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View U.S. Patent Application Publication No. US-2019-0231899 1111111111111111 IIIIII IIIII 111111111111111 1111111111 1111111111 11111 11111 1111111111 11111111 US 20190231899Al c19) United States c12) Patent Application Publication c10) Pub. No.: US 2019/0231899 Al Millette et al. (43) Pub. Date: Aug. 1, 2019 (54) VECTOR FOR GENE SILENCING AND (52) U.S. Cl. REPLACEMENT AND METHODS OF USE CPC ...... A61K 4810066 (2013.01); A61K 4810058 THEREOF (2013.01); A61P 25128 (2018.01); A61K 4810008 (2013.01); A61K 4810091 (2013.01) (71) Applicant: WISCONSIN ALUMNI RESEARCH FOUNDATION, Madison, WI (US) (57) ABSTRACT (72) Inventors: Matthew Michael Millette, Middleton, An expression cassette for gene silencing and replacement, WI (US); Erik Wolfe Dent, Madison, including, in operable communication, a promoter, an WI (US) expression attenuator, a nucleotide sequence encoding a (21) Appl. No.: 16/257,615 gene for a replacement target protein, and an shRNA sequence for knockdown of an endogenous variant of the (22) Filed: Jan. 25, 2019 target protein, wherein the promoter, the expression attenu­ ator, the nucleotide sequence encoding the gene for the Related U.S. Application Data replacement target protein, and the shRNA are expressed as (60) Provisional application No. 62/622,479, filed on Jan. a single transcript. Also included are expression vectors and 26, 2018. cells. Also included are methods of silencing and replace­ ment of a target gene in a cell in culture by transforming the Publication Classification cells with the expression vectors described herein. Also (51) Int. Cl. included are minimal expression cassettes suitable for thera­ A61K 48100 (2006.01) peutic methods. A61P 25128 (2006.01) Specification includes a Sequence Listing. ··························-\-•wwwwwwww--l- i •.... ; ii . .lit••••tiH ;; ~!~lg Patent Application Publication Aug. 1, 2019 Sheet 1 of 7 US 2019/0231899 Al Fig. 1 Target Fig. 2 ; .. Cl i i ti &. ;; ~ ct: fl::'. J:z .c ii> <I> !:I} ::: ~ 'i:: '-I 'iii U') ::: «i ! .2 (:; a.. <II 'l: 1./., ..a <ils:2..e I!! .!:: ~ q li w :. ~ e < Cl :,., :,., ;::!IS ;::!IS Cl Cle 0 0 Patent Application Publication Aug. 1, 2019 Sheet 2 of 7 US 2019/0231899 Al Fig. 3 ~ "~•/ Target Silencing Fig. 4 Patent Application Publication Aug. 1, 2019 Sheet 3 of 7 US 2019/0231899 Al .:.:.:· ..:.,::.,.:·,,,:.:.:.:. IRES Fig. 5 ·:::s ro (\; 0 ;::; ;::; u 0 0 0 u N u (~ 0 0 ,< ::::> c, rl rl 0 0 rl ...,t 0 () Z.~) z:--,,,., 0 ( \ (:1 (~' \ (,., ._, '-' (....... !"\ (....... ,, 0 (.'.} l> (Y) - u (.') < Initial /:J.G :::::. -37.20 Initial /:J.G :::::. -29.40 Initial LlG ;:;:;. -20,50 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 Fig. 6 Patent Application Publication Aug. 1, 2019 Sheet 4 of 7 US 2019/0231899 Al Rescue Gene Attenuation 150 by Graded-Stability Hairpins G)u C <l.l (.) fl) 100 2! 0 :, -I.L CL I.L ~ ........ 50 CL I.L (!) 0 Thermal Stability (-kcal/mol) Fig. 7 Patent Application Publication Aug. 1, 2019 Sheet 5 of 7 US 2019/0231899 Al 14 EB3 116 177 194 264 281 EB family homology calponin homology APC (SxlP) binding intermediate CAP-Gly domain Fig. 8 Patent Application Publication Aug. 1, 2019 Sheet 6 of 7 US 2019/0231899 Al Percent EB3 Expression Relative to Endogenous 300 200 100 Fig. 9 Patent Application Publication Aug. 1, 2019 Sheet 7 of 7 US 2019/0231899 Al EB3 Replacement with EB3Ai reduces the Percentage of Spines Invaded 20°/o 30°/o * Fig. 10 US 2019/0231899 Al Aug. 1, 2019 1 VECTOR FOR GENE SILENCING AND [0012] In yet another aspect, a method of silencing and REPLACEMENT AND METHODS OF USE replacement of a target gene in a cell in culture comprises THEREOF transforming the cell with an expression vector as described herein, and optionally applying to the cell an inducer when CROSS-REFERENCE TO RELATED the promoter is an inducible promoter to provide expression APPLICATIONS of the single transcript, wherein expression of the shRNA silences the expression of the endogenous variant of the [0001] This application claims priority to U.S. Provisional target protein, and expression of the coding sequence for the Application 62/622,479 filed on Jan. 26, 2018, which is replacement target protein provides the replacement target incorporated herein by reference in its entirety. protein in the cell. [0013] In another aspect, also included are expression STATEMENT REGARDING FEDERALLY vectors comprising the foregoing expression cassettes and SPONSORED RESEARCH & DEVELOPMENT cells comprising the expression vectors. [0002] This invention was made with govermnent support [0014] In yet another aspect, a therapeutic method com­ under NS098372 and NS080928 awarded by the National prises administering the foregoing expression vector, or Institutes of Health. The government has certain rights in the transplanting the foregoing cell into a subject, such as a invention. mammalian subject. FIELD OF THE DISCLOSURE BRIEF DESCRIPTION OF THE DRAWINGS [0003] The present disclosure is related to expression [0015] FIG. 1 shows an embodiment of an expression systems for gene silencing and replacement, and methods of cassette. An expression cassette includes a tet-ON driven, using the expression systems, particularly in developmen­ single transcript design with an RFP-membrane reporter and tally regulated proteins. rescue gene split by a P2A cleavage site to yield two separate proteins. RNAi is accomplished via the 3'UTR mir30 cas­ BACKGROUND sette, cleaved into shRNA targeting non-coding regions of endogenous gene of interest. [0004] Interrogating the roles of individual proteins in [0016] FIG. 2 shows the results of knockdown-rescue with complex biological processes requires the expression of Drebrin-GFP induced in DIV21 rat hippocampal neurons functional mutants in both gain and loss of function experi­ yields morphologically normal dendritic spines. Hippocam­ ments. However, background contribution of endogenous pal cultures were transfected with plasmid encoding either protein represents a primary confounding factor when inter­ drebrin-targeting shRNA and no rescue gene, or drebrin­ preting the consequences of these alterations. This is par­ GFP rescue gene and drebrin-targeting shRNA. Cultures ticularly the case when proteins of interest are obligate were allowed to mature for 14 days prior to tet-ON promoter dimers or multimers. One way researchers have attempted to induction with doxycycline. Cultures were fixed at 21 days, overcome this dilemma is through direct gene editing via and shRNA-only dishes were immunostained with anti­ CRISPR-Cas9. While promising, genome editing is pres­ drebrin antibody. (FIG. 2, Left) Immunostaining of neuronal ently inefficient and commonly requires the generation of cultures with drebrin antibody confirms effectiveness of stable cell lines, an expensive and time consuming endeavor RNAi. shRNA reporter-expressing cells are entirely that requires significant investment in sequencing to ensure depleted of drebrin, while adjacent non-transfected cell is permanent off-target changes have not been introduced to revealed by robust anti-drebrin fluorescence. (FIG. 2, Right) the host genome. When applied to primary, post-mitotic Knockdown-Rescue with drebrin-GFP yields morphologi­ cells, like neurons in culture, this approach becomes entirely cally normal dendritic spines. Drebrin localizes expectedly impractical. Therefore, what is needed is a high-throughput, and with specificity to dendritic spines. cost effective alternative which circumvents these caveats in [0017] FIG. 3 shows an embodiment of an expression first-line research applications. cassette. This design reorients the reporter downstream of an IRES element to provide a separate initiation site for trans­ BRIEF SUMMARY lation. This maintains reporter brightness while allowing physiologically tunable attenuation of our rescue gene by [0005] In one aspect, an expression cassette for gene interchanging 5'UTR hairpins. silencing and replacement of a target protein comprises, in [0018] FIG. 4 is an embodiment of a "minimal" expres­ operable communication, sion cassette lacking the IRES and reporter for applications [0006] a promoter, where viral packaging capacity is limited, and is designed to [0007] an expression attenuator, be easily transferable between vectors. [0008] a nucleotide sequence encoding a gene for the [0019] FIG. 5 is a diagram of the vector used to determine replacement target protein, and potency of hairpin structures under physiologically relevant, [0009] an shRNA sequence for knockdown of an endog­ live-cell conditions. enous variant of the target protein, [0020] FIG. 6 shows examples of graded-stability hairpins [0010] wherein the promoter, the expression attenuator, used to diminish protein translation of "rescue" genes. the nucleotide sequence encoding the gene for the replace­ [0021] FIG. 7 shows quantification of increasingly stable ment target protein, and the shRNA are expressed as a single hairpins compared to unattenuated (no hairpin) control. transcript. [0022] FIG. 8 shows a diagram ofEB3 protein amino acid [0011] In another aspect, also included are expression sequence with functional subdivisions outlined. vectors comprising the foregoing expression cassettes and [0023] FIG. 9 shows quantification of Western blot analy­ cells comprising the expression vectors. sis of EB3 protein content after transfection with an unat- US 2019/0231899 Al Aug. 1, 2019 2 tenuated vector containing EB3-RFP, an attenuated vector tolerated for up to three weeks prior to imaging as neurons containing EB3-RFP and two different EB3 mRNAs. mature. Loss or gain of function experiments are critical for [0024] FIG.
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