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NZ761178B2 - Modified closed-ended dna (cedna) - Google Patents

Modified closed-ended dna (cedna) Download PDF

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Publication number
NZ761178B2
NZ761178B2 NZ761178A NZ76117818A NZ761178B2 NZ 761178 B2 NZ761178 B2 NZ 761178B2 NZ 761178 A NZ761178 A NZ 761178A NZ 76117818 A NZ76117818 A NZ 76117818A NZ 761178 B2 NZ761178 B2 NZ 761178B2
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NZ
New Zealand
Prior art keywords
rule
cedna
cedna vector
substitute sheet
vector
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NZ761178A
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NZ761178A (en
Inventor
Ozan Alkan
Annaliese Jones
Douglas Anthony Kerr
Robert Michael Kotin
Ara Karl Malakian
Matthew John Simmons
Teresa L Wright
Original Assignee
Generation Bio Co
Filing date
Publication date
Application filed by Generation Bio Co filed Critical Generation Bio Co
Priority claimed from PCT/US2018/049996 external-priority patent/WO2019051255A1/en
Publication of NZ761178A publication Critical patent/NZ761178A/en
Publication of NZ761178B2 publication Critical patent/NZ761178B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • C—CHEMISTRY; METALLURGY
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/60—Vectors comprising a special translation-regulating system from viruses

Abstract

ceDNA vectors having linear and continuous structure can be produced in high yields and used for effective transfer and expression of a transgene. ceDNA vectors comprise an expression cassette and two different ITR sequences derived from AAV genomes in a specified order. Some ceDNA vectors provided herein further comprise cis-regulatory elements and provide high gene expression efficiencies. Further provided herein are methods and cell lines for reliable and efficient production of the linear, continuous and capsid-free DNA vectors. In a specific embodiment the invention is non-viral capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one nucleotide sequence positioned between asymmetric inverted terminal repeats (asymmetric ITRs), wherein at least one of the asymmetric ITRs comprises a deletion of at least a D and D' ITR region, and in at least one of an A, A', B, B', C and/or C' ITR region, and wherein at least one of the asymmetric ITRs comprises a functional terminal resolution site and a Rep binding site, and wherein the ITRs are derived from an adeno associated virus (AAV).

Claims (36)

1. A non-viral capsid-free DNA vector with covalently-closed ends (ceDNA vector), wherein the ceDNA vector comprises at least one nucleotide sequence positioned between asymmetric inverted terminal repeats (asymmetric ITRs), wherein at least one of the asymmetric ITRs comprises a on of at least a D and D’ ITR region, and in at least one of an A, A’, B, B’, C and/or C’ ITR region, and wherein at least one of the asymmetric ITRs comprises a functional terminal resolution site and a Rep binding site, and wherein the ITRs are derived from an ssociated virus (AAV).
2. The ceDNA vector of claim 1, wherein the D and D’ ITR regions contain transcription factor binding sites.
3. The ceDNA vector of claim 1, wherein the at least one asymmetric ITR comprising a deletion of at least a D and D’ ITR region further comprises a deletion of the A and/or A’ ITR regions.
4. The ceDNA vector of claim 1, n the at least one tric ITR comprising a deletion of at least a D and D’ ITR region further comprises a deletion of all of the A, A’, B, B’, C, and C’ ITR regions.
5. The ceDNA vector of claim 1 or 2, wherein the ceDNA vector displays characteristic bands of linear and continuous DNA as compared to linear and non-continuous DNA controls when digested with a restriction enzyme having a single recognition site on the ceDNA vector and analyzed by both native and denaturing gel electrophoresis.
6. The ceDNA vector of claim 1 or 2, wherein the AAV is AAV2.
7. The ceDNA vector of claim 6, wherein the asymmetric ITRs are derived from ent viral serotypes.
8. The ceDNA vector of claim 7, wherein the one or more asymmetric ITRs are d from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.
9. The ceDNA vector of any one of claims 1-6, n at least one of the asymmetric ITRs is synthetic.
10. The ceDNA vector of any one of claims 1-6 and 9, wherein at least one of the ITRs is not a wild-type ITR.
11. The ceDNA vector of any one of claims 1-10, wherein at least one of the asymmetric ITRs is modified by a deletion in at least two of the ITR regions selected from the group consisting of A, A’, B, B’, C, and C’.
12. The ceDNA vector of claim 11, wherein the deletion results in the deletion of all or part of a oop structure formed by the A, A', B, B' C, and/or C’ regions.
13. The ceDNA vector of claim 11 or claim 12, wherein one or both of the asymmetric ITRs are modified by a deletion that results in the deletion of all or part of a oop structure formed by the B and B' regions.
14. The ceDNA vector of any one of claims 11-13, wherein one or both of the tric ITRs is are modified by a on, an insertion, and/or a substitution that s in the deletion of all or part of a stem-loop structure formed by the C and C regions.
15. The ceDNA vector of claim 13 or claim 14, wherein one or both of the asymmetric ITRs is modified by a deletion that s in the deletion of all or part of a stem-loop structure formed by the B and B' regions and/or all or part of a stem-loop structure formed by the C and C regions.
16. The ceDNA vector of any one of claims 1-15, wherein one or both of the asymmetric ITRs comprise a single stem-loop ure in the region that, in a wild-type ITR, would se a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C regions.
17. The ceDNA vector of claim 16, wherein one or both of the asymmetric ITRs comprise a single stem and two loops in the region that, in a wild-type ITR, would comprise a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C regions.
18. The ceDNA vector of claim 16 or claim 17, wherein one or both of the asymmetric ITRs comprise a single stem and a single loop in the region that, in a wild-type ITR, would comprise a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C regions.
19. The ceDNA of any one of claims 1-18, n the ceDNA vector comprises an aptamer.
20. The ceDNA vector of any one of claims 1-19, wherein the ceDNA vector comprises at least one regulatory switch.
21. The ceDNA vector of claim 20, wherein the at least one regulatory switch is selected from the group consisting of: ABA; AIR; ART; BEARON, BEAROFF; BirA-tTA; BIT; Cry2-CIB1; CTA, CTS; cTA, rcTA; ne; EcR:RXR; electro-genetic switch; ER-p65-ZF; E.REX; EthR; GAL4- ER; GAL4-hPR; aps; GAL4-TR; GyrB; HEA-3; Intramer; LacI; LAD; LightOn; NICE; PPAR*; PEACE; PIT; REDOX; QuoRex; ST-TA; TIGR; TraR; TET-OFF, TET-ON; TRT; UREX; VAC; ZF-ER, ZF-RXR/EcR; s; ZF switches; ZF(TF)s; aptamer RNAi; aptamer RNAi miRNA; aptamer Splicing; aptazyme; replicon CytTS; TET-OFF-shRNA, TET-ON-shRNA; theo aptamer; 3' UTR aptazyme; 5' UTR aptazyme; Hoechst aptamer; H23 aptamer; L7Ae aptamer; MS2 aptamer; AID; ER DD; FM; HaloTag; tazyme; PROTAC; shield DD; shield LID; and TMP
22. The ceDNA vector of any one of claims 1-21, wherein the vector is in a nanocarrier.
23. The ceDNA vector of claim 22, wherein the nanocarrier comprises a lipid nanoparticle (LNP).
24. A ral capsid-free DNA vector with covalently-closed ends (ceDNA vector) of any one of claims 1-21, the ceDNA vector being obtained from a process comprising the steps of: a. incubating a population of insect cells harboring a ceDNA expression uct in the presence of at least one Rep protein, wherein the ceDNA expression construct encodes the ceDNA vector, under conditions effective and for a time ient to induce production of the ceDNA vector within the insect cells; and b. isolating the ceDNA vector from the insect cells.
25. The ceDNA vector of claim 24, wherein the ceDNA expression construct is selected from the group consisting of: a ceDNA plasmid, a ceDNA , and a ceDNA baculovirus.
26. The ceDNA vector of claim 24 or claim 25, wherein the insect cells express at least one Rep protein.
27. The ceDNA vector of claim 26, wherein the at least one Rep protein is derived from a virus selected from the group consisting of: a parvovirus, a dependovirus, and an adeno-associated virus (AAV).
28. The ceDNA vector of claim 27, wherein the at least one Rep protein is d from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.
29. A ceDNA expression construct that encodes the ceDNA vector of any one of claims 1-21.
30. The ceDNA expression construct of claim 29, ed from the group consisting of: a ceDNA plasmid, a ceDNA bacmid, and a ceDNA baculovirus.
31. An isolated or non-human host cell comprising the ceDNA expression construct of claim 29 or claim 30.
32. The host cell of claim 31, which expresses at least one Rep protein.
33. The host cell of claim 32, wherein the at least one Rep protein is from a virus selected from the group consisting of: a parvovirus, a dependovirus, and an adeno-associated virus (AAV).
34. The host cell of claim 33, wherein at least one Rep n is derived from an AAV serotype ed from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.
35. The host cell of any one of claims 31 to 34, which is an insect cell. 36. The host cell of claim 35, which is an Sf9 cell. 37. A method of producing a ceDNA vector in vitro, comprising: a. incubating the host cell of any one of claims 31-36 under conditions ive and for time sufficient to induce production of the ceDNA vector; and b. isolating the ceDNA from the host cell. 38. Use of the ceDNA vector of any one of claims 1-21 in the manufacture of a medicament for treating, preventing, rating, diagnosing or monitoring a disease or disorder, wherein the at least one nucleotide sequence is selected to treat, t, ameliorate, diagnose, or monitor the disease or disorder. 39. The use of claim 38, wherein the at least one nucleotide sequence, when transcribed or translated, es an abnormal amount of an nous protein in the subject. 40. The use of claim 38, wherein the at least one nucleotide sequence, when transcribed or translated, modifies an abnormal function or activity of an endogenous protein or pathway in the subject. 41. The use of any one of claims 38-40, wherein the at least one nucleotide sequence encodes or ses a nucleic acid molecule selected from the group consisting of: an RNAi, an siRNA, an miRNA, an IncRNA, and an antisense oligo- or polynucleotide. 42. The use of any one of claims 38-40, wherein the at least one nucleotide sequence encodes a protein. 43. The use of claim 38, wherein the at least one tide sequence s a marker protein. 44. The use of any one of claims 38-42, wherein the at least one nucleotide sequence encodes an agonist or an antagonist of an nous protein or pathway associated with the disease or disorder. 45. The use of any one of claims 38-42, wherein the at least one nucleotide sequence encodes an antibody. 46. The use of claims 38-45, wherein the ceDNA vector is to be administered in combination with a pharmaceutically acceptable carrier. 47. Use of the ceDNA vector of any of claims 1-21 in the cture of a medicament for delivering a therapeutic protein to a subject, wherein at least one nucleotide sequence encodes the therapeutic protein. 48. The use of claim 47, wherein the eutic protein is a therapeutic antibody. 49. A kit comprising the ceDNA vector of any of claims 1-21, and a nanocarrier, packaged in a container with a packet insert. 50. A kit for producing the ceDNA vector of any one of claims 1-21, the kit comprising an expression construct sing at least one restriction site for insertion of at least one nucleotide ce, the at least one restriction site operatively positioned between asymmetric inverted terminal repeats (asymmetric ITRs), wherein at least one of the asymmetric ITRs comprises a deletion in of least a D and D’ ITR region, and in at least one of an A, A’, B, B’, C and/or C’ ITR region, and wherein at least one of the tric ITRs comprises a functional terminal resolution site and a Rep binding site, and wherein the ITRs are derived from an adeno-associated virus (AAV). 51. The kit of claim 50, further comprising a tion of insect cells which is devoid of viral capsid coding sequences, that in the presence of Rep protein can induce tion of the ceDNA vector. 52. 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E3 tum-:3 U %“%i m can-mu 54mg: Kan—~94 tum-:3 54mg: Lamar: 5.4mm m m N {pm-m at 5:3 " away Mm {pm-m “I: 53mg «:1: 5-:ng 5.4mm ”Ug?uauuumm\/ GTGAT IIIIIIIIIIIQGGGATCACTA 5’"— 3’"— SUBSTITUTE SHEET (RULE 26) ?xa?agga $qu #3 Sam #2 @QO $3 a,“ “A “A “A “A “A 2 mmma Ea?amnggwEsaugagEgg£5ngamagohm E mc?cmm H A A A A A A A 3m mmmm xmhmmm 9% i 3% ?aw 0%, “Sam “Sam mgiegw ??u?? amf?m uunau mm QWE nnuuunuunnu “u PM”) u88méuwmwumm?w??u$6M8EwEuu?uwwm?wumm83m??mwmuuuwuumwmw???wu?wu?wummw mnrma SUBSTITUTE SHEET (RULE 26) Exg?g<:> \<=> am $3 $3 $9“ gum “Em an ”A “A “A “A ”A m?m: Egg$335EMWEEE@?g?gEgg”?Egg553$ m H A A A A A A A mam mmgm #3 § $3 #3 é“ 0% $0“ mmhwcm ”EumyEmu9mmm4Ew??ugnaw8%mgu?wm@8wammwuuuwwwquw???wu?wu?wuwum EEEE g m mm unun???uumu “m Egg m ”my.“ M, Twain m SUBSTITUTE SHEET (RULE 26) Qmmmxaéggm “Em Sam 0% $on Q03 3% g “A “A ”A ”A “A a“ mNm: Egg553$@533Egg?“$523Saga553$ E mgmngmm H A A A A A A A mg mmtm kmhmcm «am OE #3 $3 £2 Rug $3 w u88$8Emuw????uwmuwwmuumWEDmmBumwu mnnunununun m u wm nunnuunnumn ,mE ”Ema“ WE ”UNLESS SUBSTITUTE SHEET (RULE 26) Qmm??aéggmME 0% 9% ”E 3% gm“A ”A ”A “A a“ ”A 33Egg?“gm?gSEE553%553$ n.3,. A A A A A A A mgmncmm ?g Sam $3 0% £3 $3 $3 mam 3% 33$ E mm. /,$2a .W w .H. ,HHHUHHHHUHHHHUFHHHHU ampm inuuumnumnuu ”mm Emma ................ 6mm w88$8w SUBSTITUTE SHEET (RULE 26) «Kama a £3 Emma ?uw?wm?au mmvmamummm 33% . mummm mnxxyomnumm nm?mm? nm mmwumumcmb Emwm?mmnm @me MEN», mmmmu Tum?umm WEE ?m??mcmbmm? Ngwm?umm Wmm?g? “mummimx wmmimx ....................................................................................... EEW £me Eek 53% camusvahg .. EM «Zamu mummu Tnm?umm $me N?m?umm Emmcm Mummgm wumwmgm? ?mimw?mg Emc??cumh “umumcmm. ?gm?mrm? Emc?ga? gmmwwmmz?w mmmu?m EEMEQ .. Emmtmgm mcmuswammi?zmmu $3333 3cm cammmmaxm mmw nmmammsmb $35,333 33% gamma 33%ng WEEQEUE EMU?» mcmuswchmamm ?gmmgau cammm?axm mmgm?m EPWXQU 9ng Aminmgga SUBSTITUTE SHEET (RULE 26) 19/73 maamwm?ccu mam hmwmgmmm £39m 42%“ mmu Wm mgm?mucm ZQEUSQQmm Ewan comm?mmga $33qu Emma» Egg ““3 ccmuusnc? ......................... mw 3% “WE am m mama gm mummu ?g mgmwgahm SEES mghmm ?agm?m ”GE 33% mmnwm ................ cmm?mcmm «zmmu wamumm ngbmgeu mamumwm SUBSTITUTE SHEET (RULE 26) 13/73 mmmmmcm {2m muammmhm as Emma”: mam «$me “$3”ch Em?mm hm??m wwwumxxm Emmacu «2&3 ummrmwu?mgu WEE . comwunwa? ?mwumbxm Em mam E?cou @3wa km?tm €ng ?gmwumwahm m?mmg? gm {2m ?mwamnm @mwccumm mawmummcm WW {2mg Em?e m3 «gm aw Ea? EBEAW camumbxm «2a EB nmunnahm 333% mmmua? H Emmhma?am Emu 3 “mmamm Egg mommm?zwcmu SUBSTITUTE SHEET (RULE 26) 12/73 mmw?mm? nmmn?wmgmmmcmm $33?ne Ev Ev mmu mmmw wage gmmwumn?m QVENAW 3x31; mummemxum mctmwmgmmwwmgm?ammmgmm EEWQ EEOEQE E?cmw mmw mmst? . N am B EmEmm‘? m?mzw ?xmv Em?mmm mmm?mc? mmwm m, N m?wmx mmvcmb??som EmEmEm EmEmEm SUBSTITUTE SHEET (RULE 26) 1 3/73 ............................... I\|I\I!||1 angnaw mg 333330303354 . w ............... 83.. SEQ 3 unaSnSunguJSSsSah: r|4. 3% gunuHHuNHnum?anEN?m?um?o?mm?u . RnnnnovnunnnnNunnng?msus?unnnnnnnou E?gv?gy?nhn???ng an n 3§58§¥d033n§u .\||\Ivl4. gem .?nnguugonsbow an m@ . \LéLiiJ an 38 LI 32 msmmgmwcouggaz ... b?unnn.v?nn$k$ur&$vuhmvn3»n an @8305“ ?mm? muggycez 8% @ch ...... m?cncmwgau .10.,,. [It] an mummOmU mam _ $33 camwutwmmm mummE‘mNcm cem?mbmmm aEa w?w?m SUBSTITUTE SHEET (RULE 26) 000000000000000000000000 15i73 93129311153 {33mm WNW" aua?nj if lD?Jl’SUOD 6% E 13HJESUG3 E lD?Jl’SUOD i 133’UESUGZ} @ 6 \D RD \9 RD \9 RD CD {:3 C) {D C) {D C) ..§_ "i“ ”E“ "i“ ”E“ "i“ ”E“ LL! LL! LL! LL! LL! LL! LL! 0 0 Q Q Q Q <3 Q <3: C2 <3: C2 <3: C2 00 SD in “if M N 9'“ GQGE+00 (3“? US) SUBSTITUTE SHEET (RULE 26) 'E?i73 9312mm {'3 400m 260mg 100mg % ? E] 2§3U$?nj 913nJlSU03 SL§Q3 Z l??JESUQI} "210035—95 to \9 RD RD ‘3 c: C} C) C) CD + + + + + Lu Lu LL! LU LU 0 O Q C) O Q; Q C); Q C? 305566 m m ‘33" m N ’E G‘GOE—i—OO (33TH DEE) SUBSTITUTE SHEET (RULE 26) 17I73 33mm p$3E3J1U? KICK: 3% ??f] aua?n? i ganglsum g EJHJISUOZ) E UCl) i ZD?JISUOD L2 V" 90;; “Q ‘3’; C) 0 <13 <22) (Algaéqem 3&3) SUBSTITUTE SHEET (RULE 26) 18/73 paganism ?é??ng ?Z??ng B‘EQGng 3U35?? .................... R 8 Ej?ilSUGl) g l?nleUOZ) 17 l??jlgu?i? Z O'?? W :.,. Sq «2 “*3: “g (Ammemw) SUBSTITUTE SHEET (RULE 26) @9173 mmewm: Ammmg mm >m8m. mmwmmw .mm E 5 .. Mmamm. am g... MW. . . mmmmmw mmwmv [.37. Ewogoaa Egg. Mmmmm . “3me . UAW 3me .. 3.......I..U.......th$ mm?mmma?mm g . 3mm .. Em;?g. 53% mtg. . Egg .mmz mmmmww . . gig ma?a Mam .. 3qu ?gmmoa .. mm :33 . mm mm .Emmmmmmb 8m . .. ”mm H??mmmh ....... “ME 3%“.me m wwgwmwmmmw .... mmgum .H. . WEE. Emma Emmmmmwmwwv .wgazmwmma _. m?mmm?mmmmw .?mm?mmmw ........H...n..m%mmEmmammmmmmwww ?g Mammmv SUBSTITUTE SHEET (RULE 26) 2Qi73 mmmmmw KKNZ mm mm aSiam. : .mm ZZZ. U?mm: Z mm?mm ZZ mam. ZZZZ ZZZZZVZ .. .ZZZZZZZZZZZZZZ ZZZZZZZZZZZZZZZZZ ZZZ gnaw ZZZZZZ...WWW? ....... .. ZZZZZZEZZG .. stmmm? 3&3me ZZZZZZZZZZZZ oZZ ZZZ... ZZZZZZZ ZZZZZZZ . Eng mm- ZZZZZZ ZZZE. .. .............. aim ZZZ» ZZZZZZZZZZZZZ- ZZZZZZZZ ZmZoEoZZZ ZZZZZZZZ ZZZ ZZZZZZmZZZZmemZZZZZZZ MZZM . @un .. ZZZZZZZZZ ...ZZZZZZZ.-....ZZZZZZZZZZZZ xmm ZZZO ZZZZZZ . mmwmwv .Z aZZZ ZZZZZZZZ w ..ZZZZZZZZZZZZZWZZZZZZZZZZZZ WEE ZZZ ZZZZZZ ZZZZZZZ ..MEZZZZM Z3?. Z 0..... ....ZZZZ ZZZZZZWZ .Zaam . Z ZZZZZZ ZZZZZZZ .ZZZSZZ ZZZZ -ZZZZZZZZZZZZZZZ @mmmw Z ZZZZZZZZZ Mwmmww . ZmZZm ZZZZZZZZ ZZZZZZZ Ma?a ZZZZZZZZZ - ZZZZZZZZZ a mewg 8R8 ZZZZZZZ ZZZZZ. ZZZZZZZ 3% ZZZZZZZ SUBSTITUTE SHEET (RULE 26) emu C—G deietiee (8 pairs ii‘iSieeei (if 9) M ii ........
36. Rep g sites {iii—"2,, Left Energy 2 “72.6 Fi?. 9A SUBSTITUTE SHEET (RULE 26) ("i—"C de?etian {8 gairs d 0'? 9) Rep Einding S?tes; ETRuZ, Right Energy = ~72.6 SUBSTITUTE SHEET (RULE 26) Rep Binding sites ETR«3, Left Energy : mm SUBSTITUTE SHEET (RULE 26) ' i Rep Binding H sites BTR~3,Right j Energyx-m? .. j j&% SUBSTITUTE SHEET (RULE 26) Reg) Binding sites Energy 2 WW mag, Left SUBSTITUTE SHEET (RULE 26) Rep Binding Energy : J69 :TR~4, Right SUBSTITUTE SHEET (RULE 26) Rep g sites ETRwi Q (LE‘EE) Engrgy : ~83] Fi?, ”EZA SUBSTITUTE SHEET (RULE 26) Ran Binding Sites Energy : ~83.7 nn: 0 (Right) Fi?. ”32% SUBSTITUTE SHEET (RULE 26) Rep Binding Sites BT34 7 (Lejft) Energy : Q73 3 SUBSTITUTE SHEET (RULE 26) Rep Binding sites ETRmi 7 (Right) Energy 2 «73,3 HG i 3% SUBSTITUTE SHEET (RULE 26) 311’73 Rep Binding siteg ETR-é, Left Energy 2 “54.4 SUBSTITUTE SHEET (RULE 26) 32/73 Rep E?nd‘éng sétes , Right Energy 2 «54.4 SUBSTITUTE SHEET (RULE 26) 33/73 Rep Binding sites KR”? , LET? Energy : “74] SUBSTITUTE SHEET (RULE 26) 34/?3 E?R~"E, Right Energy 2 «74.? SUBSTITUTE SHEET (RULE 26) 35:73 10- |" 75:: Rep g Sites mus, Left Energy : ”73.4 SUBSTITUTE SHEET (RULE 26)
NZ761178A 2018-09-07 Modified closed-ended dna (cedna) NZ761178B2 (en)

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US201762556324P 2017-09-08 2017-09-08
US201762556319P 2017-09-08 2017-09-08
PCT/US2018/049996 WO2019051255A1 (en) 2017-09-08 2018-09-07 Modified closed-ended dna (cedna)

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