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(12) Patent Application Publication (10) Pub. No.: US 2009/0069256A1 Smith Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2009/0069256A1 Smith Et Al US 20090069256A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0069256A1 Smith et al. (43) Pub. Date: Mar. 12, 2009 (54) ENHANCING PROTEIN EXPRESSION Related U.S. Application Data (60) Provisional application No. 60/576,819, filed on Jun. (76) Inventors: Larry R. Smith, San Diego, CA 4, 2004. (US); Vafa Shahabi, Valley Forge, PA (US); Maninder K. Sidhu, New Publication Classification City, NY (US) (51) Int. Cl. A63L/7088 (2006.01) Correspondence Address: CI2N IS/II (2006.01) HUNTON & WILLIAMS LLP CI2N 15/87 (2006.01) INTELLECTUAL PROPERTY DEPARTMENT A6IP 43/00 (2006.01) 1900 KSTREET, N.W., SUITE 1200 CI2N IS/00 (2006.01) WASHINGTON, DC 20006-1109 (US) (52) U.S. Cl. ...... 514/44; 536/23.1; 536/23.53: 536/23.5; 536/23.6:536/23.7:536/23.72:536/23.74; 435/455; 435/320.1 (21) Appl. No.: 11/628,455 (57) ABSTRACT (22) PCT Fled: Jun. 6, 2005 Modified polynucleotide compositions providing enhanced gene expression and methods for preparing said compositions (86) PCT NO.: PCT/US05/19592 are disclosed. Methods of using the compositions, such as in screening assays, diagnostic tools, kits, etc. and for preven S371 (c)(1), tion and/or treatment of diseases and disorders are also dis (2), (4) Date: Nov. 8, 2007 closed. SWall (3733) DraI(3734) Stul (52) BclI (3512) ASCI (3496) ApaI (3482) BSInI (3370) BglII (457) 2->BGHpolyA BspEI (3271) BspMI (540) BseRI (3241) human IL-15 (BH15) Psp1406I (667) BamHI (3071) “HuigE leader PflMI (3056) kanamycin Eael (768) NsiI (791) (3737 bp) XmnI (806) BpmI (2976)1 AgeI (846) MsiI (2745) BSrFI (846) SnaBI (2722) Eam1105I (920) BSaAI (2722) DraII (1155) Asel (2390) Spel (2383) EcoRV (2291) ClaI (2285) NruI (2254) BSrBI (1965) AfilII (1896) MunI (2209) Patent Application Publication Mar. 12, 2009 Sheet 1 of 14 US 2009/006925.6 A1 s d co Co CD CD CD Co. Co. dAs as 3 N Co Ltd cro N. P. (S3LKOON3ldSoO 10.Si.) AINOWOdSEAN 09 NU0 Patent Application Publication Mar. 12, 2009 Sheet 2 of 14 US 2009/006925.6 A1 PmeI (4156) EcoRI (4167) BCI (94) Stu (55) BglII (458) PpuMI (4123) BspMI (550) BstXI (3906) Psp1406I (669) BSmI (3902) - BGirolya Tsp45I (3760) BSal (3661) kanamycin BSgI (3484) PVuII (3418) HIV-1gagp37 WLV006 (4419 bp) Sal I (3049) BSSHII (3043) BpmI (2998) HCMV promoter BSmBI (2974) Ori Seq Dral (1161) NCOI (2745) SnaBI (2725) BsaNI (2725) Afi II (1897) Ase (2392) EcoRV (2294) Muni (2210) BsrBI (1968) Nrul (2257) FIG. 2 Patent Application Publication Mar. 12, 2009 Sheet 3 of 14 US 2009/006925.6 A1 AscI (4157) BSSHII (4157) SacI (4168) kanamycin SacI (3284)\ . WLWO Ola (4397 bp) SacI (3182) SacI (3098) NCoI (3059) BSSHII (3043) SalI (3049) (7braII (1161) SacI (2953) NCOI (2745) 1842 (1841) BspLU11I (1897) EcoRV (2294) FIG. 3 Patent Application Publication Mar. 12, 2009 Sheet 4 of 14 US 2009/006925.6 A1 ASCI (5630) Bsshi I (5630) junction marker (5628) EcoRI (5478) kanamycin env 6101, less preferred COdons WLV118.gCS 1 (5870 bp) HCMV promoter ECORI (3468) EcoRI (3483) Sal (3049) NCOI junction marker (3060) BSSHII (3043) F.G. 4 Patent Application Publication Mar. 12, 2009 Sheet 5 of 14 US 2009/006925.6 A1 ASCI (5107) BclI (5722) SphI (5935) junction marker (5946) e BGH Poly A NsiI (796) Kanamycin & DraIII (1161). Econ) (4543)A • HIV-1 envgp160 from 6101 primary isolate WLW119 R (5946 bp) Ori Sequence Bsu36f(4223)N BSrBI (1968) NruI (2257) HCMV P rOmoter ClaI (2287) DraIII (3510) EcoRV (2294) NsiI (3448) Spe (2461) KpnI (3261) Sal (3126) BSmBI (3051) NcoI (2822) FIG. 5 Patent Application Publication Mar. 12, 2009 Sheet 6 of 14 US 2009/006925.6 A1 RD CELS 25OOOOO 2. C: CURRENTCLINICAL CONSTRUCT 2000000 LP:MODIFIEDIL-15 2 O: OPTIMIZED ALTERNATIVE 15000 BH: OPTIMIZED ALTERNATIVE 2 S 1000000 500000 5000 as 52 5 5 5 g H H H H 5a , , , S2 a FIG.6A COS7 CELLS 1800C 1600C C: CURRENTCLINICAL CONSTRUCT a LP:MODIFIEDIL-15 14000 O: OPTIMIZED ALTERNATIVE 2 1200c BH: OPTIMIZED ALTERNATIVE2 S 1000 is 800C ge 6000 400 2000 OOO 1 Lad a 1 4 1 S2 S , , , , is a 5 F G. 6 B Patent Application Publication Mar. 12, 2009 Sheet 7 of 14 US 2009/006925.6 A1 Hela CELS 5000 CCURRENTCNICALCONSTRUCT so-HERE : 3500 OOPTIMIZEDATERNATIVEBHOPTIMIZED ALTERNATIVE2 20 is a H e H 0. Y to 1 ha o1. 1 S2 i. i. i. i. 2 3, 2, 3, 2 E 5 = c F.G. 6C Patent Application Publication Mar. 12, 2009 Sheet 8 of 14 US 2009/006925.6 A1 1800.00 1600.00 RD - 1400.00 1200.00 100000 800.00 60000 40000 LP-15-IgE 200.00 BH-15-gE 0.00 FIG. 7A 9000.00 800000 - 700000 2 600000 500000 400000 is 300000 2000.00 100000 0.00 Patent Application Publication Mar. 12, 2009 Sheet 9 of 14 US 2009/006925.6 A1 9 OZ?E EIN00.10N.9U Patent Application Publication Mar. 12, 2009 Sheet 10 of 14 US 2009/006925.6 A1 120.00 100.00 80.00 6000 4000 2000 OOO VECTOR CURRENTL15 LS-gELEADER 0-15-gELEADER LP15-gELEADER BHS-LEADER FIG, 9 Patent Application Publication Mar. 12, 2009 Sheet 11 of 14 US 2009/0069256A1 DAY2 DAY5 40.00 35.00 O DAY9 3000 DAY.15 2500 o - 2000 3 1500 1000 i 5.00 0.00 CURRENTIL15 IL15-gELEADER 0-15-g LEADER LP15-IgELEADE FIG 10 Patent Application Publication Mar. 12, 2009 Sheet 12 of 14 US 2009/0069256A1 ASCI (3496) XhoI (3489) Ava (3489) ApaI(3482) BsgI (3430) BSaBI (3435) Swal (3733) DraI (3734). StuI (52) MscI (3317) BstEII (3219) PvuII (3210) ne2. BcgI-1 (3164) BglII (457) BcgI-2 (3164) PpuMI (3149) $7,4 2. IL-15 (P 15) Psp1406I (667) EcoO109I (3149) HugE leader kanamycin NsiI (791) BamHI (3071) -AgeI (846) PflMI (3056) BSrFI (846) (3737 bp) BSmBI (2979) Eam1105I (920) HCMV IE promoter/untranslated region SnaBI (2722) BSaAI (2722) Asel (2390) SpeI (2383) EcoRV (2291) ClaI (2285) AflIII (1896) NruI (2254) MunI (2209) FIG 11 Patent Application Publication Mar. 12, 2009 Sheet 13 of 14 US 2009/006925.6 A1 Sphl (3721) Swal (3733) DraI (3734) StuI (52) ASCI (3496) ApaI(3482) 2 4. BGHpolyA BspMI (540) PshAI (3096) human IL-15 (LP15) Psp1406I (667) BamHI (3071) Eael (768) PflMI (3056) % Hu IgEleader kanalycin XmnI (806) BSIBI (2979) Age I (846) SnaBI (2722) (3737 bp) BsrFI (846) BSaAI (2722) Eam1105I (920) AseI (2390) DraIII (1155) Spel. (2383) EcoRV (2291) ClaI (2285) Munl (2209) BsrBI (1965) FIG. 12 Patent Application Publication Mar. 12, 2009 Sheet 14 of 14 US 2009/006925.6 A1 Swal (3733) DraI(3734) StuI (52) BclI (3512) ASCI (3496) Apal (3482) BSmI (3370) BglII (457) BspEI (3271) BSOMI BseRI (3241) spMI (540) 4 human IL-15 (BH15) Psp1406I (667) BamHI (3071) HuigE leader PflMI (3056-1 kanamycin Earl's BpmI (2976)1 (3737 bp) E: 888 MsiI (2745) A BsrFI (846) E:Sa V Eam1105I (920) Dral.II (1155) Asel (2390) Spel. (2383) EcoRV (2291) Seas? Clai (2285) NruI (2254) BSrBI (1965) AfIII (1896) MunI (2209) FIG. 13 US 2009/006925.6 A1 Mar. 12, 2009 ENHANCING PROTEIN EXPRESSION B1) which limit the expression levels of gag by inhibiting nuclear export of these transcripts. FIELD OF THE INVENTION 0005 IL-15 exemplifies the problem inherent in poor gene expression. IL-15 is a pluripotent cytokine that is secreted by 0001. The present invention relates to polynucleotide antigen presenting cells Such as monocytes/macrophages and compositions that provide enhanced efficiency in the expres dendritic cells, but also a variety of nonlymphoid tissues. sion of proteins or polypeptides by genes in mammalian cells IL-15, in addition to being a growth and survival factor for (i.e., resulting in an increase in the levels of the proteins or memory CD8+ T cells, is also a potent activator of effector polypeptides encoded by the genes). Such as viral, bacterial memory CD8+ T cells, both in healthy and HIV-infected and mammalian genes, as well as methods for preparing said individuals. Because IL-15 is a prototypic Th1 cytokine, and compositions. In particular, the invention provides poly by virtue of its activity as a stimulator of T cells, NK cells, nucleotide sequences that provide enhanced gene expression LAK (lymphokine-activated killer) and TILs (tumor infiltrat over the corresponding wild-type polynucleotides. Also pro ing lymphocytes), IL-15 is a potential candidate for use as a vided are methods of using the polynucleotide compositions molecular adjuvantalong with HIV DNA vaccines to enhance in prevention and treatment of diseases and disorders (e.g., cellular immune responses. However, one major limiting fac immuno-therapeutic, immuno-prophylactic and genetic tor for its use as a genetic adjuvant, remains its poor expres therapy uses and the like), such as in DNA and RNA vaccines sion due to its complex regulation at the levels of mRNA (e.g., DNA vaccines for preventing/treating HIV/AIDS) as transcription and translation and, protein translocation and well as in biological assays, diagnostics and the like. secretion. 0006 Further, DNA vaccines, which are being studied for BACKGROUND OF THE INVENTION many diseases, including HIV, influenza, tuberculosis and malaria, usually work by injecting specially reproduced 0002 The level of protein expressed by a gene is crucial to genetic material of the organism directly into the body. This in vivo responses/effects involving the protein, as well as in genetic material encodes information that gets the individu vitro assays involving the protein. Under Some circumstances al’s own cells to make the vaccine.
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