(12) United States Patent
`Fire et al.
`
`US006506559B1
`US 6,506,559 B1
`*Jan. 14, 2003
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`(54)
`
`(75)
`
`(73)
`
`GENETIC INHIBITION BY
`DOUBLE-STRANDED RNA
`
`Inventors: Andrew Fire, Baltimore, MD (US);
`Stephen Kostas, Chicago, IL (US);
`Mary Montgomery, St. Paul, MN
`(US); Lisa Timmons, Lawrence, KS
`(US); SiQun Xu, BallWin, MO (US);
`Hiroaki Tabara, Shizuoka (JP);
`Samuel E. Driver, Providence, RI
`(US); Craig C. Mello, ShreWsbury, MA
`(Us)
`Assignee: Carnegie Institute of Washington,
`Washington, DC (US)
`
`(*)
`
`Notice:
`
`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)
`(22)
`
`(60)
`
`(51)
`(52)
`(58)
`
`(56)
`
`Appl. No.: 09/215,257
`Filed:
`Dec. 18, 1998
`
`Related US. Application Data
`Provisional application No. 60/068,562, ?led on Dec. 23,
`1997.
`
`Int. Cl.7 ......................... .. C12Q 1/68; C12N 15/85
`US. Cl. ......................... .. 435/6; 435/91.1; 435/325
`Field of Search .............................. .. 514/44; 435/6,
`435/91.1, 325
`
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`Primary Examiner—AndreW Wang
`Assistant Examiner—Karen A Lacourciere
`(74) Attorney, Agent, or Firm—Morgan, LeWis & Bockius
`LLP
`(57)
`
`ABSTRACT
`
`A process is provided of introducing an RNA into a living
`cell to inhibit gene expression of a target gene in that cell.
`The process may be practiced ex vivo or in vivo. The RNA
`has a region With double-stranded structure. Inhibition is
`sequence-speci?c in that the nucleotide sequences of the
`duplex region of the RNA and of a portion of the target gene
`are identical. The present invention is distinguished from
`prior art interference in gene expression by antisense or
`triple-strand methods.
`
`22 Claims, 5 Drawing Sheets
`
`HUD-54
`
`zmllFmllllmi u
`
`MITOCHONDRIAL
`
`Benitec - Exhibit 1006 - page 1
`
`

`
`US 6,506,559 B1
`Page 2
`
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`* cited by examiner
`
`Benitec - Exhibit 1006 - page 4
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`

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`U.S. Patent
`
`Jan. 14, 2003
`
`Sheet 1 0f 5
`
`US 6,506,559 B1
`
`96m
`
`£3
`
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`U.S. Patent
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`Sheet 2 0f 5
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`US 6,506,559 B1
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`U.S. Patent
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`Jan. 14, 2003
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`Sheet 3 0f 5
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`US 6,506,559 B1
`
`Benitec - Exhibit 1006 - page 7
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`

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`U.S. Patent
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`Jan. 14, 2003
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`US 6,506,559 B1
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`Benitec - Exhibit 1006 - page 8
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`U.S. Patent
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`Jan. 14, 2003
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`Sheet 5 0f 5
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`US 6,506,559 B1
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`

`
`US 6,506,559 B1
`
`1
`GENETIC INHIBITION BY
`DOUBLE-STRANDED RNA
`
`RELATED APPLICATION
`
`This application claims the bene?t of US. Provisional
`Appln. No. 60/068,562, ?led Dec. 23, 1997. +gi
`
`GOVERNMENT RIGHTS
`
`This invention Was made With US. government support
`under grant numbers GM-37706, GM-17164, HD-33769
`and GM-07231 aWarded by the National Institutes of Health.
`The US. government has certain rights in the invention.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`15
`
`2
`cell. Some dif?culties With antisense-based approaches
`relate to delivery, stability, and dose requirements. In
`general, cells do not have an uptake mechanism for single
`stranded nucleic acids, hence uptake of unmodi?ed single
`stranded material is extremely inef?cient. While Waiting for
`uptake into cells, the single-stranded material is subject to
`degradation. Because antisense interference requires that the
`interfering material accumulate at a relatively high concen
`tration (at or above the concentration of endogenous
`mRNA), the amount required to be delivered is a major
`constraint on ef?cacy. As a consequence, much of the effort
`in developing antisense technology has been focused on the
`production of modi?ed nucleic acids that are both stable to
`nuclease digestion and able to diffuse readily into cells. The
`use of antisense interference for gene therapy or other
`Whole-organism applications has been limited by the large
`amounts of oligonucleotide that need to be synthesiZed from
`non-natural analogs, the cost of such synthesis, and the
`dif?culty even With high doses of maintaining a suf?ciently
`concentrated and uniform pool of interfering material in
`each cell.
`
`Triple-Helix Approaches to Engineer Interference
`A second, proposed method for engineered interference is
`based on a triple helical nucleic acid structure. This
`approach relies on the rare ability of certain nucleic acid
`populations to adopt a triple-stranded structure. Under
`physiological conditions, nucleic acids are virtually all
`single- or double-stranded, and rarely if ever form triple
`stranded structures. It has been knoWn for some time,
`hoWever, that certain simple purine- or pyrimidine-rich
`sequences could form a triple-stranded molecule in vitro
`under extreme conditions of pH (i.e., in a test tube). Such
`structures are generally very transient under physiological
`conditions, so that simple delivery of unmodi?ed nucleic
`acids designed to produce triple-strand structures does not
`yield interference. As With antisense, development of triple
`strand technology for use in vivo has focused on the devel
`opment of modi?ed nucleic acids that Would be more stable
`and more readily absorbed by cells in vivo. An additional
`goal in developing this technology has been to produce
`modi?ed nucleic acids for Which the formation of triple
`stranded material proceeds effectively at physiological pH.
`
`Co-Suppression Phenomena and Their Use in
`Genetic Engineering
`A third approach to gene-speci?c interference is a set of
`operational procedures grouped under the name “co
`suppression”. This approach Was ?rst described in plants and
`refers to the ability of transgenes to cause silencing of an
`unlinked but homologous gene. More recently, phenomena
`similar to co-suppression have been reported in tWo animals:
`C. elegans and Drosophila. Co-suppression Was ?rst
`observed by accident, With reports coming from groups
`using transgenes in attempts to achieve over-expression of a
`potentially useful locus. In some cases the over-expression
`Was successful While, in many others, the result Was oppo
`site from that expected. In those cases, the transgenic plants
`actually shoWed less expression of the endogenous gene.
`Several mechanisms have so far been proposed for
`transgene-mediated co-suppression in plants; all of these
`mechanistic proposals remain hypothetical, and no de?nitive
`mechanistic description of the process has been presented.
`The models that have been proposed to explain
`co-suppression can be placed in tWo different categories. In
`one set of proposals, a direct physical interaction at the
`DNA- or chromatin-level betWeen tWo different chromo
`somal sites has been hypothesiZed to occur; an as-yet
`unidenti?ed mechanism Would then lead to de novo methy
`lation and subsequent suppression of gene expression.
`
`20
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`35
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`45
`
`1. Field of the Invention
`The present invention relates to gene-speci?c inhibition of
`gene expression by double-stranded ribonucleic acid
`(dsRNA).
`2. Description of the Related Art
`Targeted inhibition of gene expression has been a long
`felt need in biotechnology and genetic engineering.
`Although a major investment of effort has been made to
`achieve this goal, a more comprehensive solution to this
`problem Was still needed.
`Classical genetic techniques have been used to isolate
`mutant organisms With reduced expression of selected
`genes. Although valuable, such techniques require laborious
`mutagenesis and screening programs, are limited to organ
`isms in Which genetic manipulation is Well established (e.g.,
`the existence of selectable markers, the ability to control
`genetic segregation and sexual reproduction), and are lim
`ited to applications in Which a large number of cells or
`organisms can be sacri?ced to isolate the desired mutation.
`Even under these circumstances, classical genetic tech
`niques can fail to produce mutations in speci?c target genes
`of interest, particularly When complex genetic pathWays are
`involved. Many applications of molecular genetics require
`the ability to go beyond classical genetic screening tech
`niques and ef?ciently produce a directed change in gene
`expression in a speci?ed group of cells or organisms. Some
`such applications are knoWledge-based projects in Which it
`is of importance to understand What effects the loss of a
`speci?c gene product (or products) Will have on the behavior
`of the cell or organism. Other applications are engineering
`based, for example: cases in Which is important to produce
`a population of cells or organisms in Which a speci?c gene
`product (or products) has been reduced or removed. A
`further class of applications is therapeutically based in
`Which it Would be valuable for a functioning organism (e.g.,
`a human) to reduce or remove the amount of a speci?ed gene
`product (or products). Another class of applications provides
`a disease model in Which a physiological function in a living
`organism is genetically manipulated to reduce or remove a
`speci?c gene product (or products) Without making a per
`manent change in the organism’s genome.
`In the last feW years, advances in nucleic acid chemistry
`and gene transfer have inspired neW approaches to engineer
`speci?c interference With gene expression. These
`approaches are described beloW.
`
`55
`
`Use of Antisense Nucleic Acids to Engineer
`Interference
`
`Antisense technology has been the most commonly
`described approach in protocols to achieve gene-speci?c
`interference. For antisense strategies, stochiometric amounts
`of single-stranded nucleic acid complementary to the mes
`senger RNA for the gene of interest are introduced into the
`
`60
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`Benitec - Exhibit 1006 - page 10
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`

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`US 6,506,559 B1
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`3
`Alternatively, some have postulated an RNA intermediate,
`synthesized at the transgene locus, Which might then act to
`produce interference With the endogenous gene. The char
`acteristics of the interfering RNA, as Well as the nature of the
`interference process, have not been determined. Recently, a
`set of experiments With RNA viruses have provided some
`support for the possibility of RNA intermediates in the
`interference process. In these experiments, a replicating
`RNA virus is modi?ed to include a segment from a gene of
`interest. This modi?ed virus is then tested for its ability to
`interfere With expression of the endogenous gene. Initial
`results With this technique have been encouraging, hoWever,
`the properties of the viral RNA that are responsible for
`interference effects have not been determined and, in any
`case, Would be limited to plants Which are hosts of the plant
`virus.
`
`10
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`15
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`Distinction BetWeen the Present Invention and
`Antisense Approaches
`
`4
`gene expression. Viral-mediated co-suppression in plants
`appears to be quite effective, but has a number of draWbacks.
`First, it is not clear What aspects of the viral structure are
`critical for the observed interference. Extension to another
`system Would require discovery of a virus in that system
`Which Would have these properties, and such a library of
`useful viral agents are not available for many organisms.
`Second, the use of a replicating virus Within an organism to
`effect genetic changes (e.g., long- or short-term gene
`therapy) requires considerably more monitoring and over
`sight for deleterious effects than the use of a de?ned nucleic
`acid as in the present invention.
`The present invention avoids the disadvantages of the
`previously-described methods for genetic interference. Sev
`eral advantages of the present invention are discussed beloW,
`but numerous others Will be apparent to one of ordinary skill
`in the biotechnology and genetic engineering arts.
`
`SUMMARY OF THE INVENTION
`
`The present invention differs from antisense-mediated
`interference in both approach and effectiveness. Antisense
`mediated genetic interference methods have a major chal
`lenge: delivery to the cell interior of speci?c single-stranded
`nucleic acid molecules at a concentration that is equal to or
`greater than the concentration of endogenous mRNA.
`Double-stranded RNA-mediated inhibition has advantages
`both in the stability of the material to be delivered and the
`concentration required for effective inhibition. BeloW, We
`disclose that in the model organism C. elegans, the present
`invention is at least 100-fold more effective than an equiva
`lent antisense approach (i.e., dsRNA is at least 100-fol