Wl PR586A
`V.93
`N0.23
`1996
`C.01-~------SEQ: P39620000
`Tl: PROCEED[NGS OF THE
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`tl/18/96
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`

`Proc. Natl. Acad. Sci. USA
`Vol. 93, pp. 12840-12844, November 1996
`Biochemistry
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`y
`' Repair of thalassemic human /3-globin m~NA in mammalian cells
`-c-_ ... _,,,_
`by ant1sense ohgonucleot1desj
`.
`~
`(RNA splicing/gene therapy)
`
`•
`
`•
`
`•
`
`..___
`
`HALINA SIERAKOWSKA*t, MARIA J. SAMBADE*, Sum-rm AGRAWAL:J:, AND RYSZARD KOLE*§
`•tincbcrger Comprehensive Cancer Ccntci}and Department of Pharmacology,)ynivcrsity or North Carolina, Chapel 1-;iiJ, NCf27599; \,ml i1 lybridon, Inc.,
`Worcester, MA 01605
`1_.
`·-
`J (
`:. / ~; /\ r)
`
`Communicated by Y W Kan, University of California, San fi'(//zcisco, CA, September 3, 1996 (received for reFiew April 4, 1996)
`
`ABSTRACT
`r;n one form of (3-thalassemia, a genetic blood
`clisorcler, a mutation in intron 2 of the {3-globin gene (IVS2-
`654) causes aberrant splicing of {3-globin pre-mRNA and,
`consequently, (3-globin deficiency. Treatment of mammalian
`cells stably expressing the IVS2-654 human {3-globin gene
`with antisense oligonucleoticles targeted at the aberrant splice
`sites restored correct splicing in a dose-dependent fashion,
`generating correct human {3-globin mRNA and polypeptide.
`Both products persisted for up to 72 hr posttreatment. The
`oligonucleotides modified splicing by a true antisense mech(cid:173)
`anism without overt unspecific effects on cell growth and
`splicing of other pre-mRNAs. This novel approach in which
`antisense oligonucleotides are used to restore rather than to
`clown-regulate the activity of the target gene is applicable to
`other splicing mutants and is of potential clinical interes:J
`
`/3-Thalasscmia, a genetic blood disorder, affects a large num(cid:173)
`ber of people in the Mediterranean basin, Middle East, South
`East Asia, and Africa. Close to 100 thalassemic mutations
`causing defective /3-globin gene expression and /3-globin defi(cid:173)
`ciency have been identified, but no more than 10 mutations are
`responsible for =90% of cases worldwide (1). Of the fre(cid:173)
`quently occurring mutations, the ones that cause aberrant
`splicing of intron 1 of the human /3-globin gene are predom(cid:173)
`inant in South Eastern Europe, Cyprus, Lebanon (mutation
`IVSl-110), India, Malaysia, and Indonesia (IVSl-5). Addi(cid:173)
`tional splicing mutations in intron 1 (IVSl-6) as well as in
`intron 2 of the {:l-globin gene (IVS2-745) are also common in
`the above countries, while IVS2-654 is frequent among
`/3-thalassemia patients in China and Thailand (1-8). All of
`these mutations activate aberrant splice sites and change the
`splicing pathway even though the correct splice sites remain
`potentially functional. We hypothesized that blocking of the
`aberrant splice sites or other sequence elements involved in
`splicing with antisense oligonuclcotides may force the splicing
`machinery to reselect the correct splice sites and induce the
`formation of {:l-globin mRNA and polypeptide, hence restoring
`the gene function.
`Although we have previously effected correction of splicing
`by antisense oligonucleotidcs in cell-free extracts from HeLa
`cells (9), it was not at all clear whether the oligonucleotides
`delivered into the cell could enter the nucleus, hybridize to the
`aberrant splice sites in competition with the splicing factors,
`and promote the formation of the spliceosome and subsequent
`splicing at the correct splice site. Here we report that correct
`splicing was efficiently restored when phosphorothioate 2' -0-
`rnethyl-oligoribonucleotides were targeted to the aberrant
`splice sites of IVS2-654 pre-mRNA expressed in mammalian
`cells stably transformed with this mutated human /3-globin
`gene. This is a novel approach since antiscnse oligonucleotidcs
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertiJemelll" in
`accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`have been used mostly as sequence specific down-regulators of
`gene expression ( 10).
`
`MATERIALS AND METHODS
`Cells. Human /3-globin gene carrying a thalassemic mutation
`IVS2-654 was cloned under the cytomcgalovirus promoter
`(11 ). The plasmid was cotransfccted with a neomycin resis(cid:173)
`tance plasmid by lipofection with Lipofectamine (GIBCO/
`BRL) into HeLa and NIH 3T3 cells, and the cells stably
`expressing the IVS2-654 /3-globin gene were isolated by G-418
`antibiotic selection. Control cells expressing the wild-type gene
`were obtained in a similar manner. HeLa and NIH 3T3 cell
`lines were grown in MEM supplemented with 5% fetal calf and
`5% horse scra and in DMEM, high glucose, with 10% filtered
`Colorado calf serum, respectively. For all experiments, cells
`were plated in 24-well plates at 105 cells per well 24 hr before
`treatment.
`Oligonucleoticle Treatment. The phosphorothioate 2' -0-
`methyl-oligoribonucleotides (prepared and purified at Hybri(cid:173)
`don) were used. The cells were treated with oligonucleotides
`complexed with Lipofectamine for 10 and 6 hr for HeLa and
`NIH 3T3 cell lines, respectively (12, 13). In Figs. 1 Band C, 3A,
`and 4B, the cells were harvested 36 hr later and were subse(cid:173)
`quently analyzed. The oligonucleotides 5'ss-GCUAUUAC(cid:173)
`CUUAACCCAG and 3'ss-CAUUAUUGCCCUGAAAG
`were targeted to the aberrant 5' splice site and the 3' cryptic
`splice site, respectively. Oligonucleotides with random or
`scrambled sequences were used as controls. An oligonucleo(cid:173)
`tide, CCUCUUACCUCAGUUACA, targeted to positions
`696-713 of {:l-globin intron 2, encompassing thalassemic mu(cid:173)
`tation I VS2-705 (8), was used as an additional control.
`RNA Analysis. Total RNA was isolated with TRI-Reagent
`(Molecular Research Center, Cincinnati) and analyzed by
`reverse transcription-PCR (RT-PCR) using rTth DNA poly(cid:173)
`merase as suggested by the manufacturer (Perkin-Elmer).
`Forward and reverse primers spanned positions 21-43 of exon
`2 of the human {3-globin gene and positions 6-28 of cxon 3,
`respectively. The RT-PCR products were separated on 7.5%
`nondenaturing polyacrylamidc gel. To ascertain that the pro(cid:173)
`tocol is suitable for quantitative analysis, the RT-PCR was
`carried out with [ a-32P]dA TP for no more than 18-20 cycles.
`Under these conditions, the amount of the PCR product is
`proportional to the amount of input RNA as are the relative
`amounts of PCR products generated from aberrantly and
`correctly spliced RNA (ref. 14 and data not shown). No
`product is detectable without the reverse transcription step.
`Protein Analysis. Hemin (10 µM, Fluka) treatment was in
`serum-free medium for 4 hr immediately preceding the isola-
`
`Abbreviation: RT-PCR, reverse transcription-PCR.
`-ton leave from: The Institute of Biochemistry and Biophysics, War(cid:173)
`saw, Poland.
`~To whom reprint requests should be addressed at: Lineberger Com(cid:173)
`prehensive Cancer Center, University of North Carolina, CB# 7295,
`Chapel Hill, NC 27599. e-mail: kole@mcd.unc.edu.
`
`12840
`
`

`

`Biochemistry: Sierakowska et al.
`
`Proc. Natl. A cad. Sci. USA 93 ( 1996)
`
`12841
`
`A
`
`ITITI-----ITJ
`
`-'+oligo
`
`2
`
`3
`
`B
`
`5'ss
`
`5'ssH
`
`control
`
`T ~~~~ BgjRF-4 304
`
`• - · - - - o _ l== j ~ 2 3 1
`_
`1 2 3 4 5 6 7 8 9 10 11 12 13 14
`
`IW
`
`tion of RNA or protein. Blots of proteins separated on a
`Tricinc-SDS/ I 0% polyacrylamidc gel (15) were incubated
`with polyclonal affinity-purified chicken anti-human hemoglobin
`IgG as primary antibody and rabbit anti-chicken horseradish
`peroxidase-conjugated IgG as secondary antibody (Accurate
`Chemicals). Subsequently, the blots were developed with the
`Enhanced Chemiluminescence detection system (Amersham).
`All autoradiograms were captured by a DAGE-MTI (Mich(cid:173)
`igan City, IN) CCD72 video camera, and the images were
`processed using National Institutes of Health IMAGE 1.47 and
`MACDRAW PRO 1.0 software. The IMAGE 1.47 was also used for
`quantitation of the autoradiograms. The final figures were
`printed on Tektronix phaser 440 printer.
`
`RESULTS
`Since appropriate cellular or animal models of thalasscmic
`splice mutants arc not available, we have constructed two cell
`lines stably transformed with the I VS2-654 variant of the
`thalasscmic human /3-globin gene. In a HeLa-bascd cell line,
`as in thalasscmic patients (1, 3, 4), this mutation created a 5'
`splice site at nucleotide 652 of intron 2 and activated a 3'
`cryptic splice site 73 nucleotides upstream, resulting in stably
`expressed but aberrantly spliced IVS2-654 /3-globin pre(cid:173)
`mRNA (Fig. I A and B, lane 2). To restore correct splicing of
`the RNA, the cells were treated for JO hr with a complex of
`Lipofcctamine and the 18-mer phosphorothioate 2' -O-mcthyl(cid:173)
`oligoribonuclcotide (5'ss) targeted to the aberrant 5' splice
`site. The 2' -O-methyl derivatives were chosen since they
`hybridize well to their target sequences and arc very stable in
`cellular environment. Moreover, importantly, in contrast to
`commonly used oligodcoxynucleotidcs or phosphorothioate
`oligodcoxynuclcotides, they do not promote cleavage of hy(cid:173)
`bridized RNA by cellular RNase I--1 (17). The latter property is
`the key condition for the success of the experiments since
`treatment with an unmethylatcd oligonucleotide would have
`led to degradation of the /3-globin pre-mRNA and removal of
`the splicing substrate (10, 18).
`Fig. 1B shows that treatment with 2' -O-methyl phosphoro(cid:173)
`thioates was effective in blocking the aberrant splice site and
`restoring correct splicing of /3-globin pre-mRNA. Quantitative
`RT-PCR analysis (ref. 14; sec Materials and Methods) of the
`RNA showed that the amount of correctly spliced /3-globin
`mRNA increased in a dose-dependent fashion, and at 0.05, 0.1,
`and 0.2 µM oligonucleotide reached, respectively, 16, 24, and
`34% of the total (Fig. 1B, lanes 3-5 and Table 1). There was
`no further increase in the correctly spliced product at 0.4 µM
`oligonucleotide (33% ), while treatment at 0.6 µM oligonucle(cid:173)
`otide drastically lowered its amount (Fig. lB, lanes 6 and 7,
`respectively). The latter result is possibly due to the fact that
`the ratio of Lipofectamine:nuclcic acid deviated from a narrow
`range necessary for efficient cellular uptake of the complex
`( 13). The effect of the antisense oligonucleotide was sequence(cid:173)
`dependent since control oligonucleotides either with random
`or with scrambled sequences (Fig. lB, lanes 11-13) did not
`restore correct splicing. Somewhat weaker correction of ab(cid:173)
`errant splicing of IVS2-654 pre-mRNA (11 % ) was obtained
`when the cells were treated with a 17-mer oligonucleotide
`antisense to the 3' cryptic splice site activated by the IVS2-654
`mutation (see Table 1). Note that in untreated (Fig. lB, lane
`lB, lanes 11-13) cells, there was no
`2) or control (Fig.
`detectable PCR product representing the correctly spliced
`/3-globin mRNA. Therefore, in both 5'ss and 3'ss oligonucle(cid:173)
`otidc-treated cells the /3-globin mRNA must have been spliced
`de nova and the observed band could not have resulted from
`preferential RT-PCR amplification of a preexisting shorter,
`correctly spliced mRNA.
`Analysis of the total protein from oligonucleotide-trcated
`cells by immunoblotting with polyclonal antibody to human
`hemoglobin showed that the newly generated, correctly spliced
`
`C
`WT
`
`1
`
`S'ss
`
`0
`
`.05
`
`.1
`
`.2
`
`.4
`
`C :c
`0
`.6 0)
`
`-
`
`IVS2-654
`
`+hemin
`
`......... _ ... , -_. -I B-globln
`- --- -
`I B-globin
`----
`
`IVS2-654
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`(A) Splicing of human /3-globin IVS2-654 pre-mRNA in the
`F10. 1.
`presence of an antisense oligonucleotide. Boxes, exons; solid lines,
`ii1trons; dashed lines, both correct and aberrant splicing pathways;
`thick bar, oligonucleotide antisense to the aberrant 5' splice site; thin
`bars above and below exon sequences, primers used in the RT-PCR
`reaction. The aberrant 5' splice site created by IVS2-654 mutation and
`the cryptic 3' splice site activated upstream are indicated. (B) Cor(cid:173)
`rection of splicing of IVS2-654 pre-mRNA in HeLa cells by antisense
`oligonucleotide targeted to the aberrant 5 ' splice site (5'ss). Analysis
`of total RNA by RT-PCR. Lanes: I, wild-type (WT) HeLa cells; 8,
`HeLa cell line expressing normal human /3-globin (/3g); 14, RNA from
`human blood (Hb); 2-7, JVS2-654 HeLa cells treated with increasing
`concentrations of the oligonucleotide (indicated in micromolcs at the
`top); 9 and Hl, IVS2-654 HeLa cells treated with oligonucleotide
`followed by hemin (H) (16); 11-13, IVS2-654 J-IeLa cells treated with
`increasing concentrations of the scrambled oligonucleotide. The num(cid:173)
`bers on the right indicate the size, in nucleotides, of the RT-PCR
`products representing the aberrantly (304) and correctly (231) spliced
`RNAs. (C) Restoration of /3-globin expression by 5'ss oligonucleotide
`in IVS2-654 J-IeLa cells. Immunoblot of total protein with anti-human
`hemoglobin antibody. Concentration of the oligonucleotide in micro(cid:173)
`moles is indicated at the top (lanes 2-7); in lane 8, human globin
`(Sigma) was used as a marker. (Lower) Cells were treated with hemin
`preceding the isolation of proteins. The positions of human /3-globin
`and the prematurely terminated /3-globin IVS2-654 polypeptide are
`indicated. Time of exposure of the autoradiogram in Lower was ! /5th
`of that of the Upper.
`
`/3-globin mRNA was translated into full-length /3-globin. In
`agreement with the RT-PCR results shown in Fig. lB, only
`samples treated with 0.05-0.4 µM oligonucleotide contained
`significant amounts of full-length /3-globin (Fig. lC, lanes 3-6).
`
`

`

`Proc. Natl. Acad. Sci. USA 93 ( 1996)
`
`A
`
`5'ss
`Hb WT 6 24 48 96 Bg hrs
`
`_.....,
`
`•
`
`231
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`C :a
`0
`96 24H Bg BgH m hrs
`
`;_~.;.,;.iii. 1:1 :tRk~~
`
`5
`
`6
`
`7
`
`8
`
`9
`
`B
`
`S'ss
`
`0
`
`6
`
`24 48
`
`I
`
`1
`
`2
`
`3
`
`4
`
`12842 Biochemistry: Sierakowska et al.
`
`·r· r· n cif correct expression of /3-globin mRNA
`,
`Table J. Qu<1ntl d 10
`and protein
`
`Target cell
`line and
`splice site
`
`% correct /3-globin
`product
`
`34
`J-IcLa 5'ss*
`14
`HcLa S'sst
`43 (protein)
`HcLa S'ss*
`11
`HcLa 3'ss*
`49
`NIH 3T3 S'sst
`23
`NIH 3T3 3'sst
`treatment with() 7 uM antisense oligonuclcotides,
`f ti
`I
`·
`·- r
`The resu ts o 1c
`.
`.
`.
`r
`t . 1 that elicits maximal correct10n m all experiments,
`t
`the concen ra 101
`·
`.
`I
`The amount of the material in the correct P. C { prm uct. or
`.
`I
`f ti
`. . . ., b
`.,
`·t
`t
`,1rc s 1own.
`te1·11 !Janel was quant1tatcu y uens1 omc ry. o
`1c
`I I ·
`•
`/J.
`!11 ,.,-g O Jlll pro
`.
`'
`.
`. .
`. . , ,
`,
`.
`ns ·is •·'cscnbcd The results arc cxp1essed as percent of
`. ,·
`autorau1ogra1 . , u ·
`. ·
`•
`.
`. l ,
`.
`i·'tict relative to the sum of correct <1nd <1 Jcrrdnt
`t.
`t 1c corrcc pn u
`'
`I
`products.
`.
`*Treatment with oligonuclcot1dc was ~or 10 hr.
`tTrcatment with oligonucleotide was for 6 hr.
`
`There was no {3-globin in control cells (Fig. IC, la!1es l and 2)
`and only a small amount in those treat_ed. ':"1th ~.6 µ,M
`oligonuclcotide (Fig. 1 C, lane 7). Thus, the s1g111f1cant mcrea~e
`in full-length {3-globin, roughly parallel to that of the {3-globm
`mRNA, is clearly due to the effect of antisense oligonucleo(cid:173)
`tides on splicing. The quantitative analysis of the amount of the
`{3-globin polypeptide relative to the one truncated due t_o
`aberrant splicing (in the aberrant sequence the stop c?do~ 1s
`located 48 nucleotides downstream from exon 2, resultmg 111 a
`{3-globin polypeptide containing 104 {3-globin_ a~d 16 aberrant
`amino acids) shows that the amount of {3-globm 111creases from
`=30% of the total at 0.05 µ,M oligonucleotide to 43% at 0.2
`µ,M and 44% at 0.4 µ,M oligonuclcotide. ~he fac~ that the
`percentage of {3-globin protein seems to be shghtly higher than
`that of the corresponding correctly splice? mRN~ ~ay pos(cid:173)
`sibly be due to the differences in the relative stab1ht1c~ of the
`correct and aberrant polypeptides. Nevertheless, the yields of
`correct protein provide evidence that the amount o_f the
`correctly spliced {3-globin mRNA is not overrepresented 111 the
`RT-PCR assay.
`.
`The identity of the generated full-lengt? ~-g(obm ~olypep(cid:173)
`tide band was confirmed by the increase 111 its 111tens1ty upon
`posttreatment of the cells with hemin (Fig. lC Lower, lanes
`3-6) (16). Note that hemin treatment had no cffcc~ on the
`truncated IVS2-654 polypeptide or background prote111 bands
`(Fig. JC Upper and Lower, lanes 2-7). Neither ~id it affect the
`level of transcription and splicing pattern of the_ IVS2-6~4
`prc-mRNA (Fig. Ill, lanes 9 and 10). Thus, the 1~cre'.1se 111
`{3-globin band due to hcmin is not the ~esult of a_ct1vat1011 ?f
`globin gene expression, observed for fetal glob111 genes_ 111
`hematopoietic cell lines (e.g., ref. 19 and refcrenc~s the:e111).
`It seems likely that the polyclonal anti-hemoglobm ant1bo_dy
`has greater affinity for the {3-globin-heme comple~ than ~c!r
`{3-globin alone and/or that hemin treatment results 111 ~pec1f1c
`posttranslational stabilization of the full-length {3-glob111 ~~0).
`Fig. 2 shows the time course of restoration of correct spltcmg
`of {3-globin pre-mRNA and its translation to protein after
`treatment with 0.2 µ,M 5'ss oligonucleotide. Six hours after
`termination of the treatment, there was a trace, if any, of the
`correct {3-globin mRNA and protein (Fig. 2 A, lane 3, and B,
`lane 2, respectively) that increased significantly at 24 hr and
`persisted for 48 but not 96 hr (Fig. 2A, lanes 4-6, _and B, lanes
`3-5). The {3-globin signal was, as expected, stimulated by
`hem in treatment of the cells (Fig. 2B, lane 6 versus lane 3). The
`fact that correctly spliced RNA persisted for 48 hr aft~r
`termination of oligonucleotide delivery suggests that the oh(cid:173)
`gonuclcotidcs and/or the newly synthesized correctly spliced
`mRNA arc quite stable in the cellular environment. It 1s also
`
`FIG. 2. Time course of restoration of correct splicing and {3-globin
`expression in HcLa IVS2-654 cells by 0.2 µ,M S'ss oligonucleotidc. (A)
`RT-PCR assay. (!J) Immunoblot. Time after termination of oligonu(cid:173)
`clcotide treatment is indicated at the top. H, hcmin treatment of the
`cells. All other designations arc as in Fig. I.
`
`possible that the oligonucleotide is recycled after the spliced
`out intron is degraded (21 ).
`To test whether oligonucleotides are able to reverse aberrant
`splicing in other cell types, analogous experime1_1ts were per(cid:173)
`formed using NIH 3T3 cells stably transfected with the IVS2-
`654 {3-globin gene. Since lO hr incubation in the serum-free
`medium (used for HeLa cells) was damaging for the NIH 3T3
`cells, the treatment was shortened to 6 hr. Even with the
`shortened treatment, 5' ss oligonuclcotide targeted to the
`aberrant 5' splice site in IVSZ-654 pre-mRNA produced
`correctly spliced {3-globin mRNA at levels =3-fold higher than
`those observed for HeLa cells treated with the same oligonu(cid:173)
`cleotidc for the same time (Table 1 ). As expected, the effects
`of the oligonucleotide were dose- and sequence-dependent
`(Fig. 3A).
`Repair of aberrant splicing was also obtained, albeit not as
`efficiently, by targeting the 3' cryptic splice site (Fig. 3B and
`Table 1 ). This indicates that the relative accessibility of the 3'
`versus 5' splice site is similar in both HeLa and NIH 3T3 cells.
`The time course of the reaction (Fig. 3B) suggests that there
`is no major difference in the stability of the {3-globin mRNA
`and of the two oligonuclcotidcs in the two cell types.
`Although the above results clearly show that the oligonu(cid:173)
`cleotides affect splicing of their target prc-mRNAs in a
`sequence specific manner, one cannot exclude the possibility
`that they may exert other effects on the cells. The oligonucle(cid:173)
`otides may interact directly with cellular proteins (ref. 22 and
`references therein) or, possibly, inhibit gene expression by
`blocking similar splice sites in many other pre-mRNAs and
`consequently inhibit the growth of cells. However, the rcsul~s
`presented in Fig. 4 show that under our experimental condi(cid:173)
`tions no unspecific effects were detectable. First, the growth
`rate of the HeLa IVS2-654 cells treated with the Lipo(cid:173)
`fcctamine-oligonuclcotide complex was no different from that
`of cells treated with Lipofectamine alone (Fig. 4A). The
`oligonucleotides tested included 2'-O-methyl phosphorothio(cid:173)
`ates complementary to the aberrant splice sites or with a
`scrambled sequence as well as 5'ss and 3'ss 2'-O-methyl
`phosphodiestcrs. Second, the 5' ss oligonucleotide that re(cid:173)
`stored correct splicing in HeLa IVS2-654 (Fig. 4B, lane 6) had
`no effect on splicing of HeLa cells transfected with a control
`construct in which the target aberrant 5' splice site
`(GUAAUA) was modified to match the consensus splice site
`sequence (GUAAGU; ref. 23) (Fig. 4B, lanes 2-4). This
`
`

`

`Biochemistry: Sicrakowska el al.
`
`Proc. Natl. Acad. Sci. USA 93 (1996)
`
`12843
`
`A
`
`7
`
`6
`
`5
`
`4
`
`3
`
`2
`
`LO
`
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`
`)(
`UI
`
`ai u
`
`B
`
`20
`
`40
`60
`Hours
`
`80
`
`A
`0
`
`5' ss
`.2
`.1
`
`.5
`
`.05
`
`~ - - - - 304
`
`control
`
`231
`
`1
`
`2
`
`3
`
`4
`
`5
`
`B
`
`3' ss
`o 6 19 43
`
`hrs
`
`- - - - - 304
`
`231
`
`1
`
`2
`
`3
`
`4
`
`F1u. 3. Dose- and time-dependent correction of splicing in oligo(cid:173)
`nucleotide-treatcd IVS2-654 NIH 3T3 cells. RT-PCR assay. (A) Cells
`treated with increasing concentrations of the oligonuclcotide targeted
`to the aberra nt 5' spl ice site (Upper) or of the control, scrambled
`oligonucleotidc (Lower). (IJ) Time course of the correction of splicing
`after termination of treatment with 0.2 µM oligonucleotide targeted
`to the cryptic 3' splice site activated by the IVS2-654 mutation. All
`designations arc as in Figs. I and 2.
`
`modification resulted in a two nucleotide mismatch of the
`oligonucleotide with 16 nucleotides remaining complementary
`to the intron sequence. Third, splicing of I VS2-654 pre-mRNA
`was not affected in cells treated with an oligonucleotide with
`partial complementarity to the region of the aberrant 5' splice
`site (slash indicates splice site):
`
`pre-mRNA5' CUGGGUUAAG/GUAAUAGC
`
`oligo 705
`
`I I I I 11
`I I
`11 11
`3' ACAUUGACUC/CAUUCUCC
`
`Moreover, this oligonuclcotide, designed to restore correct
`splicing of IVS2-705 thalassemic mutant (ref. 8; unpublished
`work) is also complementary, with a single mismatch, to
`positions 696-713 of IVS2, 44 nucleotides downstream from
`the aberrant 5' splice site (Fig. 4B, lanes 8-10).
`Since the closely related IVS2-654 consensus splice site is
`unaffected by the 5'ss oligonucleotide, and a related oligonu(cid:173)
`clcotide, oligo 705, with partial complementarity to two sites
`in the same RNA has no effect on splicing of IVS2-654
`prc-mRNA, the likelihood that the 5'ss oligonucleotide would
`affect splicing of other pre-mRNAs with even more divergent
`sequences at and around the splice sites appears quite remote.
`This is further reinforced by the fact that we have not detected
`any changes in the level and/or the splicing patterns of two
`randomly chosen mRNAs (/3-actin and EGFR) in cells treated
`with the 5'ss oligonucleotide (not shown) and by the data from
`GenBank that show lack of complementarity of any human
`
`5'ss
`0 .2 .4 1 0
`
`.2 13g
`
`705
`.2 .4
`.1
`
`1----·:Jf-·1:::
`
`1234567 8910
`
`F 10. 4. Specificity of oligonucleoticlc treatme nts. (A) Lack of effect
`of oligonuclcotidcs on cell growth. 1-IeLa IVS2-654 cells were treated
`with Lipofectaminc-oligonuclcotidc complexes as described. Cells
`were counted at the end of the 10 hr treatment (0 hr) and at 24, 52,
`and 72 hr thereafter. Each point on the curve represents the average
`of duplicate counts of two independently treated samples; the ob(cid:173)
`served differences are within experimental error (one SD). □, Lipo(cid:173)
`fectamine alone. The remaining samples were treated with Lipo(cid:173)
`fectam inc complexed with the following 0.2 µM oligoribonuclcotides:
`2'-O-melhyl phosphorothioates, ◊ , 5'ss, x , 3'ss, ♦, scrambled; 2'-O(cid:173)
`mcthyl phosphodiestcrs, 6, 5'ss, ■, 3'ss. (IJ) Lack of effect of control
`oligonucleotides. Treatment of HeLa IVS2-654 consensus cell line
`(lanes 1-4, sec text) or HeLa IVS2-654 cell line (lanes 5 and 6, as
`positive control) with 5'ss 2'-O-mcthyl phosphorothioatc oligoribo(cid:173)
`nucleotidc. Lanes 8-10, treatment of HcLa IVS2-654 cells with
`oligonucleotidc 705 targeted 44 nucleotides downstream from the
`aberrant 5' splice site (see text). The RT-PCR assay and all designa(cid:173)
`tions arc as described in the legend to Fig. 111. Lane 7, l-leLa cell line
`expressing normal human /3-globin.
`
`sequence besides /3-globin to the 5 'ss and 3' ss oligonucleotides,
`even if two mismatches are allowed.
`
`DISCUSSION
`We showed that splicing pathways can be modified i11 vivo in
`a sequence specific manner by antisense oligonucleotides using
`cationic liposomes as a carrier. In view of the universal nature
`of the splicing mechanisms, this approach is of general appli(cid:173)
`cability because the oligonucleotides should be effective in
`different cell types, including hematopoietic cells, and against
`splice sites in a variety of pre-mRNAs.
`Although the feasibility of treatment of thalassemics with
`antisense oligonucleotides has yet to be explored, several
`observations suggest that this approach may be clinically
`promising. The optimal effect of oligonucleotides was seen at
`0.2-0.4 µM, a concentration achieved in bone marrow of
`experimental animals (24). Restoration in a patient of /3-globin
`mRNA to 20-30% of the normal level would be of therapeutic
`significance because heterozygotes with 50% of hemoglobin
`are frequently asymptomatic while the status of patients
`undergoing transfusion therapy, with even lower hemoglobin
`
`

`

`12844 Biochemistry: Sierakowska et al.
`
`Proc. Natl. Acad. Sci. USA 93 (1996)
`
`levels, is markedly improved. Furthermore, {3-globin mRNA
`and protein are very stable and so arc mature erythrocytes,
`with a lifespan of about 120 days (1 ). Thus, in principle,
`treatment with antisensc oligonucleoticles may have an ex(cid:173)
`tended effect on the in vivo levels of {3-globin mRNA and blood
`hemoglobin, reducing the need for frequent administration. In
`this context, it is encouraging that the correctly spliced {3-glo(cid:173)
`bin mRNA and protein generated by a single delivery of the
`antisensc oligonucleotide persisted in NIH 3T3 and HeLa cells
`for up to 48 hr. Moreover, the fact that it was possible to
`effectively deliver the oligonucleoticles to the nuclei of various
`cell types suggests that it should be feasible to find appropriate
`conditions and/or carriers for delivery of the oligonucleotides
`into cells of patients, including the targeted nucleated eryth(cid:173)
`roblasts. The effects of antiscnse oligonucleotides should be
`highly specific because only the latter cells contain the target
`s~qucnce.
`The restoration of correct splicing by targeting the cryptic 3'
`splice site (Fig. 3B) is of particular interest since this splice site
`is activated in other {3-thalasscmia mutations besides IVS2-
`654, i.(;:!., IVS2-745 and IYS2-705 (1, 8). Thus, a single oligo(cid:173)
`nuclcotidc should be effective in correcting splicing in all three
`mutants, which in clinical setting would translate into a larger
`nu1!1ber of patients. It is also likely that aberrant splice sites
`activated by other thalassemic mutations (1, 2) will be amc(cid:173)
`n_ahl