Antisense oligonucleotides and RNAs as modulators of pre-mRNA splicing

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Viruses, particularly adenoviruses, are likely to be extremely useful tools for the transport of ribozymes. In addition to being suitable for the administration to cultured cells, they can be utilized for the delivery of ribozyme to whole animals. They can be administered systemically or injected directly into the target organ.

[301 A n t i s e n s e Oligonucleotides and RNAs a s Modulators

of pre-mRNA Splicing By HALINA

SIERAKOWSKA,* LINDA GORMAN, SHIN-HONG KANG, a n d

RYSZARD KOLE Correction of Aberrant Splicing of H u m a n ]~-Globin pre-mRNA by Antisense Oligonucleotides Antisense oligonucleotides as sequence-specific downregulators of gene expression (for a review, see Ref. i) have become used increasingly as sequence-specific research tools 2-4 and as antiviral and anticancer agents in clinical trials and in the clinic 5-s (see also Crooke 9 and Dean 1° in this volume). We have shown that antisense oligonucleotides can also restore the expression of genes inactivated by specific mutations. 11,12The restoration of gene expression was accomplished by targeting aberrant splice sites created by mutations that cause genetic diseases such as thalassemia or cystic fibrosis. Blocking of these splice sites with antisense oligonucleotides * On leave of absence from the Institute of Biochemistry and Biophysics, Warsaw, Poland 1 S. T. Crooke and C. F. Bennett, Annu. Rev. Pharm. Tox. 36, 107 (1996). 2 G. W. Pasternak and K. M. Standifer, Trends Pharmacol. Sci. 16, 344 (1995). 3 E. Niggli, B. Schwaller and P. Lipp, Ann. N.Y. Acad. Sci. 779, 93 (1996). 4 S. Ramchandani, R. A. MacLeod, M. Pinard, E. yon Hoffe, and M. Szyf, Proc. Natl. Acad. Sci. U.S.A. 94, 684 (1997). 5 R. Zhang, J. Yan, H. Shahinian, G. Amin, Z. Lu, T. Liu, M. S. Saag, Z. Jiang, J. Temsamani, and R. R. Martin, Clin. Pharm. Ther. 58, 44 (1995). 6 M. R. Bishop, P. L. Iversen, E. Bayever, J. G. Shar, T. C. Greiner, B. L. Copple, R. Ruddon, G. Zon, J. Spinolo, M. Arneson, J. O. Armitage, and A. Kessinger, J. Clin. Oncol. 14, 1320 (1995). 7 S. Agrawal, Trends Biotech. 14, 376 (1996). 8 j. L. Tonkinson and C. A. Stein, Cancer Invest. 14, 64 (1996). 9 S. T. Crooke, Methods Enzymol. 313 [1] 1999 (this volume). 10N. Dean, this volume. 11 Z. Dominski and R. Kole, Proc. Natl. Acad. Sci. U.S.A. 911, 8673 (1993). 12H. Sierakowska, M. J. Sambade, S. Agrawal, and R. Kole, Proc. Natl. Acad. Sci. U.S.A. 93, 12840 (1996).

METHODSIN ENZYMOLOGY.VOL.313

Copyright© 1999by AcademicPress All rightsof reproductionin any formreserved. 0076-6879199 $30.00

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Thalassemic pre-mRNA

[-~"m'l-~-I/ x/

S

3' cr. 654

_''.,,

507

Thalassemic pre-mRNA + oligo .~

[--~---~'--~--IL

3' cr. 654 ' '..

~

pre-mRNA

~.d "S ~s

i 1 i 2-I 3 I ~

mRNA B-globin

FIG. 1. Splicing of human/3-globin IVS2-654 pre-mRNA in the presence of an antisense oligonucleotide. The transcribed pre-mRNA from the human/3-globin gene with the IVS2654 C to T mutation is spliced incorrectly in the absence of the antisense oligonucleotide (left). The oligonucleotide targeted to the aberrant 5' splice sites (right) prevents aberrant splicing and restores correct splicing of the mRNA, which results in transcription of the fulllength/~-globin polypeptide. Boxes, exons; solid lines, introns; dashed lines indicate both correct and aberrant splicing pathways; the aberrant 5' splice site created by the IVS2-654 mutation and the cryptic 3' splice site (3' cr.) activated upstream are indicated; heavy bar, oligonucleotide antisense to the aberrant IVS2-654 5' splice site; light bars above and below exon sequences indicate primers used in the RT-PCR reaction; heavy line below the mRNA represents the/3-globin polypeptide.

prevents aberrant splicing and, by forcing the spliceosome to reselect the original splice sites, restores correct splicing. This results in the generation of correctly spliced and translated m R N A and, therefore, restoration of the activity of the damaged gene. Methods leading to restoration of correct splicing in thalassemic mutants of the human/3-globin gene are described in detail as an example of this novel application of the antisense approach. In the IVS2-654 mutant of the human/3-globin gene, a C to T mutation at nucleotide 654 of intron 2 creates an additional aberrant 5' splice site at nucleotide 652 and activates a cryptic 3' splice site at nucleotide 579 of the fl-globin pre-mRNA. During pre-mRNA splicing, a fragment of the intron contained between the newly activated splice sites is recognized by the splicing machinery as an exon and is retained in the spliced mRNA. The aberrantly spliced m R N A appears to be relatively unstable and, moreover, due to the presence of a stop codon in the retained intron fragment, leads to translation of a truncated fl-globin polypeptide (Fig. 1). This molecular mechanism is responsible for the resultant fl-globin deficiency that causes/3-thalassemia, an inherited blood disorder in affected individuals. 13 Because the aberrant splicing of IVS2-654 pre-mRNA takes place in the 13D. J. Weatherall, in "The Molecular Basis of Blood Diseases" (G. Stamatoyannopoulos et al., eds.), p. 157. Saunders, Philadelphia, 1994.

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0.2

0.4

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0.6

8g

pMoligo

304

aberrant

231

correct

1

2

3

4

5

6

7

FIG. 2. Correction of splicing of IVS2-654 pre-mRNA in HeLa cells by an antisense oligonucleotide targeted to the aberrant 5' splice site. Analysis of total RNA by RT-PCR. Lanes 1-6, IVS2-654 HeLa cells treated with increasing concentrations of the oligonucleotide (indicated in micromoles at the top),/3 g, (lane 7) RNA from human blood. Numbers on the left indicate the size, in base pairs, of the RT-PCR products representing aberrantly (304) and correctly (231) spliced RNAs.

presence of the correct splice sites, one can anticipate that blocking of the aberrant splice sites by antisense oligonucleotides will redirect the splicing machinery from the aberrant to the correct splice sites. Indeed, the outcome of this approach is the restoration of correct splicing of/3-globin m R N A and its translation to the/3-globin polypeptide (Fig. 1). In the experiments described in this article, antisense 2'-O-methyl phosphorothioate-oligoribonucleosides were used because their duplexes with pre-mRNA are resistant to RNase H. 14 This property is essential to avoid degradation of the targeted pre-mRNA, which would have led to removal of the splicing substrate, t5 These oligonucleotides are also highly resistant to degradation by other nucleases, resulting in their stability in the cell culture environment 16 and in animal tissues) 7 Furthermore, they form very stable duplexes with R N A with Tm values higher than those of their ribo or deoxyribo analogs) 6 The IVS2-654/3-globin pre-mRNA was expressed constitutively in a HeLa cell line transfected stably with the/3-thalassemic globin gene cloned under the immediate early cytomegalovirus promoter. Cells were treated 14 C. K. Mirabelli and S. T. Crooke, in "Antisense Research and Applications" (S. T. Crooke and B. Lebleu, eds.), p. 7. CRC Press, Boca Raton, FL, 1993. 15p. F. Furdon, Z. Dominski, and R. Kole, Nucleic Acids Res. 17, 9193 (1989). 16B. S. Sproat and A. I. Lamond, in "Antisense Research and Applications" (S. T. Crooke and B. Lebleu, eds.), p. 351. CRC Press, Boca Raton, FL, 1993. 17R. Zhang, Z. Lu, H. Zhao, X. Zliang, R. B. Diasio, I. Habus, Z. Jiang, R. P. lyer, D. Yu, and S. Agrawal, Biochem. Pharm. 50, 545 (1995).

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0 0.05 0.1

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pM

B-globin 1

2

3

4

5

6

7

F~6. 3. Restoration of/3-globin expression by antisense oligonucleotide in IVS2-654 HeLa cells. Immunoblot of total protein with antihuman hemoglobin antibody. Concentration of the oligonucleotide in micromoles is indicated at the top (lanes 1-6); in lane 7, human globin (Sigma) was used as a marker. After treatment with oligonucleotides, cells were treated with hemin preceding the isolation of proteins. The positions of human/3-globin are indicated.

with LipofectamineTM in complex with antisense 18-mer phosphorothioate 2'-O-methyl oligoribonucleotides targeted to the aberrant 5' splice site created by the mutation. The total RNA was subsequently isolated and the spliced mRNA was identified by reverse transcription and polymerase chain reaction (RT-PCR) (Fig. 2). RT-PCR was carried out with [a32p]dATP for 18-20 cycles. Under these conditions the amount of the PCR product was proportional to the amount of input RNA. The relative amounts of PCR products generated from aberrantly and correctly spliced RNAs were also proportional to the input, allowing for quantitative analysis of data. Because the correctly spliced/3-globin mRNA generated by the antisense treatment underwent translation, the correction of splicing could be verified by detection in the lysate of oligonucleotide-treated cells of a full-length/3-globin polypeptide. This was accomplished by immunoblots using an antihemoglobin antibody 12 (Fig. 3). Note that correction of splicing with antisense oligonucleotides targeted to aberrant splice sites has also been accomplished for similar thalassemic mutations in the/3-globin gene, i.e., IVS2-705 and IVS2-745) 9'2° The fact that the correction of splicing was detected in oligonucleotidetreated cells indicates that several events must have taken place. Clearly, the oligonucleotides had been delivered into the cell and entered the nucleus, the site of splicing. They competed with splicing factors for the aberrant splice site, preventing aberrant splicing and promoting formation of the spliceosome and subsequent splicing at the correct sites. Apart from is p. Hawley-Nelson, V. Ciccarone, G. Gebeyehu, J. Jessee, and P. L. Felgner, Focus 15, 73 (1993). 19H. Sierakowska, M. Montague, S. Agrawal, and R. Kole, Nucleotides Nucleosides 16~ 1173 (1997). 20 H. Sierakowska, M. J. Sambade, and R. Kole, RNA, in press.

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the potential clinical applications, this system provides an excellent method for testing the efficacy of various antisense oligonucleotides. Correction of splicing may also serve as a measure of oligonucleotide uptake, its hybridization potential, and/or other parameters involved in antisense activity. Because the action of the oligonucleotides generates a new product (spliced m R N A and protein), even minor effects, difficult to discern when antisense oligonucleotides are used as downregulators of gene expression, become readily detectable (see also later). Procedures Cell Culture. Culture HeLa cells stably expressing human/3-globin thalassemic IVS2-654 pre-mRNA in a monolayer on a 75-cm 2 tissue culture plate in S-MEM to below 50% confluency. The S-MEM medium (Life Technologies) is supplemented with 5% fetal calf, 5% horse sera, 50/zg/ml gentamicin, and 200/zg/ml kanamycin. Twenty-four hours before treatment with oligonucleotides, trypsinize the cells with 1 ml trypsin-EDTA for 3-5 min, suspend them at 105 cells/ml medium, and plate in 24-well plates (2 cm 2 well area) at 1 ml per well. Oligonucleotide Treatment. Dissolve ON-654, a 2'-O-methyl phosphorothioate-oligoribonucleoside (the oligonucleotide used in this study was prepared and purified at Hybridon, Inc., Milford, MA) in water under sterile conditions at 100/zM and store at - 2 0 °. The oligonucleotide, GCUA U U A C C U U A A C C C A G , is antisense to the aberrant 5' splice site in the IVS2-654 pre-mRNA. Dilute the oligonucleotide to 5/zM or to appropriate lower concentrations in Opti-Mem I (Life Technologies) prewarmed to room temperature. Aliquot 100/zl of oligonucleotide solution into individual Eppendoff tubes. Controls should contain no oligonucleotide. Suspend 4/zl of Lipofectamine (2 mg/ml, Life Technologies) in 100 tzl of Opti-Mem I at room temperature and mix thoroughly by inversion. For multiple samples, increase the reagent volumes as appropriate. Add 100 /xl of this suspension to the oligonucleotide solution and mix the contents by pipetting up and down. Incubate the tubes at room temperature for 30 min to form the Lipofectamine-oligonucleotide complex. Subsequently add 800/xl Opti-Mem I at room temperature and mix by inverting five times. Place the 24-well plate containing IVS2-654 HeLa cells under the hood, aspirate the medium gently, and wash the cells twice for 1 min with 1 ml Opti-Mem I at 37°. This wash removes the serum that interferes with the uptake of the oligonucleotide-Lipofectamine complex. Work with no more than 6 wells per plate to prevent cooling of cells during washes. Transfer 1 ml of the oligonucleotide complex from each Eppendorf tube to the

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appropriate well and return the plate to the incubator for 10 hr. Remove the transfection medium, add to each well 1 ml of S-MEM culture medium at 37 °, and incubate the cells for 24-36 hr in a tissue culture incubator. Isolation of Total RNA. This procedure follows the recommendations of the manufacturer of TRI reagent (Molecular Research Center, Cincinnati, OH). Aspirate the medium from the wells containing oligonucleotidetreated cells and add 0.8 ml TRI reagent per well at room temperature for 10 min. Pipette the lysate five times up and down, transfer it to Eppendorf tubes, let sit for 3 min, mix by inverting the tubes several times, and touch spin. Proceed directly or store at - 8 0 °. Add 160/zl chloroform per tube, vortex vigorously for 30 sec, and let sit for 5 min. Spin at 14,000g for 20 min at 4°. Transfer 320/zl of the colorless upper aqueous phase to fresh Eppendorf tubes containing 2 ~1 nucleasefree aqueous glycogen at 20 mg/ml (Boehringer-Mannheim). Vortex the tubes vigorously and touch spin. To each tube add 400/xl 2-propanol, vortex vigorously, invert twice, store on ice for at least 30 min, and centrifuge at 14,000g for 30 min at 4 °. Pour off the supernatant and wash the pellet once with 1 ml 75% (v/v) ethanol by vortexing and subsequent centrifugation at 14,000g for 10 min at 4 °. Carefully remove as much of the supematant as possible without disturbing the now fluffy pellet. Dry the pellet, avoiding overdrying, under the hood or in a Speed-Vac. Dissolve the R N A pellet in 60/zl autoclaved distilled water by incubating it for 30-45 min at 45 ° with intermittent vortexing. The R N A solution can be stored at - 2 0 ° for at least 1 year. RNA Analysis by R T-PCR. This procedure follows the recommendation by the manufacturer of the GeneAmp thermostable R N A reverse transcriptase PCR kit (Perkin Elmer-Cetus, Norwalk, CT). All reagents for the reaction, except for the primers and radiolabeled nucleoside triphosphate, are included in the kit. Forward (5'), G G A C C C A G A G G T F C T T r GAGTCC, and reverse (3'), G C A C A C A G A C C A G C A C G T F G C C C , primers span positions 21-43 of exon 2 and positions 6-28 of exon 3 of the human/3-globin gene, respectively. Prepare the reverse transcription master mix. For one sample, use 6.4 /zl autoclaved deionized water, 2/zl 10X rTth reverse transcription buffer, 2/zl 10 mM MnC12, 1.6/zl of a mixture of 2.5 mM dGTP, dATP, dTTP, and dCTP, 1/zl (30 pmol) 3' primer, and 2/.d rTth D N A polymerase. Mix by gentle vortexing. For multiple samples, increase as appropriate. Add 15/zl of the RT-PCR master mix prewarmed to 37° to each 0.5ml PCR tube containing 5/zl R N A solution at room temperature and mix the total with the pipette tip. Overlay each sample with two drops of mineral oil and incubate the tubes in D N A thermal cycler (Perkin Elmer) at 70° for 15 min. At the end of this step the samples can be cooled to 4 °.

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In the meantime, prepare the PCR master mix. For one sample, use 63 /zl autoclaved deionized water, 8/x110X chelating buffer, 8 ~110 mM MgC12, 1/zl (32 pmol) 5' primer, and 2.5/xCi [a-32p]dATP (aqueous solution, 10 /zCi/ml, 6000 Ci/mmol, Amersham Life Science). Vortex. For multiple samples, increase as appropriate. Pipette 80 tzl of the PCR master mix to each reverse transcription reaction tube, centrifuge at 14,000g for 30 sec, and subject to PCR: 3 min at 95 ° for 1 cycle followed by 1 min at 95° and 1 min at 65 ° for 18 cycles. Analyze the PCR products by electrophoresis on a 1.5 mm x 14 cm x 14 cm nondenatauring polyacrylamide gel. The gel consists of 40 ml 7.5% acrylamide (30:1 acrylamide : bisacrylamide), 0.3 ml 10% ammonium persulfate, and 30/xl TEMED (added last) in 0.5X TEB (0.05 M Tris, 0.04 M boric acid, 0.001 M EDTA). Load 20/.d of the amplified material removed from under the mineral oil and mix with 4 /zl 10X loading dye [0.42% bromphenol blue, 0.42% xylene cyanol F.F., and 25% Ficoll (Type 400, Pharmacia, Piscataway, N J) in water] per lane. The remainder of the samples can be stored at - 2 0 ° for up to 1 week. Electrophorese at room temperature in 0.5X TEB at constant voltage for a total of 900 Vhr avoiding overheating (e.g., 45 min at 200 V and subsequently for 3 hr at 250 V or overnight at 55 V) until the xylene cyanol dye leaves the gel. Dry the gel in a gel dryer under vacuum at 80 ° and autoradiograph or analyze in Phosphoimager. Quantify correctly spliced/3-globin mRNA by densitometry of the autoradiograms. Calculate the percentage of correct product relative to the sum of correct and aberrant ones. The results need to be corrected to account for the higher [32p]dAp content of the PCR product derived from aberrantly spliced mRNA than that from correctly spliced mRNA. For IVS2-654 prem R N A the correction factor is 1.57. Separation and Blotting of Proteins. After treatment with oligonucleotides, additional incubation of cells with hemin greatly facilitates the detection of/3-globin on immunoblots. Hence, rinse the cells twice with S-MEM at 37 ° and incubate in S-MEM containing hemin for 4 hr at 37°. To prepare the latter medium, dissolve 3.2 mg hemin (Fluka, Ronkonkoma, NY) in 5 ml 20 mM NaOH and dilute 100-fold with S-MEM (without serum or antibiotics) at 37°. Wash the hemin-treated cells twice in HBSS, once with PBS, and lyse them for 15 min at room temperature in 75/.d lysis buffer [3% SDS, 63 mM Tris-HCl, pH 6.8, 7% sucrose, 1 mM EDTA, 1 m M phenylmethylsulfonyl fluoride (PMSF), 1 ~g/ml pepstatin, 5/zg/ml leupeptin, and aprotinin]. Add the proteolytic inhibitors immediately before use. Scrape the wells with fiat-tip sequencing gel-loading pipette tips and transfer the lysate with a 0.5-ml insulin syringe with a 28-gauge 1/2 needle into Eppendorf tubes.

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Homogenize the lysate by passing it five times through the syringe. Lysates can be stored at - 8 0 °. Separate proteins by electrophoresis on a 10% polyacrylamide TricineSDS gel as follows.21 The separating gel is 0.75 mm thick, 14 cm wide, and 11 cm high [3.05 ml gel stock, 5 ml gel buffer, 3.54 m150% aqueous glycerol, 3.41 ml water; 50/.d 10% ammonium persulfate, 5/zl TEMED. Gel stock: 48% acrylamide, 1.5% bisacrylamide (w/v). Gel buffer: 3 M Tris, 0.3% SDS, adjusted to pH 8.45 with HC1, stored at room temperature]. The stacking gel is 0.75 mm x 14 cm x 2 cm (0.5 ml gel stock, 1.15 ml gel buffer, 4.2 ml water; 50/.d 10% ammonium persulfate and 5 ~1 TEMED. Ammonium persulfate and TEMED are added immediately before pouring). The cathode buffer consists of 0.1 M Tris, 0.1 M Tricine, 0.1% SDS, pH 8.25. The anode buffer is 0.2 M Tris, adjusted to pH 8.9 with HCI. To each tube of cell lysate (75/~1) add 4.5/xl 1 M DTF and 6/~1 0.1% aqueous Brilliant blue G (Sigma, St. Louis, MO), heat at 100° for 5 min, and load 40/zl/lane. As markers use globin (Sigma) and S D S - P A G E low range molecular weight protein standards (Bio-Rad, Richmond, CA). To prepare the globin solution, dissolve by heating to 100° for 3 min 1 mg globin in 0.5 ml lysis buffer with 5 0 / z M DTT. Dilute sequentially with heating. Store diluted (40 ng/ml) solution at - 2 0 °. Before use, thaw the solution by heating at 45 ° for 15 min and load 10-30/xl per lane. Electrophorese the gel with anode buffer in the lower chamber and cathode buffer in the upper one for 1 hr at 30 V and subsequently for 5 hr at 150 V until the blue dye reaches 1 cm from gel bottom. Electroblot the proteins onto 0.2 /zm nitrocellulose (Schleicher and Schuel, Keene, NH) at 60 V overnight at room temperature in a Bio-Rad Trans-Blot Cell with a water-cooling coil. If desired, stain the nitrocellulose membrane with 1X Ponceau S [10× stock solution: 2 g Ponceau S (Sigma), 30 g trichloroacetic acid, 30 g sulfosalicylic acid, water to 100 ml] for 1 min at room temperature, wash with several changes of distilled water, and mark the positions of protein standards. Proceed with immunodetection of /3-globin or store the air-dried blot wrapped in Saran wrap in the refrigerator. Immunoblot Detection of [3-Globin. Perform all the immunoreactions and rinses with agitation on a clinical rotator at room temperature. Rinse blot in PBST (1% Triton X-100 in PBS) for I5 min. Block for 2 hr with Blotto (5% Carnation nonfat dry milk in PBST. Dissolve the milk in PBS, filter, and add Triton X-100). Incubate with polyclonal affinity-purified chicken antihuman hemoglobin IgG (Accurate Chemicals) diluted 1000fold with Blotto for 20 min. Wash in Blotto three times for 10 min each 21 H. Schagger and G. von Jagov, Anal. Biochem. 166, 368 (1987).

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and with PBST twice for 10 min each. Incubate for i hr with the secondary antibody, rabbit antichicken horseradish peroxidase-conjugated IgG (Accurate Chemicals), diluted 2000-fold with Blotto. Wash for 10 min each: twice with Blotto, three times with PBST containing 0.02% Tween 20, and twice with PBST. Proceed with detection of/3-globin using the E C L detection system, as recommended by the manufacturer (Amersham). Correction of A b e r r a n t Splicing of H u m a n / 3 - G l o b i n pre-mRNA b y Modified U7 snRNA The use of antisense oligonucleotides as pharmacological, therapeutic agents is very attractive due to its anticipated sequence specificity and low toxicity. However, especially in treatment of genetic disorders such as thalassemia, a significant drawback of this approach stems from the fact that the oligonucleotides do not remove the offending mutation and therefore the treated patients would require lifelong periodic administrations of these compounds. T o circumvent this problem we have developed a novel approach that allows for long-term, possibly permanent, expression of R N A antisense to aberrant thalassemic splice sites in/3-globin pre-mRNA. This was accomplished by incorporating the anti-/J-globin sequences into the gene for murine U7 small nuclear R N A (snRNA). U7 s n R N A forms a ribonucleoprotein particle (U7 snRNP) that is involved in processing of the 3' end of histone pre-mRNAs. 22-24 In addition to the 62 nucleotide RNA, the particle contains at least two U7-specific proteins and eight so-called Sm proteins, which are also found associated with other snRNAs. 25The U7 s n R N A carries out its function by hybridizing via its 5' end to a sequence within the histone pre-mRNA. This observation indicated that the 5' end of U7 s n R N A is available i n v i v o for antisense interactions. Thus it seemed likely that the insertion of appropriate sequences antisense to aberrant splice sites in the/3-globin p r e - m R N A into the U7 s n R N A will change its function and lead to correction of splicing of thalassemic/3-globin pre-mRNA. The modified U7 s n R N A was tested on the IVS2-705 mutant of the human/3-globin gene (Fig. 4A).26 This mutation, similar to IVS2-654, creates 22 G.

Galli, H. Hofstetter, H. G. Stunnenberg, and M. L. Birnstiel, Cell 34, 823 (1983). 23C. Birchmeier,D. Schiimperli,D. Sconzo,and M. L. Birnstiel,Proc. Natl. Acad. Sci. U.S.A. 81, 1057 (1984). 24M. L. Birnstiel and F. Schaufele, in "Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles" (M. L. Birnstiel, ed.), p. 155. Berlin, 1988. 25H. O. Smith, K. Tabiti, G. Schaffner, D. Soldati, U. Albrecht, and M. L. Bimstiel, Proc. Natl. Acad Sci. U.S.A. 88, 9784 (1991). 26L. Gorman, D. Suter, V. Emerick, D. Schumperli,and R. Kole, Proc. Natl. Acad. Sct U.S.A. 95, 4929 (1998).

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A

C U A C G-C U-G C-G U-A U-A U-A U-A G-C

B

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,

Anfl-histone pre-mRNA C U A C G-C U-G C-G U-A U-A U-A U-A G-C ;AG-CCCU 3'

U7.324:

Anti-3'cryptic splice site

SmOPT

FIo. 4. (A) Use of modified U7 snRNA to correct aberrant splicing. Modified U7 snRNA (U7.324) targeted to the 3' cryptic site activated by a G to T mutation in the IVS2-705/3globin gene prevents aberrant splicing and restores correct splicing of the pre-mRNA. Boxes, exons; solid lines, introns; dashed lines indicate both correct and aberrant splicing pathways; the aberrant 5' splice site created by the IVS2-705 mutation and the cryptic 3' splice site (3' cr.) activated upstream are indicated; short bars above and below exon sequences indicate primers used in the RT-PCR reaction. (B) Structure of U7 snRNA constructs. Wild-type U7 snRNA contains an antisense sequence to the 3' end of histone pre-mRNA (open boxes), the U7-specificSm-binding site (gray box), and a stem-loop structure. In the U7.324 construct, the antihistone pre-mRNA sequence has been replaced with a sequence antisense to the/3globin IVS2-705 3' cryptic splice site (open box) and the U7 Sm site has been replaced with the SmOPT sequence (gray box). a n a d d i t i o n a l 5' splice site a n d activates a 3' splice site w i t h i n i n t r o n 2 of /3-globin p r e - m R N A . A 24 n u c l e o t i d e s e q u e n c e a n t i s e n s e to the a b e r r a n t 3' splice site was i n s e r t e d i n t o the U 7 g e n e to replace the a n t i h i s t o n e s e q u e n c e of the wild-type U 7 s n R N A (Fig. 4B). A n i n t e r n a l s e q u e n c e (Sm) was also modified ( S m O P T ) to i m p r o v e the stability of the R N A a n d to

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705

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U 7 . 1 U7.2

358

231

FIG. 5. Correction of aberrant splicing in stable HeLa cell lines expressing U7.324. Total cellular RNA was isolated from HeLa cell lines that stably express IVS2-705/3-globin and U7.324 and analyzed by RT/PCR. Lane 1,/3 g, RNA from human blood. Lane 2, RNA from IVS-705 HeLa cells. Lanes 3 and 4, stable correction of aberrant splicing of/3-globin premRNA in two different clones.

increase its intranuclear concentration. 27 U7.324 snRNA was very effective in the correction of splicing of IVS2-705 pre-mRNA. Importantly, the stable transfection of cells expressing the thalassemic/3-globin gene with vectors carrying a modified U7 snRNA gene led to permanent correction of the splicing pattern of the/3-globin pre-mRNA (Fig. 5). Consequently, significant amounts of full-length/3-globin mRNA accumulated in the cells. These results suggest a possibility of gene therapy based on the antisense concept. The patients' bone marrow would be transfected ex vivo with the antisense U7 vectors and reimplanted. The correction of/3-globin premRNA splicing affected by U7.324 snRNA should increase the production of/3-globin and reduce the imbalance between the o~ and the/3 subunits of hemoglobin, promoting the maturation of erythrocytes and alleviating the symptoms of thalassemia. Procedures U7.324 s n R N A Construct. The U7 promoter and 3' sequences are included in the construct. The U7-specific Sm-binding site ( A A U U U G U 27 C. Grimm, B. Stefanovic, and D. Schiimperli, E M B O J. 12, 1229 (1993).

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CUAG) was replaced with the consensus Sm sequence ( A A U U U U U G GAG). 28 The natural 18 nucleotide sequence complementary to the 3' processing site of histone pre-mRNAs was replaced 29,3°with a 24 nucleotide sequence complementary to the 3' splice site activated by the IVS2-705 mutation (see Fig. 4). Cell Lines. Culture the HeLa cell line carrying the thalassemic IVS2-705 human/~-globin g e n e 19'2° and the cell lines stably expressing the modified U7 snRNAs (see later) under the same conditions as described in the previous section. Transient Expression of U7.324 snRNA. The procedure is essentially the same as that described in the first section. Plate HeLa IVS2-705 cells 24 hr before treatment in 24-well plates at 105 cells per 2-cm 2 well. Treat the cells for 10 hr with the modified U7.324 plasmid (0.5, 1, 2, and 4/xg/ ml) complexed with 4/zl/ml of Lipofectamine in Opti-Mem. ,Selection of Cell Lines Stably Expressing U7.324 snRNA. In 24-well plates, cotransfect HeLa IVS2-705 cells with plasmids carrying a hygromycin resistance gene (0.2 txg) and a U7 snRNA expressing plasmid (2/zg) in the presence of Lipofectamine (4 /~l/ml) as described in the first section. Twenty-four posttransfection, remove media and wash cells with 1 ml 1 x HBSS (Hanks' balanced salt solution). Add 100/zl of 1 x trypsin-EDTA (Life Technologies) to each well for approximately 5 min to detach the cells from the plate and supplement the cell suspension with 1.5 ml of serum containing culture medium per well. Mix well by pipetting up and down and plate 0.5 ml of the suspension on each of three 75-cm 2 tissue culture plates containing 10 ml of S-MEM culture medium. Incubate the cells at 37° for 24 hr. Remove the medium and replace it with the same medium containing 250/xg/ml hygromycin. Replace this medium every 3 days. Hygromycin-resistant colonies should appear in 10-14 days. To isolate the colonies wash the plates once with 5 ml 1 x HBSS. Place cloning glass rings (Bellco), with the bottoms coated with vacuum grease, over the colony and push down firmly onto the plate. Add 100 /~1 of trypsin-EDTA solution to the center of the ring. Incubate at room temperature for 2-3 min. Pipette up and down to detach the cells and transfer to 2-cm 2 wells (24-well plate) containing 1 ml culture medium without hygromycin. Once the cells are - 5 0 % confluent, wash the plates once with 1 ml 1 × HBSS and trypsinize with 100/zl of trypsin-EDTA. Add 2 ml of culture medium without hygromycin and pipette up and down to suspend 28 B. Stefanovic, W. Hackl, R. Luhrmann, and D. Schiimperli, Nucleic Acids Res. 23, 3141 (1995). 29 D. H. Jones and B. H. Howard, Biotechniques 10, 62 (1991). 30 D. H. Jones and S. C. Winistorfer, Biotechniques 12, 528 (1992).

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the cells. Remove 1 ml of cell suspension and transfer to a fresh plate. Once these cells reach -80% confluence, treat them with TRI reagent and isolate total cellular RNA as described in the first section. Analyze RNA by RT/PCR to detect correction of/3-globin splicing affected by stably expressed U7.324 snRNA. Cells remaining on the original plate are cultured for maintenance of the clonal cell line. R N A Analysis. For analysis of correction of splicing of human/3-globin pre-mRNA, carry out RT-PCR exactly as described in the previous section. To detect the expression of U7.324 snRNA, use U7.324 specific forward (GCATAAGCTTAAGCATFATTGCCCTGAA) and reverse (CGTAG A A T T C A G G G G T T I T C C G A C C G A ) primers; underlined nucleotides overlap with U7 sequences. The RT conditions are the same as described in the first section. The PCR conditions are 3 min at 95° for 1 cycle, 1 min at 95°, 1 min at 55°, and 1 min at 72° for 25 cycles. Lucfferase-Based Assay for Intracellular Activity of Antisense Oligonucleotides As pointed out in the first section, correction or modulation of splicing pathways provides a positive readout assay for antisense activity. The assay relies on a shift of the splicing machinery from one site to another caused by a block of a splice site with the antisense oligonucleotide. Therefore, the results virtually guarantee that the observed effects are sequence specific and are due to true antisense activity of the compound, which must have migrated into the nucleus, the site of pre-mRNA splicing. The sequence specificity of the effects is also supported by appropriate controls, 12'31some of which are presented in this section. We have used RT-PCR as a detection method for the correction of splicing by antisense oligonucleotides. Although the sensitivity of RT-PCR is unmatched, the procedure is relatively laborious and not easily amenable to quantitative analysis. In order to simplify the analysis of correction of splicing, we have devised a system in which the action of antisense oligonucleotides or RNAs results in an upregulation of luciferase activity. This was accomplished by interrupting the coding sequence of the luciferase gene with the IVS2-705 human/3-globin intron (Fig. 6). In cells expressing the modified Luc/705 mRNA stably, the intron is spliced incorrectly, resulting in retention of the intron fragment in the coding sequence of the luciferase mRNA and concomitant inhibition of translation of an active enzyme. Treatment of the cells with antisense oligonucleotides or RNAs targeted to the 5' splice site created by the 31S.-H. Kang, M.-J. Cho, and R. Kole,Biochemistry 37, 6235 (1998).

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Luciferase~

519

Luciferase ON-119

ON-Ran

ON-705M

FIG. 6. Splicing of Luc/705 pre-mRNA in HeLa cells. The pre-mRNA is transcribed from a pLuc/705 plasmid in which the coding sequence of luciferase (boxes) is interrupted by the /3-globin IVS2-705 intron (solid heavy line). Due to aberrant splicing of the intron (dashed lines), the luciferase is inactive. ON-705 (short bar under the intron) is targeted to the aberrant 5' splice site and restores correct splicing (solid thin line) and luciferase activity. Control oligonucleotides are indicated.

705 mutation resulted in easily detectable, dose-dependent restoration of luciferase activity, which at the optimal concentration of the oligonucleotide (0.4/zM, ON-705, CCUCUUACCUCAGUUACA, Fig. 7) increased approximately 50-fold over the background. The effect was sequence specific because neither an oligonucleotide targeted to an irrelevant site in the intron at nucleotide 119 (ON-119, U G A G A C U U C C A C A C U G A U ) nor 140,000 T

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FIG. 7. Restoration of luciferase activity by antisense oligonucleotides. The oligonucleotides used are indicated. Experiments were performed in triplicate, with the line at each bar representing standard deviation. Results are expressed in relative light units (RLU) per microgram of protein.

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the one with a random sequence (ON-Ran) exerted any effect on luciferase activity. A single mismatch (ON-705M, CCUCUUACAUCAGUUACA) resulted in an approximately 70% decrease in the antisense effectiveness of ON-705 (Fig. 7). In HeLa cells transfected with the pLuc/705 construct, RNA expression is controlled by the CMV promoter coupled to the Tet-Off system. In this system, luciferase expression is strictly regulated and is approximately 35fold higher than in cells transfected with CMV promoter/enhancer-driven luciferase expression vectors. 32 Note also that the luciferase system is approximately 1000-fold more sensitive than, for example, the CAT assay.33 The luciferase assay should be very useful in the identification of oligonucleotide carrier molecules and in investigations on the effects of oligonucleotide backbones on their antisense activity. The fact that this assay is incompatible with antisense oligonucleotides that promote cleavage of the target RNA by RNase H does not appear to be a serious limitation. The most promising examples of modified compounds include 2'-O-alkyl-oligoribonucleotides, morpholino oligonucleotides, methyl phosphonates, and the so-called peptide nucleic acids. 34 None of these compounds promote cleavage of the target RNA by RNase H and can therefore be tested in this system. Procedures Plasmid and Cell Line Construction. Intron 2 of the IVS2-705 thalassemic mutant of the/3-globin gene was inserted between nucleotides 1368 and 1369 of the luciferase cDNA sequence of the pTRE-Luc plasmid (Clontech) by PCR-based in vivo recombination.3° HeLa Tet-Off cells (Clontech) were cotransfected with the resultant recombinant plasmid, pLuc/705, and the hygromycin resistance plasmid using Lipofectamine as a carrier. Stable transfectants were obtained by selection in hygromycin (200/zg/ml) containing Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum as described earlier. Oligonucleotide Treatment. In a six-well plate treat the cells with a complex of Lipofectamine (4/zl/ml) and oligonucleotides (concentrations as in Fig. 7) in Opti-Mem for 5 hr as described earlier. Replace the medium

32D. X. Yin, L. Zhu, and R. T. Schimke,Anal. Biochem. 235, 195 (1996). 33j. R. DeWet, K. V. Wood,M. DeLuca,D. R. Helinski,and S. Subramani,MoL Cell. BioL 7, 725 (1987). 34p. H. Seeberger and M. H. Caruthers,in "AppliedAntisenseOligonucleotideTechnology" (C. A. Stein and A. M. Krieg,eds.), p. 51. Wiley-Liss,New York, 1998.

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with DMEM containing 10% fetal calf serum and, after an additional 18 hr, collect the protein for luciferase assay35 (see later for details). Luciferase Assay. Remove the culture medium. Rinse the cell monolayer in a six-well plate twice with cold PBS and place the plate on ice. Add 100 /xl of cell lysis buffer (0.1 M potassium phosphate, pH 7.8, 1% Triton X100, 1 mM DTT, 2 mM EDTA) to each well and scrape the cell monolayer with a plastic scraper (Costar). Transfer the cell lysate into Eppendorf tubes and leave it on ice for 15 rain. Subsequently centrifuge the lysate at 14,000g for 30 sec at 4°. Collect the cell extract from above the cell debris. At room temperature, mix 5-20/zl of the extract with 100/zl of ATP buffer (30 mM Tricine, 3 mM ATP, 15 mM MgSO4, 10 mM DTF, pH 7.8) in an assay cuvette. Place the cuvette in the luminometer (Analytical Luminescence Laboratory) and place up to 5 ml of 1 mM luciferin in the instrument chamber. One hundred microliters of the luciferin solution is added automatically into the cuvette. Measure luminescence for 10 sec. Protein Assay. Measure protein concentration in the cell extract using the bicinchoninic acid assay kit (Sigma). Briefly, prepare an assay solution by adding 1 part of 4% cupric sulfate solution to 50 parts of bicinchoninic acid solution. Mix 5/xl of the cell extract with 200/zl of the assay solution in a well of a 96-well plate. Incubate the plate for 30 min at 37°. Measure absorbance at 595 nm on a plate reader (Bio-Rad Model 3350). Express luciferase activity in relative light units (RLU) per microgram protein. Addendum In all the procedures described in this article, the antisense oligonucleotides and RNAs were delivered to the cells in complex with Lipofectamine. Work in this laboratory showed that other agents such as DMRIE-C, Cellfectin (Life Technologies), Superfect, Effectene (Qiagen), ExGEN 500 (Euromedex), and Cytofectin (Sigma) are also useful as carriers. Furthermore, in addition to HeLa cells, other cell types, including 3T312 and K562 cell lines (Gorman and Kole, unpublished), were subjected successfully to the antisense-mediated modification of splicing pathways. Acknowledgments We thank Dr. SudhirAgrawalfrom HybridonInc. for the oligonucleotidesused in this study and ElizabethSmithfor technicalassistance.Thisworkwas supportedin part by grants from Hybridonand National Institutesof Health to R.K.

35A. R. Brasier, J. E. Tate, and J. F. Habner, Biotechniques7, 1116 (1989).