{"search_session":{},"preferences":{"l":"en","queryLanguage":"en"},"patentId":"086-156-907-653-335","frontPageModel":{"patentViewModel":{"ref":{"entityRefId":"086-156-907-653-335","entityRefType":"PATENT"},"entityMetadata":{"linkedIds":{"empty":true},"tags":[],"collections":[{"id":8906,"type":"PATENT","title":"Univ Pennsylvania Patent Portfolio","description":"","access":"OPEN_ACCESS","displayAvatar":true,"attested":false,"itemCount":24238,"tags":[],"user":{"id":91044780,"username":"Cambialens","firstName":"","lastName":"","created":"2015-05-04T00:55:26.000Z","displayName":"Cambialens","preferences":"{\"usage\":\"public\",\"beta\":false}","accountType":"PERSONAL","isOauthOnly":false},"notes":[{"id":8221,"type":"COLLECTION","user":{"id":91044780,"username":"Cambialens","firstName":"","lastName":"","created":"2015-05-04T00:55:26.000Z","displayName":"Cambialens","preferences":"{\"usage\":\"public\",\"beta\":false}","accountType":"PERSONAL","isOauthOnly":false},"text":"
Search applicants and owners= \"Univ Pennsylvania\", \"Pennsylvania Univ\", \"Univ Penns*\", \"Penn* Univ\", \"University of Pennsylvania\", \"Pennsylvania University\".
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Search applicants and owners= \"Univ Pennsylvania\", \"Pennsylvania Univ\", \"Univ Penns*\", \"Penn* Univ\", \"University of Pennsylvania\", \"Pennsylvania University\".
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(a) a variable region 17-30 nucleotides long, consisting of a sequence, wherein said sequence is either (i) substantially random; or (ii) comprises a first sub-region and a second sub-region, wherein said first sub-region is substantially random and said second sub-region has a first sequence common to said set or library of shRNA molecules;\n
(b) a non self-complementary region 3-20 nucleotides long, consisting of a second sequence common to said library; and\n
(c) a complementary region consisting of a sequence, wherein said sequence is the reverse complement of the variable region;\n
wherein the substantially random sequence in the variable region has minimal sequence bias, wherein the variable and complementary regions form a double-stranded region and the non self-complementary region forms a loop that connects the two strands of the double-stranded region."],"number":1,"annotation":false,"claim":true,"title":false},{"lines":["The set or library of recombinant expression vectors of claim 1, wherein individual recombinant expression vectors each further comprises a promoter of an RNA polymerase."],"number":2,"annotation":false,"claim":true,"title":false},{"lines":["The set or library of recombinant expression vectors of claim 1, wherein individual recombinant expression vectors each further comprises a gene encoding an RNA inhibitory (RNAi) molecule of known function."],"number":3,"annotation":false,"claim":true,"title":false},{"lines":["The set or library of recombinant expression vectors of claim 1, wherein said variable region is 8-30 nucleotides long."],"number":4,"annotation":false,"claim":true,"title":false},{"lines":["The set or library of recombinant expression vectors of claim 1, wherein said first sub-region is 8-29 nucleotides long."],"number":5,"annotation":false,"claim":true,"title":false},{"lines":["A method for identifying an RNA molecule that is capable of affecting a biological parameter of interest, comprising the steps of:\n
a. contacting a cell population with the set or library of recombinant expression vectors of claim 1, wherein said set or library of said expression vectors, or a fraction thereof, is taken up by individual cells of said cell population; and\n
b. determining or measuring said biological parameter of interest or a readout thereof in said cell population;\n
whereby, if a particular cell in said cell population exhibits an alteration of said biological parameter of interest or readout thereof, then said particular cell carries a particular recombinant expression vector that encodes a particular RNA molecule that is capable of affecting said biological parameter of interest."],"number":6,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, wherein said biological parameter of interest or readout thereof is an expression of a protein of interest."],"number":7,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, wherein said biological parameter of interest or readout thereof is an ability of a cell to survive or maintain growth under adverse conditions."],"number":8,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, wherein said biological parameter of interest or readout thereof is a susceptibility of a cell to an intracellular pathogen or an ability of an intracellular pathogen to replicate in a cell."],"number":9,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 9, wherein said intracellular pathogen is a virus."],"number":10,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, wherein said biological parameter of interest or readout thereof is a differentiation of a cell into a cell type of interest."],"number":11,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, wherein said biological parameter of interest or readout thereof is an ability to induce long-term proliferation or sustain pluripotency of a cell."],"number":12,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, further comprising the step of isolating or amplifying said particular expression vector, its insert, or a fragment thereof, and sequencing either:\n
a. said particular expression vector; or\n
b. a fragment of said particular expression vector, wherein said fragment comprises the coding sequence for said particular RNA molecule."],"number":13,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, further comprising the steps of contacting an additional cell with said particular expression vector and determining or measuring said biological parameter of interest or readout thereof in said additional cell."],"number":14,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, further comprising the steps of generating a copy of said particular RNA molecule, contacting an additional cell with said copy of said particular RNA molecule, and determining or measuring said biological parameter of interest or readout thereof in said additional cell."],"number":15,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, further comprising the steps of\n
a. inserting into a second expression vector backbone a sequence that encodes either: (a) said particular RNA molecule or (b) a fragment of said particular RNA molecule, thereby generating a second recombinant expression vector, wherein said second recombinant expression vector encodes either:\n\ni. said particular RNA molecule; or\nii. an altered version of said particular RNA molecule, wherein said altered version of said particular RNA molecule comprises a region that shares homology with a double-stranded region of said particular RNA molecule;\n
b. contacting an additional cell with said second expression vector; and\n
c. determining or measuring said biological parameter of interest or readout thereof in said additional cell."],"number":16,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, further comprising the steps of\n
a. inserting into a second expression vector backbone a sequence that encodes said particular RNA molecule; or that encodes a fragment of said particular RNA molecule, thereby generating a second expression vector, wherein said second expression vector encodes an altered version of said particular RNA molecule, wherein said altered version of said particular RNA molecule comprises a region that shares homology with a double-stranded region of said particular RNA molecule;\n
b. producing a copy of said altered version of said particular RNA molecule;\n
c. contacting an additional cell with said copy of said altered version of said particular RNA molecule; and\n
d. determining or measuring said biological parameter of interest or readout thereof in said additional cell."],"number":17,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 6, further comprising the steps of:\n
a. isolating or amplifying either: (i) said particular expression vector; (ii) the insert of said particular expression vector; or (iii) a fragment of said particular expression vector, wherein said fragment comprises a region encoding the particular RNA molecule;\n
b. mutagenizing a fragment of said particular expression vector, thereby generating a sub-library of nucleotide molecules, said sub-library of nucleotide molecules comprising variants of said fragment of said particular expression vector;\n
c. generating a sub-library of recombinant expression vectors containing said sub-library of nucleotide molecules;\n
d. contacting a second population of cells with said sub-library of recombinant expression vectors, wherein said sub-library of recombinant expression vectors, or a fraction thereof, is taken up by individual cells of said second population of cells; and\n
e. determining or measuring said biological parameter of interest or a readout thereof in said second population of cells,\n
whereby, if said biological parameter of interest or readout thereof is further altered in a particular cell in said second set of cells, then said particular cell carries an improved expression vector, wherein said improved expression vector encodes an improved RNA molecule that affects said biological parameter of interest or readout thereof more than the particular RNA molecule of claim 6."],"number":18,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 18, wherein the step of mutagenizing comprises copying a fragment of said particular expression vector by a low-fidelity method."],"number":19,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 19, wherein said low-fidelity method is an error-prone polymerase chain reaction (PCR)."],"number":20,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 18, further comprising the step of isolating or amplifying said improved expression vector or a fragment thereof, wherein said fragment comprises the coding sequence for said improved RNA molecule, and sequencing either (a) said improved expression vector or (b) said fragment thereof."],"number":21,"annotation":false,"claim":true,"title":false},{"lines":["The method of claim 18, further comprising the step of contacting an additional cell with said improved expression vector and determining or measuring said biological parameter of interest or readout thereof in said additional cell."],"number":22,"annotation":false,"claim":true,"title":false},{"lines":["A method of identifying a drug target for a disease or disorder of interest, comprising the steps of\n
a. identifying an RNA molecule that affects a biological parameter of interest by the method of claim 6, wherein said biological parameter of interest is altered in said disease or disorder of interest; and\n
b. identifying a cellular RNA molecule whose expression is altered by said RNA molecule,\n
whereby said cellular RNA molecule is identified as a drug target for said disease or disorder of interest."],"number":23,"annotation":false,"claim":true,"title":false},{"lines":["The set or library of recombinant expression vectors of claim 1 generated by a method, said method comprising the steps of:\n
a. obtaining a nucleic acid intermediate II from a single-stranded nucleic acid intermediate I, wherein said single-stranded nucleic acid intermediate I consists of:\n\n(i) a first constant region;\n(ii) said variable region; and\n(iii) a second constant region;\n\nwherein the step of obtaining said nucleic acid intermediate II comprises the steps of (a) annealing a primer to the second constant region of the single-stranded nucleic acid intermediate I, said primer comprising one or more mismatched residues with respect to the second constant region and (b) extending said primer to obtain a double-stranded intermediate I B, and (c) obtaining the nucleic acid intermediate II from the double-stranded intermediate I B by ligating a linker nucleic acid molecule to the 3′ end of the single-stranded nucleic acid intermediate I and the 5′ end of the additional single-stranded nucleic acid molecule;\n
and wherein said nucleic acid intermediate II comprises:\n\n(i) said single-stranded nucleic acid intermediate I;\n(ii) an intervening region; and\n(iii) a region that hybridizes with said single-stranded nucleic acid intermediate I;\n
b. obtaining a double-stranded intermediate III from nucleic acid intermediate II, comprising said nucleic acid intermediate II and an additional nucleic acid molecule that hybridizes with said nucleic acid intermediate II, and wherein said double-stranded intermediate III comprises:\n\n(i) a first, double-stranded copy of said first constant region or a fragment thereof;\n(ii) a first, double-stranded copy of said variable region;\n(iii) a first, double-stranded copy of said second constant region;\n(iv) a double-stranded copy of said intervening region;\n(v) a second, inverted double-stranded copy of said second constant region;\n(vi) a second, inverted double-stranded copy of said variable region; and\n(vii) a second, inverted double-stranded copy of said first constant region or a fragment thereof;\nwherein said first, double-stranded copy of said second constant region and said second, inverted double-stranded copy of said second constant region have a restriction enzyme site asymmetry created by said mismatched primer used to obtain the double stranded intermediate II, such that:\n(i) said first, double-stranded copy of said second constant region, but not said second, inverted double-stranded copy of said second constant region, is a substrate for a first restriction enzyme, and;\n(ii) said second, inverted, double-stranded copy of said second constant region, but not said first double-stranded copy of said second constant region, is a substrate for a second restriction enzyme;\nsaid method further comprising the step of obtaining a circular intermediate IV from said double-stranded intermediate III, said circular intermediate IV comprising an expression vector backbone and, as an insert, either:\n
(a) said double-stranded intermediate III; or\n
(b) a fragment of said double-stranded intermediate III, wherein said fragment comprises said first, double-stranded copy of said variable region and said second, inverted double-stranded copy of said variable region;\nthereby generating a set or library of recombinant expression vectors, wherein said set or library of recombinant expression vectors is capable of expressing said set or library of shRNA molecules."],"number":24,"annotation":false,"claim":true,"title":false},{"lines":["The set or library of recombinant expression vectors of claim 24, wherein said set or library of recombinant expression vectors is a set or library of recombinant viruses."],"number":25,"annotation":false,"claim":true,"title":false},{"lines":["The set or library of recombinant expression vectors of claim 1, wherein the non self-complementary region has a sequence selected from (i) CCTCCCGC, (ii) CCCC, (iii) AGGC, (iv) CCTCAC, (v) CCTCAC, (vi) TCTTCCGC, (vii) GTTTAG, and (viii) CTAAAC."],"number":26,"annotation":false,"claim":true,"title":false},{"lines":["The set or library of recombinant expression vectors of claim 26, wherein the sequence of the non self-complementary region is GTTTAG or CTAAAC."],"number":27,"annotation":false,"claim":true,"title":false}]}},"filters":{"npl":[],"notNpl":[],"applicant":[],"notApplicant":[],"inventor":[],"notInventor":[],"owner":[],"notOwner":[],"tags":[],"dates":[],"types":[],"notTypes":[],"j":[],"notJ":[],"fj":[],"notFj":[],"classIpcr":[],"notClassIpcr":[],"classNat":[],"notClassNat":[],"classCpc":[],"notClassCpc":[],"so":[],"notSo":[],"sat":[]},"sequenceFilters":{"s":"SEQIDNO","d":"ASCENDING","p":0,"n":10,"sp":[],"si":[],"len":[],"t":[],"loc":[]}}