(12) United States Patent
`Graham
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,573,099 B2
`*Jun. 3, 2003
`
`US006573099B2
`
`(54) GENETIC CONSTRUCTS FOR DELAYING
`OR REPRESSING THE EXPRESSION OF A
`TARGET GENE
`
`(*) Notice:
`
`(75) Inventor: Michael Wayne Graham, St. Lucia
`(AU)
`(73) Assignee: Benitec Australia, Ltd., Queensland
`(AU)
`This patent issued on a continued pros
`ecution application ?led under 37 CFR
`1.53(d), and is subject to the tWenty year
`patent term provisions of 35 U.S.C.
`154(a)(2).
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/100,812
`(22) Filed:
`Jun. 19, 1998
`(65)
`Prior Publication Data
`
`US 2002/0168707 A1 Nov. 14, 2002
`Foreign Application Priority Data
`
`(30)
`
`Mar. 20, 1998
`
`(AU) ............................................ .. PP2492
`
`(51) Int. Cl.7 ........................ .. C12N 5/00; C12N 15/00;
`C12N 15/63; A01N 63/00; A01N 43/04;
`A61K 48/00; A61K 31/70; C07H 21/04
`
`(52) us. Cl. .................. .. 435/455; 435/320.1; 435/325;
`424/9321; 424/932; 514/44; 536/245
`
`(58) Field of Search ........................... .. 435/69.1, 320.1,
`435/325, 455; 536/231, 23.5, 24.5; 514/44;
`800/8; 424/932, 93.21
`
`(56)
`
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`W0
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`W0
`W0
`W0
`W0
`
`0560156 A2
`0242016
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`(List continued on next page.)
`
`Primary Examiner—Scott D. Priebe
`Assistant Examiner—Sumesh Kaushal
`(74) Attorney, Agent, or Firm—Scully, Scott, Murphy &
`Presser
`(57)
`
`ABSTRACT
`
`The present invention relates generally to synthetic genes for
`modifying endogenous gene expression in a cell, tissue or
`organ of a transgenic organism, in particular a transgenic
`animal or plant. More particularly, the present invention
`provides novel synthetic genes and genetic constructs Which
`are capable of repressing delaying or otherWise reducing the
`expression of an endogenous gene or a target gene in an
`organism When introduced thereto.
`
`22 Claims, 28 Drawing Sheets
`
`Benitec - Exhibit 1005 - page 1
`
`

`
`US 6,573,099 B2
`Page 2
`
`OTHER PUBLICATIONS
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`
`Benitec - Exhibit 1005 - page 2
`
`

`
`US 6,573,099 B2
`Page 3
`
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`KunZ, C., et al. (1996), “Developmentally Regulated Silenc
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`Lindbo, J .A., et al., (1993), “Induction of a Highly Speci?c
`Antiviral State in transgenic Plants—Implications for Regu
`latio of Gene Expression and Virus Resistance”, The Plant
`Cell 5(12): 1749—1759.
`MatZke, M.A., et al. (1998), “Epigenetic Silencing of Plant
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`mon Mechanisms in Plants and Fungi”, Biological Chem
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`Napoli, C., et al. (1990), Introduction of a Chimeric Chal
`cone Synthase Gene into Petunia Results in Reversible
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`
`Palauqui, J .C., et al. (1997), Systemic Acquired Silencing:
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`and Results in Meiotically Heritable Alterations in Methy
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`
`Benitec - Exhibit 1005 - page 3
`
`

`
`US 6,573,099 B2
`Page 4
`
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`
`* cited by examiner
`
`Benitec - Exhibit 1005 - page 4
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`

`
`Benitec - Exhibit 1005 - page 5
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 2 0f 28
`
`US 6,573,099 B2
`
`NheI/Ec04'7 H/BgHI/Xhol/
`SacI/HindHI/SalI/KpnI/
`ApaI/Sac II/XmaI/SmaI/BamHI
`
`pUC Ori
`
`CMV IE
`
`pCMV.cass
`3855 bp
`
`SV4O poly(A)
`
`f1 Ori
`
`HSV TK poly (A)
`
`Kan/Neo
`
`SV4O EP
`
`FIGURE 2
`
`Benitec - Exhibit 1005 - page 6
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`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 3 0f 28
`
`US 6,573,099 B2
`
`GFP stuffer
`
`Bam HI
`
`lacZ alpha
`
`f1 ori
`
`Plac
`
`pCRBgl-GFP-Bam
`4767 bp
`
`Kanamycin
`
`FIGURE 3
`
`Benitec - Exhibit 1005 - page 7
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`

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`U.S. Patent
`
`Jun. 3, 2003
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`Sheet 4 0f 28
`
`US 6,573,099 B2
`
`Sal l
`
`CO|E1
`
`Synthetac poly( )s|te
`
`A ,
`
`.
`
`Ampicillin
`
`pCR.SV4OL
`5485 bp
`
`Kanamycin
`
`SV4OL
`
`Sai |
`
`IacZ alpha
`
`f1 ori
`
`FIGURE 4
`
`Benitec - Exhibit 1005 - page 8
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`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 5 0f 28
`
`US 6,573,099 B2
`
`pUC Ori
`
`Synthetic p0Iy(A) site
`
`HSV TK poly (A)
`
`Kan/Neo
`
`pCMV.SV4OL.cass
`5415 bp
`
`$V40 EP sv40 po|y(A)
`
`FIGURE 5
`
`Benitec - Exhibit 1005 - page 9
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 6 0f 28
`
`US 6,573,099 B2
`
`Plac
`
`Bg| H
`
`pCR.BEV.1
`5285 bp
`
`Ampicillin
`
`Kanamycin
`
`FIGURE 6
`
`Benitec - Exhibit 1005 - page 10
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 7 0f 28
`
`US 6,573,099 B2
`
`Plac
`
`Bgl ll
`
`pCR.BEV.2
`5285 bp
`
`Ampicillin
`
`Kanamycin
`
`FIGURE 7
`
`Benitec - Exhibit 1005 - page 11
`
`

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`U.S. Patent
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`Jun. 3, 2003
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`Sheet 8 0f 28
`
`US 6,573,099 B2
`
`Plac
`
`B9‘ H
`
`BEV.3
`
`pCR.BEV.3
`5285 bp
`
`AmpiciHin
`
`Kanamycin
`
`FIGURE 8
`
`Benitec - Exhibit 1005 - page 12
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`

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`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 9 0f 28
`
`US 6,573,099 B2
`
`HSV TK poly (A)
`
`pEGFP.BEV.1
`6113 bp
`
`BEV.1
`
`Kan/Neo
`
`SV40 EP
`
`SV40 poly(A)
`
`FIGURE 9
`
`Benitec - Exhibit 1005 - page 13
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 10 0f 28
`
`US 6,573,099 B2
`
`pUC Ori
`
`HSV TK poly (A)
`
`Kan/Neo
`
`pCMV.BEV.2
`5235 bp
`
`BEV.2
`
`SV40 EP
`
`SV4O po!y(A)
`
`I
`
`f1 Ori
`
`FIGURE 10
`
`Benitec - Exhibit 1005 - page 14
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 11 0f 28
`
`US 6,573,099 B2
`
`pUC Ori
`
`CMV IE
`
`HSV TK poly (A)
`
`pCMV.VEB
`5235 bp
`
`BEV2
`
`sv40 EP
`
`f1 0"
`
`sv40 poly(A)
`
`FIGURE 11
`
`Benitec - Exhibit 1005 - page 15
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 12 0f 28
`
`US 6,573,099 B2
`
`pUC on
`
`CMV IE
`
`HSV TK poly (A)
`
`Kan/Neo
`
`pCMVBEVnt
`5235 bp
`
`BEV.3
`
`SV4O EP
`
`f1 Ori
`
`FIGURE 12
`
`Benitec - Exhibit 1005 - page 16
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 13 0f 28
`
`US 6,573,099 B2
`
`HSV TK poly (A)
`
`Kan/Neo
`
`SV4O EP
`
`pUC Ori
`
`CMV IE
`
`BEV.2
`
`pCMV.BEVx2
`6615 bp
`
`f1 Ori
`
`BEV.2
`
`FIGURE 13
`
`Benitec - Exhibit 1005 - page 17
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 14 0f 28
`
`US 6,573,099 B2
`
`p
`
`HSV TK poly (A)
`
`Kan/Neo
`
`BEV.2
`
`pCMV.BEV.VEB
`6615 bp
`
`SV40 EP
`
`f1 Ori
`
`SV40 po|y(A)
`
`BEV.2
`
`FIGURE 14
`
`Benitec - Exhibit 1005 - page 18
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 15 0f 28
`
`US 6,573,099 B2
`
`pUC Ori
`
`HSV TK poly (A)
`
`BEv.2
`
`Kan/Neo
`
`pCMV.BEV.GFP.VEB
`7265 bp
`
`sv40 EP
`
`GFP-STUFF
`
`SV4O po|y(A)
`
`BEV 2
`
`FIGURE 15
`
`Benitec - Exhibit 1005 - page 19
`
`

`
`U.S. Patent
`
`Jun. 3, 2003
`
`Sheet 16 0f 28
`
`US 6,573,099 B2
`
`pUC Ori
`
`HSV TK poly (A)
`
`BEV.2
`
`Kan/Neo
`
`pCMV.BEV.SV4OL -O
`6795 bp
`
`Synthetic poly(A) site
`
`SV4O EP
`
`sv40 poly(A)
`
`FIGURE 16
`
`Benitec - Exhibit 1005 - page 20
`
`

`
`Benitec - Exhibit 1005 - page 21
`
`

`
`Benitec - Exhibit 1005 - page 22
`
`

`
`Benitec - Exhibit 1005 - page 23
`
`

`
`Benitec - Exhibit 1005 - page 24
`
`

`
`Benitec - Exhibit 1005 - page 25
`
`

`
`Benitec - Exhibit 1005 - page 26
`
`

`
`Benitec - Exhibit 1005 - page 27
`
`

`
`Benitec - Exhibit 1005 - page 28
`
`

`
`Benitec - Exhibit 1005 - page 29
`
`

`
`Benitec - Exhibit 1005 - page 30
`
`

`
`Benitec - Exhibit 1005 - page 31
`
`

`
`Benitec - Exhibit 1005 - page 32
`
`

`
`Benitec - Exhibit 1005 - page 33
`
`

`
`Benitec - Exhibit 1005 - page 34
`
`

`
`Benitec - Exhibit 1005 - page 35
`
`

`
`Benitec - Exhibit 1005 - page 36
`
`

`
`Benitec - Exhibit 1005 - page 37
`
`

`
`Benitec - Exhibit 1005 - page 38
`
`

`
`Benitec - Exhibit 1005 - page 39
`
`

`
`Benitec - Exhibit 1005 - page 40
`
`

`
`Benitec - Exhibit 1005 - page 41
`
`

`
`Benitec - Exhibit 1005 - page 42
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`

`
`Benitec - Exhibit 1005 - page 43
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`

`
`Benitec - Exhibit 1005 - page 44
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`

`
`Benitec - Exhibit 1005 - page 45
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`

`
`Benitec - Exhibit 1005 - page 46
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`

`
`Benitec - Exhibit 1005 - page 47
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`

`
`Benitec - Exhibit 1005 - page 48
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`

`
`Benitec - Exhibit 1005 - page 49
`
`

`
`Benitec - Exhibit 1005 - page 50
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`

`
`Benitec - Exhibit 1005 - page 51
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`

`
`Benitec - Exhibit 1005 - page 52
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`

`
`Benitec - Exhibit 1005 - page 53
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`

`
`Benitec - Exhibit 1005 - page 54
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`

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`Benitec - Exhibit 1005 - page 55
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`

`
`Benitec - Exhibit 1005 - page 56
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`

`
`Benitec - Exhibit 1005 - page 57
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`

`
`Benitec - Exhibit 1005 - page 58
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`

`
`Benitec - Exhibit 1005 - page 59
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`

`
`Benitec - Exhibit 1005 - page 60
`
`

`
`Benitec - Exhibit 1005 - page 61
`
`

`
`Benitec - Exhibit 1005 - page 62
`
`

`
`Benitec - Exhibit 1005 - page 63