(12) United States Patent
`Zamore et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 7,691,995 B2
`Apr. 6, 2010
`
`US007691995B2
`
`(54)
`
`(75)
`
`IN VIVO PRODUCTION OF SMALL
`INTERFERING RNAS THAT MEDIATE GENE
`SILENCING
`
`Inventors: Phillip D. Zamore, Northborough, MA
`(US); Juanita McLachlan, Worcester,
`MA (US); Gyorgy Hutvagner,
`Worcester, MA (US); Alla Grishok,
`Newton, MA (US); Craig C. Mello,
`Shrewsbury, MA (US)
`
`(73)
`
`Assignee: University of Massachusetts, Boston,
`MA (US)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 891 days.
`
`(21)
`
`(22)
`
`(65)
`
`(60)
`
`(51)
`
`(52)
`(58)
`
`(56)
`
`App1.No.: 10/195,034
`
`Filed:
`
`Jul. 12, 2002
`
`Prior Publication Data
`
`US 2006/0009402 A1
`
`Jan. 12, 2006
`
`Related U.S. Application Data
`
`Provisional application No. 60/305,185, ?led on Jul.
`12, 2001.
`
`Int. Cl.
`(2006.01)
`C07H 21/04
`U.S. Cl. ................................................... .. 536/245
`
`Field of Classi?cation Search ............... .. 536/24.5
`See application ?le for complete search history.
`
`References Cited
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`(Continued)
`Primary ExamineriBrian Whiteman
`(74) Attorney, Agent, or FirmiLahive & Cock?eld, LLP;
`Debra J. Milasincic, Esq.; James H. Velema, Esq.
`
`(57)
`
`ABSTRACT
`
`The invention provides engineered RNA precursors that
`when expressed in a cell are processed by the cell to produce
`targeted small interfering RNAs (siRNAs) that selectively
`silence targeted genes (by cleaving speci?c mRNAs) using
`the cell’s own RNA interference (RNAi) pathway. By intro
`ducing nucleic acid molecules that encode these engineered
`RNA precursors into cells in vivo with appropriate regulatory
`sequences, expression of the engineered RNA precursors can
`be selectively controlled both temporally and spatially, i.e., at
`particular times and/ or in particular tissues, organs, or cells.
`
`68 Claims, 6 Drawing Sheets
`
`Benitec - Exhibit 1003 - page 1
`
`

`
`US 7,691,995 B2
`Page 2
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`Benitec - Exhibit 1003 - page 2
`
`

`
`US 7,691,995 B2
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`
`* cited by examiner
`
`Benitec - Exhibit 1003 - page 3
`
`

`
`US. Patent
`
`Apr. 6, 2010
`
`Sheet 1 of6
`
`US 7,691,995 B2
`
`FIG. 1
`
`stHNA pathway
`.._.ll
`developmentally
`regulated transcription
`
`pa Way
`
`experimentally
`introduced dsFlNA
`
`stRNA
`
`siRNA
`
`Benitec - Exhibit 1003 - page 4
`
`

`
`US. Patent
`
`Apr. 6, 2010
`
`Sheet 2 of6
`
`US 7,691,995 B2
`
`FIG. 2A
`
`G A U
`U
`5 ’ —GGCAAAUGAGGUAGUAGGUUGUAUAGUA U AU A
`'
`C
`
`3 ' —UCGUUUCUUUQE'AUCGUGUAACAUAUCAU ACUACA
`l
`/ \
`IL
`J
`stem \ loop
`unpaired
`nucleotide
`
`bulge
`
`(SEQ ID NOzl)
`
`FIG. 213
`
`G A U
`5 ' —GGCAAACGUACGCGGAAUACUUCGAUU A U AU A
`C
`3 ' -UCGUUUGCAUGCGCCUUAUGAAGCUAA U ACUACA
`
`(SEQ H) 110:2)
`
`FIG. 2C
`
`G A U
`5 ' -GGCAAAUGCUUGAAGCAGCUCUGGAGUA U AU A
`C
`3 ' -UCGUUUACGAACUUCGUCGAGACCUCAU ACUACA
`
`(SEQ ID NO:3)
`
`Benitec - Exhibit 1003 - page 5
`
`

`
`US. Patent
`
`Apr. 6, 2010
`
`Sheet 3 of6
`
`US 7,691,995 B2
`
`FIG. 2D
`
`5’—GGCAAAUGCUUGAAGCAGCUCUGGAGUAIJl??aj
`3’—UCGUUUACGAACUUCGUCGAGACCUCAU2%;H?fj
`
`G A
`
`(SEQ ID NO:4)
`
`FIG. 2E
`
`5'—GGCAAAUGCUUGAAGCAGCUCUGGAGUAGG
`3’—UCGUUUACGAACUUCGUCGAGACCUCAUGG
`
`(SEQ ID N0:5)
`
`Benitec - Exhibit 1003 - page 6
`
`

`
`U S. Patent
`
`Apr. 6, 2010
`
`Sheet 4 of6
`
`US 7,691,995 B2
`
`FIG.
`
`3
`
`buffer siRNA ESP
`030303h0urs
`
`5
`
`C u d m 0. e w V a b c
`,._,
`
`Benitec - Exhibit 1003 - page 7
`
`

`
`US. Patent
`
`Apr. 6, 2010
`
`Sheet 5 of6
`
`US 7,691,995 B2
`
`as .UHh
`
`Benitec - Exhibit 1003 - page 8
`
`

`
`Benitec - Exhibit 1003 - page 9
`
`

`
`US 7,691,995 B2
`
`1
`IN VIVO PRODUCTION OF SMALL
`INTERFERING RNAS THAT MEDIATE GENE
`SILENCING
`
`This application claims priority from US. Provisional
`PatentApplication Ser. No. 60/305,185, ?led on Jul. 12,2001,
`Which is incorporated herein by reference in its entirety.
`
`STATEMENT AS TO FEDERALLY SPONSORED
`RESEARCH
`
`This invention Was made With Government support
`GM62862-01 awarded by the National Institutes of Health.
`The Government has certain rights in the invention.
`
`TECHNICAL FIELD
`
`This invention relates to ribonucleic acid interference
`(RNAi), and more particularly to RNAi in vivo.
`
`BACKGROUND
`
`20
`
`2
`messenger RNA (mRNA) of a target gene; (ii) a second stem
`portion comprising a sequence of at least 18 nucleotides that
`is suf?ciently complementary to the ?rst stem portion to
`hybridiZe With the ?rst stem portion to form a duplex stem
`(e.g., a stem that can be processed by the enZyme Dicer); and
`(iii) a loop portion that connects the tWo stem portions. In
`another aspect, the invention features the engineered RNA
`itself. The RNA precursor targets a portion of the mRNA of
`the target gene, disrupts translation of the mRNA by cleaving
`the mRNA, and thereby prevents expression of the protein to
`be inhibited. The target genes can be, for example, human
`genes, e.g., mutant human genes, e.g., having a point muta
`tion, or they can be viral or other genes.
`In these molecules and precursors, the ?rst stem portion
`can be fully complementary (i.e., completely complemen
`tary) to the mRNA sequence. In other embodiments, the stem
`portion can be complementary, i.e., the sequence can be sub
`stantially complementary (e.g., there can be no more than one
`or tWo mismatches over a stretch of 20 nucleotides). Simi
`larly, the second stem portion can fully or substantially
`complementary to the ?rst stem portion. The ?rst stem por
`tion can be located at a 5' or 3' end of the RNA precursor.
`In these precursors, the loop portion can include at least 4,
`7, or 1 1, or more nucleotides, and the sequence of the mRNA
`is located from 100 to 300 nucleotides 3' of the start of
`translation of the mRNA. The sequence of the mRNA can be
`located in a 5' untranslated region (UTR) or a 3' UTR of the
`mRNA. The ?rst and second stem portions can each include
`about 18 to about 30 nucleotides, or about 22 to about 28
`nucleotides. The ?rst and second stem portions can each have
`the same number of nucleotides, or one of the ?rst and second
`stem portions can have 1 to 4 more nucleotides than the other
`stem portion. These overhanging nucleotides can all be
`uracils.
`In these nucleic acid molecules, the regulatory sequence
`can be a Pol III or Pol II promoter, and can be constitutive or
`inducible. In speci?c embodiments, the engineered RNA pre
`cursor can have the sequence set forth in SEQ ID NO: 1, 2, 3,
`4, 5, 8, or 9, and the nucleic acid molecule can have the
`sequence set forth in SEQ ID NO:10, 11, 17, 18, 20, or 21, or
`a complement thereof.
`In other embodiments, the invention also features vectors,
`e.g., plasmids or viral (e.g., retroviral) vectors, that include
`the neW nucleic acid molecules.
`In another aspect, the invention includes host cells, e.g.,
`mammalian cells, that contain the neW nucleic acid mol
`ecules. The invention also includes transgenes that include
`the neW nucleic acid molecules.
`In another aspect of the invention, the invention features
`transgenic, non-human animals, one or more of Whose cells
`include a trans gene containing one or more of the neW nucleic
`acid molecules, Wherein the transgene is expressed in one or
`more cells of the transgenic animal resulting in the animal
`exhibiting ribonucleic acid interference (RNAi) of the target
`gene by the engineered RNA precursor. For example, the
`transgene can be expressed selectively in one or more cardiac
`cells, lymphocytes, liver cells, vascular endothelial cells, or
`spleen cells. In these animals, the regulatory sequence can be
`constitutive or inducible, or the regulatory sequence can be
`tissue speci?c. In some embodiments, the regulatory
`sequence can a Pol III or Pol II promoter, and can be a an
`exogenous sequence. These transgenic animals can be non
`human primates or rodents, such as mice or rats, or other
`animals (e.g., othermammals, such as goats or coWs; orbirds)
`described herein.
`The invention also includes cells derived from the neW
`transgenic animals. For example, these cells can be a lym
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`RNAi is the sequence-speci?c, post-transcriptional silenc
`ing of a gene’s expression by double-stranded RNA. RNAi is
`mediated by 21 to 25 nucleotide, double-stranded RNA mol
`ecules referred to as small interfering RNAs (siRNAs) that are
`derived by enZymatic cleavage of long, double-stranded RNA
`in cells. siRNAs can also be synthesized chemically or enZy
`matically outside of cells and then delivered to cells (e.g., by
`transfection) (see, e.g., Fire et al., 1998, “Potent and speci?c
`genetic interference by double-stranded RNA in Caenorhab
`ditis elegans,” Nature, 391:806-11; Tuschl et al., 1999, “Tar
`geted mRNA degradation by double-stranded RNA in vitro,”
`Genes Dev., 13:3191-7; Zamore et al., 2000, “RNAi: double
`stranded RNA directs the ATP-dependent cleavage of mRNA
`35
`at 21 to 23 nucleotide intervals,” Cell, 101 125-33.; Elbashir et
`al., 2001, “Duplexes of 21 -nucleotide RNAs mediate RNA
`interference in mammalian cell culture,” Nature, 41 1:494
`498; and Elbashir et al., 2001 , “RNA interference is mediated
`by 21- and 22-nucleotide RNAs,” Genes Dev., 15:188-200.
`Double-stranded siRNAs mediate gene silencing by target
`ing for disruption or cleavage messenger RNAs (mRNAs)
`that contain the sequence of one strand of the siRNA. siRNAs
`introduced into mammalian cells by transfection mediate
`sequence-speci?c gene silencing, Whereas long, double
`stranded RNA induces sequence non-speci?c responses.
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`SUMMARY
`
`The invention is based on the discovery of neW arti?cial,
`engineered RNA precursors, that When expressed in a cell,
`e.g., in vivo, are processed by the cell to produce targeted
`siRNAs that selectively silence target genes (by targeting
`speci?c mRNAs for cleavage) using the cell’s oWn RNAi
`pathWay. By introducing nucleic acid molecules that encode
`these engineered RNA precursors into cells in vivo With
`appropriate regulatory sequences (e.g., a transgene in a vector
`such as a plasmid), expression of the engineered RNA pre
`cursors can be selectively controlled both temporally and
`spatially, i.e., at particular times and/or in particular tissues,
`organs, or cells.
`In general, the invention features an isolated nucleic acid
`molecule including a regulatory sequence operably linked to
`a nucleic acid sequence that encodes an engineered ribo
`nucleic acid (RNA) precursor, Wherein the precursor
`includes: (i) a ?rst stem portion comprising a sequence of at
`least 18 nucleotides that is complementary to a sequence of a
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`Benitec - Exhibit 1003 - page 10
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`US 7,691 ,995 B2
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`3
`phocyte, a hematopoietic cell, a liver cell, a cardiac cell, a
`vascular endothelial cell, or a spleen cell.
`In another aspect, the invention includes methods of induc
`ing ribonucleic acid interference (RNAi) of a target gene in a
`cell, e.g., in an animal or in culture. The neW methods include
`obtaining a transgenic animal comprising a transgene includ
`ing a nucleic acid molecule encoding an engineered RNA
`precursor and an inducible promoter; and inducing the cell to
`express the precursor to form a small interfering ribonucleic
`acid (siRNA) Within the cell, thereby inducing RNAi of the
`target gene in the animal.
`Alternatively, the methods include obtaining a host cell;
`culturing the cell; and enabling the cell to express the RNA
`precursor to form a small interfering ribonucleic acid
`(siRNA) Within the cell, thereby inducing RNAi of the target
`gene in the cell.
`A “transgene” is any nucleic acid molecule, Which is
`inserted by arti?ce into a cell, andbecomes part of the genome
`of the organism that develops from the cell. Such a transgene
`may include a gene that is partly or entirely heterologous (i.e.,
`foreign) to the transgenic organism, or may represent a gene
`homologous to an endogenous gene of the organism. The
`term “transgene” also means a nucleic acid molecule that
`includes one or more selected nucleic acid sequences, e.g.,
`DNAs, that encode one or more engineered RNA precursors,
`to be expressed in a transgenic organism, e.g., animal, Which
`is partly or entirely heterologous, i.e., foreign, to the trans
`genic animal, or homologous to an endogenous gene of the
`transgenic animal, but Which is designed to be inserted into
`the animal’s genome at a location Which differs from that of
`the natural gene. A trans gene includes one or more promoters
`and any other DNA, such as introns, necessary for expression
`of the selected nucleic acid sequence, all operably linked to
`the selected sequence, and may include an enhancer
`sequence.
`A “transformed cell” is a cell into Which (or into an ances
`tor of Which) has been introduced, by means of recombinant
`DNA techniques, a nucleic acid molecule or transgene encod
`ing an engineered RNA precursor.
`As used herein, the term “operably linked” means that a
`selected nucleic acid sequence, e.g., encoding an engineered
`RNA precursor, is in proximity With a promoter, e. g., a tissue
`speci?c promoter, to alloW the promoter to regulate expres
`sion of the selected nucleic acid sequence. In addition, the
`promoter is located upstream of the selected nucleic acid
`sequence in terms of the direction of transcription and trans
`lation.
`By “promoter” is meant a nucleic acid sequence that is
`suf?cient to direct transcription. A tissue-speci?c promoter
`affects expression of the selected nucleic acid sequence in
`speci?c cells, e.g., hematopoietic cells, or cells of a speci?c
`tissue Within an animal, e.g., cardiac, muscle, or vascular
`endothelium. The term also covers so-called “leaky” promot
`ers, Which regulate expression of a selected nucleic acid
`sequence primarily in one tissue, but cause expression in
`other tissues as Well. Such promoters also may include addi
`tional DNA sequences that are necessary for expression, such
`as introns and enhancer sequences.
`By “transgenic” is meant any cell that includes a nucleic
`acid, e.g., DNA sequence, that is inserted by arti?ce into a cell
`and becomes part of the genome of an organism that develops
`from that cell. A “transgenic animal” means an animal that
`includes a transgene that is inserted into an embryonal cell
`and becomes a part of the genome of the animal Which devel
`ops from that cell, or an offspring of such an animal. In the
`transgenic animals described herein, the transgene causes
`speci?c tissue cells to express an engineered RNA precursor.
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`Any animal that can be produced by transgenic technology is
`included in the invention, although mammals are preferred.
`Preferred mammals include non-human primates, sheep,
`goats, horses, cattle, pigs, rabbits, and rodents such as guinea
`pigs, hamsters, rats, gerbils, and, preferably, mice.
`An “isolated nucleic acid molecule or sequence” is a
`nucleic acid molecule or sequence that is not immediately
`contiguous With both of the coding sequences With Which it is
`immediately contiguous (one on the 5' end and one on the 3'
`end) in the naturally occurring genome of the organism from
`Which it is derived. The term therefore includes, for example,
`a recombinant DNA or RNA that is incorporated into a vector;
`into an autonomously replicating plasmid or virus; or into the
`genomic DNA of a prokaryote or eukaryote, or Which exists
`as a separate molecule (e.g., a cDNA or a genomic DNA
`fragment produced by PCR or restriction endonuclease treat
`ment) independent of other sequences. It also includes a
`recombinant DNA that is part of a hybrid gene encoding an
`additional polypeptide sequence.
`A “target gene” is a gene Whose expression is to be selec
`tively inhibited or “silenced.” This silencing is achieved by
`cleaving the mRNA of the target gene by an siRNA that is
`created from an engineered RNA precursor by a cell’s RNAi
`system. One portion or segment of a duplex stem of the RNA
`precursor is an anti-sense strand that is complementary, e. g.,
`fully complementary, to a section of about 18 to about 40 or
`more nucleotides of the mRNA of the target gene.
`The term “engineered,” as in an engineered RNA precur
`sor, or an engineered nucleic acid molecule, indicates that the
`precursor or molecule is not found in nature, in that all or a
`portion of the nucleic acid sequence of the precursor or mol
`ecule is created or selected by man. Once created or selected,
`the sequence can be replicated, translated, transcribed, or
`otherWise processed by mechanisms Within a cell. Thus, an
`RNA precursor produced Within a cell from a transgene that
`includes an engineered nucleic acid molecule is an engi
`neered RNA precursor.
`Unless otherWise de?ned, all technical and scienti?c terms
`used herein have the same meaning as commonly understood
`by one of ordinary skill in the art to Which this invention
`belongs. Although methods and materials similar or equiva
`lent to those described herein can be used in the practice or
`testing of the present invention, suitable methods and mate
`rials are described beloW. All publications, patent applica
`tions, patents, and other references mentioned herein are
`incorporated by reference in their entirety. In case of con?ict,
`the present speci?cation, including de?nitions, Will control.
`In addition, the materials, methods, and examples are illus
`trative only and not intended to be limiting.
`The invention provides several advantages. For example,
`the invention improves on and overcomes a signi?cant de?
`ciency in the prior art. Prior methods for inducing RNAi in
`mammalian cells using siRNAs Were restricted to cell cul
`tures. The neW methods extend RNAi to Whole animals, e. g.,
`mammals, and thus alloW RNAi to be targeted to speci?c cell
`types, organs, or tissues, and/or to speci?c developmental
`stages.
`In addition, this technology simpli?es and loWers the cost
`of siRNA construction, because DNA molecules are rela
`tively inexpensive to make. Thus, large populations of plas
`mids or other vectors can be prepared, each containing a
`nucleic acid molecule that encodes an engineered RNA pre
`cursor that targets a particular gene, can be easily prepared,
`e.g., in an array format. In addition, the neW nucleic acid
`molecules can be introduced into a variety of cells, Which can
`be cultured in vitro using knoWn techniques. Furthermore, the
`neW methods enable the long-term, e.g., permanent, reduc
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`Benitec - Exhibit 1003 - page 11
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`US 7,691,995 B2
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`5
`tion of targeted gene expression in cell lines, because siRNAs
`are transient, but a transgenic hairpin provides a long-lasting
`supply of siRNAs.
`The details of one or more embodiments of the invention
`are set forth in the accompanying draWings and the descrip
`tion beloW. Other features, objects, and advantages of the
`invention Will be apparent from the description and draWings,
`and from the claims.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a schematic diagram of the dual nature of the
`stRNA and siRNA pathWays.
`FIG. 2A is a schematic representation of a Wild-type,
`stRNA precursor (SEQ ID NO:1).
`FIGS. 2B to 2E are schematic representations of synthetic,
`engineered RNA precursors (SEQ ID NOS:2, 3, 4, and 5).
`FIG. 3 is an autoradiograph shoWing the results of an assay
`for determining Whether an engineered RNA precursor can
`promote cleavage of the target mRNA in vitro in a standard
`RNAi reaction.
`FIGS. 4A to 4C are schematic representations of synthetic
`luciferase siRNA (4A; SEQ ID NOS:6 and 7), and 5' and 3'
`synthetic, engineered RNA precursors (4B; SEQ ID NO:8;
`and 4C; SEQ ID NO:9).
`FIG. 4D is a schematic representation of a chimeric target
`mRNA f