embodiment, the expression of the target gene is increased when the aptamer ligand is present and decreased when the ligand is absent.

Ribonuclease substrates

The ribonuclease substrate sequence linked to the riboswitch can be any ribonuclease substrate that targets a ribonuclease to cleave a target RNA when inserted into a target gene as part of a gene regulatory polynucleotide cassette. In one embodiment, the ribonuclease substrate is an rnase P substrate sequence, such as a precursor tRNA or a homologous sequence. In other embodiments, the rnase P substrate sequence comprises a sequence encoding a mascRNA; and MEN β tRNA-like structures (see Sunwoo et al genome research 2009, 19:347-359; Wilusz et al Genes Dev.2012, 26(21):2392-407, both incorporated herein by reference), viral tRNA-like structures, sequences of RNase P model substrates, and homologous sequences that can initiate RNase P cleavage. Yuan and Altman describe model RNase P substrate sequences in an article published in The EMBO Journal (vol. 14, pp.159-168, incorporated herein by reference), including, for example, a D-loop or anticodon loop deleted precursor tyrosine tRNA (see, e.g., FIGS. 1-4, Table 1, and associated text for Yuan and Altman).

Ribose switch

The term "riboswitch" as used herein refers to a regulatory segment of an RNA polynucleotide, or a DNA sequence encoding a regulatory segment of an RNA polynucleotide. In the context of the present invention, a riboswitch contains a sensor region (e.g., an aptamer) and an effector region that together are responsible for sensing the presence of a ligand (e.g., a small molecule) and modulating the adaptability of a ribonuclease substrate sequence to cleavage by a ribonuclease. In one embodiment, the ribonuclease is rnase P. In one embodiment, the riboswitch is recombinant, utilizing polynucleotides from two or more sources. The term "synthetic" as used herein in the context of riboswitches refers to riboswitches that are not naturally occurring. In one embodiment, the sensor and effector region are linked by a polynucleotide linker. In one embodiment, the polynucleotide adaptor forms an RNA stem (i.e., a region of double-stranded RNA polynucleotide).

The aptamer portion of the riboswitch can be located at the 5 'end, 3' end, and/or within the stem loop of the rnase P substrate. When the aptamer is linked to the 5' end of the rnase P substrate sequence, the effector region of the riboswitch can include a sequence that is complementary to all or part of the rnase P substrate leader sequence (see, e.g., fig. 1a, 1b, and 3 b). In this configuration (i.e., the riboswitch at the 5 'end of the substrate), the effector region of the riboswitch may also include a sequence that is complementary to all or part of the 5' receptor stem sequence of the rnase P substrate (e.g., as in GM1 and GM2 structures).

When the aptamer portion of the riboswitch is located at the 3 'end of the rnase P substrate (as in the MG1-4 construct), the effector region comprises a sequence that is complementary to all or part of the 3' receptor stem sequence of the rnase P substrate. In other embodiments, the aptamer portion of the riboswitch is located in the stem-loop of the rnase P substrate sequence. In some embodiments, the aptamer portion of the riboswitch sequence is located in the D stem-loop, T stem-loop, anticodon stem-loop, or variable loop of the rnase P substrate sequence. When the riboswitch is located in the D-stem loop, the effector region can comprise all or part of the D-stem. Likewise, when the aptamer is located in a T stem loop, an anticodon stem loop, or a variable loop, the effector region may comprise all or part of the T stem, anticodon stem, or variable loop stem, respectively.

Region of effector

The effector region of the riboswitch comprises an RNA sequence that alters the sensitivity of a ribonuclease substrate to ribonuclease cleavage in response to a ligand that binds to the sensor region (e.g., an aptamer). In one embodiment, the effector region comprises all or part of a leader sequence of a rnase P substrate sequence. In this embodiment, the effector region stem comprises some or all of the leader sequence and a sequence complementary to some or all of the leader sequence. When an aptamer binds its ligand, the effector region forms a stem that includes a leader sequence, thereby preventing cleavage of the target RNA by rnase P (see, e.g., fig. 1 b). Under certain conditions (e.g., when an aptamer is not bound to its ligand), the effector region is in an environment that provides access to the leader sequence, resulting in cleavage by rnase P (see, e.g., fig. 1a and 3 b). In other embodiments, the effector stem, in addition to the leader sequence (and its complement), includes one or more nucleotides of the receptor stem for rnase P substrates, and sequences complementary to one or more nucleotides of the receptor stem (as in GM1 and GM2 structures). In other embodiments, there are 1 or more nucleotides of the 3' leader sequence of the rnase P substrate that are not part of the effector region stem (see, e.g., constructs GM6, GM7, and GM 8). In other words, in these embodiments, the sequence forming the effector stem and the sequence forming the acceptor stem of the rnase P substrate do not overlap and may be separated by 0,1, 2,3, or 4 nucleotides.

In some embodiments, the effector region comprises some or all of the acceptor stem of the rnase P substrate sequence (see, e.g., fig. 3 a). In this embodiment, the effector stem comprises some or all of the receptor stem sequence and a sequence complementary to some or all of the receptor stem sequence. When an aptamer binds its ligand, the effector region forms a stem that includes the receptor stem sequence, thereby disrupting the receptor stem structure and preventing rnase P cleavage of the target RNA. Under certain conditions (e.g., when an aptamer is not bound to its ligand), the effector region is in an environment that allows the receptor stem structure to result in cleavage by rnase P. In embodiments of the invention, the effector stem comprises 1, 2,3, 4, 5, or 6 nucleotides and complementary sequences of the acceptor stem of an rnase P substrate. In other embodiments, the effector region comprises all or part of a stem of a D stem loop, a T stem loop, a variable loop, or an anticodon stem loop.

The portion of the effector region that is capable of forming a stem upon ligand binding to the aptamer should be of sufficient length (and GC content) to substantially prevent rnase cleavage of the substrate upon ligand binding to the aptamer, while also allowing rnase substrate cleavage by rnase when the ligand is not present in sufficient quantity. In an embodiment of the invention, the stem portion of the effector region comprises a stem sequence in addition to the ribonuclease substrate sequence and its complement. In some embodiments, the additional stem sequence comprises a sequence from an aptamer stem. The length and sequence of the effector region stem can be altered using known techniques to identify a stem that allows for acceptable background expression of the target gene when no ligand is present and acceptable levels of expression of the target gene when ligand is present. If the effector region stem is, for example, too long, it may hide the acquisition of the leader sequence in the presence or absence of the ligand or otherwise prevent cleavage of the substrate by the ribonuclease. If the stem is too short, it may not form a stable stem capable of sequestering the leader sequence (or otherwise not altering the substrate conformation), in which case the target RNA will be cleaved in the presence or absence of the ligand. In one embodiment, the effector region stem has a total length of between about 7 base pairs and about 20 base pairs. In some embodiments, the stem is between about 8 base pairs to about 11 base pairs in length. In some embodiments, the stem is 8 base pairs to 11 base pairs in length. In addition to stem length, the GC base pair content of the stem can also be altered to alter stem stability.

Aptamer/ligand

In one embodiment, the sensor region comprises an aptamer. The term "ligand" refers to a molecule that specifically binds to an aptamer. In one embodiment, the ligand is a small molecule, i.e., a low molecular weight (less than about 1,000 daltons) molecule, including, for example, lipids, monosaccharides, second messengers, cofactors, metal ions, other natural products and metabolites, nucleic acids, and most therapeutic drugs. In one embodiment, the ligand is a polynucleotide having two or more nucleotide bases.

In one embodiment, the ligand is selected from the group consisting of 8-azaguanine, adenosine 5' -monophosphate monohydrate, amphotericin B, avermectin B1, azathioprine, chlormadinone acetate, mercaptopurine, moraxezine hydrochloride, N6-methyladenosine, coenzyme i (nadide), progesterone, promethazine hydrochloride, pyrimethanil pamoate (pyrvinium pamoate), sulfaguanidine, testosterone propionate, thioguanine, tyloxapol, and vorinostat.

Aptamer ligands can also be cellular endogenous components that increase significantly under specific physiological/pathological conditions, such as oncogenic transformation-these can include second messenger molecules such as GTP or GDP, calcium; fatty acids or fatty acids that are incorrectly metabolized in breast cancer, such as 13-HODE (Flaherty, JT et al, Plos One, vol 8, e63076, 2013, which is incorporated herein by reference); an amino acid or amino acid metabolite; metabolites in the glycolytic pathway, which are usually present at higher levels in cancer cells or in normal cells of metabolic diseases; and cancer-associated molecules such as Ras or mutant Ras proteins, mutant EGFR in lung cancer, indoleamine-2, 3-dioxygenase (IDO) in many types of cancer. Endogenous ligands include progesterone metabolites in breast Cancer as disclosed by JPWiebe (Endocrine-Related Cancer (2006) 13: 717-738, which is incorporated herein by reference). Endogenous ligands also include metabolites with increased levels such as lactate, glutathione, kynurenine caused by mutations in key metabolic enzymes in kidney cancer, as disclosed by Minton, DR and Nanus, DM (Nature Reviews, Urology, volume 12, 2005, which is incorporated herein by reference).

Aptamers have a binding region that is capable of forming a complex with a desired target molecule (i.e., ligand). Specificity of binding can be defined in terms of the comparative dissociation constant (Kd) of an aptamer to its ligand as compared to the dissociation constant of the aptamer to an unrelated molecule. Thus, a ligand is a molecule that binds an aptamer with a higher affinity than an unrelated material. Typically, the Kd of an aptamer with respect to its ligand will be at least about 10 times less than the Kd of an aptamer with an unrelated molecule. In other embodiments, the Kd will be at least about 20 times less, at least about 50 times less, at least about 100 times less, and at least about 200 times less. Aptamers will typically be between about 15 and about 200 nucleotides in length. More typically, aptamers will be between about 30 and about 100 nucleotides in length.

Aptamers that can be incorporated as part of a riboswitch can be naturally occurring aptamers or modified forms thereof, or aptamers that are designed de novo and/or screened by exponential enrichment of ligand system evolution (SELEX) or other screening methods. Examples of aptamers that bind small molecule ligands include, but are not limited to, theophylline, dopamine, sulforhodamine B (sulforhodamine B), cellobiose, kanamycin a, lividomycin, tobramycin, neomycin B, erythromycin, chloramphenicol, streptomycin, cytokines, cell surface molecules, and metabolites. For reviews of aptamers that recognize small molecules, see, e.g., Famulok, Science 9:324-9 (1999), and McKeague, m. and derrosa, m.c. j.nuc.aci.2012 (both of which are incorporated herein by reference). In another embodiment, the aptamer is a complementary polynucleotide.

method for identifying aptamers/ligands

In one embodiment, the aptamer is designed to bind to a specific small molecule ligand. Methods for designing and selecting aptamers that bind to a particular ligand are disclosed in WO/2018/025085, which is incorporated by reference. Other methods for screening aptamers include, for example, SELEX. Methods for designing aptamers that selectively bind small molecules using SELEX are disclosed, for example, in U.S. patent nos. 5,475,096, 5,270,163, and Abdullah Ozer et al nuc.aci.2014, which are incorporated herein by reference. Improvements to the SELEX process are described in U.S. Pat. nos. 5,580,737 and 5,567,588 (which are incorporated herein by reference).

Selection techniques for identifying aptamers typically involve preparing large pools of DNA or RNA molecules of a desired length containing regions that are randomized or mutagenized. For example, the pool of oligonucleotides used for aptamer selection may contain a region of 20-100 randomized nucleotides flanked by regions of about 15-25 nucleotides in length and available for binding to a defined sequence of PCR primers. The oligonucleotide pool is amplified using standard PCR techniques or other means that allow for amplification of the selected nucleic acid sequence. DNA pools can be transcribed in vitro when RNA aptamers are desired to produce pools of RNA transcripts. Pools of RNA or DNA oligonucleotides are then selected based on their ability to specifically bind the desired ligand. Selection techniques include, for example, affinity chromatography, although any protocol that will allow for the selection of nucleic acids based on the ability to specifically bind another molecule may be used. Selection techniques for identifying aptamers that bind to small molecules and function within cells may involve cell-based screening methods. In the case of affinity chromatography, the oligonucleotide is contacted with a target ligand immobilized on a matrix in a column or on a magnetic bead. The oligonucleotides are preferably selected for ligand binding in the presence of salt concentrations, temperatures, and other conditions that mimic normal physiological conditions. The ligand-bound oligonucleotides in the pool remain on the column or bead, while the non-binding sequences are washed away. The ligand-bound oligonucleotides are then amplified by PCR (usually after elution) (after reverse transcription if RNA transcripts are used). The selection process is repeated for the selected sequence, for a total of about three to ten iterations of the selection procedure. The resulting oligonucleotides are then amplified, cloned, and sequenced using standard procedures to identify oligonucleotide sequences capable of binding the target ligand. Once the aptamer sequence has been identified, the aptamer can be further optimized by additional selections starting from pools of oligonucleotides containing mutagenized aptamer sequences.

In vivo aptamer selection can be used after one or more rounds of in vitro selection (e.g., SELEX). For example, Konig, J.et al (RNA.2007, 13(4): 614-622, which is incorporated herein by reference) describe a combination of SELEX and yeast triple-hybrid systems for in vivo selection of aptamers.

Target genes

The gene regulatory cassette of the present invention is a platform that can be used to regulate the expression of any target gene that can be expressed in a target cell, tissue or organism. The term "target gene" refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and translated and/or expressed under appropriate conditions. Alternatively, the target gene is endogenous to the target cell, and the gene regulatory cassette of the invention is localized to the target gene (e.g., into the 5 'or 3' UTR of the endogenous target gene). An example of a target gene is a polynucleotide encoding a therapeutic polypeptide. In another embodiment, the target gene encodes RNA, such as miRNA, rRNA, small or long non-coding RNA, short hairpin RNA (shrna), and any other regulatory RNA. In one embodiment, the target gene is exogenous to the cell in which the recombinant DNA construct is to be transcribed. In another embodiment, the target gene is endogenous to the cell in which the recombinant DNA construct is to be transcribed.

The target gene according to the present invention may be a gene encoding a protein or a sequence encoding a non-protein-encoding RNA. The target gene may be, for example, a gene encoding a structural protein, an enzyme, a cell signaling protein, a mitochondrial protein, a zinc finger protein, a hormone, a transporter, a growth factor, a cytokine, an intracellular protein, an extracellular protein, a transmembrane protein, a cytoplasmic protein, a nucleoprotein, a receptor molecule, an RNA binding protein, a DNA binding protein, a transcription factor, a translation machinery, a channel protein, a motor protein, a cell adhesion molecule, a mitochondrial protein, a metabolic enzyme, a kinase, a phosphatase, an exchange factor, a chaperone protein, and a modulator of any of these. In embodiments, the target gene encodes erythropoietin (Epo), human growth hormone (hGH), transcription activator-like effector nuclease (TALEN), human insulin, CRISPR-associated protein 9 (cas9), or an immunoglobulin (or portion thereof), including, for example, a therapeutic antibody.

Expression constructs

The present invention encompasses the use of recombinant vectors for introducing a polynucleotide encoding a target gene and containing a gene regulatory cassette as described herein into a target cell. In many embodiments, the recombinant DNA constructs of the present invention comprise additional DNA elements including DNA segments that provide for the replication of the DNA in a host cell and the appropriate level of expression of a target gene in the cell. The skilled artisan understands that the expression control sequences (promoters, enhancers, etc.) are selected based on their ability to promote expression of the target gene in the target cell. By "vector" is meant a recombinant plasmid, Yeast Artificial Chromosome (YAC), minichromosome, DNA minicircle, or virus (including virus-derived sequences) comprising a polynucleotide to be delivered into a host cell in vitro or in vivo. In one embodiment, the recombinant vector is a viral vector or a combination of multiple viral vectors.

Viral vectors for aptamer-mediated expression of target genes in target cells, tissues or organisms are known in the art and include Adenovirus (AV) vectors, adeno-associated virus (AAV) vectors, retroviral and lentiviral vectors, and herpes simplex type 1 (HSV1) vectors.

Adenoviral vectors include, for example, those based on human adenovirus type 2 and human adenovirus type 5, which have been made replication-defective by deletions in the E1 and E3 regions. The transcription cassette can be inserted into the E1 region to produce a recombinant E1/E3 deletion AV vector. Adenoviral vectors also include helper-dependent high capacity adenoviral vectors (also referred to as high capacity "visless" or "eviscerated" vectors) that do not contain viral coding sequences. These vectors contain cis-acting elements necessary for viral DNA replication and packaging, mainly Inverted Terminal Repeats (ITRs) and the packaging signal (Ψ). These helper-dependent AV vector genomes have the potential to carry foreign DNA from a few hundred base pairs to about 36 kb.

Recombinant adeno-associated viral "rAAV" vectors include any vector derived from any adeno-associated viral serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7 and AAV-8, AAV-9, AAV-10, and the like. The rAAV vector may have one or more of the AAV wild-type genes deleted in whole or in part, preferably the Rep and/or Cap genes, but retains functional flanking ITR sequences. Functional ITR sequences are retained for rescue, replication, packaging and potential chromosomal integration of the AAV genome. The ITRs need not be wild-type nucleotide sequences and may be altered (e.g., by insertion, deletion, or substitution of nucleotides) so long as the sequence provides functional rescue, replication, and packaging.

Alternatively, other systems, such as lentiviral vectors, may be used in embodiments of the invention. Lentivirus-based systems can transduce non-dividing as well as dividing cells, making them useful for applications that target non-dividing cells of, for example, the CNS. Lentiviral vectors are derived from human immunodeficiency virus and, like this virus, integrate into the host genome, providing the potential for long-term gene expression.

Polynucleotides including plasmids, YACs, minichromosomes, and minicircles carrying target genes containing gene regulatory cassettes can also be introduced into cells or organisms through non-viral vector systems using, for example, cationic lipids, polymers, or both as vectors. Conjugated poly-L-lysine (PLL) polymer and Polyethyleneimine (PEI) polymer systems can also be used to deliver the vector to the cells. Other methods for delivering vectors to cells for cell cultures and organisms include hydrodynamic injection and electroporation as well as the use of ultrasound. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, n. et al (Adv Biomed res.2012; 1:27), which is incorporated herein by reference.

Method for regulating expression of target gene

In one aspect, the invention provides methods of modulating expression of a target gene (e.g., a therapeutic gene) by: (a) inserting the gene regulatory cassette of the present invention into a target gene; (b) introducing a target gene comprising a gene regulatory cassette into a cell; and (c) exposing the cells to a ligand that binds the aptamer. In one embodiment, the ligand is a small molecule. In various aspects, expression of a target gene in a target cell confers a desired property to the cell into which it is introduced, or otherwise produces a desired therapeutic result. The target cell is a eukaryotic cell, such as a mammalian cell. In embodiments, the target cell is a human cell from a target tissue including, for example, fat, Central Nervous System (CNS), muscle, heart, eye, liver, and the like.

In a preferred embodiment, one or more gene regulatory cassettes are inserted into the 5 'and/or 3' untranslated region of a target gene. In one embodiment, a single gene regulatory cassette is inserted into the target gene. In other embodiments, 2,3, 4 or more gene regulatory cassettes are inserted into the target gene. In one embodiment, two gene regulatory cassettes are inserted into the target gene. When multiple gene regulatory cassettes are inserted into a target gene, they may each contain the same aptamer, such that a single ligand may be used to modulate ribonuclease cleavage of the multiple cassettes and thereby modulate target gene expression. In other embodiments, multiple gene regulatory cassettes are inserted into a target gene, each of which may contain a different aptamer, such that exposure to multiple different small molecule ligands modulates target gene expression. In other embodiments, multiple gene regulatory cassettes are inserted into the target gene, each containing a different ribonuclease substrate sequence. This can be used to reduce recombination and improve ease of incorporation into viral vectors.

The polynucleotide cassettes of the invention may be used in combination with other mechanisms for regulating expression of a target gene. In one embodiment, the polynucleotide cassette of the invention is used in combination with a gene regulatory cassette that regulates target gene expression through aptamer-mediated regulation of alternative splicing, as described in WO 2016/126747 (which is incorporated herein by reference). The invention may also be combined with the polynucleotide constructs and methods described in PCT/US2017/016303 and PCT/US1207/016279, which are incorporated herein by reference.

Methods of treatment and pharmaceutical compositions

One aspect of the invention provides methods of modulating the level of a therapeutic protein delivered by gene therapy. In this embodiment, the "target gene" may encode a therapeutic protein. The "target gene" may encode a protein that is endogenous or exogenous to the cell.

Therapeutic gene sequences containing regulatory cassettes with aptamer-driven riboswitches are delivered to target cells in vitro or ex vivo, for example, via a vector. The cell specificity of a "target gene" may be controlled by a promoter or other element within the vector. Delivery of a vector construct containing a target gene and a polynucleotide cassette and transfection of the target tissue, stable transfection resulting in a regulated target gene, are the first steps in the production of therapeutic proteins.

However, due to the presence of the regulatory cassette within the target gene sequence, the target gene is not expressed at significant levels, i.e., it is in the "off state" in the absence of a specific ligand that binds to the aptamer contained within the riboswitch of the regulatory cassette. Target gene expression is activated only when an aptamer-specific ligand is administered (or otherwise present in a sufficient amount).

Delivery of the vector construct containing the target gene and delivery of the activating ligand are typically separated in time. Delivery of the activating ligand will control when the target gene is expressed and the level of protein expression. The ligand may be delivered by a number of routes including, but not limited to, oral, Intramuscular (IM), Intravenous (IV), intraocular, or topical.

The timing of the delivery of the ligand will depend on the activation requirements of the target gene. For example, if a therapeutic protein encoded by a target gene is in constant demand, the small molecule oral ligand may be delivered daily or multiple times a day to ensure continuous activation of the target gene and thus continuous expression of the therapeutic protein. If the target gene has a long-lasting effect, the inducing ligand may be administered less frequently.

The invention allows for temporal control of the expression of a therapeutic transgene in a manner determined by the timely administration of a ligand specific to an aptamer within a riboswitch that controls a polynucleotide cassette. Increased therapeutic transgene expression only after ligand administration increases the safety of gene therapy treatment by allowing the target gene to shut down in the absence of ligand.

Different aptamers can be used to allow different ligands to activate a target gene. In certain embodiments of the invention, each therapeutic gene containing a regulatory cassette will have a specific aptamer within the cassette that will be activated by a specific small molecule. This means that each therapeutic gene is only activated by ligands specific for the aptamer contained therein. In these embodiments, each ligand will activate only one therapeutic gene. This allows for the possibility that several different "target genes" may be delivered to one individual, and each will be activated upon delivery of a ligand specific for an aptamer contained within the regulatory cassette contained within each target gene.

The present invention allows any therapeutic protein, such as Erythropoietin (EPO) or therapeutic antibodies, whose gene can be delivered to the body to be produced by the body upon delivery of the activating ligand. This therapeutic protein delivery method can replace manufacturing such therapeutic proteins outside the body, then injecting or infusing the therapeutic protein, such as antibodies used in cancer or for blocking inflammatory or autoimmune diseases. The body containing the regulated target gene becomes the biologics manufacturing plant which is turned on when the gene specific ligand is administered.

The level and timing of administration of the therapeutic protein can be important to the therapeutic effect. For example, in the delivery of avastin (anti-VEGF antibody) for cancer. The present invention increases the ease of administration in response to monitoring therapeutic protein levels and effects.

In one embodiment, the target gene may encode a nuclease that can target and edit a specific DNA sequence. Such nucleases include Cas9, zinc-containing finger nucleases, or TALENs. In the case of these nucleases, the nuclease protein may only be required for a short period of time sufficient to edit its target endogenous gene. However, if an unregulated nuclease gene is delivered into the body, this protein may be present throughout the remaining life of the cell. In the case of nucleases, the longer the nuclease is present, the higher the risk of off-target editing. The regulation of expression of such proteins has significant safety advantages. In this case, a vector containing the nuclease gene containing the regulatory cassette can be delivered to an appropriate cell in vivo. In the absence of the cassette-specific ligand, the nuclease gene is in an "off" state, and thus no nuclease is produced. Nucleases are generated only when an activating ligand is applied. When sufficient time has elapsed to allow sufficient editing to occur, the ligand is withdrawn and no longer administered. Thus, the nuclease gene is thereafter in an "off" state, and no nuclease is produced, and editing ceases. This approach can be used to overcome genetic conditions, including many inherited retinopathies, such as LCA10 caused by mutations in CEP290 and stargardt Disease caused by mutations in ABCA 4.

The therapeutic gene can be modulated to treat many different types of diseases using the administration of a modulated target gene encoding a therapeutic protein that is activated only after administration of a specific ligand, for example, treating cancer with a therapeutic antibody, treating an immune disorder with an immunomodulatory protein or antibody, treating a metabolic disease with an anti-C5 antibody or antibody fragment as a modulated gene, treating a rare disease such as PNH or ocular angiogenesis with a therapeutic antibody, and treating dry AMD with an immunomodulatory protein.

A variety of specific target genes that allow for the treatment of a wide variety of specific diseases and conditions are suitable for use in the present invention. For example, insulin or an insulin analogue (preferably human insulin or a human insulin analogue) may be used as a target gene for the treatment of type I diabetes, type II diabetes or metabolic syndrome; human growth hormone can be used as a target gene for the treatment of children with growth disorders or adults lacking growth hormone; erythropoietin (preferably human erythropoietin) can be used as a target gene for the treatment of anemia arising from chronic kidney disease, anemia arising from myelodysplasia, or anemia arising from cancer chemotherapy.

The invention may be particularly suitable for the treatment of diseases caused by single gene defects, such as cystic fibrosis, haemophilia, muscular dystrophy, thalassemia or sickle cell anemia. Thus, human beta-globin, gamma-globin, delta-globin, or zeta-globin can be used as a target gene for the treatment of beta-thalassemia or sickle cell anemia; human factor VIII or factor IX can be used as a target gene for treatment of hemophilia a or hemophilia B.

The ligands used in the present invention are typically combined with one or more pharmaceutically acceptable carriers to form a pharmaceutical composition suitable for administration to a patient. Pharmaceutically acceptable carriers include solvents, binders, diluents, disintegrants, lubricants, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are commonly used in the pharmaceutical field. The pharmaceutical compositions can be in the form of tablets, pills, capsules, lozenges, and the like, and are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, intranasal, subcutaneous, oral, inhalation, transdermal (topical), transmucosal, and rectal.

The pharmaceutical composition comprising the ligand is administered to the patient on a dosing schedule such that an amount of the ligand sufficient to desirably modulate the target gene is delivered to the patient. When the ligand is a small molecule and the dosage form is a tablet, capsule, or the like, preferably, the pharmaceutical composition comprises 0.1 mg to 10 g of the ligand; 0.5 mg to 5 g of ligand; 1 mg to 1 g of ligand; 2 mg to 750 mg of ligand; 5 mg to 500 mg of ligand; or 10 mg to 250 mg of ligand.

The pharmaceutical composition may be administered once a day or multiple times a day (e.g., 2,3, 4, 5 or more times a day). Alternatively, the pharmaceutical composition may be administered less than once a day, e.g., once every 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or once a month or once every several months. In some embodiments of the invention, the pharmaceutical composition may be administered to the patient only a few times, e.g., once, twice, three times, etc.

The invention provides a method of treating a patient in need of increased expression of a therapeutic protein encoded by a target gene, the method comprising administering to the patient a pharmaceutical composition comprising a ligand for an aptamer, wherein the patient has previously been administered a recombinant DNA comprising the target gene, wherein the target gene comprises a gene regulatory cassette of the invention, wherein a riboswitch (comprising the aptamer) modulates rnase P cleavage of a rnase P substrate in response to the aptamer ligand.

Article and kit

Kits and articles of manufacture for use in the methods described herein are also provided. In various aspects, a kit comprises a composition described herein (e.g., a composition for delivering a vector comprising a target gene comprising a gene regulatory cassette) in a suitable package. Packaging suitable for the compositions described herein, such as injectable ophthalmic compositions, are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, canisters, flexible packaging (e.g., sealed Mylar (Mylar) or plastic bags), and the like. These articles may be further sterilized and/or sealed.

The invention also provides kits comprising the compositions described herein, and may further comprise instructions for methods of using the compositions, such as the uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for administration (including, for example, any of the methods described herein). For example, in some embodiments, a kit comprises a rAAV for expression of a target gene comprising a gene regulatory cassette of the invention, a pharmaceutically acceptable carrier suitable for injection, and one or more of: buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing injections. In some embodiments, the kit is suitable for intraocular injection, intramuscular injection, intravenous injection, and the like.

"homology" and "homologous" as used herein refer to the percentage of identity between two polynucleotide sequences or between two polypeptide sequences. The correspondence between one sequence and another can be determined by techniques known in the art. For example, homology can be determined by aligning sequence information and directly comparing two polypeptide molecules using readily available computer programs. Two polynucleotide or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, at least about 85%, at least about 90%, and at least about 95% of the nucleotides or amino acids, respectively, match over a defined length of the molecule, as determined using the methods described above, after optimal alignment with appropriate insertions or deletions.

"percent sequence identity" with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignments for the purpose of determining percent amino acid or nucleic acid sequence identity can be accomplished in a manner known to those of ordinary skill, for example, using publicly available computer software programs, including BLAST, BLAST-2, ALIGN, and Megalign (DNASTAR) software.

The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

By "heterologous" or "exogenous" is meant an entity derived from a genotype different from the rest of the entity compared to or introduced or incorporated therein. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and when expressed may encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

It will be understood and appreciated that variations can be made by those skilled in the art to the inventive principles disclosed herein, and such modifications are intended to be included within the scope of the invention. The following examples further illustrate the invention but should not be construed as in any way limiting its scope. All references cited herein are hereby incorporated in their entirety.

Examples