US 20170307608A1
`( 19 ) United States
`( 12 ) Patent Application Publication ( 10 ) Pub . No . : US 2017 / 0307608 A1
`( 43 ) Pub . Date :
`Oct . 26 , 2017
`Bettencourt
`
`( 54 ) METHODS OF TREATING
`TRANSTHYRETIN ( TTR ) MEDIATED
`AMYLOIDOSIS
`( 71 ) Applicant : Alnylam Pharmaceuticals , Inc . ,
`Cambridge , MA ( US )
`( 72 ) Inventor : Brian Bettencourt , Groton , MA ( US )
`( 73 ) Assignee : Alnylam Pharmaceuticals , Inc . ,
`Cambridge , MA ( US )
`15 / 507 , 691
`Aug . 27 , 2015
`PCT / US2015 / 047185
`
`( 21 ) Appl . No . :
`( 22 ) PCT Filed :
`( 86 ) PCT No . :
`$ 371 ( c ) ( 1 ) ,
`Feb . 28 , 2017
`( 2 ) Date :
`Related U . S . Application Data
`( 60 ) Provisional application No . 62 / 044 , 100 , filed on Aug .
`29 , 2014 , provisional application No . 62 / 150 , 596 ,
`filed on Apr . 21 , 2015 .
`
`( 51 )
`
`( 52 )
`
`Publication Classification
`
`Int . CI .
`GOIN 33 / 566
`( 2006 . 01 )
`( 2006 . 01 )
`GOIN 33 / 567
`GOIN 33 / 50
`( 2006 . 01 )
`GOIN 33 / 53
`( 2006 . 01 )
`GOIN 33 / 68
`( 2006 . 01 )
`C07D 263 / 57
`( 2006 . 01 )
`( 2006 . 01 )
`COZK 1 / 00
`U . S . CI .
`CPC . . . . . . . GOIN 33 / 566 ( 2013 . 01 ) ; GOIN 33 / 6896
`( 2013 . 01 ) ; GOIN 33 / 567 ( 2013 . 01 ) ; C07D
`263 / 57 ( 2013 . 01 ) ; GOIN 33 / 50 ( 2013 . 01 ) ;
`GOIN 33 / 53 ( 2013 . 01 ) ; COZK 1 / 00 ( 2013 . 01 )
`
`( 57 )
`ABSTRACT
`Disclosed herein are methods for reducing or arresting an
`increase in
`a Neuropathy Impairment Score ( NIS ) or a
`modified NIS ( MNIS + 7 ) in a human subject by administer
`ing an effective amount of a transthyretin ( TTR ) - inhibiting
`composition .
`
`- - - -
`
`- -
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`- - - - - - - - - - - - - - - - - - - - -
`
`SULLIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
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`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
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`IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
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`* * * * * * * *
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`AMNIS + 7 ( 0 - 6 mos . )
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`14 DAVID
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`* * *
`
`V
`
`(
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`5000
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`15000
`10
`FIR AUC ( Day 0 - 1 82
`
`N = 25
`P = 0 . 007
`
`20000
`
`ARBUTUS - EXHIBIT 2012
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation
`IPR2019-00554
`
`

`

`Patent Application Publication
`
`Oct . 26 , 2017 Sheet 1 of 2
`
`US 2017 / 0307608 A1
`
`AMNIS + 7 ( 0 - 6 mos . )
`
`EN
`
`.
`
`ANTES
`
`IIIIIIIIIIIIIIIII
`
`.
`
`5000
`
`15000
`10000
`15000
`FIR AUc ( Day 9 - 482
`
`= 25
`N
`P = 0 . 007
`
`20000
`
`FIG . 1
`
`

`

`Patent Application Publication
`
`Oct . 26 , 2017 Sheet 2 of 2
`
`US 2017 / 0307608 A1
`
`20
`
`AMNIS + 7 ( 0 - 6 mos . )
`
`, ???
`
`??
`
`. . . . .
`
`. . . . .
`
`N = 27
`P = 0 . 04
`
`60 60
`Avg . Predose Trough [ TTR ) ( ng / mL ) , Days 84 + 168
`
`FIG . 2
`
`

`

`US 2017 / 0307608 A1
`
`Oct . 26 , 2017
`
`METHODS OF TREATING
`TRANSTHYRETIN ( TTR ) MEDIATED
`AMYLOIDOSIS
`CROSS - REFERENCE TO RELATED
`APPLICATIONS
`This application claims the benefit of and priority
`[ 0001 ]
`to U . S . Provisional Patent Application No . 62 / 044 , 100 , filed
`on Aug . 29 , 2014 and to U . S . Provisional Patent Application
`No . 62 / 150 , 596 , filed on Apr . 24 , 2015 , both which are
`incorporated by reference in their entirety for all purposes .
`BACKGROUND OF THE INVENTION
`Transthyretin ( TTR ) is a tetrameric protein pro
`[ 0002 ]
`duced primarily in the liver . Mutations in the TTR gene
`destabilize the protein tetramer , leading to misfolding of
`monomers and aggregation into TTR amyloid fibrils
`( ATTR ) . Tissue deposition results in systemic ATTR amy
`loidosis ( Coutinho et al . , Forty years of experience with type
`I amyloid neuropathy . Review of 483 cases . In : Glenner et
`al . , Amyloid and Amyloidosis , Amsterdam : Excerpta Media ,
`1980 pg . 88 - 93 ; Hou et al . , Transthyretin and familial
`amyloidotic polyneuropathy . Recent progress in understand
`ing the molecular mechanism of neurodegeneration . FEBS J
`2007 , 274 : 1637 - 1650 ; Westermark et al . , Fibril in senile
`systemic amyloidosis is derived from normal transthyretin .
`Proc Natl Acad Sci USA 1990 , 87 : 2843 - 2845 ) . Over 100
`reported TTR mutations exhibit a spectrum of disease symp
`toms .
`[ 0003 ] TTR amyloidosis manifests in various forms .
`When the peripheral nervous system is affected more promi
`nently , the disease is termed familial amyloidotic polyneu
`ropathy ( FAP ) . When the heart is primarily involved but the
`nervous system is not , the disease is called familial amy
`loidotic cardiomyopathy ( FAC ) . A third major type of TTR
`amyloidosis is called leptomeningeal / CNS ( Central Nervous
`System ) amyloidosis .
`[ 0004 ] The most common mutations associated with
`familial amyloid polyneuropathy ( FAP ) and ATTR - associ
`ated cardiomyopathy , respectively , are Val30Met ( Coelho et
`al . , Tafamidis for transthyretin familial amyloid polyneu
`ropathy : a randomized , controlled trial . Neurology 2012 , 79 :
`785 - 792 ) and Val1221le ( Connors et al . , Cardiac amyloidosis
`in African Americans : comparison of clinical and laboratory
`features of transthyretin V1221 amyloidosis and immuno
`globulin light chain amyloidosis . Am Heart J 2009 , 158 :
`607 - 614 ) .
`[ 0005 ] Current treatment options for FAP focus on stabi
`lizing or decreasing the amount of circulating amyloido
`genic protein . Orthotopic liver transplantation reduces
`mutant TTR levels ( Holmgren et al . , Biochemical effect of
`liver transplantation in two Swedish patients with familial
`amyloidotic polyneuropathy ( FAP - met30 ) . Clin Genet 1991 ,
`40 : 242 - 246 ) , with improved survival reported in patients
`with early - stage FAP , although deposition of wild - type TTR
`may continue ( Yazaki et al . , Progressive wild - type transthy
`retin deposition after liver transplantation preferentially
`occurs into myocardium in FAP patients . Am J Transplant
`2007 , 7 : 235 - 242 ; Adams et al . , Rapid progression of familial
`amyloid polyneuropathy : a multinational natural history
`study Neurology 2015 Aug . 25 ; 85 ( 8 ) 675 - 82 ; Yamashita et
`al . , Long - term survival after liver transplantation in patients
`with familial amyloid polyneuropathy . Neurology 2012 , 78 :
`
`637 - 643 ; Okamoto et al . , Liver transplantation for familial
`amyloidotic polyneuropathy : impact on Swedish patients '
`survival . Liver Transpl 2009 , 15 : 1229 - 1235 ; Stangou et al . ,
`Progressive cardiac amyloidosis following liver transplan
`tation for familial amyloid polyneuropathy : implications for
`amyloid fibrillogenesis . Transplantation 1998 , 66 : 229 - 233 ;
`Fosby et al . , Liver transplantation in the Nordic countries
`An intention to treat and post - transplant analysis from The
`Nordic Liver Transplant Registry 1982 - 2013 . Scand J Gas
`troenterol . 2015 June ; 50 ( 6 ) : 797 - 808 . Transplantation , in
`press ) .
`[ 0006 ]
`Tafamidis and diflunisal stabilize circulating TTR
`tetramers , which can slow the rate of disease progression
`( Berk et al . , Repurposing diflunisal for familial amyloid
`polyneuropathy : a randomized clinical trial . JAMA 2013 ,
`310 : 2658 - 2667 ; Coelho et al . , 2012 ; Coelho et al . , Long
`term effects of tafamidis for the treatment of transthyretin
`familial amyloid polyneuropathy . J Neurol 2013 , 260 : 2802
`2814 ; Lozeron et al . , Effect on disability and safety of
`Tafamidis in
`late onset of Met30 transthyretin familial
`amyloid polyneuropathy . Eur J Neurol 2013 , 20 : 1539
`1545 ) . However , symptoms continue to worsen on treatment
`in a large proportion of patients , highlighting the need for
`new , disease - modifying treatment options for FAP .
`[ 0007 ] Description of dsRNA targeting TTR can be found
`in , for example , International patent application no . PCT /
`US2009 / 061381 ( WO2010 / 048228 ) and International patent
`application no . PCT / US2010 / 055311 ( WO2011 / 056883 ) .
`SUMMARY
`[ 0008 ] Described herein are methods for reducing or
`arresting an increase in a Neuropathy Impairment Score
`( NIS ) or a modified NIS ( MNIS + 7 ) in a human subject by
`administering an effective amount of a transthyretin ( TTR )
`inhibiting composition , wherein the effective amount
`reduces a concentration of TTR protein in serum of the
`human subject to below 50 ug / ml or by at least 80 % . Also
`described herein are methods for adjusting a dosage of a
`TTR - inhibiting composition for treatment of increasing NIS
`or Familial Amyloidotic Polyneuropathy ( FAP ) by admin
`istering the TTR - inhibiting composition to a subject having
`the increasing NIS or FAP , and determining a level of TTR
`protein in the subject having the increasing NIS or FAP . In
`some embodiments , the amount of the TTR - inhibiting com
`position subsequently administered to the subject is
`increased if the level of TTR protein is greater than 50
`ug / ml , and the amount of the TTR - inhibiting composition
`subsequently administered to the subject is decreased if the
`level of TTR protein is below 50 ug / ml . Also described
`herein are formulated versions of a TTR inhibiting siRNA .
`BRIEF DESCRIPTION OF THE DRAWINGS
`[ 0009 ]
`FIG . 1 is a graph illustrating the relationship
`between progression in ANIS or AmNIS + 7 and TTR con
`centration .
`[ 0010 ] FIG . 2 is a graph illustrating the relationship
`between progression in ANIS or AmNIS + 7 and TTR con
`centration .
`
`DETAILED DESCRIPTION
`[ 0011 ] As described in more detail below , disclosed herein
`are methods for reducing or arresting an increase in
`a
`Neuropathy Impairment Score ( NIS ) or a modified NIS
`
`

`

`US 2017 / 0307608 A1
`
`Oct . 26 , 2017
`
`( mNIS + 7 ) in a human subject by administering an effective
`amount of a transthyretin ( TTR ) - inhibiting composition ,
`such that the effective amount reduces a concentration of
`TTR protein in serum to below 50 ug / ml or by at least 80 % .
`In one embodiment the TTR - inhibiting composition is pati
`siran . Patisiran is
`a small interfering ribonucleic acid
`( siRNA ) which is specific for TTR , formulated in a hepa
`totropic lipid nanoparticle ( LNP ) for intravenous ( IV )
`administration .
`[ 0012 ] TTR - Inhibiting Compositions
`[ 0013 ] The methods described herein include administra
`tion of TTR - inhibiting composition . A TTR - inhibiting com
`position can be any compound that reduces a concentration
`of TTR protein in the serum of a human subject . Examples
`include but are not limited to RNAi , e . g . , siRNA . Examples
`of siRNA include siRNA targeting a TTR gene , e . g . , patisirin
`( described in more detail ) below and revusiran . Examples
`also include antisense RNA . Examples of antisense RNA
`targeting a TTR gene can be found in U . S . Pat . No . 8 , 697 ,
`860 .
`[ 0014 ] The TTR - inhibiting composition inhibits expres
`sion of a TTR gene . As used herein , “ transthyretin ” ( “ TTR ” )
`refers to a gene in a cell . TTR is also known as ATTR ,
`HsT2651 , PALB , prealbumin , TBPA , and transthyretin ( pre
`albumin , amyloidosis type I ) . The sequence of a human TTR
`mRNA transcript can be found at NM _ 000371 . The
`sequence of mouse TTR mRNA can be found at
`NM _ 013697 . 2 , and the sequence of rat TTR mRNA can be
`found at NM _ 012681 . 1 .
`[ 0015 ] . The terms " silence , " " inhibit the expression of , ”
`" down - regulate the expression of , " " suppress the expression
`of " and the like in as far as they refer to a TTR gene , herein
`refer to the at least partial suppression of the expression of
`a TTR gene , as manifested by a reduction of the amount of
`mRNA which may be isolated from a first cell or group of
`cells in which a TTR gene is transcribed and which has or
`have been treated such that the expression of a TTR gene is
`inhibited , as compared to a second cell or group of cells
`substantially identical to the first cell or group of cells but
`which has or have not been so treated ( control cells ) . The
`degree of inhibition is usually expressed in terms of
`
`( mRNA in control cells ) - ( mRNA in treated cells )
`reated cells ) . 100 %
`( mRNA in control cells )
`
`[ 0016 ] Alternatively , the degree of inhibition may be
`given in terms of a reduction of a parameter that is func
`tionally linked to TTR gene expression , e . g . , the amount of
`protein encoded by a TTR gene which is secreted by a cell ,
`or the number of cells displaying a certain phenotype , e . g . ,
`apoptosis . In principle , TTR gene silencing may be deter
`mined in any cell expressing the target , either constitutively
`or by genomic engineering , and by any appropriate assay .
`However , when a reference is needed in order to determine
`whether a given dsRNA inhibits the expression of a TTR
`gene by a certain degree and therefore is encompassed by the
`instant invention , the assays provided in the Examples
`below shall serve as such reference .
`[ 0017 ] RNAi
`[ 0018 ]
`In some embodiments , the methods described
`herein use a TTR - inhibiting composition that is an RNAi ,
`e . g . , an siRNA , e . g . , a dsRNA for inhibiting the expression
`of a TTR gene . In one embodiment , the siRNA is a dsRNA
`
`that targets a TTR gene . The dsRNA includes an antisense
`strand having a region of complementarity which is comple
`mentary to at least a part of an mRNA formed in the
`expression of a TTR gene , and where the region of comple
`mentarity is less than 30 nucleotides in length , generally
`19 - 24 nucleotides in length . The dsRNA of the invention can
`further include one or more single - stranded nucleotide over
`hangs . TTR - inhibiting siRNAs are described in International
`patent application no . PCT / US2009 / 061381 ( WO2010 /
`048228 ) and International patent application no . PCT /
`US2010 / 055311 ( WO2011 / 056883 ) , both incorporated by
`reference herein in their entireties .
`[ 0019 ]
`In one embodiment , the TTR - inhibiting composi
`tion is patisiran , described in more detail below . In another
`embodiment , the TTR - inhibiting composition is revusiran ,
`an siRNA specific for TTR conjugated to a Trivalent Gal
`NAc carbohydrate cluster . A complete description of revu
`siran can be found in international application number
`PCT / US2012 / 065691 and US Patent Publication No .
`US20140315835 , the contents of which are incorporated by
`reference in their entirety .
`10020 ]
`A dsRNA includes two RNA strands that are suf
`ficiently complementary to hybridize to form
`a duplex
`structure . One strand of the dsRNA ( the antisense strand )
`includes a region of complementarity that is substantially
`complementary , and generally fully complementary , to a
`target sequence , derived from the sequence of an mRNA
`formed during the expression of a TTR gene , the other strand
`( the sense strand ) includes a region that is complementary to
`the antisense strand , such that the two strands hybridize and
`form a duplex structure when combined under suitable
`conditions . The term “ antisense strand ” refers to the strand
`of a dsRNA which includes a region that is substantially
`complementary to a target sequence . As used herein , the
`term “ region of complementarity ” refers to the region on the
`antisense strand that is substantially complementary to a
`sequence , for example a target sequence , as defined herein .
`Where the region of complementarity is not fully comple
`mentary to the target sequence , the mismatches are most
`tolerated in the terminal regions and , if present , are generally
`in a terminal region or regions , e . g . , within 6 , 5 , 4 , 3 , or 2
`nucleotides of the 5 ' and / or 3 ' terminus . The term " sense
`strand , ” as used herein , refers to the strand of a dsRNA that
`includes a region that is substantially complementary to a
`region of the antisense strand . Generally , the duplex struc
`ture is between 15 and 80 , or 15 and 60 or 15 and 30 or
`between 25 and 30 , or between 18 and 25 , or between 19 and
`24 , or between 19 and 21 , or 19 , 20 , or 21 base pairs in
`length . In one embodiment the duplex is 19 base pairs in
`length . In another embodiment the duplex is 21 base pairs in
`length .
`[ 0021 ] Each strand of a dsRNA is generally between 15
`and 80 or 15 and 60 or 15 and 30 , or between 18 and 25 , or
`18 , 19 , 20 , 21 , 22 , 23 , 24 , or 25 nucleotides in length . In
`other embodiments , each is strand is 25 - 30 nucleotides in
`length . Each strand of the duplex can be the same length or
`of different lengths . When two different siRNAs are used in
`combination , the lengths of each strand of each siRNA can
`be identical or can differ .
`[ 0022 ]
`A dsRNA can include one or more single - stranded
`overhang ( s ) of one or more nucleotides . In one embodiment ,
`at least one end of the dsRNA has a single - stranded nucleo
`tide overhang of 1 to 4 , generally 1 or 2 nucleotides . In
`another embodiment , the antisense strand of the dsRNA has
`
`

`

`US 2017 / 0307608 A1
`
`Oct . 26 , 2017
`
`1 - 10 nucleotides overhangs each at the 3 ' end and the 5 ' end
`over the sense strand . In further embodiments , the sense
`strand of the dsRNA has 1 - 10 nucleotides overhangs each at
`the 3 ' end and the 5 ' end over the antisense strand .
`[ 0023 ] As used herein , and unless otherwise indicated , the
`term “ complementary , " when used to describe a first nucleo
`tide sequence in relation to a second nucleotide sequence ,
`refers to the ability of an oligonucleotide or polynucleotide
`comprising the first nucleotide sequence to hybridize and
`form a duplex structure under certain conditions with an
`oligonucleotide or polynucleotide comprising the second
`nucleotide sequence , as will be understood by the skilled
`person . Such conditions can , for example , be stringent
`conditions , where stringent conditions may include : 400
`mM NaCl , 40 mM PIPES pH 6 . 4 , 1 mM EDTA , 50° C . or
`70° C . for 12 - 16 hours followed by washing . Other condi
`tions , such as physiologically relevant conditions as may be
`encountered inside an organism , can apply . The skilled
`person will be able to determine the set of conditions most
`appropriate for a test of complementarity of two sequences
`in accordance with the ultimate application of the hybridized
`nucleotides .
`[ 0024 ] This includes base - pairing of the oligonucleotide
`or polynucleotide comprising the first nucleotide sequence
`to the oligonucleotide or polynucleotide comprising the
`second nucleotide sequence over the entire length of the first
`and second nucleotide sequence . Such sequences can be
`referred to as “ fully complementary ” with respect to each
`other herein . However , where a first sequence is referred to
`as " substantially complementary ” with respect to a second
`sequence herein , the two sequences can be fully comple
`mentary , or they may form one or more , but generally not
`more than 4 , 3 , or 2 mismatched base pairs upon hybrid
`ization , while retaining the ability to hybridize under the
`conditions most relevant to their ultimate application . How
`ever , where two oligonucleotides are designed to form , upon
`hybridization , one or more single stranded overhangs , such
`overhangs shall not be regarded as mismatches with regard
`to the determination of complementarity . For example , a
`dsRNA comprising one oligonucleotide 21 nucleotides in
`length and another oligonucleotide 23 nucleotides in length ,
`wherein the longer oligonucleotide comprises a sequence of
`21 nucleotides that is fully complementary to the shorter
`oligonucleotide , may yet be referred to as “ fully comple
`mentary ” for the purposes described herein .
`10025 ]
`“ Complementary ” sequences , as used herein , may
`also include , or be formed entirely from , non - Watson - Crick
`base pairs and / or base pairs formed from non - natural and
`modified nucleotides , in as far as the above requirements
`with respect to their ability to hybridize are fulfilled . Such
`non - Watson - Crick base pairs includes , but not limited to ,
`G : U Wobble or Hoogstein base pairing .
`[ 0026 ] The terms “ complementary , ” “ fully complemen
`tary ” and “ substantially complementary ” herein may be used
`with respect to the base matching between the sense strand
`and the antisense strand of a dsRNA , or between the
`antisense strand of a dsRNA and a target sequence , as will
`be understood from the context of their use .
`100271 . As used herein , a polynucleotide that is " substan
`tially complementary to at least part of ” a messenger RNA
`( mRNA ) refers to a polynucleotide that is substantially
`complementary to a contiguous portion of the mRNA of
`interest ( e . g . , an mRNA encoding TTR ) including a 5 ' UTR ,
`an open reading frame ( ORF ) , or a 3 ' UTR . For example , a
`
`polynucleotide is complementary to at least a part of a TTR
`mRNA if the sequence is substantially complementary to a
`non - interrupted portion of an mRNA encoding TTR .
`[ 0028 ]
`A dsRNA can be synthesized by standard methods
`known in the art as further discussed below , e . g . , by use of
`an automated DNA synthesizer , such as are commercially
`available from , for example , Biosearch , Applied Biosys
`tems , Inc .
`[ 0029 ] Modified dsRNA
`[ 0030 ]
`In some embodiments , the dsRNA used in the
`methods described herein is chemically modified to enhance
`stability . The nucleic acids featured in the invention may be
`synthesized and / or modified by methods well established in
`the art , such as those described in
`“ Current protocols in
`nucleic acid chemistry , ” Beaucage , S . L . et al . ( Eds . ) , John
`Wiley & Sons , Inc . , New York , N . Y . , USA , which is hereby
`incorporated herein by reference . Specific examples of
`dsRNA compounds useful in this invention include dsRNAS
`containing modified backbones or no natural internucleoside
`linkages . As defined in this specification , dsRNAs having
`modified backbones include those that retain a phosphorus
`atom in the backbone and those that do not have a phos
`phorus atom in the backbone . For the purposes of this
`specification , and as sometimes referenced in the art , modi
`fied dsRNAs that do not have a phosphorus atom in their
`internucleoside backbone can also be considered to be
`oligonucleosides .
`[ 0031 ] Modified dsRNA backbones include , for example ,
`phosphorothioates , chiral phosphorothioates , phosphorodi
`thioates ,
`phosphotriesters ,
`aminoalkylphosphotriesters ,
`methyl and other alkyl phosphonates including 3 ' - alkylene
`phosphonates and chiral phosphonates , phosphinates , phos
`phoramidates including 3 ' - amino phosphoramidate and
`aminoalkylphosphoramidates ,
`thionophosphoramidates ,
`thionoalkylphosphonates , thionoalkylphosphotriesters , and
`boranophosphates having normal 3 ' - 5 ' linkages , 2 - 5 ' linked
`analogs of these , and those ) having inverted polarity
`wherein the adjacent pairs of nucleoside units are linked 3 ' - 5 '
`to 5 ' - 3 ' or 2 - 5 ' to 5 ' - 2 . Various salts , mixed salts and free
`acid forms are also included .
`[ 0032 ] Representative U . S . patents that teach the prepara
`tion of the above phosphorus - containing linkages include ,
`but are not limited to , U . S . Pat . Nos . 3 , 687 , 808 ; 4 , 469 , 863 ;
`4 , 476 , 301 ; 5 , 023 , 243 ; 5 , 177 , 195 ; 5 , 188 , 897 ; 5 , 264 , 423 ;
`5 , 276 , 019 ; 5 , 278 , 302 ; 5 , 286 , 717 ; 5 , 321 , 131 ; 5 , 399 , 676 ;
`5 , 405 , 939 ; 5 , 453 , 496 ; 5 , 455 , 233 ; 5 , 466 , 677 ; 5 , 476 , 925 ;
`5 , 519 , 126 ; 5 , 536 , 821 ; 5 , 541 , 316 ; 5 , 550 , 111 ; 5 , 563 , 253 ;
`5 , 571 , 799 ; 5 , 587 , 361 ; and 5 , 625 , 050 , each of which is
`herein incorporated by reference
`[ 0033 ] Modified dsRNA backbones that do not include a
`phosphorus atom therein have backbones that are formed by
`short chain alkyl or cycloalkyl internucleoside linkages ,
`mixed heteroatoms and alkyl or cycloalkyl internucleoside
`linkages , or ore or more short chain heteroatomic or hetero
`cyclic internucleoside linkages . These include those having
`morpholino linkages ( formed in part from the sugar portion
`of a nucleoside ) ; siloxane backbones ; sulfide , sulfoxide and
`sulfone backbones ; formacetyl and thioformacetyl back
`bones ; methylene formacetyl and thioformacetyl backbones ;
`alkene containing backbones ; sulfamate backbones ; meth
`yleneimino and methylenehydrazino backbones ; sulfonate
`and sulfonamide backbones ; amide backbones ; and others
`having mixed N , O , S and CH2 component parts .
`
`

`

`US 2017 / 0307608 A1
`
`Oct . 26 , 2017
`
`[ 0034 ] Representative U . S . patents that teach the prepara
`tion of the above oligonucleosides include , but are not
`limited to , U . S . Pat . Nos . 5 , 034 , 506 ; 5 , 166 , 315 ; 5 , 185 , 444 ;
`5 , 214 , 134 ; 5 , 216 , 141 ; 5 , 235 , 033 ; 5 , 64 , 562 ; 5 , 264 , 564 ;
`5 , 405 , 938 ; 5 , 434 , 257 ; 5 , 466 , 677 ; 5 , 470 , 967 ; 5 , 489 , 677 ;
`5 , 541 , 307 ; 5 , 561 , 225 ; 5 , 596 , 086 ; 5 , 602 , 240 ; 5 , 608 , 046 ;
`5 , 610 , 289 ;
`5 , 618 , 704 ; 5 , 623 , 070 ; 5 , 663 , 312 ; 5 , 633 , 360 ;
`5 , 677 , 437 ; and , 5 , 677 , 439 , each of which is herein incor
`porated by reference .
`10035 .
`In other suitable dsRNA mimetics , both the sugar
`and the internucleoside linkage , i . e . , the backbone , of the
`nucleotide units are replaced with novel groups . The base
`units are maintained for hybridization with an appropriate
`nucleic acid target compound . One such oligomeric com
`pound , a dsRNA mimetic that has been shown to have
`excellent hybridization properties , is referred to as a peptide
`nucleic acid ( PNA ) . In PNA compounds , the sugar backbone
`of a dsRNA is replaced with an amide containing backbone ,
`in particular an aminoethylglycine backbone . The nucle
`obases are retained and are bound directly or indirectly to
`aza nitrogen atoms of the amide portion of the backbone .
`Representative U . S . patents that teach the preparation of
`PNA compounds include , but are not limited to , U . S . Pat .
`Nos . 5 , 539 , 082 ; 5 , 714 , 331 ; and 5 , 719 , 262 , each of which is
`herein incorporated by reference . Further teaching of PNA
`compounds can be found in Nielsen et al . , Science , 1991 ,
`254 , 1497 - 1500 .
`[ 0036 ] Other embodiments of the invention are dsRNAS
`with phosphorothioate backbones and oligonucleosides with
`heteroatom backbones , and in particular — CH , — NH —
`CH , —
`— CH ,
`N ( CH2 ) — 0
`CH , —
`[ known as a meth
`ylene ( methylimino ) or MMI backbone ] , CH20 - N
`( CH2 ) - CH , — ,
`CH ,
`N ( CH2 ) - N ( CH2 ) - CH ,
`and
`- N ( CH3 ) CH2CH2 – [ wherein the native phosphodi
`ester backbone is represented as O
`P
`- O
`CH - ] of
`the above - referenced U . S . Pat . No . 5 , 489 , 677 , and the amide
`backbones of the above - referenced U . S . Pat . No . 5 , 602 , 240 .
`Also preferred are dsRNAs having morpholino backbone
`structures of the above - referenced U . S . Pat . No . 5 , 034 , 506 .
`[ 0037 ] Modified dsRNAs may also contain one or more
`substituted sugar moieties . Preferred dsRNAs comprise one
`of the following at the 2 ' position : OH ; F ; O - , S - , or N - alkyl ;
`0 - , S - , or N - alkenyl ; 0 - , S - or N - alkynyl ; or O - alkyl - O
`alkyl , wherein the alkyl , alkenyl and alkynyl may be sub
`stituted or unsubstituted C , to C10 alkyl or C2 to C10 alkenyl
`and alkynyl . Particularly preferred are O [ ( CH2 ) , O ] mCHz ,
`O ( CH2 ) , OCH3 ,
`O ( CH2 ) „ NH2 ,
`O ( CH2 ) , CH3 ,
`O ( CH2 )
`FONH2 , and O ( CH2 ) ON [ ( CH2 ) , CH3 ) 2 , where n and m are
`from 1 to about 10 . Other preferred dsRNAs comprise one
`of the following at the 2 ' position : C , to C10 lower alkyl ,
`substituted lower alkyl , alkaryl , aralkyl ,
`O - alkaryl or
`O - aralkyl , SH , SCH3 , OCN , C1 , Br , CN , CF3 , OCF3 ,
`SOCH3 , SO CH3 , ONO2 , NO2 , N3 , NH2 , heterocycloalkyl ,
`heterocycloalkaryl , aminoalkylamino , polyalkylamino , sub
`stituted silyl , an RNA cleaving group , a reporter group , an
`intercalator , a group for improving the pharmacokinetic
`properties of an dsRNA , or a group for improving the
`pharmacodynamic properties of an dsRNA , and other sub
`stituents having similar properties . A preferred modification
`includes 2 ' - methoxyethoxy ( 2 - 0 CH2CH2OCHz , also
`known as 2 - O - ( 2 - methoxyethyl ) or 2 ' - MOE ) ( Martin et al . ,
`Helv . Chim . Acta , 1995 , 78 , 486 - 504 ) i . e . , an alkoxy - alkoxy
`group . A further preferred modification includes 2 ' - dimeth
`ylaminooxyethoxy , i . e . , a O ( CH2 ) 2ON ( CH3 ) 2 group , also
`
`known as 2 - DMAOE , as described in examples herein
`below , and 2 - dimethylaminoethoxyethoxy ( also known in
`the art as 2 - 0 - dimethylaminoethoxyethyl or 2 - DMAEOE ) ,
`i . e . , 2 - 0 - CH2 - 0 CH N ( CH2 ) 2 , also described in
`examples herein below .
`[ 0038 ] Other preferred modifications include 2 ' - methoxy
`( 2 ' - OCH3 ) , 2 ' - aminopropoxy ( 2 ' - OCH CH CH _ NH ) and
`2 ' - fluoro ( 2 ' - F ) . Similar modifications may also be made at
`other positions on the dsRNA , particularly the 3 ' position of
`the sugar on the 3 ' terminal nucleotide or in 2 - 5 ' linked
`dsRNAs and the 5 ' position of 5 ' terminal nucleotide .
`DsRNAs may also have sugar mimetics such as cyclobutyl
`moieties in place of the pentofuranosyl sugar . Representa
`tive U . S . patents that teach the preparation of such modified
`sugar structures include , but are not limited to , U . S . Pat .
`Nos . 4 , 981 , 957 ; 5 , 118 , 800 ; 5 , 319 , 080 ; 5 , 359 , 044 ; 5 , 393 ,
`878 ; 5 , 446 , 137 ; 5 , 466 , 786 ; 5 , 514 , 785 ; 5 , 519 , 134 ; 5 , 567 ,
`811 ; 5 , 576 , 427 ; 5 , 591 , 722 ; 5 , 597 , 909 ; 5 , 610 , 300 ; 5 , 627 ,
`053 ; 5 , 639 , 873 ; 5 , 646 , 265 ; 5 , 658 , 873 ; 5 , 670 , 633 ; and
`5 , 700 , 920 , certain of which are commonly owned with the
`instant application , and each of which is herein incorporated
`by reference in its entirety .
`[ 0039 ] DsRNAs may also include nucleobase ( often
`referred to in the art simply as " base " ) modifications or
`substitutions . As used herein , " unmodified ” or “ natural ”
`nucleobases include the purine bases adenine ( A ) and gua
`nine ( G ) , and the pyrimidine bases thymine ( T ) , cytosine ( C )
`and uracil ( U ) . Modified nucleobases include other synthetic
`and natural nucleobases such as 5 - methylcytosine ( 5 - me - C ) ,
`5 - hydroxymethyl cytosine ,
`xanthine ,
`hypoxanthine ,
`2 - aminoadenine , 6 - methyl and other alkyl derivatives of
`adenine and guanine , 2 - propyl and other alkyl derivatives of
`adenine and guanine , 2 - thiouracil , 2 - thiothymine and 2 - thio
`cytosine , 5 - halouracil and cytosine , 5 - propynyl uracil and
`cytosine , 6 - azo uracil , cytosine and thymine , 5 - uracil
`( pseudouracil ) , 4 - thiouracil , 8 - halo , 8 - amino , 8 - thiol , 8 - thio
`alkyl , 8 - hydroxyl anal other 8 - substituted adenines and
`guanines , 5 - halo , particularly 5 - bromo , 5 - trifluoromethyl
`and other 5 - substituted uracils and cytosines , 7 - methylgua
`nine and 7 - methyladenine , 8 - azaguanine and 8 - azaadenine ,
`7 - deazaguanine and 7 - daazaadenine and 3 - deazaguanine
`and 3 - deazaadenine . Further nucleobases include those dis
`closed in U . S . Pat . No . 3 , 687 , 808 , those disclosed in The
`Concise Encyclopedia Of Polymer Science And Engineer
`ing , pages 858 - 859 , Kroschwitz , J . L , ed . John Wiley &
`Sons , 1990 , these disclosed by Englisch et al . , Angewandte
`Chemie , International Edition , 1991 , 30 , 613 , and those
`disclosed by Sanghvi , Y S . , Chapter 15 , DsRNA Research
`and Applications , pages 289 - 302 , Crooke , S . T . and Lebleu ,
`B . , Ed . , CRC Press , 1993 . Certain of these nucleobases are
`particularly useful for increasing the binding affinity of the
`oligomeric compounds featured in the invention . These
`include 5 - substituted pyrimidines , 6 - azapyrimidines and
`N - 2 , N - 6 and 0 - 6 substituted purines , including 2 - amino
`propyladenine , 5 - propynyluracil and 5 - propynylcytosine .
`5 - methylcytosine substitutions have been shown to increase
`nucleic acid duplex stability by 0 . 6 - 1 . 2° C . ( Sanghvi , Y . S . ,
`Crooke , S . T . and Lebleu , B . , Eds . , DsRNA Research and
`Applications , CRC Press , Boca Raton , 1993 , pp . 276 - 278 )
`and are exemplary base substitutions , even more particularly
`when combined with 2 - O - methoxyethyl sugar modifica
`tions .
`[ 0040 ] Representative U . S . patents that teach the prepara
`tion of certain of t