TIBTECH - JULY 1990 [Vol. 8]

179

RNA enzymes (ribozymes) as antiviral therapeutic agents John J. Rossi and Nava Sarver Among the landmark discoveries of recent years are ribozymes, RNA molecules which possess enzymatic, self-cleaving activities. The concept of exploiting the ribozyme catalytic center for cleaving (inactivating) a specific RNA transcript is n o w emerging as a potential therapeutic or preventative strategy in h u m a n diseases, veterinary medicine and agriculture. Linked to the catalytic center of the ribozyme are RNA sequences w h i c h are complementary to, and thus serve to target the ribozyme to, a unique RNA sequence. Specific association of the ribozyme with its target via base pairing, cleavage of the RNA substrate and subsequent recycling of the ribozyme m a k e these catalytic RNA molecules attractive as antiviral agents. Theoretically, ribozymes can be adapted for the destruction of any RNA species, whatever its origin. Specific inhibition of viral function and replication is a major problem in medicine. This difficulty stems largely from the fact that viruses, unlike bacteria, exist intracellularly and depend upon certain host functions for replication and expression of their genetic information. Agents that block viral function often also have adverse effects on the host, especially after prolonged use. This is true even when a unique process of the viral life cycle is the target of the drug. A case in point is reverse transcriptase (RT), a viral enzyme required for the replication of all retroviruses. The nucleoside analogue azidothymidine (commonly known as AZT) is a potent inhibitor of HIV1 RT. Although RT has no enzymatic counterpart in cellular function, AZT inhibits cellular DNA polymerase and RNaseH activities, albeit

J. J. Rossi is at the Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA, and N. Sarver is at the Developmental Therapeutics Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA. © 1990, Elsevier Science Publishers Ltd (UK)

at much higher concentrations. Prolonged treatment with AZT ultimately leads to toxic side effects, thus restricting the use of the drug. Another significant problem in viral diseases, and of major concern in HIV-1 infection, is the high mutation rate of the viral genome, which results in the emergence of drug resistant strains. One of the goals in antiviral drug development is to identify an effective treatment for viral infection that is devoid of drug toxicity and drug resistance. This review describes one such strategy based on catalytic antisense RNAs, or ribozymes, and includes recent data that demonstrate the potential use of ribozymes as inhibitors of gene activity. We then address what we believe to be critical considerations in implementing ribozymes for the treatment of disease, especially for HIV-1 infection.

Trans-acting catalytic RNAs Two types of RNA catalytic domains show considerable promise for therapeutic applications. The first is the so-called 'hammerhead', identified in plant viroids and virusoids 1'2 as well as newt satellite RNA transcript 3. The hammerhead

0167 - 9430/90/$2.00

family of self-cleaving RNAs have a high degree of homology in their primary and secondary structure 1'4"5. The hammerhead structure comprises three stems and a catalytic center containing 13 conserved nucleotides (Fig. 2a). Cleavage occurs 3' to the GUX triplet (where X can be either C, U or A) generating 2'-3' cyclic phosphate and 5' OH termini at the cleavage site. A recent report by Koizumi eta]. 6 indicates a greater flexibility in the GUX requirement. In naturally occurring ribozymes, the ribozyme-mediated reactions are intramolecular, with the target and catalytic strands comprising a single molecule. Two fundamental characteristics suggest a broad scope for ribozyme application. First, specific cleavage can occur in trans, ie. intermolecularly, with separate substrate and catalytic RNA molecules 1'6-8. Only the substrate is consumed during the reaction, leaving the ribozyme catalyst free to interact with the next available target. The end result is that a large excess of target RNA substrate (relative to the amount of ribozyme) is cleaved and inactivated. Second, refinement of the sequence requirements of transacting ribozymes has shown that substrates containing only the three nucleotides comprising the cleavage site (GUX) are efficiently cleaved 9, thus expanding the range of potential targets. A second catalytic motif, distinct from the hammerhead and so far identified only in the 359 nucleotide (nt) negative strand of satellite RNA of tobacco ringspot virus [(-)sTRSV], has been designated the 'hairpin' ribozyme 7. In (-)sTRSV, the minim u m sequence required for catalytic activity consists of a 50 nt catalytic domain and a 14 nt substrate domain (Fig. 2b). The hairpin ribozyme possesses four helices believed to maintain the secondary configuration. Helices I and II form between the target and catalytic domains by virtue of base pair complementarity; helices III and IV are internal to the catalytic domain and form via intramolecular base-pairing of complementary regions. Another feature is the presence of a four base loop (AGUC) in the substrate domain. Site-directed mutagenesis studies showed that three of these bases (GUC) are invariable and

180

constitute an obligatory cleavage site, whereas the A can be any nucleotide 1°. By substituting intermolecular interaction for helices I and II, the hairpin motif has also been converted to a trans-acting ribozyme comprising separate catalyst and substrate molecules 8'1°. Target specificity is maintained by complementary base-pairing between the two reactants at helices I and II. As in the case of hammerhead-mediated reactions, hairpin-mediated cleavage also generates 2',3' cyclic phosphate and 5' OH groups at the cleavage site. The hairpin differs from the hammerhead in that the substrate is cleaved 5' to the obligatory GUC sequence. Another, less well-defined structural motif distinct from the hammerhead and the hairpin occurs in hepatitis delta virus. In vitro, a segment of the viral genome harboring this domain undergoes a selfcleavage reaction which also yields 2',3' cyclic phosphate and 5' OH termini 1~. It is possible that the catalytic domain from this RNA can be incorporated into a trans-acting ribozyme, although this has not yet been reported. RNAseP, an enzyme which processes 5' leader segments from pretRNAs, includes a catalytic RNA component which mediates in the processing reaction ~2. RNAseP is the only naturally occurring trans-acting ribozyme known to date and is also a potential candidate for an antiviral agent.

Functional trans-acting ribozymes targeted to HIV RNA Several features of ribozymes make them attractive as antiviral agents, namely, their specificity with regard to target recognition and cleavage and the catalytic nature of their inactivating action; a relatively small amount of ribozyme can destroy a molecular excess of target RNA - appropriate for an acute viral infection. Haseloff and Gerlach 9 demonstrated that hammerhead enzymes can be effectively and specifically targeted to various sites along RNA transcripts in vitro. This has subsequently been shown by several other gronps13,14.23-25. In experiments using anti-HIV-gag catalysts, cleavage efficiency of gag RNA was not affected by the addition of large

T I B T E C H - J U L Y 1990 [Vol. 8]

amounts of total cellular RNAs lacking the cleavage site. In contrast, total RNAs from HIV infected cells interfered with gag RNA cleavage 13. The conclusions drawn from this and other experiments can be summarized as follows: • long, non-complementary sequences distant from the catalytic center in either catalyst or substrate do not inhibit the cleavage reaction;

• cleavage proceeds in the presence of total RNA from uninfected cells, indicating that the association of catalyst and substrate is specific to the gag sequence; • total RNA from HIV-1 infected cells competes with input substrate for the available catalyst, as expected. We then investigated whether catalytic RNAs constitutively expressed in a mammalian cell and

targeted to HIV-1 gag RNA can inactivate viral transcripts of an invading virus 14. To this end, CD4 + HeLa cells (CD4 is the cellular receptor for HIV) were transfected with a mammalian expression vector including sequences coding for anti-gag catalytic RNA. Stable transformants expressing the catalytic RNA were isolated and challenged with HIV-1. Polymerase chain reaction (PCR) analyses were performed on total RNA to measure the relative levels of intact versus cleaved gag sequences. Indeed, these analyses showed a considerable reduction in intact gag RNA in ribozyme-expressing cells versus non-ribozyme-expressing parental cells 14. One of the processed peptides derived from GAG precursor protein is p24. Quantitative determination of soluble p24 antigen at seven days post-infection demonstrated a marked decrease (20-40 fold) in the amount

--Fig. 1

synthesis of substrate (S) and catalytic (C) RNAs*

l

o.

S association in the

OH P5'

presence of Mg +2

5'F

~

C-S complex

3'F OH P5'

cleavage of S

3'F l dissociationof 5'F and 3'F from C]

cleaved S

-4-

. HO ~ l ~ m ~

#P5'

intact C

Trans-acting ribozyme-mediated cleavage. The ribozyme (C) and substrate (S) RNAs associate through base pairing of complementary sequences surrounding the catalytic center to form a complex, thereby forming the secondary structure configuration required for cleavage. In the presence of cations, cleavage of substrate occurs, generating the two cleavage products (5'F and 3'F). The complex then dissociates, releasing the cleavage products and the ribozyme is again available to associate with the next substrate molecule. The reaction results in the generation of 2'-3' cyclic phosphate and 5'-OH termini at the cleavage site.

TIBTECH - JULY 1990 [Vol. 8]

181

~Fig. 2, a

gene inactivation can be applied for human therapy. Among these are: (1) kinetics of the reaction; (2) choice of target site and (3) intracellular delivery, functional expression and stability in the absence of toxicity. We consider possible ways of counteracting some problems that may arise in these areas.

B J

0 18 b

Cleavage site

Substrate RNA

3I

U

AUA GUG GU

U G

C A C CA A Helix IV

UU

A CUGGU

NNNNNUC

AGAGACCA

A A AG

A Helix III

/

Helix II

Helix I

Trans-acting hammerhead and hairpin ribozyme models. (a) Two motifs for trans-acting hammerhead ribozymes. In A, the catalyst is defined by the sector A - A ". In this motif, the catalyst only harbors the conserved nucleotides 5" CUGANGA 3'. The remaining conserved nucleotide active sites are derived from the substrate molecule. Loop B is a consequence of intramolecular base pairing in the target sequence, and there is no loop A, since the catalyst is supplied as a separate molecule. In B, all of the conserved nucleotides, with the exception of the cleavage site, are incorporated by the catalyst's molecule defined by the B-B" sector. Loop A is part of the catalyst, and there is no loop B, since the catalyst is supplied as a separate molecule. (b) The hairpin model ribozyme encompassing the catalytic center and the proposed helices (I, II, III and IV). The substrate RNA requires a GUC at the cleavage site and uninterrupted base pairing in helices I and II (Refs 8, 10). There is no specific sequence requirement for these helices.

of p24 secreted from anti-gag ribozyme-containing cells, compared to infected cells lacking the ribozyme a4. Since HIV-1 GAG precursor protein is required for the maturation of viral particles, this observation suggests that ribozymes can probably be used to reduce HIV-1 replication efficiency. Trans-acting ribozymes, like conventional antisense RNAs, exert their inhibitory actions in a highly specific manner and are therefore not expected to be detrimental to cell function. Long-term culturing of the HeLa cells expressing catalytic RNAs has no adverse effects on cell viability, growth rate, and overall

RNA and protein production a4. This apparent lack of (or minimal) cytotoxicity is of crucial importance in the therapeutic application of ribozymes. These results show that catalytic RNAs are expressed and are sufficiently stable in a complex intracellular environment to effect significant specific cleavage of target RNA sequences without being detrimental to cell viability. T h e u s e o f r i b o z y m e s as antiviral agents

As with all emerging technologies, there are several areas that must be addressed before ribozyme-mediated

Kinetics of the reaction Catalysis may be described in terms of the Michae]is-Menten constant (Km) and turnover number (Koat or number of substrate molecules cleaved per minute). Initial assembly of the ribozyme-substrate complex is dependent on specific base-pairing of substrate and catalyst at complementary regions. While initial assembly may occur rapidly, not all complexes formed are competent for cleavage. Cell-free studies by a number of groups illustrate that some of the assembled complexes will cleave only after a denaturation/ renaturation step, indicating that these complexes are initially in an unfavorable configuration, but may be reformed to assume a catalytically active structure 1,3. Whether this observation reflects what occurs in vivo or whether it is peculiar to & vitro conditions is, at present, unknown. For both hammerhead and hairpin ribozymes 9x° longer stems may enhance the rate of cleavagecompetent complex formation, and consequently the cleavage reaction. However, longer stems may decrease the rate of complex dissociation (due to greater stability) 9 and thus interfere with the catalytic process. A compromise must be reached to balance complex formation and cycling of the ribozyme after cleavage. Furthermore, the nucleotide composition at the cleavage site significantly affects the rate of cleavage. In the hammerhead model, GUC or GUU is preferred over GUA, with GUG being a very poor substrate 6,7,9. For the hairpin motif, only GUC will serve as a substrate 8'1°. Accumulating data also indicate that neighboring nucleotides may influence the rate of cleavage in the hairpin motif ~°. Choiee of target site Given the secondary structures associated with RNA molecules, the target site must be chosen carefully to ensure the substrate is accessible

182

for binding to the catalyst. The use of antisense oligonucleotides as inhibitors of gene expression has shown that sequences proximal to and including the AUG translation initiation codon, splice sites, and single strand loops are accessible for binding by oligodeoxyribonucleotides 25. Such regions may therefore be logical target sites for binding catalytic RNAs. Conversely, base paired (stem) regions and domains associated with binding factors are likely to be less accessible. Uhlenbeck and colleagues (unpublished) designed an elegant approach to identify accessible target sites along an RNA substrate. Using an in vitro transcription system they generated a library of catalytic RNA sequences witl~' degeneracy in sequences flanking the catalytic center. These were then incubated with the target substrate in vitro to screen for compatible cleavage sites. This 'reverse' functional screening strategy allowed potential cleavage sites within a given substrate to be screened en masse, rather than each site being tested independently with a 'tailored' catalyst. They demonstrated that (1) certain sites along the same substrate are more readily cleaved than others and (2) this approach can be used to identify cleavage sites in substrates for which sequence information is not known. Although sites identified by this method will need to be confirmed as accessible for cleavage in vivo, functional screenings should help delineate basic substrate features required for competent ribozyme-substrate complex foundation. Some of the uncertainties in substrate site accessibility or genetic variability of certain viruses, for example, HIV, may be circumvented by simultaneous use of several ribozyme molecules, each with a different substrate recognition site. Alternatively, multivalent catalysts capable of interacting with two or more different cleavage sites may be designed. Such catalysts could be targeted to several potential sites along a single substrate molecule, thus ensuring cleavage still occurs even where one site is inaccessible to binding due to structural constraints or mutational events, such as are commonly seen in HIV replication. Site-directed mutagenesis and functional screening using catalytic

TIBTECH - JULY 1990 [Vol. 8]

RNA libraries, combined with computer modeling and predictions based on secondary structure determinations, are likely to reveal other substrate targets along an RNA molecule devoid of secondary structure.

Intracellular delivery, functional expression and stability The therapeutic use of ribozymes will require high, reliable catalytic activity in an intracellular environment. Two general mechanisms exist for introducing catalytic RNA molecules into target cells: (1) exogenous delivery of a pre-formed synthetic RNA ribozyme and (2) endogenous expression from a transcriptional unit (gene therapy). Exogenous delivery of a pre-formed RNA ribozyme Several strategies, notably liposomes and lipophilic conjugates, developed for delivery of antisense DNA molecules may also be applicable to the delivery of pre-formed RNA molecules, pH-sensitive immunoliposomes conjugated to monoclonal antibodies were successfully targeted to cell surface receptors in vitro and in vivo 16. Similarly, in vitro and in vivo delivery of nucleic acids and proteins was demonstrated with proteoliposomes comprised of viral fusion proteins ~7. These modifications are believed to increase intracellular uptake, minimize clearance by the reticuloendothelial system and impede translocation of liposomes to lysosomes, where they are generally degraded ~6. Another strategy to improve delivery is to conjugate the molecule at the 3' or 5' terminus to cholesterol or other lipophi]ic conjugates. The problem of RNA stability, however, is a major impediment to the practical delivery of pre-formed catalysts to target cells. In this regard, modified oligodeoxyribonucleotide antisense molecules, such as phosphorothioates, are more resistant to nuclease degradation than unmodified oligonucleotides ~8 and are effective inhibitors of HIV-1 replication in infected cells 19. A phosphorodithioate ribonucleoside analogue (J. Nielsen and K. H. Peterson, unpublished) and a modified ribose group (2'O-methyl) 2°'21 have been synthesized and RNA oligomers containing these modifications can be made using conventional chemistry.

Chimeric molecules contaning a ribonucleotide catalytic center and deoxyribonucleotide flanking sequences may be another strategy to create more stable ribozymes (J. Rossi, B. Kaplan and P. Chang, unpublished). Recently, certain conserved nucleotides in the hammerhead catalytic center were changed to the deoxy form without preventing the catalytic activity 22. It is therefore conceivable that deoxyribonucleotide substitutions confined to sequences flanking the RNA catalytic center will enhance stability without impairing activity. Additional studies are required to ascertain the feasibility of using RNADNA chimeric ribozymes. These and other modifications that protect the RNA molecule from enzymatic degradation, coupled to lipophilic or other transport vehicles, may play an important role in the early phase of ribozyme applications to human diseases. Endogenous expression from a transcriptional unit (gene therapy) One of the major difficulties in implementing ribozymes for treating human viral diseases will be ensuring that the cells receive sufficient amounts of functional ribozymes to have an impact on the course of the disease. Exogenous delivery would require frequent infusions of preformed ribozymes to maintain an intracellular concentration sufficient to effect a significant reduction in viral RNAs. An alternative approach is gene therapy, that is, the endogenous expression of ribozyme RNAs from a DNA template permanently maintained within the cell. Endogenous expression should theoretically eliminate most of the difficulties associated with the delivery of preformed ribozyme molecules. Several groups have n o w reported endogenous expression of functional ribozymes in Xenopus o o c y t e s 23'24 and mammalian cells 14,25. Chang e t a ] . 13 and Sarver et a]. 14 have expressed a functional anti-HIV-1 ribozyme in a pol II transcriptional unit that inactivated the viral gagRNA and had an impact on p24 (GAG) antigen levels. Cameron and Jennings 25 have fused an antiCAT catalytic RNA to a transcriptional cassette driven by an SV40 promoter. This chimeric ribozyme construct caused significant inhi-

TIBTECH - J U L Y 1990 [Vol. 8]

bition of chloramphenicol acetyltransferase (CAT) activity in transfected mammalian cells. Cotten and Birnstie124 embedded a catalytic RNA within a Pol III cassette targeted to the U7 RNA sequence. Interestingly, this chimeric tRNA-ribozyme transcript remained primarily in the cell nucleus but enough leaked into the cytoplasm to inactivate the predominantly cytoplasmic U7 on snRNA sequence. Depending on the route of viral infection, different cell types need to be protected. In the case of viruses, such as HIV, that invade the immune system, it will be important to engineer the progenitor stem cells to produce protective ribozymes. Of the viral vectors (e.g. adeno-, herpesand retro-viruses) currently being adapted for potential gene therapy applications, retro-viruses are suitable for delivery of genes into the hematopoietic system 26. Since many of the cells descending from the hematopoietic lineage, namely monocytes, macrophages, T4 lymphocytes (CD4 cells) and bone marrow cells, are susceptible to HIV-1 infection, and since some are probably involved in transporting the virus to other tissues, the protection of hematopoietic cells from HIV infection is likely to have a significant impact on the course of the disease. For gene therapy, the stability, intracellular localization and potential toxicity of catalytic RNAs will be critical. A number of naturally occurring RNA modifications, such as 5' capping and 3' polyadenylation, may prolong the half-life and facilitate intracellular compartmentalization of catalytic RNAs. For example, a catalytic RNA derived from a Pol II transcriptional cassette will acquire a 5' m7GpppG cap typical of mRNAs, which may enhance its cytoplasmic stability. In contrast, a snRNA catalytic RNA transcript will acquire a 5' m32'2'TGpppG cap, and this, combined with an Sm antigen binding site found in most snRNAs (Ref. 27) should localize the ribozyme to a nuclear c o m p a r t m e n t where it could interact with unspliced pre-mRNAs. With regard to toxicity, cells expressing catalytic RNAs do not appear to be compromised in their growth properties, gene expression or longevity 14'25. If ribozymes interfere with normal cell

183

function, the effect is minimal.

Conclusions By cleaving viral genomic and messenger RNAs at specific sites, ribozymes offer a means for significantly reducing, and perhaps eliminating, viral transcripts and thus inhibiting virus replication. Theoretically, this approach is applicable to the reduction of any RNA species, provided the nucleotide sequence is known. In the immediate future, the in vivo application of ribozymes will probably rely on the delivery of preformed catalysts. Once gene therapy is an accepted strategy for the treatment of h u m a n diseases, the full potential of ribozyme-mediated therapy may be realized.

Acknowledgements N. Sarver is indebted to J. Rosenberg for his constant support. J. Rossi is supported by USPHS grants AI29329 and AI25959. Both authors would like to acknowledge Pairoj Chang, Delilah Stephens, Paula Ladne, Edouard Cantin and John Zaia for their contributions to some of the data summarized in this review.

References 1 Forster, A. C. and Symons, R. H. (1987) Cell 49, 211-220 2 Prody, G. A., Bakos, J. T., Buzayan, J. M., Schneider, I. R. and Bruening, G. (1986) Science 231, 1577-1580 3 Epstein, L. and Gall, J. (1987) Cell 48, 535-543 4 Keese, P. and Symons, R. H. (1987) in Viroids and Viroid-like Pathogens

(Semancik, J. S., ed.), pp. 1-47, CRC Press 5 Hutchins, C. J., Rathjen, P. D., Forster, A. C. and Symons, R. H. (1986) Nucleic Acids Res. 14, 3627-3640 6 Koizumi, M., Hayase, Y., Iwai, S., Kamiya, H., Inoue, H. and Ohtsuka, E. (1989) Nucleic Acids Res. 17, 7059-7071

7 Uhlenbeck, O. C. (1987) Nature 328, 596-600 8 Tritz, R. and Hampel, A. (1989) Biochemistry 28, 4929-4933 9 Haseloff, J. and Ger|ach, W. L. (1988) Nature 334, 585-591 10 Hampel, A., Tritz, R., Hicks, M. and Cruz, P. (1990) Nucleic Acids Res. 18, 299-304 11 Wu, H-N. and Lai, M. M. C. (1989) Science 243,652-654 12 Guerrier-Takada, C., Gardiner, K.,

13

14

15

16 17

18

Marsh, T., Pace, N. and Altman, S. (1983) Cell 35, 849-857 Chang, P. S., Cantin, E. M., Zaia, J. A., Ladne, P. A., Stephens, D. A., Sarver, N. and Rossi, J. J. (1990) Clin. Biotechnol. 2, 23-31 Sarver, N., Cantin, E., Chang, P., Ladne, P., Stephens, D., Zaia, J. and Rossi, J. (1990) Science 247, 1222-1224 Goodchild, J., Agrawat, S:, Civeira, M. P., Sarin, P. S., Sun, D. and Zamecnik, P. C. (1989) Proc. Natl Acad. Sci. USA 85, 5507-5511 Ho, R. S. Y., Rouse, B. T. and Huang, L. (1987) J. Biol. Chem. 262, 13973-13978 Gould-Fogerite, S., Mazurkiewicz, J. E., Raska, K. Jr., Voelkerding, K. Lehman, J. M. and Mannino, R. J. (1989) Gene 84, 429-438 Stein, C. A. and Cohen, J. S. (1990) in Oligonucleotide Antisense Inhibitors of Gene Expression (Cohen, J. S., ed.),

pp. 97-117, CRC Press 19 Matsukura, M., Zon, G., Shinozuka, K., Robert-Guroff, M., Shimada, R., Stein, C. A., Mitsuya, H., Wong-Staal, F., Cohen, J. S. and Broder, S. (1989) Proc. Natl Acad. Sci. USA 86, 4244-4248 20 Inoue, H., Inoue, H., Hayase, Y.: Imura, A., Iwai, S., Miura K. and Ohtsuka, E. (1987) Nucleic Acids Res. 15, 6131-6148 21 Shibahara, S., Mukai, S., Morisawa, H., Nakashima, H., Kobayashi, S. and Yamamoto, N. (1989) Nucleic Acids Res. 17, 239-252 22 Perreault, J. P., Talfeng, W., Cousineau, B., Ogilive, K. K. and Cedergren, R. (1990) Nature 344, 565-567 23 Cotten, M., Schaffner, G. and Birnstiel, M. L. (1989) Mol. Cell. Biol. 9, 4479-4487 24 Cotten, M. and Birnstiel, M. L. (1989) EMBO J. 8, 3861-3866 25 Cameron, F. H. and Jennings, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 9139-9143 26 Friedman, T. (1989) Science 244, 1275-1292 27 Guthrie, C. and Patterson, B. (1988) Annu. Rev. Genetics 22,421-477