MODIFIED U6 PROMOTER SYSTEM FOR TISSUE SPECIFIC EXPRESSION

Abstract

The present invention relates to a tissue-specific promoter system for expressing microRNA (miRNA) for RNA interference-based methods of gene therapy. In these systems, the miRNA will inhibit gene expression or replace natural miRNA expression using microRNA.

Claims

1-28. (canceled)

29. A method of producing a modified U6 promoter, the method comprising transfecting a host cell with a nucleic acid comprising: a modified U6 promoter sequence comprising the nucleotide sequence set forth in SEQ ID NO: 4, a mature guide strand of a miRNA comprising at least one detargeting sequence, wherein the detargeting sequence is a binding site for miRNA-122 and/or miRNA-208, and 5 or 6 thymidines at the 5′ end.

30. The method of claim 29, wherein the binding site for miRNA-122 comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 66 and/or the binding site for miRNA-208 comprises the nucleotide sequence set forth in SEQ ID NO: 6 or 67.

31. The method of claim 29, wherein the mature guide strand of the miRNA is miDUX4, miRN92, miRNA-17, miRNA-18a, miRNA-19a, miRNA-20a, miRNA-19b-1, mi-RNA-26a, miRNA-126, miRNA-335, let-7a, let-7b, miRNA-34, miR-34a, miRNA-10b, miRNA-208, miRNA-499, miRNA-195, miRNA-29a, miRNA-29b, or miRNA-29c.

32. The method of claim 29, wherein the mature guide strand of the miRNA comprises the nucleotide sequence set forth in SEQ ID NO: 8482, SEQ ID NO: 8372, SEQ ID NO: 8371, SEQ ID NO: 8370, SEQ ID NO: 8367, SEQ ID NO: 8366, SEQ ID NO: 8365, SEQ ID NO: 8219, SEQ ID NO: 8218, SEQ ID NO: 8152, SEQ ID NO: 8147, SEQ ID NO: 8145, SEQ ID NO: 7397, SEQ ID NO: 7396, SEQ ID NO: 7395, SEQ ID NO: 7108, SEQ ID NO: 7107, SEQ ID NO: 7106, SEQ ID NO: 6633, SEQ ID NO: 6631, SEQ ID NO: 6622, SEQ ID NO: 6619, SEQ ID NO: 6609, SEQ ID NO: 6608, SEQ ID NO: 6568, SEQ ID NO: 6561, SEQ ID NO: 6560, SEQ ID NO: 10971 or SEQ ID NO: 10972.

33. The method of claim 29, wherein the mature guide strand of a miRNA is miDUX4.

34. The method of claim 29, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 1, 2, or 10913-10968.

35. The method of claim 29, wherein the nucleic acid is present in a vector.

36. The method of claim 35, wherein the vector is a plasmid, adeno-associated virus, adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, pox virus, herpes virus, polio virus, sindbis virus, or vaccinia virus.

37. The method of claim 36, wherein the vector is a recombinant adeno-associated viral (AAV) vector.

38. The method of claim 37, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh.74 or AAV-B1.

39. The method of claim 37, wherein the AAV is AAV6, AAVrh.74 or AAV-B1.

40. A method of producing a modified U6 promoter, the method comprising transfecting a host cell with a nucleic acid comprising: the nucleotide sequence set forth in SEQ ID NO: 4, a mature guide strand of a miRNA comprising at least one detargeting sequence, wherein the miRNA is DUX4 and wherein the detargeting sequence is a binding site for miRNA-122 and/or miRNA-208, and 5 or 6 thymidines at the 5′ end.

41. The method of claim 40, wherein the binding site for miRNA-122 comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 66 and/or the binding site for miRNA-208 comprises the nucleotide sequence set forth in SEQ ID NO: 6 or 67.

42. The method of claim 40, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 1 or 2.

43. The method of claim 40, wherein the nucleic acid is present in a vector.

44. The method of claim 40, wherein the vector is a plasmid, adeno-associated virus, adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, pox virus, herpes virus, polio virus, sindbis virus, or vaccinia virus.

45. The method of claim 44, wherein the vector is a recombinant adeno-associated viral (AAV) vector.

46. The method of claim 45, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh.74 or AAV-B1.

47. The method of claim 45, wherein the AAV is AAV6, AAV rh.74 or AAV-B1.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0078] FIG. 1 shows the wild type U6-1 promoter (SEQ ID NO: 3) and the weakened U6-1 promoter (SEQ ID NO: 4) having mutations within the PSE region. The PSE region is underlined in FIG. 1.

[0079] FIGS. 2A and 2B set out sequences of DUX4 targeted miRNAs. In each panel, the top sequences indicate the DNA templates from which each respective miRNA is transcribed. In the top panel, the DNA template miDUX4.405 (miDUX4-1 or mi405) is SEQ ID NO: 1. In the bottom panel, the DNA template miDUX4.1155 (miDUX4-2; or mi1155) is SEQ ID NO: 2. The folded miRNA transcripts are shown as hairpin structures. The miDUX4.405 folded miRNA is SEQ ID NO: 8. The miDUX4.1155 folded miRNA is SEQ ID NO: 9. The mature miDUX4.405 and miDUX4.1155 sequences arise following processing in target cells by host miRNA processing machinery (including Drosha, DGCR8, Dicer, and Exportin-5). Sequences shaded in gray indicate restriction sites used for cloning each miRNA into the U6T6 vector. CTCGAG is an XhoI site and ACTAGT is a SpeI site (CUCGAG and ACUAGU in RNA, where the U is a uracil base). The red sequence indicates the mature miRNA antisense guide strand that ultimately helps catalyze cleavage of the DUX4 target mRNA. This sequence is also underlined in the miRNA hairpin portions of this diagram. The gray and black arrowheads indicate Drosha- and Dicer-catalyzed cleavage sites, respectively. The numbers 13, 35, 53, and 75 are provided for orientation. The sequences between (and including) positions 35-53 are derived from the natural human mir-30a sequence, except the A at position 39, which is a G is the normal mir-30a sequence. This nucleotide was changed to an A to facilitate folding of the miRNA loop, based on in silico RNA folding models. The base of the stem (5′ of position 13 and 3′ of position 75) is also derived from mir-30a structure and sequence with some modifications depending on the primary sequence of the guide strand. Specifically, the nucleotide at position 13 can vary to help facilitate a required mismatched between the position 13 and 75 nucleotides. This bulged structure is hypothesized to facilitate proper Drosha cleavage.

[0080] FIG. 3 shows an exemplary modified U6 promoter system. Panel A shows several tMCK-based systems to express miDUX4 and tested their function in human myoblasts over-expressing V5-tagged DUX4. Representative western shows our best tMCK.miDUX4 (Var1), silenced DUX4 protein at levels comparable to U6.miDUX4. Panel B shows the weakened U6 promoter miDUX4 development. The WT U6 promoter drives high levels of shRNA/miRNA expression, while a weakened version (wU6) produces ˜16-fold less transcript without significantly impacting target gene silencing. Similar results were observed with miDUX4 in a luciferase assay in which Renilla luciferase contained DUX4 sequences and could be silenced by miDUX4.

[0081] FIG. 4 shows the strategy for de-targeting miDUX4 in heart and liver. The perfect binding sites for mir-122 (liver) and mir-208 (heart) are indicated in the figure. Evidence that mir-122-modified miDUX4 is functional against a DUX4-luciferase target, and that liver cells expressing mir-122 can inhibit miDUX4 silencing when mir-122 binding sites are included in the miDUX4 sequence.

[0082] FIG. 5 shows the human DUX4 DNA sequence (SEQ ID NO: 7).

SEQUENCES

[0083] SEQ ID NO: 1 (miDUX4.405 or miDUX4-1)

[0084] SEQ ID NO: 2 (miDUX4.1155 or miDUX4-2)

[0085] SEQ ID NOS: 10-10912, 10971, 10972: Exemplary miRNA mature guide strand nucleotide sequences

[0086] SEQ ID NO: 3 wild type U6-1 promoter

[0087] SEQ ID NO: 4 weakened U6-1 promoter with mutations within the PSE region.

[0088] SEQ ID NO: 5 Binding site for miR-122 (5′ TATTTAGTGTGAT AATGGTGTTT 3′)

[0089] SEQ ID NO: 6—Binding site for miRNA-208 (5′ ACGAGCcTTTT GCTCGTCTTAT 3′)

[0090] SEQ ID NO: 8—miDUX4.405 (miDUX4-1) folded miRNA

[0091] SEQ ID NO: 9—miDUX4.1155 (miDUX4-2) folded miRNA

[0092] SEQ ID NO: 7—DUX4 gene sequence

[0093] SEQ ID NOS: 10913-10968 Exemplary nucleic acid sequences comprising the mature guide strand of miDUX4 and a binding site for miR-122 or miR-208 (also shown in Table 1)

[0094] SEQ ID NO: 10969—Binding site for miR-122 (5′ UAUUUAGU GUGAUAAUGGUGUUU 3′)

[0095] SEQ ID NO: 10970—Binding site for miR-208 (5′ ACGAGCcUUUU GCUCGUCUUAU 3′)

[0096] SEQ ID NO: 10973—miDUX4.405 (miDUX4-1) mature guide strand nucleotide sequence

[0097] SEQ ID NO: 10974—miDUX4.1155 (miDUX4-2) mature guide strand nucleotide sequence

[0098] When mature guide stand sequences are presented as DNA sequences herein, one of skill in the art understands that this DNA sequence serves as a template for transcription to RNA wherein the thymidine bases are converted to uridine bases. Examples

[0099] Thus, aspects and embodiments of the invention are illustrated by the following examples. Example 1 describes the liver and heart detargeted, weakened promoter system. Example 2 describes the luciferase assay for determining the effect of the miRNAs expression of DUX4 miRNAs. Example 3 describes rAAV vectors encoding DUX4 miRNAs.

Example 1

Liver and Heart De-Targeted, Weakened U6 Promoter System

[0100] Muscles are susceptible to damage by large overdose of miRNA vectors. Thus, a modified U6 promoter system was developed for skeletal muscle specific miRNA expression. The wild type U6 promoter was mutated in that the proximal sequence element as shown in FIG. 1. This mutation weakens U6 transcription and yields 16-fold less shRNA transcription in a AAV8 while maintaining the potency of HCV destruction for treatment of hepatitis as described in Suhy et al., Mol. Therapy 20: 1737-1749, 2012. In the present experiment, a nucleic acid molecule comprising this weakened U6 (wU6) system which drives miDUX4 and achieved significant DUX4 silencing in vitro using a luciferase assay in which Renilla luciferase contained DUX4 sequences. (FIG. 3B).

[0101] However, the proposed weakened U6 promoter system is ubiquitously active and to achieve the highest level of safety, this promoter system is further modified to limit expression to skeletal muscle as much as possible. One option for skeletal muscle specific expression is to use the AAV6 vector, as it primarily transduces skeletal muscle, liver, and heart following vascular delivery, and significantly less in other tissues. To avoid expression in liver and heart, the modified U6 promoter system detargets miDUX4 in those tissues. To do this, perfect binding sites for mir-122 and mir-208 (liver- and heart-specific natural microRNAs) are incorporated at various locations within the miDUX4 transcript as shown in FIG. 4. The de-targeted miDUX4 transcripts were destroyed by miR-122 and miR-208 RISC complexes in the liver and heart, respectively, using the DUX4-luciferase target described below in Example 2.

Example 2

Luciferase Assay for Effect of Expression of DUX4 miRNAs

[0102] Expression of the DUX4 target sequence in the presence of the DUX4 miRNAs was assayed. A lipofectamine 2000 transfection was done in 293 cells in a 96-well, white-walled assay plate. 140,000 cells were transfected with 20 ng of a Renilla-firefly plasmid containing the DUX4 target sequence and 180 ng of various DUX4 miRNA-encoding vectors, including U6T6-driven miDux4.405 or miDux4.1155 vectors from Example 1. A luciferase assay was performed 24 hours later.

[0103] The media was removed from the cells and 20 μl of lysis buffer was added per well. The plate was put on a shaker for 15 minutes at room temperature before adding 50 μl of luciferase substrate. The first reading was taken 10 minutes later. Next, 50 μl of Stop and Glo luciferase substrate was added and the second reading was taken 10 minutes later. The Renilla expression was divided by the firefly expression to calculate the relative expression. The relative expression was then normalized to the expression of cells that were transfected with a control miRNA that targets eGFP. The DUX4 miRNAs miDUX4.405 and miDUX4.1155 were the most effective at reducing luciferase protein expression in transfected cells. The de-targeted miDUX4 transcripts are destroyed by mir-122 and mir-208 RISC complexes in the liver and heart, respectively, using the DUX4-luciferase target described below in Example 1.

Example 3

Production of rAAV Encoding DUX4 MicroRNAs

[0104] Vector is produced by co-transfection in HEK293 cells of three plasmids (pAdhelper, AAV helper, and the rAAV genome containing miDUX4; described in detail below), followed by cell-harvesting, vector purification, titration, and quality control assays.

[0105] Plasmids: pAdhelper contains the adenovirus genes E2A, E4 ORF6, and VA I/11; AAV helper plasmids contain AAV rep2 and cap6 (for example, for an AAV serotype 6 preparation, the capsid gene would be called cap6); the rAAV plasmid contains AAV inverted terminal repeat (ITRs) sequences flanking the genetic elements to be packaged into the vector. For the AAV.miDUX4, this includes the U6.miDUX4 cloned upstream of the CMV.eGFP reporter gene.

[0106] Transfection: Plasmids are transfected into 293 cells (Corning 10-Stack) using CaPO.sub.4 at a 4:4:1 ratio (20 μg pAd helper: 20 μg AAV helper: 5 ug rAAV vector plasmid per plate.

[0107] Cell harvesting: Forty-eight hr post-transfection, cells are harvested and resuspended in 20 mM Tris (pH 8.0), 1 mM MgCl.sub.2 and 150 mM NaCl (T20M1N150) at a density of 5×10.sup.6 cells/ml. Cells are lysed by four sequential freeze/thaw cycles and Benzonase nuclease (AIC, Stock: 250 U/ul) added to a final concentration of 90 U/ml before cell lysate clarification.

[0108] Vector Purification and Titration: Clarified lysates are subjected to iodixanol step gradient purification as previously described (Xiao, X, et al. J. Virol 72:2224-32). The 40% iodixanol layer (containing rAAV) is diluted 5-fold with a no-salt dilution buffer (pH varying depending on serotype) and applied to a Hi-Trap HP-Q/S column. Upon elution with a NaCl salt gradient, peak 1 ml fractions (typically 3-5) are pooled, dialyzed with T20M1N200 (pH 8.0), then sterile filtered and supplemented with 0.001% Pluronic F68. Vectors are stored at −80° C. Purified virus was titered for vg using Q-PCR as previously described (Schnepp and Clark, Methods Mol. Med., 69:427-443 (2002)).