- Email: [email protected]
Ribozymes to the rescue: repairing genetically defective mRNAs
COMMENT
Ribozymes to the rescue: repairing genetically defective mRNAs JOHN J. ROSSI [email protected] DEPARTMENT OF MOLECULAR BIOLOGY, BECKMAN RESEARCH INSTITUTE OF THE CITY OF HOPE, 1450 EAST DUARTE ROAD, DUARTE, CA 91010-3011, USA.
It has been nearly 20 years untranslated region of the 5′ since the initial discoveries myotonic dystrophy proof catalytic RNAs. The first tein kinase (MDPK) trandemonstrated catalytic RNAs, script. These exciting appli5′ Splice site or ribozymes, were derived cations are built upon years from the intron of the Tetraof biochemical and genetic CUCUCU hymena large (26S) rRNA analyses of the large family GGAGGG subunit1 and the RNA comof group I introns, and ponent of Escherichia coli should pave the way for the RNAse P (Ref. 2). The use of ribozyme-mediated Internal guide sequence Tetrahymena intron was trans-splicing in the analyshown to catalyse a twosis and treatment of a wide step trans-esterification revariety of genetic disorders. 3′ action resulting in joining of 3′ the two rRNA exons and reThe group I intron GGAGGG (G) lease of the intron. The RNA cleaves and ligates in cis UCUCUC subunit of E. coli RNAse P and in trans was demonstrated to be The group I ribozyme, capable of cleaving the 5⬘ through a series of conforleader segment from premational changes, performs 5′ cursor transfer RNAs. The a two-step reaction in Tetrahymena intron is one nature: (1) cleavage of the 3′ member of a large family of phosphodiester bond at introns termed group I, the upstream exon–intron Spliced RNA CUCUCU which is classified by comboundary; and (2) ligation 5′ mon structural and sequence of the upstream exon with Released motifs3. Many, but not all, the downstream exon (Fig. 3′ intron of these are capable of 1). The sequential steps in GGAGGG 5′(G) catalysing self-splicing rethe cleavage reaction have actions. Subsequent to the been elucidated from a discoveries of group I and FIGURE 1. Diagram for cis-splicing of the Tetrahymena group I series of studies utilizing RNAse P catalytic RNAs, intron. The intron (blue) harbors the internal guide sequence that a shortened form of the five other ribozyme motifs pairs with exon I (red) as shown. The guanosine cofactor (G) is group I intron that has been have been characterized, bound within the intron and positioned for the first trans- engineered to cleave suband these are found through- esterification reaction. The guanosine becomes covalently joined strate RNAs in trans12. out bacteria, fungi, plants to the 5⬘ end of the intron, allowing the 3⬘ OH of exon I to attack the Detailed understanding 3⬘ intron–exon boundary in the second trans-esterification. The of this reaction scheme for and vertebrates4. products of this reaction are the released intron and spliced exons. There have been trethe ribozyme has resulted mendous efforts expended in several very creative towards elucidating the structures of targeted RNAs via ribozyme-medi- applications. Because it can catalyse and catalytic mechanisms of the var- ated cleavage8,9, but several of the both a cleavage and a ligation reacious RNA enzymes found in nature. ribozymes found in nature have lig- tion, the group I intron has been utiHigh-resolution three-dimensional ase activities as well. Only recently lized during in vitro evolution experstructures have been obtained for the has the ligase activity of the group I iments to generate novel substrates plant-derived hammerhead ribo- intron been exploited for potential for cleavage, such as DNA, or ribozyme5,6 and part of the catalytic core therapeutic application. Two recent zymes that can cleave under a variety of the group I self-splicing intron7. articles describe experiments that of salt, divalent metal ion or other Concurrent with structural and bio- take advantage of the group I intron conditions13. From the perspective chemical analyses of ribozymes, cleavage and ligation activities. Lan of ribozyme therapeutics, one of the these RNAs have been engineered as et al.10 have utilized trans-splicing most promising and exciting applicasite-specific RNA cleaving reagents catalysed by a group I intron to cor- tions of the group I ribozyme is its and have been utilized as surrogate rect sickle-cell transcripts, while use in trans-splicing. This reaction of genetic tools and as therapeutic Phylactou et al.11 have modeled a the ribozyme can be used to repair agents. The main efforts in this area group I intron system for correction genetically altered or truncated meshave involved site-specific destruction of a triplet repeat expansion in the 3⬘ sages, as well as to create novel TIG AUGUST 1998 VOL. 14 NO. 8 Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01530-3
295
COMMENT
(a)
(b)
Codon 6 mutation
5′ GCACCU
-Globin
X
A(n)
GGUCCUUG
␥-Globin
A(n)
CUG(5) A(n)
5′
CGUGGG 5′
(G)
A(n) A(n)
X
CUG(5) A(n)
(G) GGUCCUUG
CUG(n)
A(n)
CCAGGG 5′
CGUGGG
Trans-spliced mRNA
5′
5′ GCACCU
A(n)
GGUCCU CUG(5) A(n) Trans-spliced mRNA
5′(G) UG CGUGGG 5′
X
CUG(n)
A(n)
Release of intron and downstream cleavage product
Release of intron and downstream cleavage product
(G)
A(n)
Trans-splicing site
Trans-splicing site
GCACCU
CUG(n)
CGUGGG 5′ A(n)
FIGURE 2. Trans-splicing reactions involving the group I intron. (a) Trans-splicing of the ␥-globin message to a s-transcript. The splice site and guide sequence are derived from Lan et al.10, X is the point mutation in codon 6 of the -globin transcript at transcribed nucleotide position 70. The dark blue segment is the s transcript, and the yellow segment is the ␥-globin transcript. The intron is red. (b) Trans-splicing of the MDPK 3⬘ message containing a long CUG triplet (pale blue) with a short triplet repeat (green). The splice site and internal guide sequence are from Phylactou et al.11 The short repeat is indicated by CUG(5), and the long repeat by CUG(n). The intron is depicted in red. Abbreviation: A(n), poly(A) tail.
chimeric RNAs. Trans-splicing is possible because the internal guide sequence can be changed so that it can base pair with any given target RNA supplied in trans. Once the target RNA is bound by base pairing to the internal guide sequence it is site-specifically cleaved. This cleavage event is followed by a trans-esterification reaction in which the 3⬘ OH group of the cleaved target attacks the phosphodiester linkage at the intron–exon boundary. The net result of this twostep reaction is a trans-spliced RNA and a released intron (Fig. 2).
Demonstration of trans-splicing in vitro and in vivo Sullenger and Cech14 provided the first demonstration that a functional RNA could be generated by trans-
splicing. In these experiments, a 5⬘ exon harboring the lacZ ribosomebinding site and the first 21 nucleotides of lacZ coding sequence, including a CCCUCU/AA splice site, was trans-spliced to a 3⬘ exon encoding 67 amino acids of lacZ. The successful trans-splicing of these two transcripts produced -galactosidase in E. coli cells. Sequence analyses both of in vitro and of in vivo transcripts verified that a precise splicing event had taken place. These experiments demonstrated that the only specific sequence requirement in the target RNA is a uracil capable of base pairing with the guanosine immediately preceding the cleavage site, and the trans-splicing event can take place even during coupled transcription– translation, which occurs in bacteria. TIG AUGUST 1998 VOL. 14 NO. 8
296
The establishment of a ribozymemediated trans-splicing system has exciting implications for gene therapy in which one of the major problems is that of inappropriate expression of the therapeutic protein. Trans-splicing circumvents this problem because the functionally restored message can be created only in cells expressing the targeted transcript. Some possible uses of trans-splicing, in addition to repair of genetically defective messages, are creation of dominant-negative mutants, suicide proteins and proteins with completely novel functions. For instance, in the case of viral infection, trans-splicing of RNAs encoding some feature that would inhibit viral infection (dominant-negatives, endotoxins, pro-drug converting suicide enzymes, etc.) would only take
COMMENT place in cells infected with the virus, thereby minimizing potential toxicities from illicit expression of these proteins in non-infected cells. The two recent applications of transsplicing to disease message correction are described below.
Correction of sickle cell disease Sickle cell disease is the result of an A to T transversion in codon 6 (transcribed nucleotide 70) of the globin gene. Thus, a trans-spliced RNA would have to be spliced onto the s-globin message upstream of this mutation to create a non-mutant transcript (Fig. 2a). Individuals homozygous for this mutation accumulate long polymers of sickle hemoglobin, which leads to chronic hemolytic anemia and tissue damage15. As a potential means for diminishing this effect, Lan et al.10 chose to splice fetal ␥-globin mRNA onto the -globin transcript because ␥-globin impedes polymerization of mutant sickle-cell -globin. In order to design a transsplicing ribozyme that efficiently binds to the -globin mRNA upstream of the sixth codon, these investigators took advantage of an ingenious scheme16 to identify an optimal site for pairing to the internal guide sequence. Using a shortened version of the Tetrahymena group I intron called L-21, they created an internal guide sequence that was totally randomized except for the 5⬘ terminal guanosine (5⬘ GNNNNN 3⬘). This library of ribozymes contained a unique 3⬘ exon tag used as an RT–PCR primer site. DNA constructs expressing the ribozyme-tag library were transfected into erythrocyte precursors and total RNA was utilized for RT–PCR analyses of trans-spliced RNAs. Subsequent cloning and characterization of the trans-spliced products surprisingly yielded five out of nine clones with an identical splice junction at nucleotide 61 of the globin transcript. This ‘hotspot’ site was also identified independently during in vitro ribozyme cleavage reactions. The internal guide sequence for this site was 5⬘ GGGUGC 3⬘, which pairs with the -globin sequence 3⬘ UCCACG 5⬘. Once this optimal splice site was identified, a ribozyme with the above internal guide sequence was joined to a 3⬘ exon containing the ␥-globin sequence. Results from transfecting both umbilical cord blood and RBC
precursors from sickle-cell patients with the ribozyme-␥-globin construct demonstrated accurate splicing of the ␥-globin transcript to the position 61 splice site. Although it was not possible to measure accurately the efficiency of the splicing reaction in the RBC precursors, previous work from this group demonstrated that under the appropriate ratio of ribozyme to substrate, up to 50% of a lacZ substrate could be trans-spliced following co-transfection of target and ribozyme constructs in mammalian cells17. Lan et al.10 point out that correction of all of the -globin transcripts with the ␥-globin will probably not be required to impact the disease because sickle-cell patients that express ␥-globin at 10–20% the level of s-globin have improved clinical prognoses. Nevertheless, achieving even the 10% ratios of ribozyme to target in a gene therapy setting will require improvements in transduction, targeting and ribozyme expression.
Correction of expanded triplet repeats in myotonic dystrophy Myotonic muscular dystrophy is an autosomal dominant neuromuscular disease in adults. The disease is characterized by expansion of a trinucleotide CTG repeat in the untranslated region of the MDPK gene18. In the normal population, the number of CTG repeats is highly polymorphic, ranging from 5 to 35 units. Expansions ranging from 50 up to 2000 repeat units have been identified in patients with myotonic dystrophy. Direct evidence for how these repeat expansions cause the disease is lacking. One consequence of the expanded triplet repeats is retention of the message within the cell nucleus. This retention could have two consequences: (1) reduction in cytoplasmic MDPK levels; and (2) sequestering of cellular RNA-binding proteins that interact with the triplet-repeat structures. An example of CUG repeats that titrate a splicing factor has now been documented19. A third consequence of the repeats not directly related to mRNA levels of MDPK is that of a negative effect on the expression of the neighboring downstream gene(s). The work published by Phylactou et al.11, has begun to address the problem of the role of these repeats in the disease by using the trans-splicing capabilities of the group I intron. The hypothesis that TIG AUGUST 1998 VOL. 14 NO. 8
297
they tested assumes that correction of the repeat expansions can be achieved by trans-splicing a short triplet-repeat sequence to a MDPK message that has an expanded repeat. These investigators first demonstrated both in vitro, and in transfected human 293 cells, that a MDPK target RNA with 12 CUG repeats could be trans-spliced and converted to a sequence harboring only five repeats. In order to extend further the possibilities for trans-splicing of this message, they introduced the group I ribozyme fused to the five CUG repeats into primary human fibroblasts that also harbored five repeats in the MDPK 3⬘ untranslated region, but differed by four nucleotides in length from the transspliced RNA. The trans-spliced product was detected from these cells, demonstrating that an endogenous MDPK transcript could be used as a substrate for this reaction. This simple model for correcting repeat length needs to be substantiated by demonstrating repair of repeat expansions characteristic of the lengths found in myotonic dystrophy. The short repeats used in this study most likely have no effect on the nuclear to cytoplasmic transport of the MDPK message unlike the expanded repeats. Thus, the next critical step in this progression is to demonstrate that trans-splicing to expanded repeat transcripts can also take place in cells. To accomplish this, it might be necessary to manipulate the ribozyme transcripts so that they remain nuclear and are not transported to the cytoplasm.
Future prospects The two examples of trans-splicing cited above should generate further excitement and motivation for using this approach with other genetic diseases. Correction of genetic anomalies as well as destruction of deleterious messages are goals for ribozyme usage, and both are feasible with current technologies. The human genome project is and will continue to generate sequences that have coding potential, but are of unknown function. Ribozymes can and should be used as surrogate genetic tools for functional inhibition of the expression of ‘orphan’ transcripts. Once specific genetic alterations within a given transcribed sequence are identified, transsplicing can be used as a tool to elucidate the functional consequence of
COMMENT the mutation, as well as a potential therapeutic reagent for correction of the genetic defect. A potential limitation for effective utilization of ribozymes in an intracellular environment is lack of substantial co-localization with the target20,21. In the case of trans-splicing, Jones and Sullenger17 have demonstrated that the efficiency of transsplicing within a cellular environment can be made to approach, but not exceed 50%. This was accomplished by increasing the ribozyme : substrate ratios during co-transfection experiments. Their failure to achieve greater than 50% splicing could be due to differential sequestering or trafficking of the target and ribozyme RNAs. In this regard, it would be of interest to test the effects of incorporating strong RNA localization signals into both ribozyme and target RNAs and to determine whether or not this increases trans-splicing efficiency. This could be examined in a system where 3⬘ UTRs specify discrete trafficking and localization pathways in the cell22. Another area that needs to be addressed is mis-splicing in trans to non-targeted cellular RNAs23. This is largely a consequence of the relatively short internal guide sequence (usually six nucleotides), which is not long enough to be uniquely complementary to only the targeted sequence. Longer guide sequences are not necessarily the solution because mismatched sequences bound to the internal guide sequence will be subsequently stabilized by strong tertiary interactions with the ribozyme core, allowing mis-splicing to nontargeted RNAs to occur. Perhaps a way of correcting this problem is to introduce modifications of the ribozyme core that reduce the strength of the tertiary interactions but still allow splicing. Mismatched RNAs would
Genet work
then dissociate more readily from the guide sequence before the cleavage– splicing events. Finally, for transsplicing ribozymes to be clinically useful, they must be delivered efficiently to the target cells. This problem of gene delivery is common to all areas of gene therapy. Given the large current interest in gene therapy, there is reason to have strong optimism that efficient gene delivery approaches will be forthcoming for many different target cells and tissues. The RNA world has revealed many intriguing and important new findings. Basic studies of RNA catalytic mechanisms have lead to the development of novel RNA enzymes with altered substrate recognitions and catalytic functions. Targeted ribozyme cleavage and trans-splicing have immense potential for both genetic studies and therapeutic intervention of disease. I eagerly anticipate the use of trans-splicing in a real clinical setting, and look forward to an increasing use of this technology in the study and treatment of other inherited and acquired diseases.
Acknowledgements This work was supported by the National Institutes of Health Grants, AI29329 and AI38592. I thank K. Itakura and D. Castanotto for their critical reading of this work, and acknowledge the technical assistance of W.J. Fitzgerald in the preparation of the manuscript.
References 1 Kruger, K. et al. (1982) Cell 31, 147–157 2 Guerrier-Takada, C. et al. (1983) Cell 35, 849–869 3 Jaeger, L., Michel, F. and Westhof, E. (1996) in Nucleic Acids and Molecular Biology (Vol. 10) (Eckstein, F. and Lilley, D.M.J., eds), pp. 33–51, Springer-Verlag
4 Rossi, J.J. (1998) in Applied Antisense Oligonucleotide Technology (Stein, C.A. and Krieg, A.M., eds), pp. 511–525, Wiley–Liss 5 Pley, H.W., Flaherty, K.M. and McKay, D.B. (1994) Nature 372, 68–74 6 Scott, W.G., Finch, J.T. and Klug, A. (1995) Cell 81, 991–1002 7 Cate, J.H. et al. (1996) Science 273, 1678–1685 8 Couture, L.A. and Stinchcomb, D.T. (1996) Trends Genet. 12, 510–515 9 Rossi, J.J. (1994) Trends Biotechnol. 13, 1–9 10 Lan, N. et al. (1998) Science. 280, 1593–1596 11 Phylactou, L.A., Darrah, C. and Wood, M.J.A. (1998) Nat. Genet. 18, 378–381 12 Cech, T.R. and Herschlag, D. (1996) in Nucleic Acids and Molecular Biology (Vol. 10) (Eckstein, F. and Lilley, M.J., eds), pp. 1–17, Springer-Verlag 13 Beaudry, A.A. and Joyce, G.F. (1992) Science 257, 635–641 14 Sullenger, B.A. and Cech, T.R. (1994) Nature 371, 619–622 15 Platt, O.S. and Dover, G.J. (1993) in Hematology of Infancy and Childhood (Nathan, D.G. and Oski, F.A., eds), pp. 732–782, W.B. Saunders 16 Campbell, T.A. and Cech, T.R. (1995) RNA 1, 598–609 17 Jones, J.T. and Sullenger, B.A. (1997) Nat. Biotechnol. 15, 902–905 18 Tsilfidis, C. et al. (1992) Nat. Genet. 1, 192–195 19 Philips, A.V., Timchenko, L.T., and Cooper, T.A. (1998) Science 280, 737–740. 20 Sullenger, B.A. and Cech, T.R. (1993) Science 262, 1566–1569 21 Bertrand, E. et al. (1997) RNA 3, 75–88 22 St Johnston, D. (1995) Cell 81, 161–170 23 Jones, J.T., Lee, S.W. and Sullenger, B.A. (1996) Nat. Med. 2, 643–648
Genetwork is a regular column of news and information about Internet resources for researchers in genetics and development (pp. 332–334). Genetwork is compiled and edited with the help of Steven E. Brenner (Department of Structural Biology, Stanford University, Fairchild D-109, Stanford, CA 94305-5126, USA; [email protected]); and Fran Lewitter (Scientific Computing, Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge MA 02142-1479, USA; [email protected]). If you would like to announce or publicize an Internet resource, please contact: [email protected]
TIG AUGUST 1998 VOL. 14 NO. 8
298
Ribozymes to the rescue: repairing genetically defective mRNAs JOHN J. ROSSI [email protected] DEPARTMENT OF MOLECULAR BIOLOGY, BECKMAN RESEARCH INSTITUTE OF THE CITY OF HOPE, 1450 EAST DUARTE ROAD, DUARTE, CA 91010-3011, USA.
It has been nearly 20 years untranslated region of the 5′ since the initial discoveries myotonic dystrophy proof catalytic RNAs. The first tein kinase (MDPK) trandemonstrated catalytic RNAs, script. These exciting appli5′ Splice site or ribozymes, were derived cations are built upon years from the intron of the Tetraof biochemical and genetic CUCUCU hymena large (26S) rRNA analyses of the large family GGAGGG subunit1 and the RNA comof group I introns, and ponent of Escherichia coli should pave the way for the RNAse P (Ref. 2). The use of ribozyme-mediated Internal guide sequence Tetrahymena intron was trans-splicing in the analyshown to catalyse a twosis and treatment of a wide step trans-esterification revariety of genetic disorders. 3′ action resulting in joining of 3′ the two rRNA exons and reThe group I intron GGAGGG (G) lease of the intron. The RNA cleaves and ligates in cis UCUCUC subunit of E. coli RNAse P and in trans was demonstrated to be The group I ribozyme, capable of cleaving the 5⬘ through a series of conforleader segment from premational changes, performs 5′ cursor transfer RNAs. The a two-step reaction in Tetrahymena intron is one nature: (1) cleavage of the 3′ member of a large family of phosphodiester bond at introns termed group I, the upstream exon–intron Spliced RNA CUCUCU which is classified by comboundary; and (2) ligation 5′ mon structural and sequence of the upstream exon with Released motifs3. Many, but not all, the downstream exon (Fig. 3′ intron of these are capable of 1). The sequential steps in GGAGGG 5′(G) catalysing self-splicing rethe cleavage reaction have actions. Subsequent to the been elucidated from a discoveries of group I and FIGURE 1. Diagram for cis-splicing of the Tetrahymena group I series of studies utilizing RNAse P catalytic RNAs, intron. The intron (blue) harbors the internal guide sequence that a shortened form of the five other ribozyme motifs pairs with exon I (red) as shown. The guanosine cofactor (G) is group I intron that has been have been characterized, bound within the intron and positioned for the first trans- engineered to cleave suband these are found through- esterification reaction. The guanosine becomes covalently joined strate RNAs in trans12. out bacteria, fungi, plants to the 5⬘ end of the intron, allowing the 3⬘ OH of exon I to attack the Detailed understanding 3⬘ intron–exon boundary in the second trans-esterification. The of this reaction scheme for and vertebrates4. products of this reaction are the released intron and spliced exons. There have been trethe ribozyme has resulted mendous efforts expended in several very creative towards elucidating the structures of targeted RNAs via ribozyme-medi- applications. Because it can catalyse and catalytic mechanisms of the var- ated cleavage8,9, but several of the both a cleavage and a ligation reacious RNA enzymes found in nature. ribozymes found in nature have lig- tion, the group I intron has been utiHigh-resolution three-dimensional ase activities as well. Only recently lized during in vitro evolution experstructures have been obtained for the has the ligase activity of the group I iments to generate novel substrates plant-derived hammerhead ribo- intron been exploited for potential for cleavage, such as DNA, or ribozyme5,6 and part of the catalytic core therapeutic application. Two recent zymes that can cleave under a variety of the group I self-splicing intron7. articles describe experiments that of salt, divalent metal ion or other Concurrent with structural and bio- take advantage of the group I intron conditions13. From the perspective chemical analyses of ribozymes, cleavage and ligation activities. Lan of ribozyme therapeutics, one of the these RNAs have been engineered as et al.10 have utilized trans-splicing most promising and exciting applicasite-specific RNA cleaving reagents catalysed by a group I intron to cor- tions of the group I ribozyme is its and have been utilized as surrogate rect sickle-cell transcripts, while use in trans-splicing. This reaction of genetic tools and as therapeutic Phylactou et al.11 have modeled a the ribozyme can be used to repair agents. The main efforts in this area group I intron system for correction genetically altered or truncated meshave involved site-specific destruction of a triplet repeat expansion in the 3⬘ sages, as well as to create novel TIG AUGUST 1998 VOL. 14 NO. 8 Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01530-3
295
COMMENT
(a)
(b)
Codon 6 mutation
5′ GCACCU
-Globin
X
A(n)
GGUCCUUG
␥-Globin
A(n)
CUG(5) A(n)
5′
CGUGGG 5′
(G)
A(n) A(n)
X
CUG(5) A(n)
(G) GGUCCUUG
CUG(n)
A(n)
CCAGGG 5′
CGUGGG
Trans-spliced mRNA
5′
5′ GCACCU
A(n)
GGUCCU CUG(5) A(n) Trans-spliced mRNA
5′(G) UG CGUGGG 5′
X
CUG(n)
A(n)
Release of intron and downstream cleavage product
Release of intron and downstream cleavage product
(G)
A(n)
Trans-splicing site
Trans-splicing site
GCACCU
CUG(n)
CGUGGG 5′ A(n)
FIGURE 2. Trans-splicing reactions involving the group I intron. (a) Trans-splicing of the ␥-globin message to a s-transcript. The splice site and guide sequence are derived from Lan et al.10, X is the point mutation in codon 6 of the -globin transcript at transcribed nucleotide position 70. The dark blue segment is the s transcript, and the yellow segment is the ␥-globin transcript. The intron is red. (b) Trans-splicing of the MDPK 3⬘ message containing a long CUG triplet (pale blue) with a short triplet repeat (green). The splice site and internal guide sequence are from Phylactou et al.11 The short repeat is indicated by CUG(5), and the long repeat by CUG(n). The intron is depicted in red. Abbreviation: A(n), poly(A) tail.
chimeric RNAs. Trans-splicing is possible because the internal guide sequence can be changed so that it can base pair with any given target RNA supplied in trans. Once the target RNA is bound by base pairing to the internal guide sequence it is site-specifically cleaved. This cleavage event is followed by a trans-esterification reaction in which the 3⬘ OH group of the cleaved target attacks the phosphodiester linkage at the intron–exon boundary. The net result of this twostep reaction is a trans-spliced RNA and a released intron (Fig. 2).
Demonstration of trans-splicing in vitro and in vivo Sullenger and Cech14 provided the first demonstration that a functional RNA could be generated by trans-
splicing. In these experiments, a 5⬘ exon harboring the lacZ ribosomebinding site and the first 21 nucleotides of lacZ coding sequence, including a CCCUCU/AA splice site, was trans-spliced to a 3⬘ exon encoding 67 amino acids of lacZ. The successful trans-splicing of these two transcripts produced -galactosidase in E. coli cells. Sequence analyses both of in vitro and of in vivo transcripts verified that a precise splicing event had taken place. These experiments demonstrated that the only specific sequence requirement in the target RNA is a uracil capable of base pairing with the guanosine immediately preceding the cleavage site, and the trans-splicing event can take place even during coupled transcription– translation, which occurs in bacteria. TIG AUGUST 1998 VOL. 14 NO. 8
296
The establishment of a ribozymemediated trans-splicing system has exciting implications for gene therapy in which one of the major problems is that of inappropriate expression of the therapeutic protein. Trans-splicing circumvents this problem because the functionally restored message can be created only in cells expressing the targeted transcript. Some possible uses of trans-splicing, in addition to repair of genetically defective messages, are creation of dominant-negative mutants, suicide proteins and proteins with completely novel functions. For instance, in the case of viral infection, trans-splicing of RNAs encoding some feature that would inhibit viral infection (dominant-negatives, endotoxins, pro-drug converting suicide enzymes, etc.) would only take
COMMENT place in cells infected with the virus, thereby minimizing potential toxicities from illicit expression of these proteins in non-infected cells. The two recent applications of transsplicing to disease message correction are described below.
Correction of sickle cell disease Sickle cell disease is the result of an A to T transversion in codon 6 (transcribed nucleotide 70) of the globin gene. Thus, a trans-spliced RNA would have to be spliced onto the s-globin message upstream of this mutation to create a non-mutant transcript (Fig. 2a). Individuals homozygous for this mutation accumulate long polymers of sickle hemoglobin, which leads to chronic hemolytic anemia and tissue damage15. As a potential means for diminishing this effect, Lan et al.10 chose to splice fetal ␥-globin mRNA onto the -globin transcript because ␥-globin impedes polymerization of mutant sickle-cell -globin. In order to design a transsplicing ribozyme that efficiently binds to the -globin mRNA upstream of the sixth codon, these investigators took advantage of an ingenious scheme16 to identify an optimal site for pairing to the internal guide sequence. Using a shortened version of the Tetrahymena group I intron called L-21, they created an internal guide sequence that was totally randomized except for the 5⬘ terminal guanosine (5⬘ GNNNNN 3⬘). This library of ribozymes contained a unique 3⬘ exon tag used as an RT–PCR primer site. DNA constructs expressing the ribozyme-tag library were transfected into erythrocyte precursors and total RNA was utilized for RT–PCR analyses of trans-spliced RNAs. Subsequent cloning and characterization of the trans-spliced products surprisingly yielded five out of nine clones with an identical splice junction at nucleotide 61 of the globin transcript. This ‘hotspot’ site was also identified independently during in vitro ribozyme cleavage reactions. The internal guide sequence for this site was 5⬘ GGGUGC 3⬘, which pairs with the -globin sequence 3⬘ UCCACG 5⬘. Once this optimal splice site was identified, a ribozyme with the above internal guide sequence was joined to a 3⬘ exon containing the ␥-globin sequence. Results from transfecting both umbilical cord blood and RBC
precursors from sickle-cell patients with the ribozyme-␥-globin construct demonstrated accurate splicing of the ␥-globin transcript to the position 61 splice site. Although it was not possible to measure accurately the efficiency of the splicing reaction in the RBC precursors, previous work from this group demonstrated that under the appropriate ratio of ribozyme to substrate, up to 50% of a lacZ substrate could be trans-spliced following co-transfection of target and ribozyme constructs in mammalian cells17. Lan et al.10 point out that correction of all of the -globin transcripts with the ␥-globin will probably not be required to impact the disease because sickle-cell patients that express ␥-globin at 10–20% the level of s-globin have improved clinical prognoses. Nevertheless, achieving even the 10% ratios of ribozyme to target in a gene therapy setting will require improvements in transduction, targeting and ribozyme expression.
Correction of expanded triplet repeats in myotonic dystrophy Myotonic muscular dystrophy is an autosomal dominant neuromuscular disease in adults. The disease is characterized by expansion of a trinucleotide CTG repeat in the untranslated region of the MDPK gene18. In the normal population, the number of CTG repeats is highly polymorphic, ranging from 5 to 35 units. Expansions ranging from 50 up to 2000 repeat units have been identified in patients with myotonic dystrophy. Direct evidence for how these repeat expansions cause the disease is lacking. One consequence of the expanded triplet repeats is retention of the message within the cell nucleus. This retention could have two consequences: (1) reduction in cytoplasmic MDPK levels; and (2) sequestering of cellular RNA-binding proteins that interact with the triplet-repeat structures. An example of CUG repeats that titrate a splicing factor has now been documented19. A third consequence of the repeats not directly related to mRNA levels of MDPK is that of a negative effect on the expression of the neighboring downstream gene(s). The work published by Phylactou et al.11, has begun to address the problem of the role of these repeats in the disease by using the trans-splicing capabilities of the group I intron. The hypothesis that TIG AUGUST 1998 VOL. 14 NO. 8
297
they tested assumes that correction of the repeat expansions can be achieved by trans-splicing a short triplet-repeat sequence to a MDPK message that has an expanded repeat. These investigators first demonstrated both in vitro, and in transfected human 293 cells, that a MDPK target RNA with 12 CUG repeats could be trans-spliced and converted to a sequence harboring only five repeats. In order to extend further the possibilities for trans-splicing of this message, they introduced the group I ribozyme fused to the five CUG repeats into primary human fibroblasts that also harbored five repeats in the MDPK 3⬘ untranslated region, but differed by four nucleotides in length from the transspliced RNA. The trans-spliced product was detected from these cells, demonstrating that an endogenous MDPK transcript could be used as a substrate for this reaction. This simple model for correcting repeat length needs to be substantiated by demonstrating repair of repeat expansions characteristic of the lengths found in myotonic dystrophy. The short repeats used in this study most likely have no effect on the nuclear to cytoplasmic transport of the MDPK message unlike the expanded repeats. Thus, the next critical step in this progression is to demonstrate that trans-splicing to expanded repeat transcripts can also take place in cells. To accomplish this, it might be necessary to manipulate the ribozyme transcripts so that they remain nuclear and are not transported to the cytoplasm.
Future prospects The two examples of trans-splicing cited above should generate further excitement and motivation for using this approach with other genetic diseases. Correction of genetic anomalies as well as destruction of deleterious messages are goals for ribozyme usage, and both are feasible with current technologies. The human genome project is and will continue to generate sequences that have coding potential, but are of unknown function. Ribozymes can and should be used as surrogate genetic tools for functional inhibition of the expression of ‘orphan’ transcripts. Once specific genetic alterations within a given transcribed sequence are identified, transsplicing can be used as a tool to elucidate the functional consequence of
COMMENT the mutation, as well as a potential therapeutic reagent for correction of the genetic defect. A potential limitation for effective utilization of ribozymes in an intracellular environment is lack of substantial co-localization with the target20,21. In the case of trans-splicing, Jones and Sullenger17 have demonstrated that the efficiency of transsplicing within a cellular environment can be made to approach, but not exceed 50%. This was accomplished by increasing the ribozyme : substrate ratios during co-transfection experiments. Their failure to achieve greater than 50% splicing could be due to differential sequestering or trafficking of the target and ribozyme RNAs. In this regard, it would be of interest to test the effects of incorporating strong RNA localization signals into both ribozyme and target RNAs and to determine whether or not this increases trans-splicing efficiency. This could be examined in a system where 3⬘ UTRs specify discrete trafficking and localization pathways in the cell22. Another area that needs to be addressed is mis-splicing in trans to non-targeted cellular RNAs23. This is largely a consequence of the relatively short internal guide sequence (usually six nucleotides), which is not long enough to be uniquely complementary to only the targeted sequence. Longer guide sequences are not necessarily the solution because mismatched sequences bound to the internal guide sequence will be subsequently stabilized by strong tertiary interactions with the ribozyme core, allowing mis-splicing to nontargeted RNAs to occur. Perhaps a way of correcting this problem is to introduce modifications of the ribozyme core that reduce the strength of the tertiary interactions but still allow splicing. Mismatched RNAs would
Genet work
then dissociate more readily from the guide sequence before the cleavage– splicing events. Finally, for transsplicing ribozymes to be clinically useful, they must be delivered efficiently to the target cells. This problem of gene delivery is common to all areas of gene therapy. Given the large current interest in gene therapy, there is reason to have strong optimism that efficient gene delivery approaches will be forthcoming for many different target cells and tissues. The RNA world has revealed many intriguing and important new findings. Basic studies of RNA catalytic mechanisms have lead to the development of novel RNA enzymes with altered substrate recognitions and catalytic functions. Targeted ribozyme cleavage and trans-splicing have immense potential for both genetic studies and therapeutic intervention of disease. I eagerly anticipate the use of trans-splicing in a real clinical setting, and look forward to an increasing use of this technology in the study and treatment of other inherited and acquired diseases.
Acknowledgements This work was supported by the National Institutes of Health Grants, AI29329 and AI38592. I thank K. Itakura and D. Castanotto for their critical reading of this work, and acknowledge the technical assistance of W.J. Fitzgerald in the preparation of the manuscript.
References 1 Kruger, K. et al. (1982) Cell 31, 147–157 2 Guerrier-Takada, C. et al. (1983) Cell 35, 849–869 3 Jaeger, L., Michel, F. and Westhof, E. (1996) in Nucleic Acids and Molecular Biology (Vol. 10) (Eckstein, F. and Lilley, D.M.J., eds), pp. 33–51, Springer-Verlag
4 Rossi, J.J. (1998) in Applied Antisense Oligonucleotide Technology (Stein, C.A. and Krieg, A.M., eds), pp. 511–525, Wiley–Liss 5 Pley, H.W., Flaherty, K.M. and McKay, D.B. (1994) Nature 372, 68–74 6 Scott, W.G., Finch, J.T. and Klug, A. (1995) Cell 81, 991–1002 7 Cate, J.H. et al. (1996) Science 273, 1678–1685 8 Couture, L.A. and Stinchcomb, D.T. (1996) Trends Genet. 12, 510–515 9 Rossi, J.J. (1994) Trends Biotechnol. 13, 1–9 10 Lan, N. et al. (1998) Science. 280, 1593–1596 11 Phylactou, L.A., Darrah, C. and Wood, M.J.A. (1998) Nat. Genet. 18, 378–381 12 Cech, T.R. and Herschlag, D. (1996) in Nucleic Acids and Molecular Biology (Vol. 10) (Eckstein, F. and Lilley, M.J., eds), pp. 1–17, Springer-Verlag 13 Beaudry, A.A. and Joyce, G.F. (1992) Science 257, 635–641 14 Sullenger, B.A. and Cech, T.R. (1994) Nature 371, 619–622 15 Platt, O.S. and Dover, G.J. (1993) in Hematology of Infancy and Childhood (Nathan, D.G. and Oski, F.A., eds), pp. 732–782, W.B. Saunders 16 Campbell, T.A. and Cech, T.R. (1995) RNA 1, 598–609 17 Jones, J.T. and Sullenger, B.A. (1997) Nat. Biotechnol. 15, 902–905 18 Tsilfidis, C. et al. (1992) Nat. Genet. 1, 192–195 19 Philips, A.V., Timchenko, L.T., and Cooper, T.A. (1998) Science 280, 737–740. 20 Sullenger, B.A. and Cech, T.R. (1993) Science 262, 1566–1569 21 Bertrand, E. et al. (1997) RNA 3, 75–88 22 St Johnston, D. (1995) Cell 81, 161–170 23 Jones, J.T., Lee, S.W. and Sullenger, B.A. (1996) Nat. Med. 2, 643–648
Genetwork is a regular column of news and information about Internet resources for researchers in genetics and development (pp. 332–334). Genetwork is compiled and edited with the help of Steven E. Brenner (Department of Structural Biology, Stanford University, Fairchild D-109, Stanford, CA 94305-5126, USA; [email protected]); and Fran Lewitter (Scientific Computing, Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge MA 02142-1479, USA; [email protected]). If you would like to announce or publicize an Internet resource, please contact: [email protected]
TIG AUGUST 1998 VOL. 14 NO. 8
298